Final Report Summary - PROMYSE (Products from methanol by synthetic cell factories)
Executive Summary:
There is a high societal demand for a sustainable production of special, fine, bulk, and fuel chemicals, including food and health care compounds. Biotechnological processes will play a prominent role in the coming Bio economy era by gradually complementing and substituting petrochemical synthesis. In biotechnology, microorganisms are widely used as cell factories and, in particular for high-volume products, raw material costs make up a large part of process costs. Industrial biotechnology mainly uses sugars and molasses as carbon source, and these raw materials are derived from plants demanding cultivable land which is more and more needed for human nutrition. The possibility to utilize non-food raw materials, such as one-carbon (C1) substrates like methane (CH4) and methanol (CH3OH), as alternative feedstock has therefore gained high scientific interest but is not yet implemented at commercial scale. Major reasons for this are that methanol is still a more expensive substrate than sugar (worldwide methanol prizes at Methanex: https://www.methanex.com/our-business/pricing) and that methanol fermentations can be technically challenging; for example high O2 requirements and concomitant heat production may cause increased cooling requirements, and careful substrate feeding is needed to avoid toxic formaldehyde accumulation in the cells. Methanol is a pure and non-food chemical that is soluble in water and it is completely utilized during microbial fermentations. Methanol should thus represent an attractive alternative raw material for biotechnological processes, both from an economic, ecologic, and process point of view. With a worldwide production capacity of more than 53 million tons per year, methanol is one of the most important raw chemicals on earth. The supply of methanol can base either upon fossil or renewable resources, rendering it a highly flexible and sustainable raw material. Today, almost all methanol worldwide is produced from syngas, a fuel gas mixture consisting of H2, CO and CO2, obtained from incomplete combustion of natural gas. In addition, new ways for methanol production directly from natural gas, carbon dioxide or biogas are being developed, and mega-methanol production facilities (5,000 tons per day) are now being constructed in regions rich in natural gas. This is expected to allow the price of methanol to remain at a relatively low level compared to raw sugar. A methanol-based economy as an alternative fuel and feedstock concept is being discussed in the scientific community, and the Nobel Prize laureate George Olah has been among its most prominent propagators.
Methylotrophy, the ability of certain specialized microorganisms to utilize reduced C1 compounds l as their sole carbon source for growth, bears the potential to build value from methanol through production of key chemicals. In nature, methylotrophic bacteria can synthesize all their cell constituents from C1 compounds and use them as both carbon and energy source. This is in contrast to most autotrophs that perform total biosynthesis from carbon dioxide, but require a separate energy source. Methylotrophic bacteria have been the key organisms in this project.
The PROMYSE vision is a viable methanol-based European bio-economy, which we will promote by for the first time applying synthetic biology principles for cell factory development harnessing methanol as a general feedstock for the manufacturing of special and fine chemicals. PROMYSE combines two frontline research topics: orthogonal modularization of methylotrophy within a Synthetic Biology concept and employing methanol as a feedstock for biotechnological production. The PROMYSE project has demonstrated for the first time bioproduction of several industrially valuable products, including platform chemicals and fine chemicals, from methanol. In addition, the PROMYSE project has provided extensive and new insight (genetic, regulatory, biochemical) into bacterial methylotrophy and made the first critical successful steps towards the engineering of this highly novel and complex metabolic trait into non-methylotrophic bacteria. The results and achievements have been presented in so far 22 peer review scientific publications, numerous conference presentations, and one patent.
Project Context and Objectives:
Project context
Microorganisms are widely used as cell factories for production of special, fine, bulk, and fuel chemicals and they typically use sugars and other food derived sources as raw materials. Sugars originating from plants demand cultivable land which is more and more needed for human nutrition. Methanol - with a worldwide production capacity of more than 46 million tons per year - is regarded as an alternative raw material in microbial bioprocesses. The supply of methanol can base upon both fossil and renewable resources, rendering it a highly flexible and sustainable raw material. In nature, methylotrophic microorganisms can utilize one-carbon compounds such as methanol as their sole source of carbon and energy. However, methylotrophic bacteria typically lack certain favourable traits needed by industry for application as microbial cell factories, and in the project PROMYSE the main objective is to engineer methylotrophy to non-methylotrophic bacteria by using Synthetic Biology. More specifically, the chosen host strains are the biotechnologically well-established bacteria Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum.
Methylotrophy is a complex metabolic trait involving a large number of dedicated genes and enzymes and it proceeds via the cell-toxic intermediate formaldehyde. Accordingly, PROMYSE is a challenging and ambitious project. Synthetic Biology is an enabling technology which is expected to contribute to the knowledge-based bio-economy. The cell factory concept, which has been supported by the EU in several frameworks and key actions, lends itself ideal as field of application to capitalize on the potential of Synthetic Biology. PROMYSE combines two frontline research topics: orthogonal modularization of methylotrophy within a Synthetic Biology concept and harnessing methanol as general feedstock for biotechnological production. Functional modules encoding metabolic and production path¬ways are combined to form novel production hosts that are optimised to convert methanol to high value products. The project thus generates knowledge in methylotrophy, the required bio¬chemical components, and reactions. In addition, novel production hosts are created that are based on either established industrial producers or on natural methylo-trophs.
PROMYSE brings together leading European expertise in methylotrophy and biotechnology; moreover, one SME and two industrial partners among its participants secure and evaluate the commercialization potential of synthetic methylotrophic cell factories. Altogether, PROMYSE involves researchers from 3 universities, 3 research institutes, 1 SME and 2 industrial partners. The major product focus in PROMYSE is on terpenoids because they constitute the largest class of natural products covering a multitude of interesting physiochemical properties and biological activities. In addition, we will also focus on producing diaminopentane and dicarboxylic acids from methanol. The PROMYSE project is organized into totally eight Work packages (WPs); 6 RTD WPs (WP1-WP6), as well as dissemination WP7 and management WP8.
Project objectives
The main objective of PROMYSE is to follow a Synthetic Biology approach to cell factory development for harnessing methanol for biotechnological production of value-added chemicals, thus paving the way for our vision of a viable methanol-based European bio-economy. In order to achieve this ambitious goal, the following project objectives were made:
Functional characterization and establishment of methylotrophy modules: to select and characterize methylotrophic pathway modules from native methylotrophic bacteria and transfer them into heterologous hosts
- Assembly and demonstration of functional expression of methanol oxidation modules in Gram-positive and Gram-negative bacterial hosts
- Expression of natural and synthetic formaldehyde dissimilation modules enabling in vivo formaldehyde oxidation in heterologous hosts.
- Expression of natural and synthetic formaldehyde assimilation modules leading to formaldehyde fixation into cell constituents necessary for cell growth of heterologous hosts.
- Physiological analysis of the recombinant strains under controlled conditions.
To functionally integrate methylotrophy modules into the biotechnologically relevant bacteria B. subtilis, C. glutamicum and E. coli
- Evaluation and improvement of engineered strains
- Platform organisms – engineering and integration of synthetic methylotrophy islands
In silico analysis of metabolic networks in the bacterial hosts: to iteratively combine experimental and in silico strategies for the rational design and implementation of synthetic metabolic modules, ensuring controlled and efficient conversion of methanol into key precursors and onwards to selected products, but without disturbing the host system as a whole.
- Development of orthogonal metabolic modules for the efficient conversion of methanol
- Identification of the auxiliary metabolic modifications ensuring high precursors supply from methanol for high products formation in non-methylotrophic hosts (B. subtilis, C. glutamicum, E. coli) and in methylotrophic hosts (B. methanolicus, M. extorquens)
- Iterative optimization of targeted metabolic processes in both natural and synthetic methylotrophs
- Adapting hosts for metabolic efficiency
Design, construct and functionally express synthetic pathways of enzymes catalyzing the conversion of methanol into useful products in native methylotrophs: To design and construct novel microbial cell factories based on the native methylotrophs B. methanolicus and M. extorquens.
- Characterization of the putative MEP pathway in B. methanolicus and elucidation of its potential for terpenoid production from methanol at elevated temperatures.
- Exploitation of the ethylmalonyl-CoA pathway (EMCP) for the production of dicarboxylic acids with M. extorquens
- Engineering the mevalonate (MVA) pathway into M. extorquens for enhancement of the native terpene production capacity
Functional expression of synthetic pathways for conversion of methanol into useful products in synthetic methylotrophic hosts
- Assessment of methanol-based production by synthetic hosts as proof-of-principle
- Proof –of-concept of reciprocity: assessment of methanol-based diamine production by synthetic C. glutamicum
- Assessment of methanol-based terpenoid production by synthetic methylotrophs
- Assessment of methanol-based production of EMCP intermediates by synthetic methylotrophs
Demonstration of potential industrial relevance
- Establishing first fermentation protocols in the bioreactor
- Evaluation of methanol based bioprocess under industrial conditions
Dissemination, demonstration and implementation
- Identification of novel inventions and patent protection
- Provide data on high yield of methanol to product conversion
- To boost the awareness og high ecological and economical value of methanol based synthetic cell factories
Management
- To manage administrative aspects of the project
- Technical management of the project
- Strategic management of the project
Project Results:
The PROMYSE project is now completed and all the efforts, results and achievements are carefully and detailed described in the Periodic Report 1 and Periodic Report 2. Hereunder is a brief presentation summarizing the major results and findings in the totally 8 WPs of PROMYSE. In addition, we emphasise that the PROMYSE results have been presented in so far 22 accepted peer review publications and one patens, as well as broadly presented in a high number of channels and events (see complete dissemination activities listed in the PROMYSE Final Report submitted in parallel to this report). The potential impact and use of the results can be separated in various segments; first, PROMYSE has contributed to extensive and valuable new insight and knowledge on synthetic biology and methylotrophy in general, highly useful for the biotechnology industry and the scientific community. We have experienced increased interest and direct contact from both industry and academia towards end of project, as high frequency of publications has been published by our consortium. A patent established at start of project has attracted increased commercial interest as we have demonstrated usefulness of the key enzymes methanol dehydrogenase for engineering of methylotrophy. In the era of bio economy, efforts to establish alternative raw materials such as methanol for biotechnology usage are attractive, and interest on this is rapidly growing internationally. The project has also educated a number of young and talented PhD candidates and researchers into the field synthetic biology.
PROMYSE is composed of six RTD work packages (WP1 – WP6); For WP1 –WP4 we continue with basis in what was reported in previous Periodic report 1, while for WP5 and WP6 this is naturally then the complete reporting. We hereunder summarize the main achievements and results obtained during the PROMYSE project.
WP1: Functional characterization and establishment of methylotrophy modules
Within WP1 the aim is to characterize and demonstrate biochemical functionality of the core methylotrophy modules at the enzyme level, as a basis for their functional integration into the three hosts E. coli, B. subtilis and C. glutamicum. Methylotrophy as a metabolic trait can be divided into three distinct principle modules, module 1, methanol oxidation into formaldehyde; module 2, formaldehyde dissimilation into CO2; and module 3, formaldehyde assimilation to generate cell mass. In the previous periodic report we had initiated work on the methanol dehydrogenase enzymes and ACT (totally 8 enzymes) and this work has now been completed and published in the second period. In particular we also have completed the successful transfer of all these enzymes into our three non-methylotrophic hosts – E. coli, B. subtilis and C. glutamicum – and demonstrated and further optimized methanol oxidation (the key step for methylotrophy). In particular for E. coli this has been successful and also with strong iterations with work performed in WP2 and WP33 (below) has resulted in significant step towards engineering of methylotrophy into this bacterium (published and patented results). Also, the two initial genes of the RuMP pathway hps and phi are central both for formaldehyde dissimilation (module 2) and assimilation (module 3). Both enzymes could be functionally expressed in all three host organisms, as was validated by in vitro and in vivo assays. We also in this period has performed extensive biochemical and regulatory characterization of a majority of genes and enzymes involved in the central methylotrophic RuMP pathway; the work has been published in several peer review papers and also carefully review in another publication from the PROMYSE consortium at end of project (see publication list in Final Report on this). In total, this project has generated extensive and solid characterization of genes and modules representing all three methylotrophy modules and in close iteration with WP2 and WP3 used to systematically test out their functionality and importance for the engineering of methylotrophy (see below).
WP2: Integration of methylotrophy into non-methylotrophic hosts
The focus in WP2 has been on using biological materials derived from WP1 for the functional integration of all three methylotrophy modules into the heterologous hosts, i.e. module 1 for methanol oxidation to formaldehyde, module 2 to dissimilate into CO2 for energy generation, and module 3 to allow for formaldehyde assimilation to generate cell biomass. Work initiated on transfer of methanol dehydrogenases and act, as well as the two genes phi and phs for formaldehyde fixation was well initiated in periodic report 1 and has been completed and published in periodic report 2. 13C labelling experiments (see WP3) were used as the principal tool to evaluate functionality of modules in the three hosts. The experiments demonstrated that the presence of the minimum set of genes representing the modules leads to incorporation of methanol and formaldehyde into biomass in all three hosts and thus the achievement of the deliverables within the reporting period. Multiple labels in central metabolites were also observed indicative of a fully operable RuMP cycle, and in particular for E. coli host the work has been complete and extensive and was recently published in the high-rank journal Metabolic Engineering (see publication list). Also for the two other hosts, extensive progress has been made and a number of RuMP pathway genes characterized under WP1 has been tested alone and in combinations. The strategies for engineering design has been constantly evaluated as result of extensive proteomic and transcriptome analysis of Bacillus methanolicus growing under relevant conditions, also published work in this second period.
WP3: In silico analysis of metabolic networks
The major focus in WP3 has been to complete the in silico design of metabolic modules to be expressed in the heterologous hosts, as to build metabolic network models of the methylotrophic model strains, in silico optimization of product formation in the non-methylotrophic hosts and systematic integration of wet lab data to improve the Insilico models. These developments included an iterative process between modelling and experiment, in which systems-level investigations of the engineered strains were performed to guide the integration of synthetic methylotrophy and production modules. Genome-scale models of the natural methylotrophic hosts M. extorquens and B. methanolicus were completed, and the capability of methylotrophic growth has been verified for both models. Synthetic production modules that allow for the formation of compounds which are in the focus of WP4 and WP5 were also included in these models. The first genome-scale model for B. methanolicus has been reconstructed on the basis genome data which was provided by the project partner SINTEF. These models were used to define the auxiliary metabolic modifications ensuring high supply of precursors from methanol for efficient product formation in non-methylotrophic hosts. Using 13C labelling experiments we demonstrated that methanol indeed can be incorporated into cell constituents in all three hosts; this is a major milestone in this project and demonstrates that it should be possible to engineer methylotrophy.
WP4: Design, construction and functional expression of synthetic pathways for conversion of methanol into useful products in native methylotrophs
The promising work initiated in WP-4.1 on the construction of expression vectors carrying lysine decarboxylase for 1,5-diaminopentane (cadaverine) production in the gram-positive native methylotrophic bacterium B. methanolicus has been completed and published during this second period. In particular, several additional strains have been constructed in order to understand and improve bottlenecks for cadaverine production in B. methanolicus; the work has solely been performed in shake flask experiments and the best strains were successfully tested under HCDC in WP6. The work also included development of new genetic tools for B. methanolicus. In WP-4.2 we in previous report demonstrated for the first time terpenoid production from methanol by recombinant B. methanolicus strains, and this work has been extended in the reporting period and a more systematic investigation of the genes involved has been made. In WP4.3 the work initiated in previous report on the construction of deletion strains for genes coding for enzymes of the ethylmalonyl-CoA-pathway (EMCP) in the gram-negative native methylotrophic bacterium Methylobacterium extorquens, has been completed and published. In WP4-4 we have constructed recombinant M. extorquens strains that can produce monoterpenoids and sesquiterpenoids giving a proof-of-concept for terpenoid production from methanol. In total, WP4 has demonstrated for the first time production of all targeted product classes from methanol by genetically modified methylotrophic bacteria.
WP5: Functional expression of synthetic pathways for conversion of methanol into useful products in synthetic methylotrophic hosts
This WP was initiated in the reporting period and focused on the feasibility of methanol-based production at different levels of complexity by establishing (1) functional integration of methylotrophy modules available from WP-1 into existing non-methylotrophic production hosts E. coli, B. subtilis and C. glutamicum and (2) functional integration of production modules into synthetic methylotrophs generated in WP-2. Since methanol conversion could be achieved for the three hosts, while growth on methanol as sole carbon source could not (s. other WPs), the work within WP-5 primarily aimed at functional integration of production modules into the hosts E. coli, B. subtilis and C. glutamicum. As test cases, strains for the production of L-lysine, cadaverine and amylase were constructed and are ready to be integrated into (synthetic) methylotrophic host backgrounds. However, emphasis was on the production of terpenoids as well as on the production of intermediates of the EMCP. Synthetic pathways leading to production of terpenoids, a diamine, and organic acids have been implemented into the three hosts. In all three heterologous hosts methylotrophy modules could be established and their operation demonstrated in vivo; besides methanol incorporation into biomass could be shown for all three heterologous hosts, albeit at low rate. The overall aim of this WP to establish methanol-based production based on synthetic non-methylotrophic microorganisms is ambitious; however, if successful it will provide strategies to new routes to produce added-value products from methanol in the context of synthetic biology. The work resulted in several publications (see publication list).
WP6: Demonstration of potential industrial relevance.
This WP was initiated in this reporting period. The aim of this WP was to establish upscale proven methanol based production in shake flask scale (see WP4) to laboratory bioreactor scale including controlled substrate feeding and first downstream processing experiments. Recombinant B. methanolicus strains producing cadaverine were run under HCDC and we obtained for the best strain tested up to 17 g/l – which is a very good production yield – under such conditions; the results have been published in the reporting period. Also for similar testing of recombinant M. extorquens strains producing monoterpenoids and sesquiterpenoids we obtained good production results; we also should mention that such cultivations under carefully controlled conditions offer valuable data used for the evaluation of the strains as well. In addition a theoretical evaluation of methanol based bioprocess under industry relevant conditions has been made.
WP7: Dissemination, demonstration and implementation
In total, dissemination output from PROMYSE has been extensive and of high quality and complete overview of all PROMYSE dissemination activities are listed in the electronic report (Template A1, Template A2, and Template B1). As much of the results have been published in high-rank peer review scientific journals I choose here to provide a complete publication lists (solely accepted publications!) very precisely summarizing the main dissemination achievements for entire project period:
1) Irla M, Neshat A, Brautaset T, Ruckert C, Kalinowski J, and Wendisch VF. (2015). Transcriptome analysis of the thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape. In Press BMC Genomics.
2) Jonas E.N. Müller, Fabian Meyer, Boris Litsanov, Patrick Kiefer, Eva Potthoff, Stephanie Heux, Wim J. Quax, Volker Wendisch, Trygve Brautaset, Jean-Charles Portais, Julia A. Vorholt. (2015). Engineering Escherichia coli for methanol conversion. Metab Eng Jan 14. [Epub ahead of print]
3) Nærdal I., Pfeifenschneider J., Brautaset T. & Wendisch VF. (2015). Methanol-based production of cadaverine by metabolically engineered strains of Bacillus methanolicus. Microb biotechnol. 2015 Jan 23. [Epub ahead of print]
4) Müller JE, Heggeset TMB, Vorholt JA, Wendisch VF, Brautaset T. (2014). Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity productions from methanol. Appl Microbiol biotechnol. Nov 28. [Epub ahead of print]
5) Ochsner AM, Sonntag F, Buchhaupt M, Schrader J, Vorholt JA. (2014). Methylobacterium extorquens: methylotrophy and biotechnological applications. Appl Microbiol Biotechnol. 2014 Nov 30. [Epub ahead of print]
6) Irla M, Neshat A, Winkler A, Albersmeier A, Heggeset TM, Brautaset T, Kalinowski J, Wendisch VF, Rückert C. (2014). Complete genome sequence of Bacillus methanolicus MGA3, a thermotolerant amino acid producing methylotroph. J Biotechnol. Epub ahead of print.
7) Heider SA, Peters-Wendisch P., Beekwilder J. & Wendisch VF (2014). IdsA is the major geranylgeranyl pyrophosphate synthase involved in carotenogenesis in Corynebacterium glutamicum. FEBS J. Epub ahead of print.
8) Ochsner AM, Müller JE, Mora CA, Vorholt JA. (2014). In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed. FEBS lett, Epub 2014 Jun 10.
9) Heider SA, Wolf N., Hofemeier A., Peters-Wendisch P. and Volker F. Wendisch (2014). Optimization of the IPP precursor supply for the production of lycopene, decaprenoxanthin and astaxanthin by Corynebacterium glutamicum. Frontiers in Bioengineering and Biotechnology.
10) Wendisch V.F. (2014) Microbial production of amino acids and derived chemicals: Synthetic biology approaches to strain development. Curr. Opin. Biotechnol. 30: 51-58.
11) Frohwitter J, Heider SA, Peters-Wendisch P, Beekwilder J, Wendisch VF. (2014). Production of the sesquiterpene (+)-valencene by metabolically engineered Corynebacterium glutamicum. J Biotechnol. June 6. Epub ahead of print.
12) Sonntag F, Buchhaupt M, Schrader J. (2014). Thioesterases for ethylmalonyl-CoA pathway derived dicarboxylic acid production in Methylobacterium extorquens AM1. Appl Microbiol Biotechnol. 98(10):4533-44.
13) Müller JE, Litsanov B, Bortfeld-Miller M, Trachsel C, Grossmann J, Brautaset T, Vorholt JA. (2014). Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3. Proteomics. 14(6):725-37.
14) Markert B, Stolzenberger J, Brautaset T, Wendisch VF. (2014). Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. BMC Microbiol. 14:7.
15) Heider SAE, Peters-Wendisch P, Wendisch VF, Beekwilder J, Brautaset T. (2014). Metabolic engineering for the microbial production of carotenoids and related products with a focus on the rare C50 carotenoids. Appl Microbiol Biotechnol. 98(10):4355-68.
16) Stolzenberger J., Lindner S., Persicke M., Brautaset T. & Wendisch V.F. (2013) Characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. J. Bacteriol. 195: 5112-5122.
17) Stolzenberger J., Lindner S. & Wendisch V.F. (2013) The methylotrophic Bacillus methanolicus MGA3 possesses two distinct fructose 1,6-bisphosphate aldolases. Microbiol. 159: 1170-1781.
18) Heider S.A.E. Peters-Wendisch P., Netzer R., Stafnes M., Brautaset T. & Wendisch V.F. (2013). Production and glucosylation of C50 and C40 carotenoids by metabolically engineered Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 98(3):1223-35.
19) Krog A, Heggeset TM, Müller JE, Kupper CE, Schneider O, Vorholt JA, Ellingsen TE, Brautaset T. (2013). Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties. PLoS One. 8(3):e59188.
20) Lessmeier L, Hoefener M, Wendisch VF. (2013). Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase. Microbiology. 159:2651-62.
21) Heider SA, Peters-Wendisch P, Wendisch VF. (2012). Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum. BMC Microbiol. 10; 12:198.
In addition, PROMYSE results and efforts have been extensively communicated in also other publication channels (conferences, workshops, meetings, radio, popular scientific journals, public meetings, industry workshops). We also have one patent with an invention at the core of the project – engineering of methylotrophy.
WP8: Management
The Operational Management Team (OMT) in PROMYSE – responsible for both scientific and administrative/economic coordination of the project - was formed from regular offices at SINTEF and consisted of the following representatives:
Name Beneficiary Role
Trygve Brautaset SINTEF Coordinator
Marthe Hagerup Indal SINTEF In charge of adm, legal and financial aspects
Tove L Hunstad SINTEF Management coordinator
The General Assembly (GA) is the highest decision-making body in PROMYSE project and consisted of one representative from each partner:
Name Beneficiary
Trygve Brautaset SINTEF
Volker Wendisch UNIBI
Julia Vorholt ETHZ
Wim Quax RUG
Robert Thummer BASF
Joachim Schmid INSILICO
Jean-Charles Portais INSAT
Audun Goksøyr PROMAR
Jens Schrader DFS /DECHEMA
The Technical Committee (TC) has been responsible for the daily execution of the project and consists of the individual WP leaders as follows:
Name Beneficiary
Tonje MB Heggeset (WP1) SINTEF
Julia Vorholt (WP2) ETHZ
Stephanie Heux (WP3) INSAT
Jens Schrader (WP4) DFS/DECHEMA
Volker Wendisch (WP5) UNIBI
Robert Thummer (WP6) BASF
Wim Quax (WP7) RUG
Trygve Brautaset (WP8) SINTEF
The IPR and Exploitation group (IEG) in PROMYSE, including the GA and the administrative manager is constituted to deal with the strategic issues of dissemination strategy, exploitation, IPR and knowledge management, consists of the following representatives:
Name Beneficiary
Wim Quax (leader) RUG
Trygve Brautaset SINTEF
Volker Wendisch UNIBI
Julia Vorholt ETHZ
Robert Thummer BASF
Joachim Schmid INSILICO
Jean-Charles Portais INSAT
Audun Goksøyr PROMAR
Jens Schrader DFS/DECHEMA
Before the start of the project all partners signed a Consortium Agreement (CA) regulating the work between the parties, the management of the project and the rights and the obligations of the parties concerning inter alia liability, access rights and dispute resolution.
PROMYSE management in general:
The most important forum for planning and monitoring the status of the project are the PROMYSE project meetings which are arranged every 6 months; totally 7 meetings (including the kick-off meeting). These 2 day meetings include presentations among the project members of progress and plans for all on-going activities in each WP, for both plenary and bilateral discussions as well as in the TC. Minutes are written after these meetings and all presentations are put on the e-room available for all project partners. In addition to these meetings there have been several discussions and meetings between two or more partners on the working and WP levels. These include face-to-face meetings, research visits, and telephone and Skype meetings. We have put high emphasis in PROMYSE to establish close and direct contact between the researchers representing the different partners. We have established an internal web-site, a so-called eRoom, at the initiation of the project. This eRoom was recognized at the project start to facilitate the needs for a well-structured common place for storage of all kinds of electronic documents related to the project, like articles, project reports, presentations, minutes of meetings, etc. Document integrity is well assured due to the high degree of security of the eRoom service as well as the ownership and detailed role-based visible/read/write access rights associated with folders and individual documents. The PROMYSE project web-site (http://www.sintef.no/Projectweb/PROMYSE/) was established at the start of the project and has been updated with relevant information since then. Due to IPR issues we have chosen to carefully limit the publication of project results on this public available web site. In addition we have established an internal web-site, a so-called eRoom, see point 3.2.3.5 above.
Co-operation with other projects/programmes:
PROMYSE partners 1, 2, 3, 4 and 5 constituted the consortium of the ESF funded EuroCORE project SynMet (2010 – 2013).There were totally five EuroCORE projects working with synthetic biology and during the 3 year project period there were several highly useful cross project activities arranged, including EuroCORE meetings in Cannes (May 2011), Groningen (November 2012), and Elmau (May 2013) with PROMYSE partners well represented. The PROMYSE Coordinator is member of the EuroCORE scientific boardTogether, this has extended our European network within synthetic biology and new project proposal collaborations are currently initiated. In addition, two PROMYSE partners 1 and 3 are members of the scientific board ERASynBio Scientific advisory board (SAB) and has participated on several workshops and meetings (including participation on the ERASynBio US synthetic biology tour October 2012) to discuss European synthetic biology development, including shaping of the ERASynBio call topic. This has also contributed to new funding possibilities for cross-Atlantic research on synthetic biology. Importantly also; four of the PROMYSE partners have recently received funding for a new ERASysApp project (“MetApp”) with strong basis in parts of the PROMYSE project; MetApp is a 3-year project 2015 – 2017.
Potential Impact:
Industrial biotechnology offers alternative and sustainable production routes for chemicals and this is a rapidly growing sector worldwide. Typically sugar-based raw materials used are in unwarranted competition with the food sector. Accordingly, there is a high interest in using alternative non-sugar based substrates that do not require cultivable land. Today, there is massive research in the field bio refinery aiming at utilizing lignocellulose materials from wood and plants as substrates. Methanol represents an additional attractive alternative and in this regard methylotrophic bacteria are of great interest. Methylotrophy is a complex metabolic trait involving a number of enzymes and pathways that are interlinked with each other. Methylotrophy is modular in its nature consisting of module 1 (methanol oxidation into formaldehyde), module 2 (formaldehyde dissimilation into CO2), and module 3 (formaldehyde assimilation into cell constituents).
The PROMYSE project was divided into totally 6 different RTD WPs – although they consisted of separate tasks, deliverables and milestones, they were much integrated. The work of the 6 RTD work packages is performed in an integrated way by the project partners and most work packages depend on deliverables from other work packages. WP-1 delivers methylotrophy modules for WP-2 which in turn delivers synthetic strains to be used in WP-5. Strains used and knowledge acquired in WP-1 were needed for the implementation of WP-4. Activities in WP-3 were central in the project by linking WP-1 and WP-2 to WP-4 and WP-5. Synthetic strains produced in WP-4 and WP-5 were candidates for the up-scaling and demonstration activities in WP-6.
Within WP1 to WP3 we explore Synthetic Biology strategies as a combination of engineering and testing on the one hand coupled with analysis, predictions and modelling and on the other hand, to generate novel engineered methylotrophs for the generation valuable products from methanol using well established biotechnological relevant hosts. The work in these three work packages is tightly connected: In WP3 we have designed and predicted methylotrophic models in silico for the host bacteria as basis for the engineering efforts made in WP1. Genetic engineering and biochemistry results generated in WP1 were tested in the chosen host strains in WP2 with the common goal of understanding and engineering of methylotrophy. In WP3 we have also performed experimental evaluation of selected strains representing all three hosts from WP1 and WP2 by using labelled methanol and in this way demonstrated that we have succeeded in the construction of recombinant strains with a new feature, i.e. consuming and incorporating methanol into cell constituents. Together, these important results demonstrate that we reached a solid basis to further optimize the engineered hosts to build new methylotrophic bacteria. The work conducted up to now in WP1 to WP3 has provided new and fundamental understanding of the genetic and enzymatic basis for methylotrophy in both gram-positive and gram-negative bacteria. Together, this knowledge will be highly useful for future utilization of methylotrophic bacteria in white biotechnology. In particular, we have made substantial progress on module 1, the key initial methylotrophy step of methanol oxidation. This has to be very efficient and also tightly co-regulated with the remaining modules 2 and 3 to ensure efficient growth and energy generation and at the same time to avoid toxic formaldehyde accumulation. We have in PROMYSE biochemically characterized six different methanol dehydrogenases and their activator proteins, and transferred each of them to the three selected non-methylotrophic hosts. We have unravelled that the three host bacteria displayed different preferences with respect to optimum choice of enzymes representing module 1. These results have been patented and published (see below). In addition, we have combined integration of module 1 with key enzymes representing modules 2 and 3, and in this way managed to construct recombinant strains representing all three hosts that are capable of oxidizing methanol and further incorporating the formaldehyde into downstream cell components. We expect that the knowledge and technology generated in this work package should be a highly valuable basis for all efforts aiming at improving and/or transferring methylotrophy in bacteria, for the overall goal of utilizing methanol as raw material for white biotechnology. Important publications achieved under the combined efforts of WP1 – WP3 are (se complete publication list PROMYSE above): Irla et al 2015; Müller et al 2015; Irla et al 2014; Ochsner et al 2014B; Müller et al 2014; Markert et al 2014; Stolzenberger et al 2013A and B; Krog et al 2013; Lessmeier et al 2012. Of particular notice I would like to highlight the publication Müller et al 2015; this one was accepted for publication in the journal Metabolic Engineering on the 31.12.2104 – the very last day of the PROMYSE project – and it describes the generation of synthetic Escherichia coli cells that can truly metabolize methanol – a key publication for entire PROMYSE project and with co-authors from several PROMYSE partners.
The WP4 activities differ from the work in WP1-WP3 as we are here engineering in native methylotrophic hosts. We have constructed several recombinant B. methanolicus strains that secrete the important platform chemical cadaverine; this is to our knowledge the first demonstration of cadaverine production from methanol. We are currently focusing on improving cadaverine production yields, including also improved export out of the cells. We have also demonstrated for the first time that methylotrophic B. methanolicus has a functional MEP pathway. By introducing a synthetic operon including heterologous genes we have demonstrated that this MEP pathway can be a basis for producing C30 terpenoids from methanol. To our knowledge, terpenoid production from methanol has not previously been described in the scientific literature. We are currently in progress engineering lycopene production into this bacterium as well; lycopene is a key precursor for a high number of commercially interesting carotenoids. In parallel we have also made analogous engineering of the gram-negative native methylotroph M. extorquens and demonstrated that the EMCP is an excellent starting point for the bioproduction of special dicarboxylic acids from methanol. The expected results of the engineering efforts on the native methylotrophs will have high impact on the future bioproduction of platform and fine chemicals from methanol. Important publications achieved under WP4 are (se complete publication list PROMYSE above): Nærdal et al 2015; Sonntag et al 2014
In WP5 the major focus has naturally been on the product modules; in particular we have made very extensive achievements in host C. glutamicum on the exploitation of this industrially important bacterium as a platform cell factory for sustainable production of various carotenoids. We have unravelled the biosynthetic pathway (genes, enzymes, regulation) for the C50 carotenoid decaprenoxanthin, and also demonstrated that this bacterium can be engineered to produce other known C50 carotenoids. We have optimized upstream pathways for higher production yields and also shown that it can produce alternative length C30 and C40 carotenoids. Important publications achieved under WP5 are (se complete publication list PROMYSE above): Heider et al 2014A, B, and C; Frohwitter et al 2014; Heider et al 2013; Heider et al 2012.
In WP6 – which was leaded by industry partner BASF - we have demonstrated up scaled production of chemicals from methanol using genetically engineered methylotrophic bacteria constructed in WP1-WP4 as cell factories. Of particular importance has been to design good cultivation strategies using methanol as C source and to achieve production yields higher than those obtained under small-scale laboratory conditions. In particular, we managed to produce up to 17 g/l of the important platform chemical cadaverine from methanol and at elevated temperatures by using GMO B. methanolicus strains constructed in WP4. Parts of the results of WP6 have been published in Nærdal et al 2015 and Sonntag et al 2014. Finally; the summarized efforts and achievements obtained during the PROMYSE project resulted in substantial new and valuable information and insight into the two model methylotrophic bacteria bacillus methanolicus and Methylobacterium extorquens. Therefore, it was very valuable to summarize all this good work into two back-to-back review articles at the end of PROMYSE project with authors representing PROMYSE partners broad; see references Muller et al 2014 and Ochsner et al 2014A.
Methylotrophy is the ability of certain specialized microorganisms to utilize reduced carbon substrates containing no carbon-carbon bonds as sole sources of carbon and energy. These organisms have been known since the late nineteenth century, and substantial knowledge has accumulated, in particular in the past 50 years. The knowledge in the field can be used to separate methylotrophs according to different aspects such as (i) phylogeny of organisms, (ii) obligate versus facultative methylotrophy, (iii) methylotrophic substrate range, and in particular (iv) operation of sets of enzymes and pathways allowing methylotrophic growth. Phylogenetically, methylotrophs belong to a rather small number of genera within Alpha- (e.g. Methylobacterium), Beta- (e.g. Methylobacillus), and Gammaproteobacteria (e.g. Methylococcus), as well as within the Gram-positives (e.g. Bacillus), and Verrucomicrobia (e.g. Methylacidiphilum). Many of these are facultative methylotrophs that are able to grow on one carbon but also on a generally limited number of multi-carbon compounds. Methylobacterium extorquens and Bacillus methanolicus are representatives of facultative methylotrophs and have the advantage that alternative substrates can be used to identify genes essential for methylotrophic growth. Both bacteria use methanol as reduced one carbon source but not methane which is used by a number of obligate (with few exceptions) methanotrophs of the Alpha- and Gammaproteobacteria as well as members of a genus within Verrucomicrobia. These two bacteria have been the major model organisms used in PROMYSE. A key question to understand methylotrophy is how methylotrophic organisms generate energy and how they convert one carbon substrates to carbon compounds with carbon-carbon bonds required for their own living. The knowledge has been gathered from enzymatic characterizations, pathway elucidation via labelling experiments, genetic and genomic approaches. Taking a level of abstraction, methylotrophy can be regarded as a set of discrete specialized functional modules that are ultimately linked to central metabolism. This is concluded from the presence of alternative enzymes or pathways for specific metabolic goals in different methylotrophic bacteria and also the existence of different combinations of these systems. These specific “metabolic goals” are (1) oxidation of the primary substrate (e.g. methanol) to formaldehyde; (2) oxidation of formaldehyde to CO2, and (3) assimilation of one carbon compounds, the latter taking place either at the level of formaldehyde (as free formaldehyde or as methylene tetrahydrofolate (H4F)) or CO2 or a combination thereof.
Synthetic Biology is regarded as an emerging enabling technology which will contribute to fostering the knowledge-based bio-economy. The cell factory concept, which has been supported by the EU in several frameworks and key actions, lends itself ideal as field of application to capitalize on the potential of Synthetic Biology. The main objective of PROMYSE has been to follow a Synthetic Biology approach to cell factory development for harnessing methanol for biotechnological production of value-added chemicals, thus paving the way for our vision of a viable methanol-based European bio-economy. PROMYSE combined two frontline research topics: orthogonal modularization of methylotrophy within a Synthetic Biology concept and harnessing methanol as general feedstock for biotechnological production. The potential of natural methylotrophs for use in industrial biotechnology remains to be fully explored. Still, established bioprocesses in this industry typically employ non-methylotrophic organisms as cell factories and carbohydrates as feedstock. The emerging concept of Synthetic Biology offers the possibility for a rational advancement of the field paving the way to methanol-based biotechnology. In PROMYSE, orthogonal methylotrophy modules has been identified, characterized, optimized and transferred to non-methylotrophic production strains. In parallel, methylotrophic hosts have been endowed with production modules derived from established producing hosts by functional integration into the metabolic network. The synthetic cell factories developed within PROMYSE have been designed to enable utilization of methanol for the production of fine and bulk chemicals. Systems Biology is in demand to understand the biological meaning and interconnectivity of metabolic or other networks. The Synthetic Biology approaches we have used in PROMYSE was combined with systems level understanding in a way that has totally changed the perspective we are currently having on methylotrophy. Within this project we used modelling as the starting point to analyze metabolism, and we used in silico models to find optimal solutions for maximum growth and later precursor formation for product formation. Feeding the in silico models with naturally existing parts allowed us to explore and learn from this process what optimal solutions are and where the limits are. In particular, we have learned from comparing predictions generated through modelling with experiments.
Using methanol as an alternative non-food feedstock for biotechnological production offers several advantages in line with a methanol-based bioeconomy. There is a high societal demand for a sustainable production of special, fine, bulk, and fuel chemicals, including food and health care compounds. Biotechnological processes will play a prominent role in the coming bioeconomy era by gradually complementing and substituting petrochemical synthesis. In biotechnology, microorganisms are widely used as cell factories and, in particular for high-volume products, raw material costs make up a large part of process costs. In White Biotechnology mainly sugars and molasses are used as carbon sources, and these raw materials are derived from plants demanding cultivable land which is more and more needed for human nutrition. The possibility to utilize non-food raw materials, such as one-carbon (C1) substrates like methane (CH4) and methanol (CH3OH), as alternative feedstock has therefore gained high scientific interest but is not yet implemented at commercial scale. Major reasons for this are that methanol is still a more expensive substrate than sugar (worldwide methanol prizes at Methanex: https://www.methanex.com/our-business/pricing) and that methanol fermentations can be technically challenging; for example high O2 requirements and concomitant heat production may cause increased cooling requirements, and careful substrate feeding is needed to avoid toxic formaldehyde accumulation in the cells (see below). However, substantial progress on fermentation technology and construction of production strains should enable commercialization of methanol based bioprocesses in the near future, as reviewed here and elsewhere.
Methanol is a pure and non-food chemical that is soluble in water and it is completely utilized during microbial fermentations. Methanol should thus represent an attractive alternative raw material for biotechnological processes, both from an economic, ecologic, and process point of view. With a worldwide production capacity of more than 53 million tons per year, methanol is one of the most important raw chemicals on earth. The supply of methanol can base either upon fossil or renewable resources, rendering it a highly flexible and sustainable raw material. Today, almost all methanol worldwide is produced from syngas, a fuel gas mixture consisting of H2, CO and CO2, obtained from incomplete combustion of natural gas. In addition, new ways for methanol production directly from natural gas, carbon dioxide or biogas are being developed, and mega-methanol production facilities (5,000 tons per day) are now being constructed in regions rich in natural gas.
The products we have focused on in PROMYSE are terpenoids (including carotenoids), platform chemicals and some fine chemicals – all with high commercial interest. There is today a rapid transformation globally from chemical to biotechnological production processes for a wide range of chemicals; and in parallel with this there is a growing interest and need in finding alternative non-food raw materials for the biotechnology sector; PROMYSE has delivered solutions, technology, knowledge and innovations to both these two challenges.
PROMYSE has brought together a balanced and well-functioning consortium from many different European countries, including complementary competence and infrastructures – all needed to achieve the project goals. Training of young scientists, PhDs and postdoc has been good as they have been part of a multidisciplinary project consortium and ideas and insight has been shared via meetings and e-mail correspondence, co-publications and guest visits. Also, the involvement of industry partners has been important to constantly also look into the commercial and innovative sides of biotechnology research. One important added-value of this collaboration is that 5 of the project partners have recently received funding for a new ERASysAPP project in systems biology, the new project denoted “MetApp” has a solid basis in much of the results, knowledge and biological materials generated in PROMYSE.
All dissemination activities of the PROMYSE project has been listed and described in the electronic final report – much of the peer review publication achievements are also described and explained above. In total, we believe that the output has been very good in terms of publications, one key patent, and various dissemination activities. The project has attracted broad international interest as we have been contacted from both academia and industry requesting for protocols and genetic/biological materials and strains. Also, researchers have been established at the international arena as important contributors to the field synthetic biology; we have been requested as members of various scientific advisory boards, invited to popular scientific meetings and workshops in synthetic biology / bioeconomy, and also invited specifically to write book chapters in synthetic biology, and also contacted for interviews in radio and newspapers. The PROMYSE project has been popular also among master students to take their thesis associated with the science going on in this key project for the partners involved.
List of Websites:
PROMYSE public web site: http://www.sintef.no/Projectweb/PROMYSE/
There is a high societal demand for a sustainable production of special, fine, bulk, and fuel chemicals, including food and health care compounds. Biotechnological processes will play a prominent role in the coming Bio economy era by gradually complementing and substituting petrochemical synthesis. In biotechnology, microorganisms are widely used as cell factories and, in particular for high-volume products, raw material costs make up a large part of process costs. Industrial biotechnology mainly uses sugars and molasses as carbon source, and these raw materials are derived from plants demanding cultivable land which is more and more needed for human nutrition. The possibility to utilize non-food raw materials, such as one-carbon (C1) substrates like methane (CH4) and methanol (CH3OH), as alternative feedstock has therefore gained high scientific interest but is not yet implemented at commercial scale. Major reasons for this are that methanol is still a more expensive substrate than sugar (worldwide methanol prizes at Methanex: https://www.methanex.com/our-business/pricing) and that methanol fermentations can be technically challenging; for example high O2 requirements and concomitant heat production may cause increased cooling requirements, and careful substrate feeding is needed to avoid toxic formaldehyde accumulation in the cells. Methanol is a pure and non-food chemical that is soluble in water and it is completely utilized during microbial fermentations. Methanol should thus represent an attractive alternative raw material for biotechnological processes, both from an economic, ecologic, and process point of view. With a worldwide production capacity of more than 53 million tons per year, methanol is one of the most important raw chemicals on earth. The supply of methanol can base either upon fossil or renewable resources, rendering it a highly flexible and sustainable raw material. Today, almost all methanol worldwide is produced from syngas, a fuel gas mixture consisting of H2, CO and CO2, obtained from incomplete combustion of natural gas. In addition, new ways for methanol production directly from natural gas, carbon dioxide or biogas are being developed, and mega-methanol production facilities (5,000 tons per day) are now being constructed in regions rich in natural gas. This is expected to allow the price of methanol to remain at a relatively low level compared to raw sugar. A methanol-based economy as an alternative fuel and feedstock concept is being discussed in the scientific community, and the Nobel Prize laureate George Olah has been among its most prominent propagators.
Methylotrophy, the ability of certain specialized microorganisms to utilize reduced C1 compounds l as their sole carbon source for growth, bears the potential to build value from methanol through production of key chemicals. In nature, methylotrophic bacteria can synthesize all their cell constituents from C1 compounds and use them as both carbon and energy source. This is in contrast to most autotrophs that perform total biosynthesis from carbon dioxide, but require a separate energy source. Methylotrophic bacteria have been the key organisms in this project.
The PROMYSE vision is a viable methanol-based European bio-economy, which we will promote by for the first time applying synthetic biology principles for cell factory development harnessing methanol as a general feedstock for the manufacturing of special and fine chemicals. PROMYSE combines two frontline research topics: orthogonal modularization of methylotrophy within a Synthetic Biology concept and employing methanol as a feedstock for biotechnological production. The PROMYSE project has demonstrated for the first time bioproduction of several industrially valuable products, including platform chemicals and fine chemicals, from methanol. In addition, the PROMYSE project has provided extensive and new insight (genetic, regulatory, biochemical) into bacterial methylotrophy and made the first critical successful steps towards the engineering of this highly novel and complex metabolic trait into non-methylotrophic bacteria. The results and achievements have been presented in so far 22 peer review scientific publications, numerous conference presentations, and one patent.
Project Context and Objectives:
Project context
Microorganisms are widely used as cell factories for production of special, fine, bulk, and fuel chemicals and they typically use sugars and other food derived sources as raw materials. Sugars originating from plants demand cultivable land which is more and more needed for human nutrition. Methanol - with a worldwide production capacity of more than 46 million tons per year - is regarded as an alternative raw material in microbial bioprocesses. The supply of methanol can base upon both fossil and renewable resources, rendering it a highly flexible and sustainable raw material. In nature, methylotrophic microorganisms can utilize one-carbon compounds such as methanol as their sole source of carbon and energy. However, methylotrophic bacteria typically lack certain favourable traits needed by industry for application as microbial cell factories, and in the project PROMYSE the main objective is to engineer methylotrophy to non-methylotrophic bacteria by using Synthetic Biology. More specifically, the chosen host strains are the biotechnologically well-established bacteria Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum.
Methylotrophy is a complex metabolic trait involving a large number of dedicated genes and enzymes and it proceeds via the cell-toxic intermediate formaldehyde. Accordingly, PROMYSE is a challenging and ambitious project. Synthetic Biology is an enabling technology which is expected to contribute to the knowledge-based bio-economy. The cell factory concept, which has been supported by the EU in several frameworks and key actions, lends itself ideal as field of application to capitalize on the potential of Synthetic Biology. PROMYSE combines two frontline research topics: orthogonal modularization of methylotrophy within a Synthetic Biology concept and harnessing methanol as general feedstock for biotechnological production. Functional modules encoding metabolic and production path¬ways are combined to form novel production hosts that are optimised to convert methanol to high value products. The project thus generates knowledge in methylotrophy, the required bio¬chemical components, and reactions. In addition, novel production hosts are created that are based on either established industrial producers or on natural methylo-trophs.
PROMYSE brings together leading European expertise in methylotrophy and biotechnology; moreover, one SME and two industrial partners among its participants secure and evaluate the commercialization potential of synthetic methylotrophic cell factories. Altogether, PROMYSE involves researchers from 3 universities, 3 research institutes, 1 SME and 2 industrial partners. The major product focus in PROMYSE is on terpenoids because they constitute the largest class of natural products covering a multitude of interesting physiochemical properties and biological activities. In addition, we will also focus on producing diaminopentane and dicarboxylic acids from methanol. The PROMYSE project is organized into totally eight Work packages (WPs); 6 RTD WPs (WP1-WP6), as well as dissemination WP7 and management WP8.
Project objectives
The main objective of PROMYSE is to follow a Synthetic Biology approach to cell factory development for harnessing methanol for biotechnological production of value-added chemicals, thus paving the way for our vision of a viable methanol-based European bio-economy. In order to achieve this ambitious goal, the following project objectives were made:
Functional characterization and establishment of methylotrophy modules: to select and characterize methylotrophic pathway modules from native methylotrophic bacteria and transfer them into heterologous hosts
- Assembly and demonstration of functional expression of methanol oxidation modules in Gram-positive and Gram-negative bacterial hosts
- Expression of natural and synthetic formaldehyde dissimilation modules enabling in vivo formaldehyde oxidation in heterologous hosts.
- Expression of natural and synthetic formaldehyde assimilation modules leading to formaldehyde fixation into cell constituents necessary for cell growth of heterologous hosts.
- Physiological analysis of the recombinant strains under controlled conditions.
To functionally integrate methylotrophy modules into the biotechnologically relevant bacteria B. subtilis, C. glutamicum and E. coli
- Evaluation and improvement of engineered strains
- Platform organisms – engineering and integration of synthetic methylotrophy islands
In silico analysis of metabolic networks in the bacterial hosts: to iteratively combine experimental and in silico strategies for the rational design and implementation of synthetic metabolic modules, ensuring controlled and efficient conversion of methanol into key precursors and onwards to selected products, but without disturbing the host system as a whole.
- Development of orthogonal metabolic modules for the efficient conversion of methanol
- Identification of the auxiliary metabolic modifications ensuring high precursors supply from methanol for high products formation in non-methylotrophic hosts (B. subtilis, C. glutamicum, E. coli) and in methylotrophic hosts (B. methanolicus, M. extorquens)
- Iterative optimization of targeted metabolic processes in both natural and synthetic methylotrophs
- Adapting hosts for metabolic efficiency
Design, construct and functionally express synthetic pathways of enzymes catalyzing the conversion of methanol into useful products in native methylotrophs: To design and construct novel microbial cell factories based on the native methylotrophs B. methanolicus and M. extorquens.
- Characterization of the putative MEP pathway in B. methanolicus and elucidation of its potential for terpenoid production from methanol at elevated temperatures.
- Exploitation of the ethylmalonyl-CoA pathway (EMCP) for the production of dicarboxylic acids with M. extorquens
- Engineering the mevalonate (MVA) pathway into M. extorquens for enhancement of the native terpene production capacity
Functional expression of synthetic pathways for conversion of methanol into useful products in synthetic methylotrophic hosts
- Assessment of methanol-based production by synthetic hosts as proof-of-principle
- Proof –of-concept of reciprocity: assessment of methanol-based diamine production by synthetic C. glutamicum
- Assessment of methanol-based terpenoid production by synthetic methylotrophs
- Assessment of methanol-based production of EMCP intermediates by synthetic methylotrophs
Demonstration of potential industrial relevance
- Establishing first fermentation protocols in the bioreactor
- Evaluation of methanol based bioprocess under industrial conditions
Dissemination, demonstration and implementation
- Identification of novel inventions and patent protection
- Provide data on high yield of methanol to product conversion
- To boost the awareness og high ecological and economical value of methanol based synthetic cell factories
Management
- To manage administrative aspects of the project
- Technical management of the project
- Strategic management of the project
Project Results:
The PROMYSE project is now completed and all the efforts, results and achievements are carefully and detailed described in the Periodic Report 1 and Periodic Report 2. Hereunder is a brief presentation summarizing the major results and findings in the totally 8 WPs of PROMYSE. In addition, we emphasise that the PROMYSE results have been presented in so far 22 accepted peer review publications and one patens, as well as broadly presented in a high number of channels and events (see complete dissemination activities listed in the PROMYSE Final Report submitted in parallel to this report). The potential impact and use of the results can be separated in various segments; first, PROMYSE has contributed to extensive and valuable new insight and knowledge on synthetic biology and methylotrophy in general, highly useful for the biotechnology industry and the scientific community. We have experienced increased interest and direct contact from both industry and academia towards end of project, as high frequency of publications has been published by our consortium. A patent established at start of project has attracted increased commercial interest as we have demonstrated usefulness of the key enzymes methanol dehydrogenase for engineering of methylotrophy. In the era of bio economy, efforts to establish alternative raw materials such as methanol for biotechnology usage are attractive, and interest on this is rapidly growing internationally. The project has also educated a number of young and talented PhD candidates and researchers into the field synthetic biology.
PROMYSE is composed of six RTD work packages (WP1 – WP6); For WP1 –WP4 we continue with basis in what was reported in previous Periodic report 1, while for WP5 and WP6 this is naturally then the complete reporting. We hereunder summarize the main achievements and results obtained during the PROMYSE project.
WP1: Functional characterization and establishment of methylotrophy modules
Within WP1 the aim is to characterize and demonstrate biochemical functionality of the core methylotrophy modules at the enzyme level, as a basis for their functional integration into the three hosts E. coli, B. subtilis and C. glutamicum. Methylotrophy as a metabolic trait can be divided into three distinct principle modules, module 1, methanol oxidation into formaldehyde; module 2, formaldehyde dissimilation into CO2; and module 3, formaldehyde assimilation to generate cell mass. In the previous periodic report we had initiated work on the methanol dehydrogenase enzymes and ACT (totally 8 enzymes) and this work has now been completed and published in the second period. In particular we also have completed the successful transfer of all these enzymes into our three non-methylotrophic hosts – E. coli, B. subtilis and C. glutamicum – and demonstrated and further optimized methanol oxidation (the key step for methylotrophy). In particular for E. coli this has been successful and also with strong iterations with work performed in WP2 and WP33 (below) has resulted in significant step towards engineering of methylotrophy into this bacterium (published and patented results). Also, the two initial genes of the RuMP pathway hps and phi are central both for formaldehyde dissimilation (module 2) and assimilation (module 3). Both enzymes could be functionally expressed in all three host organisms, as was validated by in vitro and in vivo assays. We also in this period has performed extensive biochemical and regulatory characterization of a majority of genes and enzymes involved in the central methylotrophic RuMP pathway; the work has been published in several peer review papers and also carefully review in another publication from the PROMYSE consortium at end of project (see publication list in Final Report on this). In total, this project has generated extensive and solid characterization of genes and modules representing all three methylotrophy modules and in close iteration with WP2 and WP3 used to systematically test out their functionality and importance for the engineering of methylotrophy (see below).
WP2: Integration of methylotrophy into non-methylotrophic hosts
The focus in WP2 has been on using biological materials derived from WP1 for the functional integration of all three methylotrophy modules into the heterologous hosts, i.e. module 1 for methanol oxidation to formaldehyde, module 2 to dissimilate into CO2 for energy generation, and module 3 to allow for formaldehyde assimilation to generate cell biomass. Work initiated on transfer of methanol dehydrogenases and act, as well as the two genes phi and phs for formaldehyde fixation was well initiated in periodic report 1 and has been completed and published in periodic report 2. 13C labelling experiments (see WP3) were used as the principal tool to evaluate functionality of modules in the three hosts. The experiments demonstrated that the presence of the minimum set of genes representing the modules leads to incorporation of methanol and formaldehyde into biomass in all three hosts and thus the achievement of the deliverables within the reporting period. Multiple labels in central metabolites were also observed indicative of a fully operable RuMP cycle, and in particular for E. coli host the work has been complete and extensive and was recently published in the high-rank journal Metabolic Engineering (see publication list). Also for the two other hosts, extensive progress has been made and a number of RuMP pathway genes characterized under WP1 has been tested alone and in combinations. The strategies for engineering design has been constantly evaluated as result of extensive proteomic and transcriptome analysis of Bacillus methanolicus growing under relevant conditions, also published work in this second period.
WP3: In silico analysis of metabolic networks
The major focus in WP3 has been to complete the in silico design of metabolic modules to be expressed in the heterologous hosts, as to build metabolic network models of the methylotrophic model strains, in silico optimization of product formation in the non-methylotrophic hosts and systematic integration of wet lab data to improve the Insilico models. These developments included an iterative process between modelling and experiment, in which systems-level investigations of the engineered strains were performed to guide the integration of synthetic methylotrophy and production modules. Genome-scale models of the natural methylotrophic hosts M. extorquens and B. methanolicus were completed, and the capability of methylotrophic growth has been verified for both models. Synthetic production modules that allow for the formation of compounds which are in the focus of WP4 and WP5 were also included in these models. The first genome-scale model for B. methanolicus has been reconstructed on the basis genome data which was provided by the project partner SINTEF. These models were used to define the auxiliary metabolic modifications ensuring high supply of precursors from methanol for efficient product formation in non-methylotrophic hosts. Using 13C labelling experiments we demonstrated that methanol indeed can be incorporated into cell constituents in all three hosts; this is a major milestone in this project and demonstrates that it should be possible to engineer methylotrophy.
WP4: Design, construction and functional expression of synthetic pathways for conversion of methanol into useful products in native methylotrophs
The promising work initiated in WP-4.1 on the construction of expression vectors carrying lysine decarboxylase for 1,5-diaminopentane (cadaverine) production in the gram-positive native methylotrophic bacterium B. methanolicus has been completed and published during this second period. In particular, several additional strains have been constructed in order to understand and improve bottlenecks for cadaverine production in B. methanolicus; the work has solely been performed in shake flask experiments and the best strains were successfully tested under HCDC in WP6. The work also included development of new genetic tools for B. methanolicus. In WP-4.2 we in previous report demonstrated for the first time terpenoid production from methanol by recombinant B. methanolicus strains, and this work has been extended in the reporting period and a more systematic investigation of the genes involved has been made. In WP4.3 the work initiated in previous report on the construction of deletion strains for genes coding for enzymes of the ethylmalonyl-CoA-pathway (EMCP) in the gram-negative native methylotrophic bacterium Methylobacterium extorquens, has been completed and published. In WP4-4 we have constructed recombinant M. extorquens strains that can produce monoterpenoids and sesquiterpenoids giving a proof-of-concept for terpenoid production from methanol. In total, WP4 has demonstrated for the first time production of all targeted product classes from methanol by genetically modified methylotrophic bacteria.
WP5: Functional expression of synthetic pathways for conversion of methanol into useful products in synthetic methylotrophic hosts
This WP was initiated in the reporting period and focused on the feasibility of methanol-based production at different levels of complexity by establishing (1) functional integration of methylotrophy modules available from WP-1 into existing non-methylotrophic production hosts E. coli, B. subtilis and C. glutamicum and (2) functional integration of production modules into synthetic methylotrophs generated in WP-2. Since methanol conversion could be achieved for the three hosts, while growth on methanol as sole carbon source could not (s. other WPs), the work within WP-5 primarily aimed at functional integration of production modules into the hosts E. coli, B. subtilis and C. glutamicum. As test cases, strains for the production of L-lysine, cadaverine and amylase were constructed and are ready to be integrated into (synthetic) methylotrophic host backgrounds. However, emphasis was on the production of terpenoids as well as on the production of intermediates of the EMCP. Synthetic pathways leading to production of terpenoids, a diamine, and organic acids have been implemented into the three hosts. In all three heterologous hosts methylotrophy modules could be established and their operation demonstrated in vivo; besides methanol incorporation into biomass could be shown for all three heterologous hosts, albeit at low rate. The overall aim of this WP to establish methanol-based production based on synthetic non-methylotrophic microorganisms is ambitious; however, if successful it will provide strategies to new routes to produce added-value products from methanol in the context of synthetic biology. The work resulted in several publications (see publication list).
WP6: Demonstration of potential industrial relevance.
This WP was initiated in this reporting period. The aim of this WP was to establish upscale proven methanol based production in shake flask scale (see WP4) to laboratory bioreactor scale including controlled substrate feeding and first downstream processing experiments. Recombinant B. methanolicus strains producing cadaverine were run under HCDC and we obtained for the best strain tested up to 17 g/l – which is a very good production yield – under such conditions; the results have been published in the reporting period. Also for similar testing of recombinant M. extorquens strains producing monoterpenoids and sesquiterpenoids we obtained good production results; we also should mention that such cultivations under carefully controlled conditions offer valuable data used for the evaluation of the strains as well. In addition a theoretical evaluation of methanol based bioprocess under industry relevant conditions has been made.
WP7: Dissemination, demonstration and implementation
In total, dissemination output from PROMYSE has been extensive and of high quality and complete overview of all PROMYSE dissemination activities are listed in the electronic report (Template A1, Template A2, and Template B1). As much of the results have been published in high-rank peer review scientific journals I choose here to provide a complete publication lists (solely accepted publications!) very precisely summarizing the main dissemination achievements for entire project period:
1) Irla M, Neshat A, Brautaset T, Ruckert C, Kalinowski J, and Wendisch VF. (2015). Transcriptome analysis of the thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape. In Press BMC Genomics.
2) Jonas E.N. Müller, Fabian Meyer, Boris Litsanov, Patrick Kiefer, Eva Potthoff, Stephanie Heux, Wim J. Quax, Volker Wendisch, Trygve Brautaset, Jean-Charles Portais, Julia A. Vorholt. (2015). Engineering Escherichia coli for methanol conversion. Metab Eng Jan 14. [Epub ahead of print]
3) Nærdal I., Pfeifenschneider J., Brautaset T. & Wendisch VF. (2015). Methanol-based production of cadaverine by metabolically engineered strains of Bacillus methanolicus. Microb biotechnol. 2015 Jan 23. [Epub ahead of print]
4) Müller JE, Heggeset TMB, Vorholt JA, Wendisch VF, Brautaset T. (2014). Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity productions from methanol. Appl Microbiol biotechnol. Nov 28. [Epub ahead of print]
5) Ochsner AM, Sonntag F, Buchhaupt M, Schrader J, Vorholt JA. (2014). Methylobacterium extorquens: methylotrophy and biotechnological applications. Appl Microbiol Biotechnol. 2014 Nov 30. [Epub ahead of print]
6) Irla M, Neshat A, Winkler A, Albersmeier A, Heggeset TM, Brautaset T, Kalinowski J, Wendisch VF, Rückert C. (2014). Complete genome sequence of Bacillus methanolicus MGA3, a thermotolerant amino acid producing methylotroph. J Biotechnol. Epub ahead of print.
7) Heider SA, Peters-Wendisch P., Beekwilder J. & Wendisch VF (2014). IdsA is the major geranylgeranyl pyrophosphate synthase involved in carotenogenesis in Corynebacterium glutamicum. FEBS J. Epub ahead of print.
8) Ochsner AM, Müller JE, Mora CA, Vorholt JA. (2014). In vitro activation of NAD-dependent alcohol dehydrogenases by Nudix hydrolases is more widespread than assumed. FEBS lett, Epub 2014 Jun 10.
9) Heider SA, Wolf N., Hofemeier A., Peters-Wendisch P. and Volker F. Wendisch (2014). Optimization of the IPP precursor supply for the production of lycopene, decaprenoxanthin and astaxanthin by Corynebacterium glutamicum. Frontiers in Bioengineering and Biotechnology.
10) Wendisch V.F. (2014) Microbial production of amino acids and derived chemicals: Synthetic biology approaches to strain development. Curr. Opin. Biotechnol. 30: 51-58.
11) Frohwitter J, Heider SA, Peters-Wendisch P, Beekwilder J, Wendisch VF. (2014). Production of the sesquiterpene (+)-valencene by metabolically engineered Corynebacterium glutamicum. J Biotechnol. June 6. Epub ahead of print.
12) Sonntag F, Buchhaupt M, Schrader J. (2014). Thioesterases for ethylmalonyl-CoA pathway derived dicarboxylic acid production in Methylobacterium extorquens AM1. Appl Microbiol Biotechnol. 98(10):4533-44.
13) Müller JE, Litsanov B, Bortfeld-Miller M, Trachsel C, Grossmann J, Brautaset T, Vorholt JA. (2014). Proteomic analysis of the thermophilic methylotroph Bacillus methanolicus MGA3. Proteomics. 14(6):725-37.
14) Markert B, Stolzenberger J, Brautaset T, Wendisch VF. (2014). Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. BMC Microbiol. 14:7.
15) Heider SAE, Peters-Wendisch P, Wendisch VF, Beekwilder J, Brautaset T. (2014). Metabolic engineering for the microbial production of carotenoids and related products with a focus on the rare C50 carotenoids. Appl Microbiol Biotechnol. 98(10):4355-68.
16) Stolzenberger J., Lindner S., Persicke M., Brautaset T. & Wendisch V.F. (2013) Characterization of fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase from the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. J. Bacteriol. 195: 5112-5122.
17) Stolzenberger J., Lindner S. & Wendisch V.F. (2013) The methylotrophic Bacillus methanolicus MGA3 possesses two distinct fructose 1,6-bisphosphate aldolases. Microbiol. 159: 1170-1781.
18) Heider S.A.E. Peters-Wendisch P., Netzer R., Stafnes M., Brautaset T. & Wendisch V.F. (2013). Production and glucosylation of C50 and C40 carotenoids by metabolically engineered Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 98(3):1223-35.
19) Krog A, Heggeset TM, Müller JE, Kupper CE, Schneider O, Vorholt JA, Ellingsen TE, Brautaset T. (2013). Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties. PLoS One. 8(3):e59188.
20) Lessmeier L, Hoefener M, Wendisch VF. (2013). Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase. Microbiology. 159:2651-62.
21) Heider SA, Peters-Wendisch P, Wendisch VF. (2012). Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum. BMC Microbiol. 10; 12:198.
In addition, PROMYSE results and efforts have been extensively communicated in also other publication channels (conferences, workshops, meetings, radio, popular scientific journals, public meetings, industry workshops). We also have one patent with an invention at the core of the project – engineering of methylotrophy.
WP8: Management
The Operational Management Team (OMT) in PROMYSE – responsible for both scientific and administrative/economic coordination of the project - was formed from regular offices at SINTEF and consisted of the following representatives:
Name Beneficiary Role
Trygve Brautaset SINTEF Coordinator
Marthe Hagerup Indal SINTEF In charge of adm, legal and financial aspects
Tove L Hunstad SINTEF Management coordinator
The General Assembly (GA) is the highest decision-making body in PROMYSE project and consisted of one representative from each partner:
Name Beneficiary
Trygve Brautaset SINTEF
Volker Wendisch UNIBI
Julia Vorholt ETHZ
Wim Quax RUG
Robert Thummer BASF
Joachim Schmid INSILICO
Jean-Charles Portais INSAT
Audun Goksøyr PROMAR
Jens Schrader DFS /DECHEMA
The Technical Committee (TC) has been responsible for the daily execution of the project and consists of the individual WP leaders as follows:
Name Beneficiary
Tonje MB Heggeset (WP1) SINTEF
Julia Vorholt (WP2) ETHZ
Stephanie Heux (WP3) INSAT
Jens Schrader (WP4) DFS/DECHEMA
Volker Wendisch (WP5) UNIBI
Robert Thummer (WP6) BASF
Wim Quax (WP7) RUG
Trygve Brautaset (WP8) SINTEF
The IPR and Exploitation group (IEG) in PROMYSE, including the GA and the administrative manager is constituted to deal with the strategic issues of dissemination strategy, exploitation, IPR and knowledge management, consists of the following representatives:
Name Beneficiary
Wim Quax (leader) RUG
Trygve Brautaset SINTEF
Volker Wendisch UNIBI
Julia Vorholt ETHZ
Robert Thummer BASF
Joachim Schmid INSILICO
Jean-Charles Portais INSAT
Audun Goksøyr PROMAR
Jens Schrader DFS/DECHEMA
Before the start of the project all partners signed a Consortium Agreement (CA) regulating the work between the parties, the management of the project and the rights and the obligations of the parties concerning inter alia liability, access rights and dispute resolution.
PROMYSE management in general:
The most important forum for planning and monitoring the status of the project are the PROMYSE project meetings which are arranged every 6 months; totally 7 meetings (including the kick-off meeting). These 2 day meetings include presentations among the project members of progress and plans for all on-going activities in each WP, for both plenary and bilateral discussions as well as in the TC. Minutes are written after these meetings and all presentations are put on the e-room available for all project partners. In addition to these meetings there have been several discussions and meetings between two or more partners on the working and WP levels. These include face-to-face meetings, research visits, and telephone and Skype meetings. We have put high emphasis in PROMYSE to establish close and direct contact between the researchers representing the different partners. We have established an internal web-site, a so-called eRoom, at the initiation of the project. This eRoom was recognized at the project start to facilitate the needs for a well-structured common place for storage of all kinds of electronic documents related to the project, like articles, project reports, presentations, minutes of meetings, etc. Document integrity is well assured due to the high degree of security of the eRoom service as well as the ownership and detailed role-based visible/read/write access rights associated with folders and individual documents. The PROMYSE project web-site (http://www.sintef.no/Projectweb/PROMYSE/) was established at the start of the project and has been updated with relevant information since then. Due to IPR issues we have chosen to carefully limit the publication of project results on this public available web site. In addition we have established an internal web-site, a so-called eRoom, see point 3.2.3.5 above.
Co-operation with other projects/programmes:
PROMYSE partners 1, 2, 3, 4 and 5 constituted the consortium of the ESF funded EuroCORE project SynMet (2010 – 2013).There were totally five EuroCORE projects working with synthetic biology and during the 3 year project period there were several highly useful cross project activities arranged, including EuroCORE meetings in Cannes (May 2011), Groningen (November 2012), and Elmau (May 2013) with PROMYSE partners well represented. The PROMYSE Coordinator is member of the EuroCORE scientific boardTogether, this has extended our European network within synthetic biology and new project proposal collaborations are currently initiated. In addition, two PROMYSE partners 1 and 3 are members of the scientific board ERASynBio Scientific advisory board (SAB) and has participated on several workshops and meetings (including participation on the ERASynBio US synthetic biology tour October 2012) to discuss European synthetic biology development, including shaping of the ERASynBio call topic. This has also contributed to new funding possibilities for cross-Atlantic research on synthetic biology. Importantly also; four of the PROMYSE partners have recently received funding for a new ERASysApp project (“MetApp”) with strong basis in parts of the PROMYSE project; MetApp is a 3-year project 2015 – 2017.
Potential Impact:
Industrial biotechnology offers alternative and sustainable production routes for chemicals and this is a rapidly growing sector worldwide. Typically sugar-based raw materials used are in unwarranted competition with the food sector. Accordingly, there is a high interest in using alternative non-sugar based substrates that do not require cultivable land. Today, there is massive research in the field bio refinery aiming at utilizing lignocellulose materials from wood and plants as substrates. Methanol represents an additional attractive alternative and in this regard methylotrophic bacteria are of great interest. Methylotrophy is a complex metabolic trait involving a number of enzymes and pathways that are interlinked with each other. Methylotrophy is modular in its nature consisting of module 1 (methanol oxidation into formaldehyde), module 2 (formaldehyde dissimilation into CO2), and module 3 (formaldehyde assimilation into cell constituents).
The PROMYSE project was divided into totally 6 different RTD WPs – although they consisted of separate tasks, deliverables and milestones, they were much integrated. The work of the 6 RTD work packages is performed in an integrated way by the project partners and most work packages depend on deliverables from other work packages. WP-1 delivers methylotrophy modules for WP-2 which in turn delivers synthetic strains to be used in WP-5. Strains used and knowledge acquired in WP-1 were needed for the implementation of WP-4. Activities in WP-3 were central in the project by linking WP-1 and WP-2 to WP-4 and WP-5. Synthetic strains produced in WP-4 and WP-5 were candidates for the up-scaling and demonstration activities in WP-6.
Within WP1 to WP3 we explore Synthetic Biology strategies as a combination of engineering and testing on the one hand coupled with analysis, predictions and modelling and on the other hand, to generate novel engineered methylotrophs for the generation valuable products from methanol using well established biotechnological relevant hosts. The work in these three work packages is tightly connected: In WP3 we have designed and predicted methylotrophic models in silico for the host bacteria as basis for the engineering efforts made in WP1. Genetic engineering and biochemistry results generated in WP1 were tested in the chosen host strains in WP2 with the common goal of understanding and engineering of methylotrophy. In WP3 we have also performed experimental evaluation of selected strains representing all three hosts from WP1 and WP2 by using labelled methanol and in this way demonstrated that we have succeeded in the construction of recombinant strains with a new feature, i.e. consuming and incorporating methanol into cell constituents. Together, these important results demonstrate that we reached a solid basis to further optimize the engineered hosts to build new methylotrophic bacteria. The work conducted up to now in WP1 to WP3 has provided new and fundamental understanding of the genetic and enzymatic basis for methylotrophy in both gram-positive and gram-negative bacteria. Together, this knowledge will be highly useful for future utilization of methylotrophic bacteria in white biotechnology. In particular, we have made substantial progress on module 1, the key initial methylotrophy step of methanol oxidation. This has to be very efficient and also tightly co-regulated with the remaining modules 2 and 3 to ensure efficient growth and energy generation and at the same time to avoid toxic formaldehyde accumulation. We have in PROMYSE biochemically characterized six different methanol dehydrogenases and their activator proteins, and transferred each of them to the three selected non-methylotrophic hosts. We have unravelled that the three host bacteria displayed different preferences with respect to optimum choice of enzymes representing module 1. These results have been patented and published (see below). In addition, we have combined integration of module 1 with key enzymes representing modules 2 and 3, and in this way managed to construct recombinant strains representing all three hosts that are capable of oxidizing methanol and further incorporating the formaldehyde into downstream cell components. We expect that the knowledge and technology generated in this work package should be a highly valuable basis for all efforts aiming at improving and/or transferring methylotrophy in bacteria, for the overall goal of utilizing methanol as raw material for white biotechnology. Important publications achieved under the combined efforts of WP1 – WP3 are (se complete publication list PROMYSE above): Irla et al 2015; Müller et al 2015; Irla et al 2014; Ochsner et al 2014B; Müller et al 2014; Markert et al 2014; Stolzenberger et al 2013A and B; Krog et al 2013; Lessmeier et al 2012. Of particular notice I would like to highlight the publication Müller et al 2015; this one was accepted for publication in the journal Metabolic Engineering on the 31.12.2104 – the very last day of the PROMYSE project – and it describes the generation of synthetic Escherichia coli cells that can truly metabolize methanol – a key publication for entire PROMYSE project and with co-authors from several PROMYSE partners.
The WP4 activities differ from the work in WP1-WP3 as we are here engineering in native methylotrophic hosts. We have constructed several recombinant B. methanolicus strains that secrete the important platform chemical cadaverine; this is to our knowledge the first demonstration of cadaverine production from methanol. We are currently focusing on improving cadaverine production yields, including also improved export out of the cells. We have also demonstrated for the first time that methylotrophic B. methanolicus has a functional MEP pathway. By introducing a synthetic operon including heterologous genes we have demonstrated that this MEP pathway can be a basis for producing C30 terpenoids from methanol. To our knowledge, terpenoid production from methanol has not previously been described in the scientific literature. We are currently in progress engineering lycopene production into this bacterium as well; lycopene is a key precursor for a high number of commercially interesting carotenoids. In parallel we have also made analogous engineering of the gram-negative native methylotroph M. extorquens and demonstrated that the EMCP is an excellent starting point for the bioproduction of special dicarboxylic acids from methanol. The expected results of the engineering efforts on the native methylotrophs will have high impact on the future bioproduction of platform and fine chemicals from methanol. Important publications achieved under WP4 are (se complete publication list PROMYSE above): Nærdal et al 2015; Sonntag et al 2014
In WP5 the major focus has naturally been on the product modules; in particular we have made very extensive achievements in host C. glutamicum on the exploitation of this industrially important bacterium as a platform cell factory for sustainable production of various carotenoids. We have unravelled the biosynthetic pathway (genes, enzymes, regulation) for the C50 carotenoid decaprenoxanthin, and also demonstrated that this bacterium can be engineered to produce other known C50 carotenoids. We have optimized upstream pathways for higher production yields and also shown that it can produce alternative length C30 and C40 carotenoids. Important publications achieved under WP5 are (se complete publication list PROMYSE above): Heider et al 2014A, B, and C; Frohwitter et al 2014; Heider et al 2013; Heider et al 2012.
In WP6 – which was leaded by industry partner BASF - we have demonstrated up scaled production of chemicals from methanol using genetically engineered methylotrophic bacteria constructed in WP1-WP4 as cell factories. Of particular importance has been to design good cultivation strategies using methanol as C source and to achieve production yields higher than those obtained under small-scale laboratory conditions. In particular, we managed to produce up to 17 g/l of the important platform chemical cadaverine from methanol and at elevated temperatures by using GMO B. methanolicus strains constructed in WP4. Parts of the results of WP6 have been published in Nærdal et al 2015 and Sonntag et al 2014. Finally; the summarized efforts and achievements obtained during the PROMYSE project resulted in substantial new and valuable information and insight into the two model methylotrophic bacteria bacillus methanolicus and Methylobacterium extorquens. Therefore, it was very valuable to summarize all this good work into two back-to-back review articles at the end of PROMYSE project with authors representing PROMYSE partners broad; see references Muller et al 2014 and Ochsner et al 2014A.
Methylotrophy is the ability of certain specialized microorganisms to utilize reduced carbon substrates containing no carbon-carbon bonds as sole sources of carbon and energy. These organisms have been known since the late nineteenth century, and substantial knowledge has accumulated, in particular in the past 50 years. The knowledge in the field can be used to separate methylotrophs according to different aspects such as (i) phylogeny of organisms, (ii) obligate versus facultative methylotrophy, (iii) methylotrophic substrate range, and in particular (iv) operation of sets of enzymes and pathways allowing methylotrophic growth. Phylogenetically, methylotrophs belong to a rather small number of genera within Alpha- (e.g. Methylobacterium), Beta- (e.g. Methylobacillus), and Gammaproteobacteria (e.g. Methylococcus), as well as within the Gram-positives (e.g. Bacillus), and Verrucomicrobia (e.g. Methylacidiphilum). Many of these are facultative methylotrophs that are able to grow on one carbon but also on a generally limited number of multi-carbon compounds. Methylobacterium extorquens and Bacillus methanolicus are representatives of facultative methylotrophs and have the advantage that alternative substrates can be used to identify genes essential for methylotrophic growth. Both bacteria use methanol as reduced one carbon source but not methane which is used by a number of obligate (with few exceptions) methanotrophs of the Alpha- and Gammaproteobacteria as well as members of a genus within Verrucomicrobia. These two bacteria have been the major model organisms used in PROMYSE. A key question to understand methylotrophy is how methylotrophic organisms generate energy and how they convert one carbon substrates to carbon compounds with carbon-carbon bonds required for their own living. The knowledge has been gathered from enzymatic characterizations, pathway elucidation via labelling experiments, genetic and genomic approaches. Taking a level of abstraction, methylotrophy can be regarded as a set of discrete specialized functional modules that are ultimately linked to central metabolism. This is concluded from the presence of alternative enzymes or pathways for specific metabolic goals in different methylotrophic bacteria and also the existence of different combinations of these systems. These specific “metabolic goals” are (1) oxidation of the primary substrate (e.g. methanol) to formaldehyde; (2) oxidation of formaldehyde to CO2, and (3) assimilation of one carbon compounds, the latter taking place either at the level of formaldehyde (as free formaldehyde or as methylene tetrahydrofolate (H4F)) or CO2 or a combination thereof.
Synthetic Biology is regarded as an emerging enabling technology which will contribute to fostering the knowledge-based bio-economy. The cell factory concept, which has been supported by the EU in several frameworks and key actions, lends itself ideal as field of application to capitalize on the potential of Synthetic Biology. The main objective of PROMYSE has been to follow a Synthetic Biology approach to cell factory development for harnessing methanol for biotechnological production of value-added chemicals, thus paving the way for our vision of a viable methanol-based European bio-economy. PROMYSE combined two frontline research topics: orthogonal modularization of methylotrophy within a Synthetic Biology concept and harnessing methanol as general feedstock for biotechnological production. The potential of natural methylotrophs for use in industrial biotechnology remains to be fully explored. Still, established bioprocesses in this industry typically employ non-methylotrophic organisms as cell factories and carbohydrates as feedstock. The emerging concept of Synthetic Biology offers the possibility for a rational advancement of the field paving the way to methanol-based biotechnology. In PROMYSE, orthogonal methylotrophy modules has been identified, characterized, optimized and transferred to non-methylotrophic production strains. In parallel, methylotrophic hosts have been endowed with production modules derived from established producing hosts by functional integration into the metabolic network. The synthetic cell factories developed within PROMYSE have been designed to enable utilization of methanol for the production of fine and bulk chemicals. Systems Biology is in demand to understand the biological meaning and interconnectivity of metabolic or other networks. The Synthetic Biology approaches we have used in PROMYSE was combined with systems level understanding in a way that has totally changed the perspective we are currently having on methylotrophy. Within this project we used modelling as the starting point to analyze metabolism, and we used in silico models to find optimal solutions for maximum growth and later precursor formation for product formation. Feeding the in silico models with naturally existing parts allowed us to explore and learn from this process what optimal solutions are and where the limits are. In particular, we have learned from comparing predictions generated through modelling with experiments.
Using methanol as an alternative non-food feedstock for biotechnological production offers several advantages in line with a methanol-based bioeconomy. There is a high societal demand for a sustainable production of special, fine, bulk, and fuel chemicals, including food and health care compounds. Biotechnological processes will play a prominent role in the coming bioeconomy era by gradually complementing and substituting petrochemical synthesis. In biotechnology, microorganisms are widely used as cell factories and, in particular for high-volume products, raw material costs make up a large part of process costs. In White Biotechnology mainly sugars and molasses are used as carbon sources, and these raw materials are derived from plants demanding cultivable land which is more and more needed for human nutrition. The possibility to utilize non-food raw materials, such as one-carbon (C1) substrates like methane (CH4) and methanol (CH3OH), as alternative feedstock has therefore gained high scientific interest but is not yet implemented at commercial scale. Major reasons for this are that methanol is still a more expensive substrate than sugar (worldwide methanol prizes at Methanex: https://www.methanex.com/our-business/pricing) and that methanol fermentations can be technically challenging; for example high O2 requirements and concomitant heat production may cause increased cooling requirements, and careful substrate feeding is needed to avoid toxic formaldehyde accumulation in the cells (see below). However, substantial progress on fermentation technology and construction of production strains should enable commercialization of methanol based bioprocesses in the near future, as reviewed here and elsewhere.
Methanol is a pure and non-food chemical that is soluble in water and it is completely utilized during microbial fermentations. Methanol should thus represent an attractive alternative raw material for biotechnological processes, both from an economic, ecologic, and process point of view. With a worldwide production capacity of more than 53 million tons per year, methanol is one of the most important raw chemicals on earth. The supply of methanol can base either upon fossil or renewable resources, rendering it a highly flexible and sustainable raw material. Today, almost all methanol worldwide is produced from syngas, a fuel gas mixture consisting of H2, CO and CO2, obtained from incomplete combustion of natural gas. In addition, new ways for methanol production directly from natural gas, carbon dioxide or biogas are being developed, and mega-methanol production facilities (5,000 tons per day) are now being constructed in regions rich in natural gas.
The products we have focused on in PROMYSE are terpenoids (including carotenoids), platform chemicals and some fine chemicals – all with high commercial interest. There is today a rapid transformation globally from chemical to biotechnological production processes for a wide range of chemicals; and in parallel with this there is a growing interest and need in finding alternative non-food raw materials for the biotechnology sector; PROMYSE has delivered solutions, technology, knowledge and innovations to both these two challenges.
PROMYSE has brought together a balanced and well-functioning consortium from many different European countries, including complementary competence and infrastructures – all needed to achieve the project goals. Training of young scientists, PhDs and postdoc has been good as they have been part of a multidisciplinary project consortium and ideas and insight has been shared via meetings and e-mail correspondence, co-publications and guest visits. Also, the involvement of industry partners has been important to constantly also look into the commercial and innovative sides of biotechnology research. One important added-value of this collaboration is that 5 of the project partners have recently received funding for a new ERASysAPP project in systems biology, the new project denoted “MetApp” has a solid basis in much of the results, knowledge and biological materials generated in PROMYSE.
All dissemination activities of the PROMYSE project has been listed and described in the electronic final report – much of the peer review publication achievements are also described and explained above. In total, we believe that the output has been very good in terms of publications, one key patent, and various dissemination activities. The project has attracted broad international interest as we have been contacted from both academia and industry requesting for protocols and genetic/biological materials and strains. Also, researchers have been established at the international arena as important contributors to the field synthetic biology; we have been requested as members of various scientific advisory boards, invited to popular scientific meetings and workshops in synthetic biology / bioeconomy, and also invited specifically to write book chapters in synthetic biology, and also contacted for interviews in radio and newspapers. The PROMYSE project has been popular also among master students to take their thesis associated with the science going on in this key project for the partners involved.
List of Websites:
PROMYSE public web site: http://www.sintef.no/Projectweb/PROMYSE/