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Ultra High-throughput platform for the selection of thermostable proteins by thermophilic in vitro transcription-translation and microfluidics

Final Report Summary - HOTDROPS (Ultra High-throughput platform for the selection of thermostable proteins by thermophilic in vitro transcription-translation and microfluidics)

EXECUTIVE SUMMARY
The HOTDROPS project was an EC-funded Marie-Curie program initiative aimed at the training of industry and academic researchers around the collaborative development of a new platform for the high-throughput selection of thermostable enzymes using microfluidics. Four academic institutions (Universities of Coruña, Autónoma de Madrid, Vigo and Cambridge) and four industrial partners (Novozymes, Droptech, Biochemize, Galchimia) participated in this initiative through a total of 98 months of intersectoral secondments which involved 14 researchers and the recruitment of 8 Marie Curie postdoctoral fellows. The first part of the project was focused on training and standardization of assays at high temperatures in microfluidic format, development of libraries, in vitro transcription translation systems derived from thermophiles, construction of metagenomic libraries in fosmids from thermal effluents and of variants libraries in folding interference fluorescence-selectable vectors. During the second period, three thermostable enzymes derived from the metagenomic libraries were produced at lab-scale and biochemically characterized. The potential use of one of the selected enzymes, in an industrial context was studied by the industrial partners of the project. In addition to the personnel specifically trained in this action, the overall results of this project have allowed the development of a selection platform for thermostable enzymes both in a thermophilic host and also in a thermostable in vitro transcription translation system for its further use in future screenings.

PROJECT CONTEXT AND OBJECTIVES
Industrial processes for the manufacture or modification of chemicals and raw stuffs through “classic” chemistry and catalysis often involve the use of hazardous substances and substantial amounts of energy. In contrast, natural enzymes are able to carry out these reactions under “mild” reaction conditions with exquisite selectivity and velocity. Therefore, the application of enzymes to catalyze industrial reactions, known as “biocatalysis”, has the potential to make chemical processes less contaminant and more sustainable. Unfortunately, reaction conditions in industrial biocatalysis are usually far from those found in nature by most common enzymes, frequently using non-natural substrates at high concentrations, in the presence of cosolvents, and at high temperatures. Therefore, enzyme discovery in organisms withstanding extreme conditions and/or thermostabilization by engineering is needed to obtain stable biocatalysts for each specific process under industrial operational conditions.
Thermal environments are currently viewed as potential sources of enzymes for biocatalysis because enzymes of the resident microbiota (thermophiles) are adapted to harsh living conditions also present in industrial processes, such as high temperatures. Moreover, thermostability affords not only resistance against thermal denaturation, but also against denaturation caused by organic cosolvents. However, the vast majority of thermophiles corresponds to unculturable microbes. To cope with this, metagenomic libraries are constructed. These libraries consist in a collection of DNA fragments, obtained from the environment, corresponding to both cultivable and non-cultivable microbes, which are cloned into a DNA vector that replicates in a bacterial of yeast host. The whole metagenome can be directly sequenced through massive sequencing (NGS) for further search by bioinformatics tools for the biocatalyst. However, such screens rely on comparisons to previously known enzymes thus yielding biocatalysts that are similar to what is already known. In contrast in the HOTDROPS project, functional screenings are used, allowing for the discovery of completely novel enzymes. In order to find these new “rare” activities, high-throughput functional screening methods are required.
The expression of thermophilic enzymes in mesophilic host systems is not always possible, due either to the absence of the appropriate chaperone or an excessively fast folding rate compared to that at the natural temperature of expression. In addition, the promoters of genes from thermophiles are in many cases poorly recognized by the RNA polymerase of conventional hosts such as E. coli. Due to this, alternative thermophilic hosts have been proposed and used for the expression of thermozymes. Thermus thermophilus is by far the best laboratory adapted thermophile for this purpose, as it grows fast both under aerobic and anaerobic conditions and shows a highly efficient natural competence system that has allowed the development of a quite complete genetic toolbox. For this reason, in the HOTDROPS project it was a major aim the use of this organism and of its gene expression machinery for the screening of thermozymes from metagenomes of thermal environments.
However, a thermophilic counterpart for every industrially interesting enzyme is likely not extant in the restricted natural diversity of thermal environments. An alternative consists in the isolation of more thermostable variants of an already known enzyme through protein engineering. Although successful in a few cases, the rational prediction of thermostability is a complex task, and directed evolution on a protein scaffold is preferred. However, this strategy implies the accumulation of mutations in a single individual leading to an exponential increase in the size of the library to be screened in order to find these rare variants (10e8 to 10e9 individuals).
The throughput to screen depends strictly on the equipment available: usual screens are in the order of 103 assays/day, and with the help of robotics 104-105 assays/day depending on the complexity of the assay. Thus, the screening of libraries of natural or man-made diversity has been particularly hindered by the lack of ultrahigh-throughput screening methods that are independent of complex and expensive equipment. This is the gap that the HOTDROPS project intended to fill by using microfluidics and microdroplets-based assays for screening.
Microfluidics entails the manipulation of fluids in micrometric-scale, allowing for the miniaturization and versatility of the assays. The miniaturization of assays to the size of picoliter droplets results in an increase in throughput as well as the reduction of assay costs and time for screening by roughly 1000 fold. By using microfluidcs devices, it is possible to generate monodisperse emulsions of water-in-oil microdroplets at rates of thousands per second, each of them acting as individual assays. Once the enzyme reaction develops, the positive reactions can be sorted either in chip or by FACS also at high rates (thousands per second). In fact, a library of 10e7 individuals can be encapsulated in 30 minutes and screened in 1 hour, although faster screening times have been reported in the literature.
Consequently, the scientific objective of the HOTDROPS project aimed to demonstrate the unequaled capacity of droplet microfluidics to substitute conventional enzyme assays in order to create a screening platform to identify new enzymes or new variants of enzymes at high temperatures. In addition, as a Marie Curie program, this project wants to promote a tight collaboration between academic and industrial partners and to promote the transfer of knowledge between both sectors through intersectorial training and exchange programs.

DESCRIPTION OF THE MAIN RESULTS IN THE HOTDROPS PROJECT
1. Development of thermostable in vitro transcription and translation system. - The use of in vitro transcription and translation system alleviates the limitations on the size of the libraries imposed by the reduced efficiency of natural competence, and decreases the putative toxicity that could be produced by the expression of specific enzymes in living cells. On the other hand, the sensitivity for the detection of activities from a single gene copy or the recovery of positive clones are hurdles in the development of a fully in vitro screening platform.
A pure-component system derived from Thermus spp was attempted following published work (Zhou et al 2012 , Nucleic Acids Res. 40:7932-45). Along the development of the system 34 proteins corresponding to the translation apparatus of T. thermophilus and an energy regeneration system were overproduced. Also, tRNA and ribosomes from T. thermophilus were purified to reconstitute a thermostable IVT system. Transcription was coupled through the adding of a recombinant Thermus aquaticus RNA polymerase that was also produced and purified. Evidence of the functionality of most of these components was obtained either by assaying their enzymatic activity or by replacement of the corresponding components in E. coli Pure© IVTT extracts. However, the RNA polymerase showed insufficient activity and two recombinant amonoacyl-tRNA synthetases where not active at all. These enzymes are currently being produced recombinantly using different strategies to implement a complete pure-component system.
In parallel, we succeeded in obtaining an active IVT system based on S30 extracts from T. thermophilus. Coupling of this IVT to the recombinant transcription machinery from T. aquaticus (RNA polymerase) was not successful, whereas a native RNA polymerase produced significant IVTT activity at 50 °C. Nevertheless the efficiency of the coupled IVTT using native RNA polymerase was insufficient for a screening platform. Due to this, we replaced the bacterial RNA polymerase by a thermostable variant of a viral polymerase, allowing the coupled IVTT system to reach a high activity at 50 and 55 °C, thus setting the basis for the in vitro selection at high temperatures in this project and in future work.
2. Metagenomic and variant libraries. - Metagenomic libraries from water and mud were obtained from the Rio Caldo hotspring (77 °C, pH8). Metagenomic libraries were constructed in the bifunctional Thermus-E. coli pCT3FK fosmid (Angelov, et al, 2009. Syst. Appl. Microbiol. 32, 177–185) with about 130,000 clones each with average DNA insertions of 35 kb and 43 kb. The library has been transferred to a mutant strain of T. thermophilus defective in the main esterases and in a major beta-galactosidase (Angelov, et al, 2009. Syst. Appl. Microbiol. 32, 177–185), to reach 112,000 clones (mud) and 75,000 clones (water).
In addition, the metagenome of Río Caldo soil samples was sequenced for diversity studies by using Illumina Hiseq1500 technology. Metagenome sequences consisted of 94,562,020 sequences with a meant length of 100 bps. Over 74% of these reads were assembled into 117,315 contigs with from 200 bp to 272.5 kbp, N50 length of 2.8 kb, and total contig length of 148 Mbp with a 61% G + C content. Community analysis revealed that 95.4% of sequences belonged to bacteria, while 3.9% were derived from Archaea, 0.4% from Eukaryota and 0.05% from viruses. Chloroflexi (23%), Firmicutes (19%) and Proteobacteria (19%) were the most abundant bacterial phyla. Euryarchaeota (46%) dominated sequences classified to Archaea. The most abundant genera were Thermomicrobium (8.1%), Thermus (6.6%), Sphaerobacter (5.9%), Roseiflexus (3.1%), Rhodothermus (2.8%), Thermaerobacter (2.4%), Bacillus (2.1%) and Paenibacillus (2.0%), consisting 33% of all classified sequences in the metagenome. Plasmid-based libraries were also constructed as further selection makes them easier to recover.
Semi-rational and fully randomized variants libraries of a yeast beta-galactosidase and a bacterial esterase were also constructed for selection of activity and thermostability through the microfluidics platform both in vivo and in vitro.
3. Development of folding interference vectors for the selection of thermostable protein variants. - Folding interference allows for the selection of thermostable variants of a given protein fused to a reporter that can be selected for. In the HOTDROPS project, a series of folding interference vectors in which the reporter gene was a thermostable fluorescent protein were constructed. These vectors permit the discrimination of thermostable variants of a bacterial esterase used as model in thermophilic IVTT system, and can be used for the high-throughput selection of variants of other proteins in the microfluidic platform.
4. Workflow for microfluidics enzyme assays at high temperatures. - Enzyme assays at high temperatures in water in oil microdroplets represent a complicated challenge due to factors such as the stability of the microdroplets at high temperatures and also to the leakage of substrates and products through the water-oil interface. An additional challenge was also the lysis of thermophilic host cells at high temperatures.
In this context, we checked for a series of fluorinated oils and surfactants to check the range of temperatures at which the microdroplets were stable under prolonged heating. Basic protocols have been defined using hyperfluorinated HFE7500 oil and specific surfactants in single water-in oil droplets. However, specific details have to be adapted to each specific experiment, because factors such as inhibition of the enzyme activity by components used in the emulsion require further specific adaptations. The thermostable single emulsion droplet system developed is compatible with both thermophilic and mesophilic extracts (such as IVTT), and is not toxic for the cells, allowing the growth of thermophilic bacteria for 24 hours at 70 °C to form colonies inside the droplets. The activity of thermostable model lipases (BTL2) and beta-galactosidase (BglA), were used to demonstrate that the production of fluorescent products (fluorescein), which can be followed for long time at 70 °C in microdroplets .
5. Workflow for thermozyme screening at high temperatures. - Screening protocols have been stablished within the HOTDROPS project for the selection of thermozymes in vivo in microdroplet format using T. thermophilus cells as expression hosts. The main conclusion from the in vivo screening methods for esterases and beta-galactosidases is that detection of these activities from a single cell containing a single copy of the searched gene integrated into the genome (bifunctional fosmid libraries) seems not feasible due to the low activity level detected compared to the background of the cells. Background activity can be decreased by using as specific mutants of T. thermophilus lacking the major interfering activities as host, whereas the increase of the signal-to-noise ratio from a single cell requires expression from a shuttle multicopy plasmid, such as pMK184. Also, the recovery of positive hits is easier with plasmid-based libraries. Thus, workflows for selection of lipolytic and beta-glycolytic activities in microdroplet format were stablished for enzymes expressed from a multicopy plasmid.
Protocols for the detection of IVTT-expressed enzymes were also stablished, but detection required between at least 100 copies of the gene to be expressed. This drawback implies that a prior step of DNA amplification inside the droplets was needed for the selection of single variants of a given gene in vitro. The low compatibility of the amplification methods (RCA, emulsion-based) with the IVTT extracts have yet to be overcome to make this selection a real possibility.
6. Selected enzymes and putative applications. - New thermozymes were selected from the metagenomic libraries. A thermostable esterase/lipase (LipD11) active both in E. coli and in T. thermophilus was one of the most promising enzymes discovered. Also a putative Xylanase (XylA3) and endoglucanase (CelB4) were functionally identified from the metagenomes of thermal origin. Amino acid homology of LipD11 showed maximum identity (68%) with the esterase/lipase EstC23 derived from a soil metagenome in China. Amino acid homology of XylA3 showed maximum identity (72%) with the thermostable xylanase genes of Paenibacillus campinasensis. Amino acid homology of CelB4 showed maximum identity (74%) with the thermostable beta-1,4-glucanase genes of Clostridium cellulolyticum.
Further characterization has been carried out mainly with LipD11, for which a scalable and highly efficient system for the production and purification was developed. Also, reactions with methyl/ethyl esters in biphasic systems or organic media supported the potential utilization of LipD11 in organic synthesis. Differential Scanning Fluorimetry (DSF), revealed a moderate to high thermal stability, with melting point (Tm) at 69 °C. Some additives such as DMSO, an organic solvent routinely used in organic synthesis reactions, or detergents, increased the melting point towards higher temperatures. A panel of substrates synthesized by HOTDROPS industrial partners showed for LipD11 significant activity towards industrially and pharmacologically relevant substrates.
In collaboration with external groups (Dr. JA Hermoso, CSIC, Madrid) crystals of LipD11 have been obtained at 1Å resolution. Results obtained from these crystals will be of enormous value to understand the catalytic mechanism of LipD11 and conformational changes involved in the catalysis for its further industrial use. Additionally, amino acid residues which are important for specificity and stability will be revealed, and could open a new window for a protein engineering and modifications of LipD11 in order to enhance its industrial potential.

POTENTIAL IMPACT OF THE HOTDROPS PROJECT
Droplet microfluidics has the potential to be a truly disruptive technology and the new gold standard for screening due to its unprecedented throughput and relatively low cost compared with expensive robotic screening. However, two major roadblocks prevent the realization of its full potential, both of which we have addressed in this project: the lack of a fully in vitro workflow that reproduces the central dogma of biology in droplets and the lack of workflows adapted to the peculiarities of screening for thermozymes, which are coveted for biocatalysis due to their intrinsic stability. To the best of our knowledge, neither of the two concepts has been published yet. Furthermore, through our strategic academy-industry collaborations, we have access to both academic researchers and companies at the forefront of microfluidics and metagenomics.
Demonstrating the utility of our approach towards enzymes involved in this project targets growing markets such as industrial biocatalysis. The robust system to screen thermostable variants of enzymes of their interest is of strategic value to European companies such as Novozymes. Specifically, panels of thermozymes will generally exhibit superior tolerance to not only higher temperatures, but also many organic solvents and extreme pH values frequently employed in industrial biocatalytic processes.

MAIN DISSEMINATION ACTIVITIES
The HOTDROPS website is publically accessible at http://hotdrops.cbm.uam.es/. Besides, information on HOTDROPS project has been posted in two websites (Sociedad Española de Microbiología and Center for Molecular Biology Severo Ochoa), and the seminars and workshops organized through the HOTDROPS project were announced in different physical and digital platforms.
One of the most societal and scientifically rewarding outreach activities of the HOTDROPS project was the organization of four workshops along the project activity. The first workshop was hosted by UAM on May 19th-23rd 2014 in the form of a theoretical-practical summer school, and focused on genetics of thermophiles. Students and early researchers of HOTDROPS partners, as well as external students attended this course. In 2015, further workshops were held at the University of Cambridge (“Microfluidics fundamentals and applications” and “Innovation and entrepreneurship for scientis”) and the University of Vigo (“Spring waters as a source of new enzymes for the industry”), the latter integrated in a wider meeting, with attendance of several Ph. D. students.
HOTDROPS has been represented by several researchers in scientific events, as both oral communication or poster. Our presence in these events has allowed us to promote the HOTDROPS project among a heterogeneous audience, including the scientific and entrepreneurial sector as well as many representatives from companies and research institutions. During the first period HOTDROPS partners attended conferences in Portugal (ML Rúa, 1 contribution), Spain (AL Ribeiro, 2 contributions; K Knapik, 1 contribution), Rusia (AL Ribeiro, 1 contribution) and USA (A Hidalgo, 1 contribution).
Among others, the results generated in the second half of the project were presented in 5 communications in Spain (J Berenguer, November 2015; AL Ribeiro et al., July 2015; L van Vliet, November 2015) and Chile (A Blesa et al., September 2015) during 2015; 7 communications in Germany (F Hollfelder, April 2016; AL Ribeiro et al., September 2016), Spain (J Rajkovic et al., October 2016; M Becerra, September 2016; E Beneventi, October 2016) and Denmark (ML Rúa, July 2016; M Becerra, July 2016) during 2016, and 1 in Slovenia in 2016 (F Hollfelder, April 2017). Additionally, and as a result of the activities during the second period, there are already 10 articles published and four sections in edited books.

EXPECTED EXPLOITATION OF RESULTS
Preparing for a future valorization of results, we are considering commercialization and licensing some of the results, specially the enzymes and the platform itself, and the IPR issues for these accomplishments are under study. In addition, the results obtained along the HOTDROPS project constitute the basis of the H2020 METAFLUIDICS project coordinated by UAM, aimed to further develop this screening platform and to extend it to additional extremophilic environments for the selection of extremozymes for bioenergy and agricultural waste valorization.