Final Report Summary - ATRIEM (Advanced Training in Industrial Enzyme Manufacturing)
To ensure that the European biotech industry has access to graduates with the appropriate skills and expertise, it is essential to foster strong collaborations between industry and academia, and this is the objective of this doctoral training programme. Newcastle University (UK) has partnered with the Royal DSM N.V. biotech company (The Netherlands) to deliver a European Industrial Doctorate (EID) programme entitled: Advanced Training in Industrial Enzyme Manufacturing (ATRIEM). The Early Stage Researchers (ESR) employed on the ATRIEM project have both obtained high-quality training in molecular and cellular microbiology techniques and relevant work and research experience in an industrial environment. The project was focussed at two important limitations encountered during industrial enzyme production: 1. Differences in the productivity of individual cells during fermentation (cellular heterogeneity) and; 2. Bottlenecks in the secretion of heterologous enzymes.
Scientific results
Overall the research has gone according to plan, and the work programme has been exceeded in a number of areas. A technical issue prevented the use of a new protein tagging system to monitor the synthesis and release of secreted enzymes by microscopy in real time and this was replace by an existing and well established pulse-chase technology. Both ESRs have written and submitted their theses and will be examined in January 2017.
Project 1. Cellular heterogeneity. ESR1, Chris Sauer
ESR1 began his project by constructing an array of fluorescent promoter reporter fusion to investigate how individual cells respond to high-level enzyme production. Eight promoters were chosen based on stress indicators that have been published in the literature and transcriptome data provided by DSM. The promoters were associated with different physiological and differentiation pathways. The reporter fusions were tested at DSM under production-like conditions in a highly parallel fashion, using a microfermentation system. Cell-to-cell differences in their expression were also studied. This facilitated the monitoring of gene expression in the population as a whole and on a cell-by-cell basis by fluorescent microscopy and by flow cytometry. Changes in the the expression profiles and population heterogeneity were determined in response to the production of two model industrial enzymes, one native to the Bacillus subtilis host, the other a heterologous proteins from another bacterium. The data showed that, in the case of the native enzyme, no significant differences in Gfp expression were observed at the population level of the eight physiological and developmental pathways between the producing and non-producing strains, and this was confirmed at the single-cell level. In contrast, the strains expressing the heterologous enzyme showed very clear differences with respect to a key production bottleneck, both at the population and single-cell levels. The stress brought about by the heterologous enzyme was studied in detail in strains with a variety of genetic backgrounds and using a combination of reporter genes. This allowed a direct link to be established for individual cells between enzyme production and secretion stress.
Project 1 was extended by developing two additional lines of research. Together with DSM a randomized promoter library was constructed to find optimal promoters. By cloning these promoters upstream of a gfp reporter gene, the ESR was able to select clones encoding promoters with a very wide range of activities. Their expression was monitored at the population level and at the single cell level using FACScan technology. DSM is still evaluating the relationships between the promoter sequences and their activity.
It has been known for some time that recombinant gene expression is influenced by their location on the chromosome. In the second extension to Project 1, ESR1 used a Newcastle-developed a gene cassette that randomly integrates reporter genes to different locations on the chromosome. Using the divergently located reporters, the expression levels at different chromosomal locations were compared directly, and this showed 5-fold differences in expression that was based on the cassette integration site. This work has been published in the journal ASC Synthetic Biology.
Project 2. Production bottlenecks.
We have previously identified secretion stress and the presence of quality control proteases to be a major production bottleneck for the synthesis and secretion of heterologous enzymes. Using the same model enzymes as in Project 1, the impact of secretion stress on productivity has been quantified in detail, by studying the impact of enzyme production on secretion stress and the impact of mutations in the quality control enzymes on both enzyme production and secretion stress. The results again showed that the native enzyme had little impact on secretion stress compared with the non-producing strains, even during high-level production. In contrast, high level heterologous enzyme production increased section stress dramatically. The data show the importance of co-evolution between a native enzyme and its natural host, and point to novel strategies for improving productivity. Project 2 was extended by the use of a Synthetic Biology approach to engineer the genes encoding the components of the secretory translocase, the molecular machine responsible for transporting the enzymes across the cell membrane, into single operons. Several synthetic inducible expression cassettes were designed and constructed, and their impact on the secretion of the model enzymes evaluated.
The Atrium project achieved all of its goals: Both ESRs have completed their PhD programmes and submitted their theses for examination in January 2017. Both ESRs have developed expertise and knowledge that is relevant to the European Industrial Biotechnology and Biomanufacturing Industries A great deal of industrially-relevant information about production bottlenecks has been identified, leading to potentially novel strategies for improving productivity.
Scientific results
Overall the research has gone according to plan, and the work programme has been exceeded in a number of areas. A technical issue prevented the use of a new protein tagging system to monitor the synthesis and release of secreted enzymes by microscopy in real time and this was replace by an existing and well established pulse-chase technology. Both ESRs have written and submitted their theses and will be examined in January 2017.
Project 1. Cellular heterogeneity. ESR1, Chris Sauer
ESR1 began his project by constructing an array of fluorescent promoter reporter fusion to investigate how individual cells respond to high-level enzyme production. Eight promoters were chosen based on stress indicators that have been published in the literature and transcriptome data provided by DSM. The promoters were associated with different physiological and differentiation pathways. The reporter fusions were tested at DSM under production-like conditions in a highly parallel fashion, using a microfermentation system. Cell-to-cell differences in their expression were also studied. This facilitated the monitoring of gene expression in the population as a whole and on a cell-by-cell basis by fluorescent microscopy and by flow cytometry. Changes in the the expression profiles and population heterogeneity were determined in response to the production of two model industrial enzymes, one native to the Bacillus subtilis host, the other a heterologous proteins from another bacterium. The data showed that, in the case of the native enzyme, no significant differences in Gfp expression were observed at the population level of the eight physiological and developmental pathways between the producing and non-producing strains, and this was confirmed at the single-cell level. In contrast, the strains expressing the heterologous enzyme showed very clear differences with respect to a key production bottleneck, both at the population and single-cell levels. The stress brought about by the heterologous enzyme was studied in detail in strains with a variety of genetic backgrounds and using a combination of reporter genes. This allowed a direct link to be established for individual cells between enzyme production and secretion stress.
Project 1 was extended by developing two additional lines of research. Together with DSM a randomized promoter library was constructed to find optimal promoters. By cloning these promoters upstream of a gfp reporter gene, the ESR was able to select clones encoding promoters with a very wide range of activities. Their expression was monitored at the population level and at the single cell level using FACScan technology. DSM is still evaluating the relationships between the promoter sequences and their activity.
It has been known for some time that recombinant gene expression is influenced by their location on the chromosome. In the second extension to Project 1, ESR1 used a Newcastle-developed a gene cassette that randomly integrates reporter genes to different locations on the chromosome. Using the divergently located reporters, the expression levels at different chromosomal locations were compared directly, and this showed 5-fold differences in expression that was based on the cassette integration site. This work has been published in the journal ASC Synthetic Biology.
Project 2. Production bottlenecks.
We have previously identified secretion stress and the presence of quality control proteases to be a major production bottleneck for the synthesis and secretion of heterologous enzymes. Using the same model enzymes as in Project 1, the impact of secretion stress on productivity has been quantified in detail, by studying the impact of enzyme production on secretion stress and the impact of mutations in the quality control enzymes on both enzyme production and secretion stress. The results again showed that the native enzyme had little impact on secretion stress compared with the non-producing strains, even during high-level production. In contrast, high level heterologous enzyme production increased section stress dramatically. The data show the importance of co-evolution between a native enzyme and its natural host, and point to novel strategies for improving productivity. Project 2 was extended by the use of a Synthetic Biology approach to engineer the genes encoding the components of the secretory translocase, the molecular machine responsible for transporting the enzymes across the cell membrane, into single operons. Several synthetic inducible expression cassettes were designed and constructed, and their impact on the secretion of the model enzymes evaluated.
The Atrium project achieved all of its goals: Both ESRs have completed their PhD programmes and submitted their theses for examination in January 2017. Both ESRs have developed expertise and knowledge that is relevant to the European Industrial Biotechnology and Biomanufacturing Industries A great deal of industrially-relevant information about production bottlenecks has been identified, leading to potentially novel strategies for improving productivity.