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The creation of a synthetic methylotrophic Bacillus subtilis for the sustainable production of high value chemicals
Abstracts / New Biotechnology 33S (2016) S1–S213
nology for the production of both fine and commodity chemicals. Indeed, Nature has evolved a number of methods to produce such conditions, most notably through the evolution of bacterial microcompartments where metabolic enzymes are encased within a semi-permeable protein membrane. We have developed recombinant bacterial microcompartments to allow for the design of specific nano-bioreators, which contain specific pathways of choice. We have also deconstructed the bacterial microcompartment to allow for the development of protein scaffolds onto which metabolic enzymes can be tethered. These and methods for the production of both internal and external lipid vesicles will be discussed. http://dx.doi.org/10.1016/j.nbt.2016.06.812
O7-2 Chassis organism from Corynebacterium glutamicum – Genome reduction as a tool toward improved strains for synthetic biology and industrial biotechnology Meike Baumgart ∗ , Simon Unthan, Andreas Radek, Marius Herbst, Daniel Siebert, Natalie Brühl, Anna Bartsch, Michael Bott, Wolfgang Wiechert, Kay Marin, Stephan Hans, Reinhard Krämer, Gerd Seibold, Jörn Kalinowski, Christian Rückert, Volker F. Wendisch, Stephan Noack, Julia Frunzke Forschungszentrum Juelich, Germany Synthetic biology projects for the rational design of novel biotechnological production strains require a robust and predictable strain background to allow the successful integration of new functions. These chassis organisms can either be built by de novo synthesis or derived by top-down approaches by sequential deletion of non-essential genes. Here we describe the construction of a chassis based on the industrial amino acid producer Corynebacterium glutamicum. The aim of this study was the definition of a so called “relevant gene set” for C. glutamicum which maintains its fast growth and high cell density in defined medium. Sequential deletion of the non-relevant genes should lead to a C. glutamicum chassis with a significantly reduced genome that still maintains the growth behavior and application range of the respective wild-type strain. Deletions were performed by double crossover and the resulting strains were analyzed using high-throughput cultivation and phenotyping. Besides the three prophages and the IS-elements, 41 gene clusters ranging from 3.7 to 49.7 kbp were chosen as targets. In the prophage-free strain, 36 deletions were successful and 26 thereof showed no negative phenotype during growth on defined medium which classifies them as irrelevant. Theoretically, a combinatory deletion of all these gene clusters would decrease the genome size of C. glutamicum by 22%. To date strains possessing an up to 12% reduced genome and still growing comparable to the wild type have been constructed. This study demonstrates the applicability of the relevant gene concept to construct a chassis of a given model organism. http://dx.doi.org/10.1016/j.nbt.2016.06.814
S25
O7-3 The creation of a synthetic methylotrophic Bacillus subtilis for the sustainable production of high value chemicals Linda Dijkshoorn University of Groningen, Netherlands Our rapidly increasing understanding of methylotrophy has resulted in newly designed purposes for methylotrophy augmenting the development of a C1 economy. One of the developed strategies is the creation of a synthetic transferrable methylotrophy module. Our knowledge of aerobic methylotrophs has inspired us to create a transferrable, single-vector methylotrophic module for Bacillus subtilis. B. subtilis is the most utilized organism with the highest output for industrial production of proteins, chemicals and pharmaceuticals globally. Products produced by Bacillus subtilis are fit for human consumption since it has the generally recognized as safe (GRAS) status. The created inducible synthetic methylotrophy module contains methanol dehydrogenase 3 (mdh3) and its activator protein (act) genes, responsible for methanol conversion to formaldehyde, along with 7 genes from the Ribulose monophosphate (RuMP) pathway of Bacillus methanolicus and B. subtilis coding for key components of formaldehyde assimilation and dissimilation routes. This single vector transforms B. subtilis into a methylotroph. C13 labelling experiments, using C13 labelled methanol, have shown the assimilation of methanol into central metabolites like phosphoenolpyruvic acid (PEP). To improve the effectiveness of the methylotrophy module a whole genome scale model and kinetic model of the methylotrophic B. subtilis have been built providing quantitative insight allowing the creation of possible optimization strategies. Furthermore, production modules for B. subtilis are being created to link the methylotrophy with a chemical production module. An example of these modules is a amorphadiene production module, allowing the production of the precursor of anti-malarial artemisinin, using the naturally present MEPP pathway. http://dx.doi.org/10.1016/j.nbt.2016.06.815
O7-4 Building effective microbial cell factories for production of fuels and chemicals with the aid of transport proteins Frank Baganz 1,∗ , Christopher Grant 1 , Phattaraporn Morris 1 , Kalim Akhtar 1 , Michael Sadowski 2 1 2
University College London, United Kingdom Synthace Ltd, United Kingdom
Despite progressive efforts to produce biological routes for the synthesis of hydrophobic chemicals of interest, such as alkanes and fatty acids, often little is understood about how production of the molecules of interest is effected by transport of substrates, products and/or intermediates. Hence, efforts to engineer this aspect of the cell factory have been limited in many cases, particularly for hydrophobic products. This work demonstrates a systematic multifactorial approach to characterising the effect of supected transport proteins on both an alkane synthesis pathway and the reverse case of a whole-cell alkane oxidation reaction. We demonstrate here benefits of the transporter plug-in approach for: (i) Facilitated delivery of alkane substrates to improve whole-cell biocatalysis rates by > 60 fold. (ii) Reducing byproduct formation in whole-cell bioconversion of alkanes to alkanols. (iii) Improving bioalkane synthesis yields from glycerol by up to 5 fold and reducing intermediate
nology for the production of both fine and commodity chemicals. Indeed, Nature has evolved a number of methods to produce such conditions, most notably through the evolution of bacterial microcompartments where metabolic enzymes are encased within a semi-permeable protein membrane. We have developed recombinant bacterial microcompartments to allow for the design of specific nano-bioreators, which contain specific pathways of choice. We have also deconstructed the bacterial microcompartment to allow for the development of protein scaffolds onto which metabolic enzymes can be tethered. These and methods for the production of both internal and external lipid vesicles will be discussed. http://dx.doi.org/10.1016/j.nbt.2016.06.812
O7-2 Chassis organism from Corynebacterium glutamicum – Genome reduction as a tool toward improved strains for synthetic biology and industrial biotechnology Meike Baumgart ∗ , Simon Unthan, Andreas Radek, Marius Herbst, Daniel Siebert, Natalie Brühl, Anna Bartsch, Michael Bott, Wolfgang Wiechert, Kay Marin, Stephan Hans, Reinhard Krämer, Gerd Seibold, Jörn Kalinowski, Christian Rückert, Volker F. Wendisch, Stephan Noack, Julia Frunzke Forschungszentrum Juelich, Germany Synthetic biology projects for the rational design of novel biotechnological production strains require a robust and predictable strain background to allow the successful integration of new functions. These chassis organisms can either be built by de novo synthesis or derived by top-down approaches by sequential deletion of non-essential genes. Here we describe the construction of a chassis based on the industrial amino acid producer Corynebacterium glutamicum. The aim of this study was the definition of a so called “relevant gene set” for C. glutamicum which maintains its fast growth and high cell density in defined medium. Sequential deletion of the non-relevant genes should lead to a C. glutamicum chassis with a significantly reduced genome that still maintains the growth behavior and application range of the respective wild-type strain. Deletions were performed by double crossover and the resulting strains were analyzed using high-throughput cultivation and phenotyping. Besides the three prophages and the IS-elements, 41 gene clusters ranging from 3.7 to 49.7 kbp were chosen as targets. In the prophage-free strain, 36 deletions were successful and 26 thereof showed no negative phenotype during growth on defined medium which classifies them as irrelevant. Theoretically, a combinatory deletion of all these gene clusters would decrease the genome size of C. glutamicum by 22%. To date strains possessing an up to 12% reduced genome and still growing comparable to the wild type have been constructed. This study demonstrates the applicability of the relevant gene concept to construct a chassis of a given model organism. http://dx.doi.org/10.1016/j.nbt.2016.06.814
S25
O7-3 The creation of a synthetic methylotrophic Bacillus subtilis for the sustainable production of high value chemicals Linda Dijkshoorn University of Groningen, Netherlands Our rapidly increasing understanding of methylotrophy has resulted in newly designed purposes for methylotrophy augmenting the development of a C1 economy. One of the developed strategies is the creation of a synthetic transferrable methylotrophy module. Our knowledge of aerobic methylotrophs has inspired us to create a transferrable, single-vector methylotrophic module for Bacillus subtilis. B. subtilis is the most utilized organism with the highest output for industrial production of proteins, chemicals and pharmaceuticals globally. Products produced by Bacillus subtilis are fit for human consumption since it has the generally recognized as safe (GRAS) status. The created inducible synthetic methylotrophy module contains methanol dehydrogenase 3 (mdh3) and its activator protein (act) genes, responsible for methanol conversion to formaldehyde, along with 7 genes from the Ribulose monophosphate (RuMP) pathway of Bacillus methanolicus and B. subtilis coding for key components of formaldehyde assimilation and dissimilation routes. This single vector transforms B. subtilis into a methylotroph. C13 labelling experiments, using C13 labelled methanol, have shown the assimilation of methanol into central metabolites like phosphoenolpyruvic acid (PEP). To improve the effectiveness of the methylotrophy module a whole genome scale model and kinetic model of the methylotrophic B. subtilis have been built providing quantitative insight allowing the creation of possible optimization strategies. Furthermore, production modules for B. subtilis are being created to link the methylotrophy with a chemical production module. An example of these modules is a amorphadiene production module, allowing the production of the precursor of anti-malarial artemisinin, using the naturally present MEPP pathway. http://dx.doi.org/10.1016/j.nbt.2016.06.815
O7-4 Building effective microbial cell factories for production of fuels and chemicals with the aid of transport proteins Frank Baganz 1,∗ , Christopher Grant 1 , Phattaraporn Morris 1 , Kalim Akhtar 1 , Michael Sadowski 2 1 2
University College London, United Kingdom Synthace Ltd, United Kingdom
Despite progressive efforts to produce biological routes for the synthesis of hydrophobic chemicals of interest, such as alkanes and fatty acids, often little is understood about how production of the molecules of interest is effected by transport of substrates, products and/or intermediates. Hence, efforts to engineer this aspect of the cell factory have been limited in many cases, particularly for hydrophobic products. This work demonstrates a systematic multifactorial approach to characterising the effect of supected transport proteins on both an alkane synthesis pathway and the reverse case of a whole-cell alkane oxidation reaction. We demonstrate here benefits of the transporter plug-in approach for: (i) Facilitated delivery of alkane substrates to improve whole-cell biocatalysis rates by > 60 fold. (ii) Reducing byproduct formation in whole-cell bioconversion of alkanes to alkanols. (iii) Improving bioalkane synthesis yields from glycerol by up to 5 fold and reducing intermediate