- Email: [email protected]
Metabolic engineering of Saccharomyces cerevisiae microbial cell factories for succinic acid production
Abstracts / Journal of Biotechnology 131S (2007) S196–S210
S205
20. Metabolic engineering of Saccharomyces cerevisiae microbial cell factories for succinic acid production
21. Recombineering with Red® /ET—Modification of the bacterial chromosome
Jose Manuel Otero ∗ , Lisbeth Olsson, Jens Nielsen
Tim Zeppenfeld ∗ , Harald Kranz
Technical University of Denmark, Center for Microbial Biotechnology, Building 223, Office 213, DK-2800 Kgs. Lyngby, Denmark
Gene Bridges GmbH, Im Neuenheimer Feld 584, 69120 Heidelberg, Germany
Saccharomyces cerevisiae is a proven, robust, industrial production platform used for expression of a wide range of therapeutic agents, high added-value chemicals, and commodities. Central carbon metabolism in S. cerevisiae has been extensively investigated using a wide variety of substrates for determination of how glycolytic flux is distributed across C1 (CO2,g ), C2 (ethanol, acetate), and C3 (glycerol, pyruvate) products. For the S. cerevisiae CEN.PK113-7D strain cultivated under carbon-limited, aerobic, well-controlled batch fermentations, the distribution of carbon across biomass, C1 , C2 , and C3 products is 18, 14, 54, and 9 C-mol/C-mol-glucose, respectively, with <5 C-mol/C-mol glucose unaccounted for. A class of high added-value chemicals being targeted for biotechnology production is C4 organic acids, encompassing fumaric, malic, and succinic acid. Succinic acid is a key building block molecule for further conversion to precursor molecules such as tetrahydrofuran, 1,4-butanediol, and butyrolactone. Succinic acid has the potential to become a commodity chemical, with world-wide annual demand exceeding $2 billion USD and over 160 million kg currently produced from petrochemical conversion of maleic anhydride. There are several biomass platforms, all prokaryotic, for succinic acid production; however, overproduction of succinic acid in S. cerevisiae offers distinct process advantages. For example, S. cerevisiae has been awarded GRAS status for use in human consumables, grows well at low pH significantly minimizing purification and acidification costs associated with organic acid production, and can utilize diverse carbon substrates in chemically defined medium. S. cerevisiae offers the unique advantage of being the most well characterized eukaryotic expression system. Here we describe the use of systems biology tools to drive C6 carbon flux to succinic acid by enhancement of the two native pathways for succinic acid production: the TCA and glyoxylate cycles. S. cerevisiae does not naturally accumulate succinic acid; however, through the use of in silico metabolic predictions guiding targeted gene deletions and over-expression, mutants that overproduce succinic acid have been engineered and thoroughly characterised. Metabolic engineering approaches developed promise to have broad applicability to industrial biotechnology platforms, as well as enhancing fundamental understanding of central carbon metabolism in S. cerevisiae. doi:10.1016/j.jbiotec.2007.07.367
Metabolic engineering to design and construct microorganisms suitable for the production of industrial products like ethanol or aromatic amino acids requires the disruption of specific genes on the bacterial chromosome. Regulatory circuits, the uptake of carbon and amino acids, the glycolytic and pentose phosphate pathway, as well as the common aromatic amino acid pathway have to be manipulated. The complexity of the necessary modifications requires a tool allowing the precise knock-out or alteration of multiple genes without leaving antibiotic selection markers. Red/ET recombination (Zhang et al., 1998) also known as recombineering is an easy to use modification system for prokaryotic functional genomics. There is proven evidence that Red/ET works not only in E. coli, but also in Salmonella, Shigella, Yersinia, Serratia and Citrobacter. Here we demonstrate the easy and precise knock-out/knockin of genes as well as the possibility to use Red/ET as a powerful subcloning-tool. Reference Zhang, Y., et al., 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–138.
doi:10.1016/j.jbiotec.2007.07.368 22. Solvent tolerant Pseudomonas putida S12 as platform for the production of chemicals from renewable feedstock Harald Ruijssenaars TNO Quality of Life, Julianalaan 67, 2628BC, Delft, Netherlands In our laboratory, the solvent tolerant bacterium Pseudomonas putida S12 is developed as a platform organism for the production of chemicals from renewable feedstock. Its extreme solvent tolerance makes this organism especially suited for the production of toxic chemicals such as substituted aromatics. As a platform, P. putida S12 is modified such that chemicals are produced from renewables via central metabolite precursors, e.g., aromatic amino acids. In demonstrator cases, phenol and t-cinnamate were produced from glucose and glycerol, via tyrosine, respectively, phenylalanine. These strains initially showed modest product yield and accumulation. Various mutant strains were obtained, via random mutagenesis and subsequent high-throughput screening, which showed greatly production characteristics (Nijkamp et al., 2005; Wierckx et al., 2005). The transcriptomes of the optimized phenol producer P. putida S12TPL3 and the parent strain were compared to gain
S205
20. Metabolic engineering of Saccharomyces cerevisiae microbial cell factories for succinic acid production
21. Recombineering with Red® /ET—Modification of the bacterial chromosome
Jose Manuel Otero ∗ , Lisbeth Olsson, Jens Nielsen
Tim Zeppenfeld ∗ , Harald Kranz
Technical University of Denmark, Center for Microbial Biotechnology, Building 223, Office 213, DK-2800 Kgs. Lyngby, Denmark
Gene Bridges GmbH, Im Neuenheimer Feld 584, 69120 Heidelberg, Germany
Saccharomyces cerevisiae is a proven, robust, industrial production platform used for expression of a wide range of therapeutic agents, high added-value chemicals, and commodities. Central carbon metabolism in S. cerevisiae has been extensively investigated using a wide variety of substrates for determination of how glycolytic flux is distributed across C1 (CO2,g ), C2 (ethanol, acetate), and C3 (glycerol, pyruvate) products. For the S. cerevisiae CEN.PK113-7D strain cultivated under carbon-limited, aerobic, well-controlled batch fermentations, the distribution of carbon across biomass, C1 , C2 , and C3 products is 18, 14, 54, and 9 C-mol/C-mol-glucose, respectively, with <5 C-mol/C-mol glucose unaccounted for. A class of high added-value chemicals being targeted for biotechnology production is C4 organic acids, encompassing fumaric, malic, and succinic acid. Succinic acid is a key building block molecule for further conversion to precursor molecules such as tetrahydrofuran, 1,4-butanediol, and butyrolactone. Succinic acid has the potential to become a commodity chemical, with world-wide annual demand exceeding $2 billion USD and over 160 million kg currently produced from petrochemical conversion of maleic anhydride. There are several biomass platforms, all prokaryotic, for succinic acid production; however, overproduction of succinic acid in S. cerevisiae offers distinct process advantages. For example, S. cerevisiae has been awarded GRAS status for use in human consumables, grows well at low pH significantly minimizing purification and acidification costs associated with organic acid production, and can utilize diverse carbon substrates in chemically defined medium. S. cerevisiae offers the unique advantage of being the most well characterized eukaryotic expression system. Here we describe the use of systems biology tools to drive C6 carbon flux to succinic acid by enhancement of the two native pathways for succinic acid production: the TCA and glyoxylate cycles. S. cerevisiae does not naturally accumulate succinic acid; however, through the use of in silico metabolic predictions guiding targeted gene deletions and over-expression, mutants that overproduce succinic acid have been engineered and thoroughly characterised. Metabolic engineering approaches developed promise to have broad applicability to industrial biotechnology platforms, as well as enhancing fundamental understanding of central carbon metabolism in S. cerevisiae. doi:10.1016/j.jbiotec.2007.07.367
Metabolic engineering to design and construct microorganisms suitable for the production of industrial products like ethanol or aromatic amino acids requires the disruption of specific genes on the bacterial chromosome. Regulatory circuits, the uptake of carbon and amino acids, the glycolytic and pentose phosphate pathway, as well as the common aromatic amino acid pathway have to be manipulated. The complexity of the necessary modifications requires a tool allowing the precise knock-out or alteration of multiple genes without leaving antibiotic selection markers. Red/ET recombination (Zhang et al., 1998) also known as recombineering is an easy to use modification system for prokaryotic functional genomics. There is proven evidence that Red/ET works not only in E. coli, but also in Salmonella, Shigella, Yersinia, Serratia and Citrobacter. Here we demonstrate the easy and precise knock-out/knockin of genes as well as the possibility to use Red/ET as a powerful subcloning-tool. Reference Zhang, Y., et al., 1998. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–138.
doi:10.1016/j.jbiotec.2007.07.368 22. Solvent tolerant Pseudomonas putida S12 as platform for the production of chemicals from renewable feedstock Harald Ruijssenaars TNO Quality of Life, Julianalaan 67, 2628BC, Delft, Netherlands In our laboratory, the solvent tolerant bacterium Pseudomonas putida S12 is developed as a platform organism for the production of chemicals from renewable feedstock. Its extreme solvent tolerance makes this organism especially suited for the production of toxic chemicals such as substituted aromatics. As a platform, P. putida S12 is modified such that chemicals are produced from renewables via central metabolite precursors, e.g., aromatic amino acids. In demonstrator cases, phenol and t-cinnamate were produced from glucose and glycerol, via tyrosine, respectively, phenylalanine. These strains initially showed modest product yield and accumulation. Various mutant strains were obtained, via random mutagenesis and subsequent high-throughput screening, which showed greatly production characteristics (Nijkamp et al., 2005; Wierckx et al., 2005). The transcriptomes of the optimized phenol producer P. putida S12TPL3 and the parent strain were compared to gain