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Modular design of metabolic network for robust production of n-butanol from galactose-glucose mixtures.

Lim HG, Lim JH, Jung GY - Biotechnol Biofuels (2015)

Bottom Line: Specifically, the engineered strain showed dramatically increased n-butanol production (3.3-fold increased to 6.2 g/L after 48-h fermentation) compared to the parental strain (1.9 g/L) in galactose-supplemented medium.Collectively, modular pathway engineering of metabolic network can be an effective approach in strain development for optimal biofuel production with cost-effective fermentable sugars.Moreover, robust production of n-butanol with sugar mixtures with variable composition would facilitate the economic feasibility of the microbial process using a mixture of sugars from cheap biomass in the near future.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Pohang, 37673 Gyeongbuk Korea.

ABSTRACT

Background: Refactoring microorganisms for efficient production of advanced biofuel such as n-butanol from a mixture of sugars in the cheap feedstock is a prerequisite to achieve economic feasibility in biorefinery. However, production of biofuel from inedible and cheap feedstock is highly challenging due to the slower utilization of biomass-driven sugars, arising from complex assimilation pathway, difficulties in amplification of biosynthetic pathways for heterologous metabolite, and redox imbalance caused by consuming intracellular reducing power to produce quite reduced biofuel. Even with these problems, the microorganisms should show robust production of biofuel to obtain industrial feasibility. Thus, refactoring microorganisms for efficient conversion is highly desirable in biofuel production.

Results: In this study, we engineered robust Escherichia coli to accomplish high production of n-butanol from galactose-glucose mixtures via the design of modular pathway, an efficient and systematic way, to reconstruct the entire metabolic pathway with many target genes. Three modular pathways designed using the predictable genetic elements were assembled for efficient galactose utilization, n-butanol production, and redox re-balancing to robustly produce n-butanol from a sugar mixture of galactose and glucose. Specifically, the engineered strain showed dramatically increased n-butanol production (3.3-fold increased to 6.2 g/L after 48-h fermentation) compared to the parental strain (1.9 g/L) in galactose-supplemented medium. Moreover, fermentation with mixtures of galactose and glucose at various ratios from 2:1 to 1:2 confirmed that our engineered strain was able to robustly produce n-butanol regardless of sugar composition with simultaneous utilization of galactose and glucose.

Conclusions: Collectively, modular pathway engineering of metabolic network can be an effective approach in strain development for optimal biofuel production with cost-effective fermentable sugars. To the best of our knowledge, this study demonstrated the first and highest n-butanol production from galactose in E. coli. Moreover, robust production of n-butanol with sugar mixtures with variable composition would facilitate the economic feasibility of the microbial process using a mixture of sugars from cheap biomass in the near future.

No MeSH data available.


Related in: MedlinePlus

Schematic diagram of the assembled modules to convert galactose to n-butanol. In galactose utilization module, β-d-galactose is converted into α-d-glucose-6-phosphate through the Leloir pathway, which is a more complicated enzymatic pathway than that required for glucose. Acetoacetyl-CoA from α-d-glucose-6-phosphate is transformed into n-butanol by the n-butanol production module. Four moles of NADH generated from one mole of galactose are used in the n-butanol biosynthesis. In NADH supplementation module, additional NADH is produced by converting formate to carbon dioxide. The red arrows indicate the homologous and heterologous overexpression of genes. The blue arrow represents controllable expression of fdh1 for optimal n-butanol production
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Fig1: Schematic diagram of the assembled modules to convert galactose to n-butanol. In galactose utilization module, β-d-galactose is converted into α-d-glucose-6-phosphate through the Leloir pathway, which is a more complicated enzymatic pathway than that required for glucose. Acetoacetyl-CoA from α-d-glucose-6-phosphate is transformed into n-butanol by the n-butanol production module. Four moles of NADH generated from one mole of galactose are used in the n-butanol biosynthesis. In NADH supplementation module, additional NADH is produced by converting formate to carbon dioxide. The red arrows indicate the homologous and heterologous overexpression of genes. The blue arrow represents controllable expression of fdh1 for optimal n-butanol production

Mentions: The galactose metabolizing pathway, also known as the “Leloir pathway”, involves several enzymes that catalyze galactose catabolism in addition to glucose metabolism. As shown in Fig. 1, galactose is imported by galactose transporters (encoded by galP, and mglBAC) and is subsequently converted into glucose-1-phosphate through multiple enzymes encoded by the galactose operon (galETKM). Finally, phosphoglucomutase (encoded by pgm) converts glucose-1-phosphate to glucose-6-phosphate to enter glycolysis. In the presence of glucose, however, galactose metabolism is regulated by several factors, including a Gal repressor (GalR), Gal iso-repressor (GalS), and intracellular cyclic AMP, which induces CCR, enabling the preferential utilization of glucose. Although multiple enzymatic steps and the regulatory hierarchy in the pathway limit the rate of galactose utilization and the simultaneous fermentation of many carbohydrates [21], the reconstruction of galactose pathway on chromosome enhanced the galactose utilization rate and the simultaneous assimilation of galactose and glucose [10].Fig. 1


Modular design of metabolic network for robust production of n-butanol from galactose-glucose mixtures.

Lim HG, Lim JH, Jung GY - Biotechnol Biofuels (2015)

Schematic diagram of the assembled modules to convert galactose to n-butanol. In galactose utilization module, β-d-galactose is converted into α-d-glucose-6-phosphate through the Leloir pathway, which is a more complicated enzymatic pathway than that required for glucose. Acetoacetyl-CoA from α-d-glucose-6-phosphate is transformed into n-butanol by the n-butanol production module. Four moles of NADH generated from one mole of galactose are used in the n-butanol biosynthesis. In NADH supplementation module, additional NADH is produced by converting formate to carbon dioxide. The red arrows indicate the homologous and heterologous overexpression of genes. The blue arrow represents controllable expression of fdh1 for optimal n-butanol production
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4559943&req=5

Fig1: Schematic diagram of the assembled modules to convert galactose to n-butanol. In galactose utilization module, β-d-galactose is converted into α-d-glucose-6-phosphate through the Leloir pathway, which is a more complicated enzymatic pathway than that required for glucose. Acetoacetyl-CoA from α-d-glucose-6-phosphate is transformed into n-butanol by the n-butanol production module. Four moles of NADH generated from one mole of galactose are used in the n-butanol biosynthesis. In NADH supplementation module, additional NADH is produced by converting formate to carbon dioxide. The red arrows indicate the homologous and heterologous overexpression of genes. The blue arrow represents controllable expression of fdh1 for optimal n-butanol production
Mentions: The galactose metabolizing pathway, also known as the “Leloir pathway”, involves several enzymes that catalyze galactose catabolism in addition to glucose metabolism. As shown in Fig. 1, galactose is imported by galactose transporters (encoded by galP, and mglBAC) and is subsequently converted into glucose-1-phosphate through multiple enzymes encoded by the galactose operon (galETKM). Finally, phosphoglucomutase (encoded by pgm) converts glucose-1-phosphate to glucose-6-phosphate to enter glycolysis. In the presence of glucose, however, galactose metabolism is regulated by several factors, including a Gal repressor (GalR), Gal iso-repressor (GalS), and intracellular cyclic AMP, which induces CCR, enabling the preferential utilization of glucose. Although multiple enzymatic steps and the regulatory hierarchy in the pathway limit the rate of galactose utilization and the simultaneous fermentation of many carbohydrates [21], the reconstruction of galactose pathway on chromosome enhanced the galactose utilization rate and the simultaneous assimilation of galactose and glucose [10].Fig. 1

Bottom Line: Specifically, the engineered strain showed dramatically increased n-butanol production (3.3-fold increased to 6.2 g/L after 48-h fermentation) compared to the parental strain (1.9 g/L) in galactose-supplemented medium.Collectively, modular pathway engineering of metabolic network can be an effective approach in strain development for optimal biofuel production with cost-effective fermentable sugars.Moreover, robust production of n-butanol with sugar mixtures with variable composition would facilitate the economic feasibility of the microbial process using a mixture of sugars from cheap biomass in the near future.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Pohang, 37673 Gyeongbuk Korea.

ABSTRACT

Background: Refactoring microorganisms for efficient production of advanced biofuel such as n-butanol from a mixture of sugars in the cheap feedstock is a prerequisite to achieve economic feasibility in biorefinery. However, production of biofuel from inedible and cheap feedstock is highly challenging due to the slower utilization of biomass-driven sugars, arising from complex assimilation pathway, difficulties in amplification of biosynthetic pathways for heterologous metabolite, and redox imbalance caused by consuming intracellular reducing power to produce quite reduced biofuel. Even with these problems, the microorganisms should show robust production of biofuel to obtain industrial feasibility. Thus, refactoring microorganisms for efficient conversion is highly desirable in biofuel production.

Results: In this study, we engineered robust Escherichia coli to accomplish high production of n-butanol from galactose-glucose mixtures via the design of modular pathway, an efficient and systematic way, to reconstruct the entire metabolic pathway with many target genes. Three modular pathways designed using the predictable genetic elements were assembled for efficient galactose utilization, n-butanol production, and redox re-balancing to robustly produce n-butanol from a sugar mixture of galactose and glucose. Specifically, the engineered strain showed dramatically increased n-butanol production (3.3-fold increased to 6.2 g/L after 48-h fermentation) compared to the parental strain (1.9 g/L) in galactose-supplemented medium. Moreover, fermentation with mixtures of galactose and glucose at various ratios from 2:1 to 1:2 confirmed that our engineered strain was able to robustly produce n-butanol regardless of sugar composition with simultaneous utilization of galactose and glucose.

Conclusions: Collectively, modular pathway engineering of metabolic network can be an effective approach in strain development for optimal biofuel production with cost-effective fermentable sugars. To the best of our knowledge, this study demonstrated the first and highest n-butanol production from galactose in E. coli. Moreover, robust production of n-butanol with sugar mixtures with variable composition would facilitate the economic feasibility of the microbial process using a mixture of sugars from cheap biomass in the near future.

No MeSH data available.


Related in: MedlinePlus