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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

a Production of n-butanol by GAL_080 harboring pCOLADuet, GAL_081~084 harboring fdh1 variants after cultivation for 48 h in galactose-supplemented modified TB medium. b Time-course of fermentation profiles of the optimized strain (GAL_083), showing maximum n-butanol production. The pH was adjusted to around 7.2 at 6-h intervals. The lefty-offset and righty-axis represent galactose and metabolite (n-butanol, ethanol, and butyrate) concentrations (g/L), respectively. The lefty-axis represents OD600. The x-axis represents time in culture (h). The error bars indicate standard deviations of measurements from two independent cultures. Symbols: open circle OD600; open rectangle galactose; closed up trianglen-butanol; closed diamond ethanol; closed down triangle butyrate
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Fig4: a Production of n-butanol by GAL_080 harboring pCOLADuet, GAL_081~084 harboring fdh1 variants after cultivation for 48 h in galactose-supplemented modified TB medium. b Time-course of fermentation profiles of the optimized strain (GAL_083), showing maximum n-butanol production. The pH was adjusted to around 7.2 at 6-h intervals. The lefty-offset and righty-axis represent galactose and metabolite (n-butanol, ethanol, and butyrate) concentrations (g/L), respectively. The lefty-axis represents OD600. The x-axis represents time in culture (h). The error bars indicate standard deviations of measurements from two independent cultures. Symbols: open circle OD600; open rectangle galactose; closed up trianglen-butanol; closed diamond ethanol; closed down triangle butyrate

Mentions: In this manner, the commitment of expression cassettes for various levels of FDH1, termed as NADH supplementation module, was expected to effectively coordinate galactose-utilizing and n-butanol producing modules by optimization of intracellular redox state. To find the optimal redox state for n-butanol production equipped with galactose module, fdh1 variants showing different levels of expression [18] were used to transform the GAL_061 strain, finally resulting in the GAL_080, 081, 082, 083, and 084 strains (Additional file 1: Table S1). After fermentation of the variants for 48 h, the relationship between n-butanol and fdh1 expression level titer exhibited a concave curve (Fig. 4a). The carbon flux towards n-butanol was enhanced up to 22 % depending on the enzymatic activity of FDH1, indicating that the GAL_083 strain was redox balanced.Fig. 4


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

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

a Production of n-butanol by GAL_080 harboring pCOLADuet, GAL_081~084 harboring fdh1 variants after cultivation for 48 h in galactose-supplemented modified TB medium. b Time-course of fermentation profiles of the optimized strain (GAL_083), showing maximum n-butanol production. The pH was adjusted to around 7.2 at 6-h intervals. The lefty-offset and righty-axis represent galactose and metabolite (n-butanol, ethanol, and butyrate) concentrations (g/L), respectively. The lefty-axis represents OD600. The x-axis represents time in culture (h). The error bars indicate standard deviations of measurements from two independent cultures. Symbols: open circle OD600; open rectangle galactose; closed up trianglen-butanol; closed diamond ethanol; closed down triangle butyrate
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: a Production of n-butanol by GAL_080 harboring pCOLADuet, GAL_081~084 harboring fdh1 variants after cultivation for 48 h in galactose-supplemented modified TB medium. b Time-course of fermentation profiles of the optimized strain (GAL_083), showing maximum n-butanol production. The pH was adjusted to around 7.2 at 6-h intervals. The lefty-offset and righty-axis represent galactose and metabolite (n-butanol, ethanol, and butyrate) concentrations (g/L), respectively. The lefty-axis represents OD600. The x-axis represents time in culture (h). The error bars indicate standard deviations of measurements from two independent cultures. Symbols: open circle OD600; open rectangle galactose; closed up trianglen-butanol; closed diamond ethanol; closed down triangle butyrate
Mentions: In this manner, the commitment of expression cassettes for various levels of FDH1, termed as NADH supplementation module, was expected to effectively coordinate galactose-utilizing and n-butanol producing modules by optimization of intracellular redox state. To find the optimal redox state for n-butanol production equipped with galactose module, fdh1 variants showing different levels of expression [18] were used to transform the GAL_061 strain, finally resulting in the GAL_080, 081, 082, 083, and 084 strains (Additional file 1: Table S1). After fermentation of the variants for 48 h, the relationship between n-butanol and fdh1 expression level titer exhibited a concave curve (Fig. 4a). The carbon flux towards n-butanol was enhanced up to 22 % depending on the enzymatic activity of FDH1, indicating that the GAL_083 strain was redox balanced.Fig. 4

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