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Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli.

Munjal N, Mattam AJ, Pramanik D, Srivastava PS, Yazdani SS - Microb. Cell Fact. (2012)

Bottom Line: However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity.The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity.This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.

View Article: PubMed Central - HTML - PubMed

Affiliation: Synthetic Biology and Biofuel Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India.

ABSTRACT

Background: E. coli is a robust host for various genetic manipulations and has been used commonly for bioconversion of hexose and pentose sugars into valuable products. One of the products that E. coli make under fermentative condition is ethanol. However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity. Here, we have constructed an E. coli strain to produce high yield of ethanol from hexose and pentose sugars by modulating the expression of pyruvate dehydrogenase and acetate kinase and by deleting pathways for competing co-products.

Results: The availability of reducing equivalence in E. coli was increased by inducing the expression of the pyruvate dehydrogenase (PDH) operon under anaerobic condition after replacement of its promoter with the promoters of ldhA, frdA, pflB, adhE and gapA. The SSY05 strain, where PDH operon was expressed under gapA promoter, demonstrated highest PDH activity and maximum improvement in ethanol yield. Deletion of genes responsible for competing products, such as lactate (ldhA), succinate (frdA), acetate (ack) and formate (pflB), led to significant reduction in growth rate under anaerobic condition. Modulation of acetate kinase expression in SSY09 strain regained cell growth rate and ethanol was produced at the maximum rate of 12 mmol/l/h from glucose. The resultant SSY09(pZSack) strain efficiently fermented xylose under microaerobic condition and produced 25 g/l ethanol at the maximum rate of 6.84 mmol/l/h with 97% of the theoretical yield. More importantly, fermentation of mixture of glucose and xylose was achieved by SSY09(pZSack) strain under microaerobic condition and ethanol was produced at the maximum rate of 0.7 g/l/h (15 mmol/l/h), respectively, with greater than 85% of theoretical yield.

Conclusions: The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity. This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.

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Fermentation profiles of E. coli B (A and B) and SSY09(pZSack) (C and D) grown in the bioreactor in defined medium with glucose (A and C) and xylose (B and D) as carbon source. Competing products of ethanol are produced at significant level during fermentation of both glucose and xylose in E. coli B while SSY09(pZSack) primarily produced ethanol. SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).
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Figure 4: Fermentation profiles of E. coli B (A and B) and SSY09(pZSack) (C and D) grown in the bioreactor in defined medium with glucose (A and C) and xylose (B and D) as carbon source. Competing products of ethanol are produced at significant level during fermentation of both glucose and xylose in E. coli B while SSY09(pZSack) primarily produced ethanol. SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).

Mentions: When grown in a bioreactor, wild type E. coli B produced ethanol at the yield of 0.65 and 0.61 mmol per mmol of glucose and xylose, respectively, in defined medium under anaerobic condition as against the theoretical maximum yield of 2 and 1.67 mmol per mmol of these sugars due to generation of competing co-products (Figure 4A and 4B, Table 2). The promoter engineered SSY05 strain showed 10% higher ethanol yield as compared to wild type strain, indicating favourable redox balance towards ethanol production (Table 2). Successive deletion made in the competing pathways to generate SSY06, SSY07 and SSY09(pZSack) strains resulted in corresponding increase in ethanol and decrease in co-product yield (Table 2). SSY09(pZSack) strain grown in defined medium with 20 g/l glucose or xylose at the bioreactor level produced ethanol at 83% or 68% of theoretical maximum yield, respectively (Figure 4C and 4D, Table 2). However, ~25% of substrate remained unutilized at ~200 hrs of fermentation and ethanol was produced at the low volumetric productivity of 1.6-1.9 mmol/l/h. This observation indicated that the engineered PDH pathway possibly could not fully complement the loss the PFL pathway.


Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli.

Munjal N, Mattam AJ, Pramanik D, Srivastava PS, Yazdani SS - Microb. Cell Fact. (2012)

Fermentation profiles of E. coli B (A and B) and SSY09(pZSack) (C and D) grown in the bioreactor in defined medium with glucose (A and C) and xylose (B and D) as carbon source. Competing products of ethanol are produced at significant level during fermentation of both glucose and xylose in E. coli B while SSY09(pZSack) primarily produced ethanol. SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539902&req=5

Figure 4: Fermentation profiles of E. coli B (A and B) and SSY09(pZSack) (C and D) grown in the bioreactor in defined medium with glucose (A and C) and xylose (B and D) as carbon source. Competing products of ethanol are produced at significant level during fermentation of both glucose and xylose in E. coli B while SSY09(pZSack) primarily produced ethanol. SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).
Mentions: When grown in a bioreactor, wild type E. coli B produced ethanol at the yield of 0.65 and 0.61 mmol per mmol of glucose and xylose, respectively, in defined medium under anaerobic condition as against the theoretical maximum yield of 2 and 1.67 mmol per mmol of these sugars due to generation of competing co-products (Figure 4A and 4B, Table 2). The promoter engineered SSY05 strain showed 10% higher ethanol yield as compared to wild type strain, indicating favourable redox balance towards ethanol production (Table 2). Successive deletion made in the competing pathways to generate SSY06, SSY07 and SSY09(pZSack) strains resulted in corresponding increase in ethanol and decrease in co-product yield (Table 2). SSY09(pZSack) strain grown in defined medium with 20 g/l glucose or xylose at the bioreactor level produced ethanol at 83% or 68% of theoretical maximum yield, respectively (Figure 4C and 4D, Table 2). However, ~25% of substrate remained unutilized at ~200 hrs of fermentation and ethanol was produced at the low volumetric productivity of 1.6-1.9 mmol/l/h. This observation indicated that the engineered PDH pathway possibly could not fully complement the loss the PFL pathway.

Bottom Line: However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity.The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity.This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.

View Article: PubMed Central - HTML - PubMed

Affiliation: Synthetic Biology and Biofuel Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India.

ABSTRACT

Background: E. coli is a robust host for various genetic manipulations and has been used commonly for bioconversion of hexose and pentose sugars into valuable products. One of the products that E. coli make under fermentative condition is ethanol. However, availability of limited reducing equivalence and generation of competing co-products undermine ethanol yield and productivity. Here, we have constructed an E. coli strain to produce high yield of ethanol from hexose and pentose sugars by modulating the expression of pyruvate dehydrogenase and acetate kinase and by deleting pathways for competing co-products.

Results: The availability of reducing equivalence in E. coli was increased by inducing the expression of the pyruvate dehydrogenase (PDH) operon under anaerobic condition after replacement of its promoter with the promoters of ldhA, frdA, pflB, adhE and gapA. The SSY05 strain, where PDH operon was expressed under gapA promoter, demonstrated highest PDH activity and maximum improvement in ethanol yield. Deletion of genes responsible for competing products, such as lactate (ldhA), succinate (frdA), acetate (ack) and formate (pflB), led to significant reduction in growth rate under anaerobic condition. Modulation of acetate kinase expression in SSY09 strain regained cell growth rate and ethanol was produced at the maximum rate of 12 mmol/l/h from glucose. The resultant SSY09(pZSack) strain efficiently fermented xylose under microaerobic condition and produced 25 g/l ethanol at the maximum rate of 6.84 mmol/l/h with 97% of the theoretical yield. More importantly, fermentation of mixture of glucose and xylose was achieved by SSY09(pZSack) strain under microaerobic condition and ethanol was produced at the maximum rate of 0.7 g/l/h (15 mmol/l/h), respectively, with greater than 85% of theoretical yield.

Conclusions: The E. coli strain SSY09(pZSack) constructed via endogenous pathway engineering fermented glucose and xylose to ethanol with high yield and productivity. This strain lacking any foreign gene for ethanol fermentation is likely to be genetically more stable and therefore should be tested further for the fermentation of lignocellulosic hydrolysate at higher scale.

Show MeSH
Related in: MedlinePlus