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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 profile of E. coli B (A and B) and SSY09(pZSack) (C and D) grown in the bioreactor in complex medium with glucose (A and C) and xylose (B and D) as carbon source. Only small fraction of carbon has been used by the E. coli B cells to produce ethanol. SSY09(pZSack) strain utilized glucose and produced ethanol at a significantly high rate. Xylose utilization rate, however, was still slow. Strain description: SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).
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Figure 5: Fermentation profile of E. coli B (A and B) and SSY09(pZSack) (C and D) grown in the bioreactor in complex medium with glucose (A and C) and xylose (B and D) as carbon source. Only small fraction of carbon has been used by the E. coli B cells to produce ethanol. SSY09(pZSack) strain utilized glucose and produced ethanol at a significantly high rate. Xylose utilization rate, however, was still slow. Strain description: SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).

Mentions: To improve the growth, substrate utilization and ethanol production rate, we grew the engineered cells in LB medium with 50 g/l substrate. The wild type strain produced lactate and acetate as major metabolic products with ethanol produced only at the yield of 0.31 and 0.79 mmol per mmol glucose and xylose, respectively (Figure 5A and 5B, Table 2). Remarkable improvement in growth rate was observed in the case of SSY09(pZSack) strain when grown in complex medium. The growth profile indicated that complex media served as nutrient supplement for achieving initial cell growth, and sugar consumption occurred after the growth cycle (Figure 5C and 5D). This strategy allowed the cells to overcome the growth limitation arose due to pflB deletion. Ethanol was produced at the rate of 12.34 mmol/l/h with 95% of the theoretical yield using glucose as carbon source (Figure 5C, Table 2). The final ethanol concentration achieved was 21 g/l (457 mM) from 46 g/l (255 mM) glucose in 80 h (Figure 5C).


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 profile of E. coli B (A and B) and SSY09(pZSack) (C and D) grown in the bioreactor in complex medium with glucose (A and C) and xylose (B and D) as carbon source. Only small fraction of carbon has been used by the E. coli B cells to produce ethanol. SSY09(pZSack) strain utilized glucose and produced ethanol at a significantly high rate. Xylose utilization rate, however, was still slow. Strain description: 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 5: Fermentation profile of E. coli B (A and B) and SSY09(pZSack) (C and D) grown in the bioreactor in complex medium with glucose (A and C) and xylose (B and D) as carbon source. Only small fraction of carbon has been used by the E. coli B cells to produce ethanol. SSY09(pZSack) strain utilized glucose and produced ethanol at a significantly high rate. Xylose utilization rate, however, was still slow. Strain description: SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).
Mentions: To improve the growth, substrate utilization and ethanol production rate, we grew the engineered cells in LB medium with 50 g/l substrate. The wild type strain produced lactate and acetate as major metabolic products with ethanol produced only at the yield of 0.31 and 0.79 mmol per mmol glucose and xylose, respectively (Figure 5A and 5B, Table 2). Remarkable improvement in growth rate was observed in the case of SSY09(pZSack) strain when grown in complex medium. The growth profile indicated that complex media served as nutrient supplement for achieving initial cell growth, and sugar consumption occurred after the growth cycle (Figure 5C and 5D). This strategy allowed the cells to overcome the growth limitation arose due to pflB deletion. Ethanol was produced at the rate of 12.34 mmol/l/h with 95% of the theoretical yield using glucose as carbon source (Figure 5C, Table 2). The final ethanol concentration achieved was 21 g/l (457 mM) from 46 g/l (255 mM) glucose in 80 h (Figure 5C).

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