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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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Central metabolic pathways of E. coli functional under anaerobic condition during glucose and xylose fermentation. Relevant genes and corresponding enzymes are shown. Pyruvate dehydrogenase (PDH) operon was expressed under anaerobic condition via promoter replacement and is represented as thick line. The competing pathways to ethanol were blocked as shown by two parallel bars. Broken arrows represent multiple reactions of a pathway. Extracellular metabolites are placed in boxes. Abbreviations are as follows: ADH, acetaldehyde/alcohol dehydrogenase; ACK, acetate kinase; FHL, formate hydrogen-lyase; FRD, fumarate reductase; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PFL, pyruvate formate-lyase; PTA, phosphate acetyltransferase; PYK, pyruvate kinase.
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Figure 1: Central metabolic pathways of E. coli functional under anaerobic condition during glucose and xylose fermentation. Relevant genes and corresponding enzymes are shown. Pyruvate dehydrogenase (PDH) operon was expressed under anaerobic condition via promoter replacement and is represented as thick line. The competing pathways to ethanol were blocked as shown by two parallel bars. Broken arrows represent multiple reactions of a pathway. Extracellular metabolites are placed in boxes. Abbreviations are as follows: ADH, acetaldehyde/alcohol dehydrogenase; ACK, acetate kinase; FHL, formate hydrogen-lyase; FRD, fumarate reductase; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PFL, pyruvate formate-lyase; PTA, phosphate acetyltransferase; PYK, pyruvate kinase.

Mentions: Under anaerobic condition, E. coli produces ethanol through a pathway that involves pyruvate formate lyase (PFL), which converts pyruvate into acetyl-CoA and formate (Figure 1) [9]. However, this pathway is not redox balanced because in the process of metabolizing one mole of glucose into ethanol, four moles of NADH are consumed while only two moles of NADH are produced (Reaction (i)-(iii)).


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)

Central metabolic pathways of E. coli functional under anaerobic condition during glucose and xylose fermentation. Relevant genes and corresponding enzymes are shown. Pyruvate dehydrogenase (PDH) operon was expressed under anaerobic condition via promoter replacement and is represented as thick line. The competing pathways to ethanol were blocked as shown by two parallel bars. Broken arrows represent multiple reactions of a pathway. Extracellular metabolites are placed in boxes. Abbreviations are as follows: ADH, acetaldehyde/alcohol dehydrogenase; ACK, acetate kinase; FHL, formate hydrogen-lyase; FRD, fumarate reductase; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PFL, pyruvate formate-lyase; PTA, phosphate acetyltransferase; PYK, pyruvate kinase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Central metabolic pathways of E. coli functional under anaerobic condition during glucose and xylose fermentation. Relevant genes and corresponding enzymes are shown. Pyruvate dehydrogenase (PDH) operon was expressed under anaerobic condition via promoter replacement and is represented as thick line. The competing pathways to ethanol were blocked as shown by two parallel bars. Broken arrows represent multiple reactions of a pathway. Extracellular metabolites are placed in boxes. Abbreviations are as follows: ADH, acetaldehyde/alcohol dehydrogenase; ACK, acetate kinase; FHL, formate hydrogen-lyase; FRD, fumarate reductase; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PFL, pyruvate formate-lyase; PTA, phosphate acetyltransferase; PYK, pyruvate kinase.
Mentions: Under anaerobic condition, E. coli produces ethanol through a pathway that involves pyruvate formate lyase (PFL), which converts pyruvate into acetyl-CoA and formate (Figure 1) [9]. However, this pathway is not redox balanced because in the process of metabolizing one mole of glucose into ethanol, four moles of NADH are consumed while only two moles of NADH are produced (Reaction (i)-(iii)).

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