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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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Functional characterization of promoter engineered E. coli B strains (SSY01-05, see Table1for genotype). Effect of PDH operon promoter replacement on (A) pyruvate dehydrogenase activity and (B) ethanol production was monitored. Cells were grown anaerobically in completely filled Hungate tubes and were harvested and permeabilized to measure PDH activity. The supernatant of the culture was used to analyze metabolite concentration via HPLC. Strain description for changed PDH promoter: SSY01 – PldhAPDH, SSY02 –PfrdAPDH, SSY03 – PpflBPDH, SSY04 – PadhEPDH, SSY05 – PgapAPDH.
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Figure 2: Functional characterization of promoter engineered E. coli B strains (SSY01-05, see Table1for genotype). Effect of PDH operon promoter replacement on (A) pyruvate dehydrogenase activity and (B) ethanol production was monitored. Cells were grown anaerobically in completely filled Hungate tubes and were harvested and permeabilized to measure PDH activity. The supernatant of the culture was used to analyze metabolite concentration via HPLC. Strain description for changed PDH promoter: SSY01 – PldhAPDH, SSY02 –PfrdAPDH, SSY03 – PpflBPDH, SSY04 – PadhEPDH, SSY05 – PgapAPDH.

Mentions: The engineered cells were grown under anaerobic condition in defined medium for different time intervals and used for measuring pyruvate dehydrogenase (PDH) activity. The results indicated maximum PDH activity between 18–24 h of anaerobic growth (Figure 2A). Except SSY01 strain (PldhAPDH), all promoter engineered strains showed significant improvement in PDH activity over wild type strain. This result was intriguing since pdh gene in Geobacillus thermoglucosidasius under the control of ldh promoter was shown to have positive effect on ethanol production [12], indicating higher flux through PDH pathway in this strain due to higher PDH activity. We therefore compared ethanol production capabilities of all the engineered strains to assess flux towards PDH 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)

Functional characterization of promoter engineered E. coli B strains (SSY01-05, see Table1for genotype). Effect of PDH operon promoter replacement on (A) pyruvate dehydrogenase activity and (B) ethanol production was monitored. Cells were grown anaerobically in completely filled Hungate tubes and were harvested and permeabilized to measure PDH activity. The supernatant of the culture was used to analyze metabolite concentration via HPLC. Strain description for changed PDH promoter: SSY01 – PldhAPDH, SSY02 –PfrdAPDH, SSY03 – PpflBPDH, SSY04 – PadhEPDH, SSY05 – PgapAPDH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Functional characterization of promoter engineered E. coli B strains (SSY01-05, see Table1for genotype). Effect of PDH operon promoter replacement on (A) pyruvate dehydrogenase activity and (B) ethanol production was monitored. Cells were grown anaerobically in completely filled Hungate tubes and were harvested and permeabilized to measure PDH activity. The supernatant of the culture was used to analyze metabolite concentration via HPLC. Strain description for changed PDH promoter: SSY01 – PldhAPDH, SSY02 –PfrdAPDH, SSY03 – PpflBPDH, SSY04 – PadhEPDH, SSY05 – PgapAPDH.
Mentions: The engineered cells were grown under anaerobic condition in defined medium for different time intervals and used for measuring pyruvate dehydrogenase (PDH) activity. The results indicated maximum PDH activity between 18–24 h of anaerobic growth (Figure 2A). Except SSY01 strain (PldhAPDH), all promoter engineered strains showed significant improvement in PDH activity over wild type strain. This result was intriguing since pdh gene in Geobacillus thermoglucosidasius under the control of ldh promoter was shown to have positive effect on ethanol production [12], indicating higher flux through PDH pathway in this strain due to higher PDH activity. We therefore compared ethanol production capabilities of all the engineered strains to assess flux towards PDH 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