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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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Improvement in cell growth upon modulation of expression of ack gene in the engineered SSY09 strain. SSY09 strain having plasmid pZSack was grown in Hungate tube completely filled with defined media + 2.5g/l glucose (A) or 2.5g/l xylose (B) at 37°C for 24 hr. Acetate kinase expression was induced with 0, 0.1, 100 ng/ml of anhydrotetracycline. E. coli B and SSY09 bearing pZS*mcs plasmid were used as positive and negative control, respectively. Results indicate improvement in growth of PZSack transformed cells as compared to control plasmid and less acetate production as compared to wild type strain. Strain description: SSY09 - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB.
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Figure 3: Improvement in cell growth upon modulation of expression of ack gene in the engineered SSY09 strain. SSY09 strain having plasmid pZSack was grown in Hungate tube completely filled with defined media + 2.5g/l glucose (A) or 2.5g/l xylose (B) at 37°C for 24 hr. Acetate kinase expression was induced with 0, 0.1, 100 ng/ml of anhydrotetracycline. E. coli B and SSY09 bearing pZS*mcs plasmid were used as positive and negative control, respectively. Results indicate improvement in growth of PZSack transformed cells as compared to control plasmid and less acetate production as compared to wild type strain. Strain description: SSY09 - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB.

Mentions: Though the promoter engineered SSY05 strain exhibited significant enhancement in the ethanol level as compared to the wilde type strain, it still produced considerable amount of competing co-products such as lactate, succinate, acetate and formate (Table 2). To further improve ethanol yield, we introduced deletion in the genes for lactate dehydrogenase (ldhA), fumarate reductase (frdA), acetate kinase (ack) and pyruvate formate lyase (pflB) responsible for the formation of lactate, succinate, acetate and formate, respectively, to obtain SSY06 (PgapA PDH ΔldhA), SSY07 (PgapA PDH ΔldhA ΔfrdA), SSY08 (PgapA PDH ΔldhA ΔfrdA Δack) and SSY09 (PgapA PDH ΔldhA ΔfrdA Δack ΔpflB) strains (Table 1). When grown in defined medium under anaerobic condition, SSY06 and SSY07 grew normally (Table 2), SSY08 grew very slowly and SSY09 did not grow at all. This observation indicated that deletion of ack had deleterious impact on cell growth, possibly due to corresponding depletion of ATP pool, and further deletion of pflB had a cumulative effect on adverse impact even after the expression of parallel PDH pathway. Deletion of ack and pflB leading to adverse impact on cell growth under anaerobic condition has also been observed earlier [9,13]. To regain the cell growth, we introduced ack gene through a very low copy plasmid in SSY09 strain. We found significant improvement in cell growth upon transformation with the plasmid containing ack gene (pZSack) as compared to the strain transformed with the control plasmid (pZS*mcs) even without addition of an inducer, indicating minor leaky expression of ack gene (Figure 3A and 3B). Acetate level in uninduced SSY09(pZSack) strain was less than 50% of the wild type strain. We further tested ethanol-producing capability of the engineered strains at the bioreactor level under controlled environmental condition.


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)

Improvement in cell growth upon modulation of expression of ack gene in the engineered SSY09 strain. SSY09 strain having plasmid pZSack was grown in Hungate tube completely filled with defined media + 2.5g/l glucose (A) or 2.5g/l xylose (B) at 37°C for 24 hr. Acetate kinase expression was induced with 0, 0.1, 100 ng/ml of anhydrotetracycline. E. coli B and SSY09 bearing pZS*mcs plasmid were used as positive and negative control, respectively. Results indicate improvement in growth of PZSack transformed cells as compared to control plasmid and less acetate production as compared to wild type strain. Strain description: SSY09 - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Improvement in cell growth upon modulation of expression of ack gene in the engineered SSY09 strain. SSY09 strain having plasmid pZSack was grown in Hungate tube completely filled with defined media + 2.5g/l glucose (A) or 2.5g/l xylose (B) at 37°C for 24 hr. Acetate kinase expression was induced with 0, 0.1, 100 ng/ml of anhydrotetracycline. E. coli B and SSY09 bearing pZS*mcs plasmid were used as positive and negative control, respectively. Results indicate improvement in growth of PZSack transformed cells as compared to control plasmid and less acetate production as compared to wild type strain. Strain description: SSY09 - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB.
Mentions: Though the promoter engineered SSY05 strain exhibited significant enhancement in the ethanol level as compared to the wilde type strain, it still produced considerable amount of competing co-products such as lactate, succinate, acetate and formate (Table 2). To further improve ethanol yield, we introduced deletion in the genes for lactate dehydrogenase (ldhA), fumarate reductase (frdA), acetate kinase (ack) and pyruvate formate lyase (pflB) responsible for the formation of lactate, succinate, acetate and formate, respectively, to obtain SSY06 (PgapA PDH ΔldhA), SSY07 (PgapA PDH ΔldhA ΔfrdA), SSY08 (PgapA PDH ΔldhA ΔfrdA Δack) and SSY09 (PgapA PDH ΔldhA ΔfrdA Δack ΔpflB) strains (Table 1). When grown in defined medium under anaerobic condition, SSY06 and SSY07 grew normally (Table 2), SSY08 grew very slowly and SSY09 did not grow at all. This observation indicated that deletion of ack had deleterious impact on cell growth, possibly due to corresponding depletion of ATP pool, and further deletion of pflB had a cumulative effect on adverse impact even after the expression of parallel PDH pathway. Deletion of ack and pflB leading to adverse impact on cell growth under anaerobic condition has also been observed earlier [9,13]. To regain the cell growth, we introduced ack gene through a very low copy plasmid in SSY09 strain. We found significant improvement in cell growth upon transformation with the plasmid containing ack gene (pZSack) as compared to the strain transformed with the control plasmid (pZS*mcs) even without addition of an inducer, indicating minor leaky expression of ack gene (Figure 3A and 3B). Acetate level in uninduced SSY09(pZSack) strain was less than 50% of the wild type strain. We further tested ethanol-producing capability of the engineered strains at the bioreactor level under controlled environmental condition.

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