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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 SSY09(pZSack) strain grown under microaerobic condition in the bioreactor in complex medium with (A) xylose and (B) mixture of glucose and xylose as carbon source. The profile indicated efficient utilization of xylose and mixture of glucose and xylose under microaerobic condition and production of ethanol with high yield and productivity. Strain description: SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).
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Figure 6: Fermentation profile of SSY09(pZSack) strain grown under microaerobic condition in the bioreactor in complex medium with (A) xylose and (B) mixture of glucose and xylose as carbon source. The profile indicated efficient utilization of xylose and mixture of glucose and xylose under microaerobic condition and production of ethanol with high yield and productivity. Strain description: SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).

Mentions: The xylose utilization rate, however, was still slow in the SSY09(pZSack) strain with 25% xylose remained unutilized after 260 h of fermentation and ethanol produced only at the rate of 1.67 mmol/l/h (Figure 5C, Table 2). This was because xylose fermentation into ethanol leads to generation of only 0.67 ATP per xylose as against glucose fermentation where 2 ATPs per glucose are generated. There was a slight increase in xylose utilization rate when pH of the cultivation was maintained at 6.3 (data not shown), possibly due to higher activity of a xylose/proton symporter [14]. To further enhance the growth rate of the cells, we introduced a microaerobic condition by passing compressed air in the headspace of bioreactor at a very slow flow rate as mentioned in the methods section. Microaerobic condition would allow extra ATP for cell growth through partial activation of TCA cycle. Cell growth and xylose utilization rate improved significantly with 50 g/l xylose utilized in 115 h and 25 g/l ethanol produced at the rate of 6.84 mmol/l/h with 97% of the maximum theoretical yield (Figure 6A). This yield of ethanol from xylose was higher than those reported in the literature from the engineered E. coli without the foreign genes [7,8].


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 SSY09(pZSack) strain grown under microaerobic condition in the bioreactor in complex medium with (A) xylose and (B) mixture of glucose and xylose as carbon source. The profile indicated efficient utilization of xylose and mixture of glucose and xylose under microaerobic condition and production of ethanol with high yield and productivity. 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 6: Fermentation profile of SSY09(pZSack) strain grown under microaerobic condition in the bioreactor in complex medium with (A) xylose and (B) mixture of glucose and xylose as carbon source. The profile indicated efficient utilization of xylose and mixture of glucose and xylose under microaerobic condition and production of ethanol with high yield and productivity. Strain description: SSY09(pZSack) - PgapAPDH ΔldhA ΔfrdA Δack ΔpflB (pZSack).
Mentions: The xylose utilization rate, however, was still slow in the SSY09(pZSack) strain with 25% xylose remained unutilized after 260 h of fermentation and ethanol produced only at the rate of 1.67 mmol/l/h (Figure 5C, Table 2). This was because xylose fermentation into ethanol leads to generation of only 0.67 ATP per xylose as against glucose fermentation where 2 ATPs per glucose are generated. There was a slight increase in xylose utilization rate when pH of the cultivation was maintained at 6.3 (data not shown), possibly due to higher activity of a xylose/proton symporter [14]. To further enhance the growth rate of the cells, we introduced a microaerobic condition by passing compressed air in the headspace of bioreactor at a very slow flow rate as mentioned in the methods section. Microaerobic condition would allow extra ATP for cell growth through partial activation of TCA cycle. Cell growth and xylose utilization rate improved significantly with 50 g/l xylose utilized in 115 h and 25 g/l ethanol produced at the rate of 6.84 mmol/l/h with 97% of the maximum theoretical yield (Figure 6A). This yield of ethanol from xylose was higher than those reported in the literature from the engineered E. coli without the foreign genes [7,8].

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