Modulation of endogenous pathways enhances bioethanol yield and productivity in Escherichia coli

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.</p

Engineered Production of Short Chain Fatty Acid in <i>Escherichia coli</i> Using Fatty Acid Synthesis Pathway

<div><p>Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from <i>Anaerococcus tetradius</i>, TesBF from <i>Bryantella formatexigens</i> and TesBT from <i>Bacteroides thetaiotaomicron</i>, for production of SCFAs in <i>Escherichia coli</i> utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. <i>E</i>. <i>coli</i> strain expressing <i>tesBT</i> gene yielded maximum butyric acid titer at 1.46 g L^-1, followed by <i>tesBF</i> at 0.85 g L^-1 and <i>tesAT</i> at 0.12 g L^-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the <i>fadD</i> and <i>fadE</i> that initiates the fatty acid degradation cycle and overexpression of <i>fadR</i> that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. <i>In vitro</i> enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L^-1 butyric acid and 17.5 g L^-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in <i>E</i>. <i>coli</i> through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.</p></div

Impact of cerulenin on thioesterase mediated butyric acid production.

<p><i>E</i>. <i>coli</i> MG1655 transformed with either (A) pQE-tesBF for expression on thioesterase TesBF or (B) pZA-tesBT for expression of thioesterase TesBT were grown in presence of different concentration of cerulenin and extracellular metabolites were analyzed using HPLC.</p

Effect of oxygen availability on production of butyric acid.

<p>(A) <i>E</i>. <i>coli</i> MG1655 transformed with pZA-tesBT plasmid was grown in 100 ml flask containing different volume of culture medium to vary oxygen availability and checked for butyric acid production. (B) <i>E</i>. <i>coli</i> MG1655 transformed with pZA-tesBT plasmid was grown in the bioreactor containing 350 ml culture medium with different oxygen saturation level and checked for butyric acid production.</p

Engineered Production of Short Chain Fatty Acid in <i>Escherichia coli</i> Using Fatty Acid Synthesis Pathway - Fig 4

<p><b>Impact of deletion of genes having inhibitory effect on host fatty acid biosynthesis on production of free fatty acid using thioesterase TesBF (A) and TesBT (B).</b> Gene function: <i>fadD</i>—fatty acyl-CoA synthetase; <i>fadE</i>–acyl-CoA dehydrogenase; <i>fabR–</i>transcriptional repressor of FASII.</p

Impact of media composition and nutrient limitation on butyric acid production.

<p>Impact of media composition and nutrient limitation on butyric acid production.</p

Impact of plasmid type on butyric acid production in <i>E</i>. <i>coli</i> MG1655 using different thioesterases.

<p>(A) Genes for thioesterase TesAT, TesBF and TesBT were cloned in low (pZS21mcs), medium (pZA31mcs) and high (pQE30) copy number plasmid, transformed in <i>E</i>. <i>coli</i> MG1655 and evaluated for production of butyric acid. Western blot was performed with the soluble (B) and insoluble (C) fraction of the lysate of the cells expressing genes for TesAT, TesBF and TesBT. Protein band of desired molecular weight was observed for each construct in either one or both of the cellular fractions. M indicates protein marker in kilodalton.</p