| Biodiesel, as a renewable energy, has been received more and more attention all over the world due to the increasing need for clean energy. Biodiesel, defined as fatty acid esters, is mainly produced from plant oils by transesterification with alcohols in the presence of a base, or an enzyme catalyst. Methanol is mostly used because of its lower cost compared with other alcohols, so biodiesel most commonly refers to fatty acid methyl esters (FAMEs). However, the high cost and limited availability of plant oils has become a rising problem for large-scale cummercial viability of biodiesel production. In order to make biodiesel an economically suitable fuel and increase its marketability, the biodiesel should be produced from more wide range of low-cost feed stocks, such as glucose or cellulose. On the other hand, the poor cold flow properties of biodiesel hinders its use in winter, so we need to develop new biodiesel molecules with better properties.The first direct microbial production of fatty acid ethyl esters (FAEEs), as a microdiesel, in engineered Escherichia coli was performed by Steinbuchel’s group through combination of the ethanol and fatty acid pathways in2006. This process is based on a novel wax ester synthase/fatty acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) from Acinetobacter baylyi strain ADP1, which can synthesize FAEEs from ethanol and fatty acyl-CoAs. Based on the above research, we can produce a variety of fatty acid esters to expand biodiesel molecular species by replacing ethanol with other alcohols. These short-chain alcohols, including isobutanol,1-butanol,2-methyl-l-butanol, and3-methyl-1-butanol, can be easily produced from the2-keto acid pathway by the expression of a2-keto acid decarboxylase and an alcohol dehydrogenase. Therefore, we hypothesized that these corresponding fatty acids short-chain esters (FASEs) could be biosynthesized by short-chain alcohol-producing E. coli through the expression of WS/DGAT that catalyzes the esterification of short-chain alcohols and fatty acyl-coenzyme A.Here, we present de novo biosynthesis of FASEs from glycerol in engineered E. coli for the first time through combination of the fatty acid and2-keto acid pathways. The engineered E. coli co-expresses aro10(2-keto acid decarboxylase) and adh2 (alcohol dehydrogenase) genes from Saccharomyces cerevisiae YPH499for the production of short-chain alcohols and the ws/dgat gene for catalytic esterification of short-chain alcohols and fatty acyl-coenzyme A. In addition, overexpression of the tesA’and fadD genes, and knockout of the fadE gene resulted in greater production of FASEs due to increased fatty acyl-CoA availability. Fed-batch cultivation of the engineered TL101/pDG102/pMSD15strain resulted in a titer of1008mg/L FASEs.All biodiesel fuels regardless of its source shows a high crystallization temperature which is one of the most critical obstacles against the widespread biodiesel usage. FAMEs or FAEEs has considerably higher crystallization temperatures than diesel fuel, so they crystallize at temperatures below0℃often experienced in wintertime operation. These formed crystals can cause operation problems because they can plug the fuel lines and filters. Crystallization involves the arrangement of molecules in an orderly pattern. When branches are introduced into linear, long-chain esters, intramolecular associations should be attenuated and crystallization temperatures reduced. Thus, the high crystallization temperatures property of biodiesel can by replacing the methyl or ethyl ester with a branched moiety.Here, we showed the de novo biosynthesis of fatty acid isobutyl esters (FAIBEs) and fatty acid isoamyl esters (FAIAEs) from glucose in engineered E. coli by co-expressing the genes alsS (acetolactate synthase from Bacillus subtilis), ilvC (ketol-acid reductoisomerase from E. coli), ilvD (dihydroxyacid dehydratase from E. coli) genes, aro10and adh2(2-keto acid decarboxylase and alcohol dehydrogenase from S. cerevisiae YPH499) gens for the production of branched-chain alcohols (isobutanol and isoamylol), the fadD gene encoding fatty acyl-CoA synthetase from E. coli for catalytic the formation of fatty acyl-CoA from corresponding fatty acids and CoA, and the ws/dgat gene encoding acyltransferase from Acinetobacter. baylyi strain ADP1for catalytic esterification of branched-chain alcohols and fatty acyl-CoA. On the other hand, we further showed the de novo biosynthesis of fatty acid isopropyl esters (FAIPEs) from glucose in engineered E. coli by co-expressing the atoB (acetyl-CoA acetyltransferase from E. coli), atoAD (acetoacetyl-CoA transferase encoded from E. coli), adc (acetoacetate decarboxylase from Clostridium acetobutylicum), adh (NADP-dependent alcohol dehydrogenase from Clostridium beijerinckii) genes for the production of isopropanol, and the ws/dgat gene for catalytic esterification of isopropanol and fatty acyl-coenzyme A. Similarly, we further enhanced the fatty acid branched-chain esters biosynthetic ability of E. coli by combining several genotypic changes:overexpression of tesA’and fadD, and knockout of the fadE gene.Having previously demonstrated the capacity for incorporation of branched-chain alcohols into the fatty acid branched-chain esters, we further designed a branched-chain fatty acids biosynthetic module that would generate branched fatty acids substrates. Here, we showed the denovo biosynthesis of branched fatty acids from glucose in engineered E. coli by co-expressing the fabHB and the bckd gene from B. subtilis. To determine if it were possible to convert branched fatty acids into branched fatty acids short-chain esters, both the branched fatty acids biosynthesis module and fatty acids short-chain esters biosynthesis module were simultaneously expressed in E. coli. This combination resulted in the formation of branched fatty acids short-chain esters in the engineered E. coli. The results therefore clearly demonstrate the feasibility of engineering artificial pathways for branched fatty acids short-chain esters biosynthesis in a microbial host.After succeeding in E. coli demonstrated the biosynthesis of various fatty acid esters, we hope to further establish the fatty acid esters synthesis in yeast. The well-studied ethanol-producing microorganism Saccharomyces cerevisiae offers a number of advantages for producing fatty acid ethyl esters (FAEEs) due to the convenient cultivation and genetic manipulation, and the thick cell walls and good resistance to contamination. Thus it will be a good choice to produce FAEEs in S. cerevisiae. Here we showed the de novo biosynthesis of FAEEs from glucose in S. cerevisiae based on the heterologous expression of WS/DGAT, which could synthesize FAEEs from ethanol and fatty acyl-coA with a titer of6.7mg/L in shake flasks. Furthermore, this study provides some attractive strategies for increasing FAEEs production in S. cerevisiae. As an initial attempt to increase FAEEs production in S. cerevisiae, we sought to the overexpression of acc1gene to increase the malonyl-CoA availability. The overexpression of the accl gene resulted in an approximately65%increase in FAEEs production up to11.1mg/L. Coupled with deletion of pox1to block degradation of fatty acyl-CoA, which demonstrated another43%increase in FAEEs production up to15.9mg/L. Compared with the similar engineering of recombinant E. coli, S. cerevisiae FAEEs yield is not ideal.The Pichia pastoris is a powerful host for the heterologous expression of proteins, so we hope to further establish the efficient synthesis of fatty acid esters in P. pastoris. Since the fatty acid and2-keto acid pathways are native microbial synthesis pathways, this strategy can be implemented in P. pastoris to produce FASEs. In order to verify the feasibility of this strategy, we further established the biosynthesis of FASEs in recombinant P. pastoris by combining expression of the genes aro10, adh2and ws/dgat with a titer of201.5mg/L in shake flasks. |
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