High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties

Abstract

BackgroundMicrobial synthesis of oleochemicals derived from native fatty acid (FA) metabolism has presented significant advances in recent years. Even so, native FA biosynthetic pathways often provide a narrow variety of usually linear hydrocarbons, thus yielding end products with limited structural diversity. To overcome this limitation, we took advantage of a polyketide synthase-based system from Mycobacterium tuberculosis and developed an Escherichia coli platform with the capacity to synthesize multimethyl-branched long-chain esters (MBE) with novel chemical structures.ResultsWith the aim to initiate the characterization of these novel waxy compounds, here, we describe the chassis optimization of the MBE producer E. coli strain for an up-scaled oil production. By carrying out systematic metabolic engineering, we improved the final titer to 138.1 ± 5.3 mg MBE L−1 in batch cultures. Fed-batch microbial fermentation process was also optimized achieving a maximum yield of 790.2 ± 6.9 mg MBE L−1 with a volumetric productivity of 15.8 ± 1.1 mg MBE (L h)−1. Purified MBE oil was subjected to various physicochemical analyses, including differential scanning calorimetry (DSC) and pressurized-differential scanning calorimetry (P-DSC) studies.ConclusionsThe analysis of the pour point, DSC, and P-DSC data obtained showed that bacterial MBE possess improved cold flow properties than several plant oils and some chemically modified derivatives, while exhibiting high oxidation stability at elevated temperatures. These encouraging data indicate that the presence of multiple methyl branches in these novel esters, indeed, conferred favorable properties which are superior to those of linear esters.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0631-x) contains supplementary material, which is available to authorized users.

Metabolic pairing of aerobic and anaerobic production in a one-pot batch cultivation

“…As an example, the yeast S. cerevisiae has been recently reported to produce very long chain WEs from glucose with the yield of 0.75 mg/g glucose [ 27 ]. Another example is the production of branched chain WEs with engineered E. coli from glucose (with additional supplementation with propionate, n-octanol and oleic acid), with the yield of 2.5 mg/g glucose [ 28 ]. In our earlier study, we produced WEs with engineered A. baylyi from glucose with the yield of 40 mg/g glucose [ 26 ].…”

Escherichia coli coculture for de novo production of esters derived of methyl-branched alcohols and multi-methyl branched fatty acids

“…For improving MBE titers, as reported previously, the expression system for the M. tuberculosis fadD28, mas and papA5 genes is clearly relevant, i.e. the inducible promoter used for controlling their expression and the copy number of the vector that harbors these genes [ 13 ]. In this sense, plasmid pMB07, constructed to contain fadD28 , mas and papA5 genes cloned in an operon configuration downstream the T7 promoter in a pBR322 derivative vector, was selected as the starting expression system [ 13 ].…”

“…[ 12 ], who took advantage of the in-depth genetic and biochemical understanding of FA synthesis in the model organism E. coli to engineer a mycocerosic acid (MA) polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched esters (MBE). These MBE, with novel chemical structures and properties, could be used, for example, for the development of new and improved bio-based molecules with high added value (e.g., specialty chemicals) [ 12 , 13 ]. The E. coli platform for the production of these compounds contained three heterologous enzymes of the MA pathway that enabled the biosynthesis of multi-methyl-branched-fatty acids (MBFA): (1) the acyl-AMP ligase FadD28, that loads the long-chain FA onto the mycocerosic acid polyketide synthase Mas, (2) the Mas enzyme that elongates the linear-chain FA loaded in this multidomain protein with four elongation cycles, using methylmalonyl-CoA as extender unit, and (3) the polyketide-associated protein A5 (PapA5) which catalyzes the release and transesterification of the MBFA with an alcohol.…”

Development of a cyanobacterial heterologous polyketide production platform

“…The PKS MAS system has been previously repurposed for the production of multimethyl-branched wax esters (MBEs) in E. coli 8 . MBEs have been shown to have exceptional physicochemical properties as bio-lubricants 12 . This system relies on the expression of FadD28 (acyl-AMP ligase), the PKS Mas (mycocerosic acid synthase), and PapA5 (polyketide-associated protein A5) 8 .…”

“…Therefore, as an alternative to studying these polyketides and their encoding gene clusters from the native producers, the heterologous production of polyketide molecules has proven to be a valuable strategy. Since its early days 10,11 , the heterologous expression of the PKS biosynthetic gene clusters has greatly facilitated our understanding of the basic enzymatic mechanisms involved in polyketide biosynthesis and enabled the manipulation of polyketide-encoding pathways either to improve their production or in order to generate novel molecules with novel properties 1,8,12–15 .…”