Modification of PapA5 acyltransferase substrate selectivity for optimization of short-chain alcohol-derived multimethyl-branched ester production in Escherichia coli.

Plant waxes are interesting substitutes of fossil-derived compounds; however, their limited sources and narrow structural diversity prompted the development of microbial platforms to produce esters with novel chemical structures and properties. One successful strategy was the heterologous expression of the mycocerosic polyketide synthase-based biosynthetic pathway (MAS-PKS, PapA5 and FadD28 enzymes) from Mycobacterium tuberculosis in Escherichia coli. This recombinant strain has the ability to produce a broad spectrum of multimethyl-branched long-chain esters (MBE) with novel chemical structures and high oxidation stability. However, one limitation of this microbial platform was the low yields obtained for MBE derived of short-chain alcohols. In an attempt to improve the titers of the short-chain alcohol-derived MBE, we focused on the PapA5 acyltransferase-enzyme that catalyzes the ester formation reaction. Specific amino acid residues located in the two-substrate recognition channels of this enzyme were identified, rationally mutated, and the corresponding mutants characterized both in vivo and in vitro. The phenylalanine located at 331 position in PapA5 (F331) was found to be a key residue that when substituted by other bulky and aromatic or bulky and polar amino acid residues (F331W, F331Y or F331H), gave rise to PapA5 mutants with improved bioconversion efficiency; showing in average, 2.5 higher yields of short-chain alcohol-derived MBE compared with the wild-type enzyme. Furthermore, two alternative pathways for synthetizing ethanol were engineered into the MBE producer microorganism, allowing de novo production of ethanol-derived MBE at levels comparable with those obtained by the external supply of this alcohol. KEY POINTS: • Mutation in channel 2 changes PapA5 acyltransferase bioconversion efficiency. • Improved production of short-chain alcohol derived multimethyl-branched esters. • Establishing ethanologenic pathways for de novo production of ethanol derived MBE. • Characterization of a novel phenylethanol-derived MBE.

Development of a cyanobacterial heterologous polyketide production platform.

The development of new heterologous hosts for polyketides production represents an excellent opportunity to expand the genomic, physiological, and biochemical backgrounds that better fit the sustainable production of these valuable molecules. Cyanobacteria are particularly attractive for the production of natural compounds because they have minimal nutritional demands and several strains have well established genetic tools. Using the model strain Synechococcus elongatus, a generic platform was developed for the heterologous production of polyketide synthase (PKS)-derived compounds. The versatility of this system is based on interchangeable modules harboring promiscuous enzymes for PKS activation and the production of PKS extender units, as well as inducible circuits for a regulated expression of the PKS biosynthetic gene cluster. To assess the capability of this platform, we expressed the mycobacterial PKS-based mycocerosic biosynthetic pathway to produce multimethyl-branched esters (MBE). This work is a foundational step forward for the production of high value polyketides in a photosynthetic microorganism.

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

BACKGROUND: Microbial 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. RESULTS: With 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. CONCLUSIONS: The 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.