Abstract
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.
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Acknowledgments
We are grateful to Dr. Christopher R. French and Marco Valenzuela for kindly providing pSB1K2 plasmids. We thank Guillermo Marcuzzi for technical assistance in the GC-MS experiments. MS, AA, and HG are members of the Research Career; VC is a member of the FBIOyF Faculty; VG is a doctoral fellow of CONICET; and JR and JL were doctoral fellows of CONICET.
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This work was supported by PID-2013-0042 grant to HG and PICT-2017-2184 to AA.
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AA and HG conceived and designed the research. JR, VG, AA, and HG designed the experiments and analyzed the data. JR and VG performed all the experiments. JL collaborated with the structural analysis. VC and MS helped in specific experimental methods. AA and HG wrote the manuscript. All the authors read and approved the final version.
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Roulet, J., Galván, V., Lara, J. et al. Modification of PapA5 acyltransferase substrate selectivity for optimization of short-chain alcohol-derived multimethyl-branched ester production in Escherichia coli. Appl Microbiol Biotechnol 104, 8705–8718 (2020). https://doi.org/10.1007/s00253-020-10872-w
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DOI: https://doi.org/10.1007/s00253-020-10872-w