Genetic engineering of the branched fatty acid metabolic pathway of Bacillus subtilis for the overproduction of surfactin C₁₄ isoform

Abstract

Surfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl-CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP-HPLC and the surfactin isoforms were characterized using LC-MS-MS and GC-MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C FA chain.

Rational strain improvement for surfactin production: enhancing the yield and generating novel structures

“…Compared with the NRPS-catalyzed synthesis of the peptide structure, the biosynthesis of the fatty acyl structure of the surfactin molecule still needs further research. The fatty acid chain length of surfactin is generally C 13 –C 15 , whereby C 14 and C 15 are usually predominant (60–80%) [34]. The length and isomerism of the FA chain were known to have an impact on the physicochemical properties and biological activity [86].…”

“…The relative proportions of C 13 and C 14 in the control strain were 39.7% and 21.2%, respectively, which changed to 26.5% and 40.6% after knocking out codY . Later, Coutte et al further studied the branched-chain amino acid metabolic pathway and found that the proportion of surfactin with linear C 14 was increased 2.5 times after knocking out the lpdV gene responsible for the final degradation of branched-chain amino acids [34].…”

Enhancing surfactin production by using systematic CRISPRi repression to screen amino acid biosynthesis genes in Bacillus subtilis

“…Others achieved srfA overexpression by upregulating the quorum sensing system ComQXPA [11, 13–15] or downregulating the negative factors [13, 14, 16–19]. Dhali et al [20]. enhanced the production to 1556 ± 123 mg of surfactin per g dry weight of cell biomass by knocking out the global regulation factor codY , which had negative effects on srfA expression.…”

Surfactin as a Green Agent Controlling the Growth of Porous Calcite Microstructures

“…Surfactin is a lipopeptide biosurfactant composed of a cyclic ring of amino acids linked to hydroxylic fatty acids by a lactone bond [ 41 , 42 ]. The cyclic peptide part contains two hydrophilic residues of Glu and Asp acid because of free groups of COO − at a pH above pK a = 5.8 [ 43 , 44 ].…”