Genetic engineering of the precursor supply pathway for the overproduction of the nC14-surfactin isoform with promising MEOR applications

Abstract

Background Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. Results In this study, the fatty acid precursor supply pathway in Bacillus subtilis 168 for surfactin production was strengthened through two steps. Firstly, pathways competing for the precursors were eliminated with inactivation of pps and pks. Secondly, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed. As a result, the surfactin titer after 24 h of cultivation improved by 34%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC–MS showed that the proportion of nC14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. Conclusion The obtained results identified that enhancing the supply of fatty acid precursor is very essential for the synthesis of surfactin. At the same time, this study also proved that thioesterase BTE can promote the production of nC14-surfactin and experimentally demonstrated its higher surface activity and better performance in oil-washing. These results are of great significance for the MEOR application of surfactin. Graphic abstract

Genetic engineering of the branched‐chain fatty acid biosynthesis pathway to enhance surfactin production from Bacillus subtilis

“…The proportion of the C 16 isoform was also increased, which contributed to the whole yield of surfactin from strain TPS8. Many studies have also demonstrated that the proportion of surfactin isoforms can be altered by genetically modifying the fatty acid biosynthesis pathway 26,30 . Hu et al 30 .…”

“…Many studies have also demonstrated that the proportion of surfactin isoforms can be altered by genetically modifying the fatty acid biosynthesis pathway. 26,30 Hu et al 30 reported a 6.4-fold increase in the proportion of nC14 surfactin after the elimination of pathways competing for the precursors and overexpression of BTE and exhibited higher surface activity and better performance in oil washing. In this study, surfactin may exhibit different properties due to the increased proportion of the C 16 isoform.…”

“…26 The proportion of nC 14 surfactin increased by 6.4 times after the elimination of pathways competing for the precursors and overexpression of the plant medium-chain acyl-ACP thioesterase (BTE). 30 However, the reported modification of the fatty acid biosynthesis pathway was mostly carried out in the model strains, in which only a few genes were involved. 26,30 Despite success in achieving genetically engineered strains for surfactin production, it remains a formidable challenge in the genetic engineering of the branched-chain fatty acid biosynthesis pathway for improving surfactin production from B. subtilis.…”

“…By knocking out the gene lpdV , located in the bkd operon (made up of genes lpdV , bkdAA , bkdAB , and bkdB ) and responsible for the synthesis of branched‐chain α‐ketoacyl‐CoA, the proportion of surfactin with the linear chain C 14 was increased by 2.5 times 26 . The proportion of n C 14 surfactin increased by 6.4 times after the elimination of pathways competing for the precursors and overexpression of the plant medium‐chain acyl‐ACP thioesterase (BTE) 30 . However, the reported modification of the fatty acid biosynthesis pathway was mostly carried out in the model strains, in which only a few genes were involved 26,30 .…”

A new bacterial-derived biosurfactant for biotechnological applications in the oil industry: production, optimization, biosurfactant functional and physicochemical characterization

“…By way of comparison, we note, for example, that the MI27-derived BioS is more effective than surfactin isoforms produced by Bacillus subtilis isolate LSFM-05 (de Faria et al 2011), those produced by B. subtilis SL having a CMC value of 154 mg/L and a ST of 28.68 mN/m (Wu et al 2022) and surfactin isoforms produced by Bacillus nealsonii S2MT (Phulpoto et al 2020). In means of the ST reduction power, it is more efficient than surfactin isoforms produced by B. subtilis BS-37 displaying a CMC value of mg/L (Liu et al 2014), Bacillus amyloliquefaciens MO13-derived surfactin isoforms displaying a CMC value of 36 mg/L and an ST of 27 mN/m (Moro et al 2022), engineered surfactin isoforms produced by B. subtilis 168 having a CMC value of 38-36 mg/L with potential ST of 28 mN/m (Hu et al 2021) and surfactin isoforms derived from B. subtilis #309 having a CMC value of 15 mg/L with potential ST of 28 mN/m (Janek et al 2021).…”