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

Surfactin, a representative biosurfactant of lipopeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a [beta]-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. 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 nC.sub.14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC.sub.14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC.sub.14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. 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 nC.sub.14-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.