Construction Of The Bacillus Subtilis Strains For High Surfactin Production

Surfactin is a lipopeptide that contains two acidic amino acid residues(glutamate and aspartate),five nonpolar amino acid residues(leucine and valine)and a C₁₂-C₁₉?-hydroxy fatty acid chain.It is expected to be used in many industrial fields including oil recovery,pharmaceuticals,due to its extraordinary surface activity and antimicrobial,antiviral,and antitumor activities.However,the large-scale production and industrial application of surfactin remained restricted by its low production of natural Bacillus subtilis strains.Surfactin biosynthesis could be improved by increasing the supply of fatty acids,however,the effect of the regulation of amino acid metabolism on surfactin production was not yet clear.In this study,we selected the wild-type B.subtilis 168 strain as start strains,which had a clear genetic background.Firstly,we integrated the complete sfp gene into the genomic ydeO site of the wild-type B.subtilis 168 strain,which generated the recombinant BS168NU-S strain.The surfactin production by the BS168NU-S strain was 0.45 g/L,which was determined at 24 h after inoculation in semi-defined fermentation medium when no xylose was added.Then,we interfered 20 potential genes which related to the four kinds of amino acids metabolism for surfactin production by CRISPRi.The surfactin titre of BS168NU-Sd was only 0.17 g/L at 24 h in fermentation medium with the addition of 4 g/L xylose inducer after inoculation.This negative impact on surfactin production resulted from the toxicity of the highly expressed d Ca S9 protein.Among the 20 recombinant strains,16 strains obtained higher surfactin titres than that produced by the parent BS168NU-Sd strain(the surfactin production of BS168NU-Sd with only d Cas9 but no sg RNA expression was 0.17 g/L).In particular,the strains in which the yrp C,rac E or mur C genes were inhibited individually produced 0.54,0.41,or 0.42 g/L surfactin,respectively.All three genes are related to the metabolism of L-glutamate,whose acylation is the first step in the surfactin condensation reaction.It is worth mentioning that the inhibition of the cluster of bkd AA and bkd AB which is related to the metabolism of L-leucine and L-valine,is not only increased the surfactin production,but also improved the proportions of C₁₄isoforms from 25.7%(BS168NU-S)or 18.7%(BS168NU-Sd)to 82.4%or 85.1%,respectively.We final combined these three genes which improved the surfactin production obviously in single gene inhibition.In four multiple genes strains,the surfactin production of three strains which inhibiting two genes at the same time improved,and the highest reached 0.75 g/L when inhibited yrp C and rac E,which was over 4.4-fold of the control.This study,for the first time to our best knowledge,regulated the amino acid metabolism to improve surfactin production,which proved to be an effective strategy and could provide a new perspective for the surfactin production in B.subtilis.