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Background  Surfactin is a cyclic lipopeptide that is of great industrial use owing to its extraordinary surfactant power and antimicrobial, antiviral, and antitumor activities. Surfactin is synthesized by a condensation reaction in microbes, which uses fatty acids and four kinds of amino acids ( l -glutamate,  l -aspartate,  l -leucine and  l -valine) as precursors. 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.    Results  In this study, we aimed to improve surfactin production in  B. subtilis  by repressing the genes on the branch metabolic pathways of amino acid biosynthesis using CRISPRi technology. First, 20 genes were inhibited individually, resulting in 2.5- to 627-fold decreases in transcriptional level as determined by RT-qPCR. 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 dCas9 but no sgRNA expression was 0.17 g/L). In particular, the strains in which the  yrpC ,  racE  or  murC  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. Furthermore, these three genes were repressed in combination, and the strain with co-inhibition of  yrpC  and  racE  produced 0.75 g/L surfactin, which was 4.69-fold higher than that of the parent strain. In addition, the inhibition of  bkdAA  and  bkdAB,  which are related to the metabolism of  l -leucine and  l -valine, not only improved surfactin production but also increased the proportion of the C 14  isoform.    Conclusions  This study, to the best of our knowledge for the first time, systematically probed the regulatory effect of increasing the supply of amino acids on surfactin production. It provided an effective strategy and a new perspective for systematic studies on surfactin and other amino acid-derived chemicals.    Electronic supplementary material  The online version of this article (10.1186/s12934-019-1139-4) contains supplementary material, which is available to authorized users.

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