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

Abstract

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.

Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine

“…So et al developed a CRISPR-derived genome engineering technique to efficiently generate large genomic deletions in B. subtilis without the introduction of counter-selectable markers such as antibiotic-resistance genes, which had previously limited the application of B. subtilis in food engineering [ 25 ]. This method has wide applicability for various types of site-directed mutagenesis in B. subtilis [ 24 , 26 , 27 ]. However, CRISPR/Cas9 has low efficiency in multi-gene editing.…”

“…The industrial application of B. subtilis has developed rapidly in the last decades, and it has become the major microbial cell factory for many industrial products [ 44 , 45 ], including enzymes [ 46 ], heterologous proteins [ 31 ], antibiotics [ 47 ], vitamins [ 48 ], and amino acids [ 26 ]. Chemicals produced by B. subtilis also play an important role in various fields, such as food, feed, cosmetics, chemicals, and pharmaceuticals.…”

Impact of CRISPR interference on strain development in biotechnology

“…In bacilli, CRISPRi was used to produce the bacterial cyclic lipopeptide surfactin [130], which is a powerful surfactant commonly used as an antibiotic [131]. This was achieved by targeting the genes yrpC , racE , encoding MurI‐type glutamate racemases and murC , encoding acetylmuramate‐ l ‐alanine ligase in B. subtilis , leading to enhanced fatty acids supply as consequence to surfactin production.…”

A combination of genome reduction and promoter engineering can enhance surfactin production by Bacillus amyloliquefaciens LL3

“…Use of genome-reduced strain GR167 as a starting strain for surfactin production Surfactin is synthesized by the non-ribosomal peptide synthetase (NRPS) encoded by srfA operon (srfAA, srfAB, srfAC, srfAD) in microbes, which uses four amino acids ( L-glutamate, L-leucine, L-valine, and Laspartate) and fatty acids as precursors to form cyclic lipopeptide surfactin via a complex mechanism (condensation reaction) [34]. The biosynthesis pathway of surfactin was presented in Figure S4, which mainly contains three parts: the synthesis and activation of branched-chain fatty acids; the synthesis of the four amino acids; and the assembly of peptide chain.…”