The Construction Of Bacillus Subtilis Gene Editing System Based On CRISPR-Cpf1 And Metabolic Engineering Application Of Hyaluronic Acid Synthesis

The rapid development of CRISPR technology has provided many molecular biological tools for B.subtilis.Therefore,the establishment and expansion of B.subtilis CRISPR system is important.Hyaluronic acid,as macromolecular polysaccharide,is widely used in the field of medicine,food,and cosmetics.B.subtilis is regarded as an ideal host for hyaluronic acid production.It is important to study industrial application of hyaluronic acid in B.subtilis.In this study,we first constructed the CRISPR-AsCpf1 system containing two plasmids pHT01-AsCpf1 and pJOE-DRN23DR for genome editing of B.subtilis.In this system,the plasmid pJOE-DRN23DR containing the temperature-sensitive replicon rep pE194ts could be efficiently cured,which optimized the genome editing process.Using this system,the highest editing efficiencies of gene deletion,gene insertion,and large genomic deletion were evaluated as 100%,92%,and 87%,respectively.Then,metabolic engineering of B.subtilis was implemented for hyaluronic acid production using the CRISPR-AsCpf1 system.First,the strain B.subtilis-HA03 with a hyaluronic acid yield of 0.96 g/L was obtained by introducing the hyaluronic acid synthesis pathway(hasA insertion)and enhancing UDP-glucuronic acid synthesis pathway(tuaD insertion).Second,the strain B.subtilis-HA05 with a hyaluronic acid yield of 1.55 g/L was obtained by orderly deleting exopolysaccharide gene cluster(epsA-O)and key autolysis gene(lytC).Thirdly,the synthesis pathway of hyaluronic acid was optimized by UDP-acetylglucosamine synthesis pathway enhancement,glycolysis and pentose phosphate pathway regulation.And the hyaluronic acid yield of the strain B.subtilis-HA07,which was obtained by enhancing UDP-acetylglucosamine synthesis,was further increased to 2.27 g/L.Finally,the fermentation optimization of strain B.subtilis-HA07 was carried out.Through Plackett-Burman experimental design,sucrose and yeast extract were selected as the most significant factors for the production of hyaluronic acid.The yield of hyaluronic acid was increased to 4.64 g/L by optimizing sucrose and yeast extract contents in the steepest climbing experiment.Finally,the concentration and add time of inducer were optimized to increase the hyaluronic acid yield to 4.72 g/L,and the hyaluronic acid molecular weight was 0.54×105 Da.The yield of HA in 3 L batch fermentation was 5.12 g/L.