Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis

Hyaluronic acid (HA) is a high-value biopolymer that is produced in large scales using attenuated strains ofgroup C streptococci. However, due to the pathogenicity and fastidious nature of these bacteria, the development of bioprocesses for HA production centered on robust 'Generally Recognized as Safe (GRAS)' organisms, such as Bacillus subtilis, is of increased interest. Here, we report metabolic engineering of novel B. subtilis strains in which the carbon flux has been partially diverted from central metabolism, i.e. the pentose phosphate pathway (PPP) and glycolysis, into HA biosynthesis. First, an improved base strain of B. subtilis was engineered for more effective HA production with less susceptibility to catabolite repression when expressing genes from a xylose-inducible promoter. Subsequently, Clustered Regularly Interspaced Palindromic Repeats interference (CRISPRi) was applied to reduce the expression of individual pfkA or zwf in the base strain, leading to substantial improvements to the HA titer with a concomitant decrease in the molecular weight (MW). On the other hand, multiplexed repression of both pfkA and zwf expression resulted in increases to the HA titer of up to 108% and enhancements to the MW, compared to the base strain. Moreover, the addition of exogenous HA monomers, i.e. glucuronic acid (GlcUA) and N-acetyl-glucosamine (GlcNAc), to B. subtilis cultures markedly improved the HA MW but decreased the HA titer, providing insights into the mechanism of HA biosynthesis by streptococcal hyaluronan synthase (SeHAS) in B. subtilis. Our study demonstrates the successful application of metabolic engineering strategies to establish B. subtilis as an effective platform for high-level HA production.