Reduced Genetic Heterogeneity for Stable Bioproduction by Harnessing the Bias and Mechanism of Mutation.

Microbial bioproduction is an important approach to realising green biomanufacturing. However, poor bioproduction stability caused by genetic heterogeneity is one of the important factors limiting its industrial-scale applications. Here, two methods have been developed to reduce genetic heterogeneity in Bacillus subtilis. SiteMuB (the site-dependent mutation bias) was proposed to enable stable genome integration expression by analysing the spontaneous mutation rate of the same DNA sequences integrated at different genome sites. Additionally, robustly growing chassis with low mutation rates (ChassisLMR) were developed by deleting unstable elements and enhancing DNA repair. These methods were then employed to improve the production stability of small molecule metabolites and proteins. In N-acetylneuraminic acid production, after 76 generations of cell division, corresponding to the number of cell generations required for > 200-m industrial-scale production, strains with SiteMuB and ChassisLMR achieved 15.9-fold and 11.1-fold higher titres than that of the starting strain, respectively. Moreover, by improving the genetic stability of burdensome T7RNAP, combining SiteMuB with ChassisLMR stably maintained the T7 expression system for up to 74 generations, representing a 2.1-fold improvement. Furthermore, ChassisLMR improved the production stability of GFP on the plasmids by 1.38-fold. Overall, SiteMuB and ChassisLMR provide broadly applicable and highly efficient ways to achieve stable bioproduction by reducing genetic heterogeneity.