Engineering Zymomonas mobilis for improving genetic transformation and stability of multi-gene biosynthetic pathways

Zymomonas mobilis holds significant promise for metabolic engineering but suffers from inefficient transformation and instability of plasmids over 8 kb. In this study, an element library containing promoters and terminators was constructed by analyzing the promoter activities of Z. mobilis ZM4. Using these regulatory elements, the β-carotene gene cluster (crtEXYIB) and individual genes (crtE, crtXYI, crtB) were regulated to construct pEZ-crt1 and pEZ-crt2 plasmid series. Screening identified top-yielding strains CRT1-29 (0.93 mg/g DCW) and CRT2-22 (1.25 mg/g DCW). Additionally, three key genes determining both the genetic transformation and stability of exogenous plasmids were identified by inactivating the genes encoding Restriction-Modification (R-M) or DNA-repair systems in Z. mobilis ZM4. The knockout of mrr and hsdM not only enhanced the transformation efficiency of a 3.95-kb methylated regular plasmid but also enabled the successful electroporation of a 9.5-kb reporter plasmid harboring a β-carotene expression cassette, which could not be transformed into Z. mobilis ZM4 and Mrr via electroporation previously. Moreover, the deletion of tatD prompted the stability of exogenous plasmid DNAs. The resultant triple-deficiency mutant MHT (Δmrr-hsdM-tatD) maintained the β-carotene expression plasmid in Z. mobilis, producing 2.09 mg/g DCW of β-carotene. Our study will prompt the application of Z. mobilis in metabolic engineering and synthetic biology.