Determination of Nucleotide Sequences within Promoter Regions Affecting Promoter Compatibility between Zymomonas mobilis and Escherichia coli .

A promoter plays a crucial role in controlling the expression of the target gene in cells, thus being one of the key biological parts for synthetic biology practices. Although significant efforts have been made to identify and characterize promoters with different strengths in various microorganisms, the compatibility of promoters within different hosts still lacks investigation. In this study, we chose the native P gap promoter of Zymomonas mobilis to investigate nucleotide sequences within promoter regions affecting promoter compatibility between Escherichia coli and Z. mobilis . P gap is one of the strongest promotors in Z. mobilis that has many excellent characteristics to be developed as microbial cell factories. Using EGFP as a reporter, a Z. mobilis -derived P gap mutant library was constructed and sorted in E. coli , with candidate promoters exhibiting high fluorescence intensity collected. A total of 53 variants were finally selected and sequenced by Sanger sequencing. The sequencing results grouped these variants into 12 different P gap variant types, among which seven types presented higher promoter strength than native P gap in E. coli . The next-generation sequencing technique was then employed to identify key mutations within the P gap promoter region that affect the promoter compatibility. Finally, six important sites were identified and confirmed to help increase P gap strength in E. coli while keeping similar strength of native P gap in Z. mobilis . Compared to native P gap , synthetic promoters combining these sites had enhanced strength; especially, P gap -6M combining all six sites exhibited 20-fold greater strength than native P gap in E. coli . This study thus not only determined six important sites affecting promoter compatibility but also confirmed a series of P gap promoter variants with strong promoter activity in both E. coli and Z. mobilis . In addition, a strategy was established in this study to investigate and determine nucleotide sequences in promoter regions affecting promoter compatibility, which can be applied in other microorganisms to help reveal universal factors affecting promoter compatibility and design promoters with desired strengths among different microbial cell factories.