Exploitation of strong constitutive and stress-driven promoters from Acetobacter pasteurianus for improving acetic...

Acetobacter pasteurianus is an excellent cell factory for production of highly-strength acetic acid, and attracts an increasing attention in metabolic engineering. However, the available well-characterized constitutive and inducible promoters are rather limited to adjust metabolic fluxes in A. pasteurianus. In this study, we screened a panel of constitutive and acid stress-driven promoters based on time-series of RNA-seq data and characterized in A. pasteurianus and Escherichia coli. Nine constitutive promoters ranged in strength from 1.7-fold to 100-fold that of the well-known strong promoter P_adh under non-acetic acid environment. Subsequently, an acetic acid-stable red fluorescent visual reporting system was established and applied to evaluate acid stress-driven promoter in A. pasteurianus during highly-acidic fermentation environment. P_groES was identified as acid stress-driven strong promoters, with expression outputs varied from 100% to 200% when acetic acid treatment. To assess their application potential, ultra-strong constitutive promoter P_tuf and acid stress-driven strong promoter P_groES were selected to overexpress acetyl-CoA synthase and greatly improved acetic acid tolerance. Notably, the acid stress-driven promoter displayed more favorable for regulating strain robustness against acid stress by overexpressing tolerance gene. In summary, this is the first well-characterized constitutive and acid stress-driven promoter library from A. pasteurianus, which could be used as a promising toolbox for metabolic engineering in acetic acid bacteria and other gram-negative bacteria.