Design and construction of shuttle expression vectors for non- conventional yeasts and bacteria

Abstract

Abstract Synthetic biology seeks to engineer microbial cells for sustainable efficient production of value-added biofuels and bioproducts from low-cost renewable feedstocks. In order to resolve the conflicts of carbon flux between cell growth and bioproducts synthesis, the dynamic up-regulation on the bioproduct synthesis pathways and down-regulation on the competitive pathways simultaneously could be adjusted by promoter sets with diverse strengths. The development of broad-spectrum promoter libraries comprising promoters of varying strengths for different hosts without tedious reconstruction processes are attractive for biosynthetic engineers. In this study, we observed that five K. marxianus promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) can all express genes in Y. lipolytica and that five Y. lipolytica promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) can all express genes in K. marxianus with variable expression strengths. Interestingly, we also found two yeast promoters could shuttle express reporter genes in P. pastoris, E. coli and C. glutamicum. The yl.TEF1 promoter can also strongly express amylase and RFP in yeast P. pastoris and the eukaryotic promoter km.TEF1 can constitutively strong express RFP in bacterium E. coli and C. glutamicum. The RFP expression strength of the promoter km.TEF1 reached ∼20% to that of the T7 promoter in E. coli and was much stronger (more than 10 times) than in K. marxianus. Our work will expand the future development of broad host acceptable dynamic regulated systems with these broad-spectrum promoters for dynamically orchestrate the carbon flux to maximize target bioproduct synthesis.