Screening of broad-host expression promoters for shuttle expression vectors in non-conventional yeasts and bacteria

Abstract Background Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers. Results In this study, five Yarrowia lipolytica constitutive promoters ( yl.hp4d , yl.FBA1in , yl.TEF1 , yl.TDH1 , yl.EXP1 ) and five Kluyveromyces marxianus constitutive promoters ( km.PDC1 , km.FBA1 , km.TEF1 , km.TDH3 , km.ENO1 ) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y . lipolytica , K. marxianus , Pichia pastoris , Escherichia coli , and Corynebacterium glutamicum . We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y . lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris , E. coli , and C. glutamicum . In yeast P. pastoris , the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum , the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli , The RFP expression strength of the km . TEF1 promoter reached ∼20% of the T7 promoter. Conclusion Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts. Graphical Abstract