Standard Intein Gene Expression Ramps (SIGER) for protein-independent expression control

Abstract

AbstractCoordination of multi-gene expression is one of the key challenges of metabolic engineering for the development of cell factories. Constraints on translation initiation and early ribosome kinetics of mRNA are imposed by features at the start of the gene, referred to as the “gene ramp”, such as rare codons and mRNA secondary structures forming in relation with the 5’UTR. These features strongly influence translation yield and protein quality by regulating ribosome distribution on mRNA strands. The utilization of genetic expression sequences, such as promoters and 5’UTRs in combination with different target genes leads to a wide variety of gene ramp compositions with irregular translation rates leading to unpredictable levels of protein yield and quality. Here, we present the Standard Intein Gene Expression Ramps (SIGER) system for controlling protein expression. The SIGER system uses inteins to uncouple a characterized gene ramp from a target protein. We generated sequence-specific gene expression sequences for two inteins (DnaB and DnaX) that display defined levels of protein expression. Additionally, we used inteins that possess the ability to release the C-terminal fusion protein in vivo to avoid impairment of protein functionality by the fused intein. Overall, our results show that SIGER systems are unique tools to mitigate the undesirable effects of gene ramp variation and to control the relative ratios of enzymes involved in molecular pathways.Graphical abstract