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

Abstract

Coordination 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.

Standard Intein Gene Expression Ramps (SIGER) for Protein-Independent Expression Control

“…In order to test the accuracy of SIGER systems in controlling multienzyme expression, we coupled the DnaB-mCherry and DnaX-GFP cassettes on the same plasmid. The two intein cassettes were coupled on a level 2 vector (Lv2), containing a chloramphenicol resistance marker, by using the level assembly method developed by Fages-Lartaud et al for pathway assembly 67 (see Materials and Methods and Figure S1 ). In brief, the DnaB and DnaX gene cassettes were constructed on a level 1 plasmid (Lv1), bearing an ampicillin marker, and containing BbsI restriction sites on each side of the gene cassettes.…”

“…Coupling of Intein Cassettes. The gene cassettes described above with DnaB-GOI and DnaX-GOI were assembled on pUC19 backbones from the previously described pathway assembly method (Fages-Lartaud et al 67 ). The principles of the method utilized in this study are described hereafter.…”

“…Different combinations of DnaB-mCherry and DnaX-GFP with various GES intensities were tested. The respective Lv1 plasmids were mixed together with an Lv2 backbone and subjected to 50 Golden Gate-like assembly cycles for BbsI restriction (2 min, 37 °C) and T4 ligase ligation (2 min, 16 °C). , The resulting assembly mixtures were transformed into E. coli cells that were plated on LB-agar plates containing 30 μg/mL chloramphenicol and incubated overnight at 37 °C. Positive clones were grown in triplicate, overnight at 37 °C with 800 rpm agitation, in 96-well plates in LB supplemented with 30 μg/mL chloramphenicol.…”