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

Coordination of multigene 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 of the 5'UTR in combination with the start of the gene, referred to as the "gene ramp", such as rare codons and mRNA secondary structures. These features strongly influence the translation yield and protein quality by regulating the 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 Ramp (SIGER) system for controlling protein expression. The SIGER system makes use of inteins to decouple the translation initiation features from the gene of 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 the 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. As a proof of concept of the potential of the system, we also used a SIGER system to express two difficult-to-produce proteins, GumM and CBM73.

A universal approach to gene expression engineering.

In this study, we provide a universal approach to Gene Expression Engineering (GeneEE) for creating artificial expression systems. GeneEE leads to the generation of artificial 5' regulatory sequences (ARES) consisting of promoters and 5' untranslated regions. The ARES lead to the successful recruitment of RNA polymerase, related sigma factors and ribosomal proteins that result in a wide range of expression levels. We also demonstrate that by engaging native transcription regulators, GeneEE can be used to generate inducible promoters. To showcase the universality of the approach, we demonstrate that 200-nucleotide (nt)-long DNA with random composition can be used to generate functional expression systems in six bacterial species, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Thermus thermophilus, Streptomyces albus and Streptomyces lividans, and the eukaryote yeast Saccharomyces cerevisiae.

Mechanisms governing codon usage bias and the implications for protein expression in the chloroplast of Chlamydomonas reinhardtii.

Chloroplasts possess a considerably reduced genome that is decoded via an almost minimal set of tRNAs. These features make an excellent platform for gaining insights into fundamental mechanisms that govern protein expression. Here, we present a comprehensive and revised perspective of the mechanisms that drive codon selection in the chloroplast of Chlamydomonas reinhardtii and the functional consequences for protein expression. In order to extract this information, we applied several codon usage descriptors to genes with different expression levels. We show that highly expressed genes strongly favor translationally optimal codons, while genes with lower functional importance are rather affected by directional mutational bias. We demonstrate that codon optimality can be deduced from codon-anticodon pairing affinity and, for a small number of amino acids (leucine, arginine, serine, and isoleucine), tRNA concentrations. Finally, we review, analyze, and expand on the impact of codon usage on protein yield, secondary structures of mRNA, translation initiation and termination, and amino acid composition of proteins, as well as cotranslational protein folding. The comprehensive analysis of codon choice provides crucial insights into heterologous gene expression in the chloroplast of C. reinhardtii, which may also be applicable to other chloroplast-containing organisms and bacteria.

mCherry contains a fluorescent protein isoform that interferes with its reporter function.

Fluorescent proteins are essential reporters in cell and molecular biology. Here, we found that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform in both prokaryotes and eukaryotes. The short isoform creates significant background fluorescence that biases the outcome of expression studies. In this study, we identified the short protein isoform, traced its origin, and determined the extent of the issue within the family of red fluorescent protein. Our analysis showed that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. We provided a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

Overview of tRNA Modifications in Chloroplasts.

The chloroplast is a promising platform for biotechnological innovation due to its compact translation machinery. Nucleotide modifications within a minimal set of tRNAs modulate codon-anticodon interactions that are crucial for translation efficiency. However, a comprehensive assessment of these modifications does not presently exist in chloroplasts. Here, we synthesize all available information concerning tRNA modifications in the chloroplast and assign translation efficiency for each modified anticodon-codon pair. In addition, we perform a bioinformatics analysis that links enzymes to tRNA modifications and aminoacylation in the chloroplast of Chlamydomonas reinhardtii. This work provides the first comprehensive analysis of codon and anticodon interactions of chloroplasts and its implication for translation efficiency.