Overexpression inEscherichia coliof the AT-richtrpAandtrpBgenes from the hyperthermophilic archaeonPyrococcus furiosus

Abstract

Expression of AT-rich genes from microorganisms such as archaea is often inefficient in Escherichia coli. The trpA and trpB genes encoding the tryptophan synthase subunits were cloned from the hyperthermophilic archaeon Pyrococcus furiosus. No apparent difference in codon bias was found between the genes. However, using a conventional cloning vector having the lac promoter, the trpB gene was expressed poorly in E. coli, whereas the trpA gene was overexpressed. The expression of the trpB gene was remarkably enhanced (>12-fold) by the introduction of an overlapping leader open reading frame. The expression of the trpA gene was also improved ( approximately 1.5-fold). This approach may be useful for overexpressing various kinds of AT-rich genes.

Characteristics of Escherichia coli Isolated from Intestinal Microbiota Children of 0–5 Years Old in the Commune of Abomey-Calavi

“…In addition, it may be due to inefficient translation initiation. Inefficient translation initiation in E. coli occurs in many adenine- and thymine-rich genes [ 43 ].…”

High-efficiency expression and secretion of human FGF21 in Bacillus subtilis by intercalation of a mini-cistron cassette and combinatorial optimization of cell regulatory components

“…Previous studies showed the 5′ Untranslated Region (5′UTR) of a poorly expressed heterologous protein may form a stable and complex mRNA secondary structure, which can bury the ribosome binding site (RBS) region and the ATG start codon of the target protein, resulting in a decrease in translation initiation efficiency [33]. Structure of mini-cistron cassettes could reduce locally stabilized mRNA secondary structures of the downstream target gene and result in an improvement of translation initiation efficiency [34–37]. Thus, to destabilize any potential stem-loop secondary structures in the rhFGF21 mRNA sequence, we designed seven auxiliary mini-cistron cassettes and introduced them by inserting mini open reading frames (ORF) upstream of the rhFGF21 gene.…”

“…Ishida et al reported that poor expression of genes can be due to inefficient translation initiation, and they introduced an overlapping leader ORF to activate the translation of a downstream gene through efficient translation re-initiation to improve the expression of the target protein [37]. In order to further increase the production of rhFGF21, different types of auxiliary mini-cistron cassettes were designed to disrupt the locally stabilized mRNA secondary structure around the RBS and initiation codon, and to enable efficient enhancement of translation initiation.…”

Comparative Genomic Analysis of Soil Dwelling Bacteria Utilizing a Combinational Codon Usage and Molecular Phylogenetic Approach Accentuating on Key Housekeeping Genes

“…Our results thus further corroborates the finding that the housekeeping gene trpB demonstrates an aberrant codon usage profile in comparison to the rest of the housekeeping genes considered in this study. The trpB gene encodes the β subunit of the enzyme tryptophan synthase, which is a pyridoxal 5 ′ -phosphate-dependent αββα multi enzyme complex, responsible for catalyzing the final two steps of tryptophan biosynthesis (Yanofsky, 2001;Ishida et al, 2002;Dunn et al, 2008;Raboni et al, 2009). This enzyme is bestowed with certain unique features, such as dual catalytic ability, substrate channeling (Leopoldseder et al, 2006;Raboni et al, 2009) and the trpB gene might have been subjected to gene duplication, fusion, loss as well as parallel evolution in some archaea and eubacteria (Xie et al, 2001;Leopoldseder et al, 2006).…”