Directed Evolution of Ribosomal Protein S1 for Enhanced Translational Efficiency of High GC Rhodopseudomonas palustris DNA in Escherichia coli

Abstract

The expression of foreign DNA in Escherichia coli is important in biotechnological applications. However, the translation of genes from GC-rich organisms is inefficient in E. coli. To overcome this problem, we applied directed evolution to E. coli ribosomal protein S1. Two selected mutants enabled 12-and 8-fold higher expression levels from GC-rich DNA targets. General improvements in translation efficiency over a range of genes from Rhodopseudomonas palustris and E. coli was achieved using an S1 mutant selected against multiple genes from R. palustris. This method opens new opportunities for the expression of GC-rich genes in E. coli.The recombinant expression of DNA in Escherichia coli is important for biotechnological applications, enabling the extension of pathways for the production of non-native metabolites with a growing economic and social impact (1-5). Heterologous expression of GC-rich genes in E. coli poses a unique set of challenges, in comparison with other DNA. Overcoming these problems will generate increased flexibility in the production of proteins and metabolites in this host. Although most efforts for recombinant expression in this host are achieved using E. coli promoters and optimal ribosome-binding sites (RBS), 2 it would be advantageous to express large pathways, consisting of many genes, without needing to change each promoter and RBS. Such a strategy would enable hybrid functionalities from multiple organisms using technology such as the fusing of two genomes (6) or transformation-associated recombination (7). Here we assess the ability of E. coli to express native DNA, including transcription and translation initiation regions, from the high GC ␣-bacterium Rhodopseudomonas palustris (8), and we propose a novel method for enhancing expression of foreign transcripts, altering E. coli ribosomal protein S1 by directed evolution.

An efficient design strategy for a whole-cell biosensor based on engineered ribosome binding sequences

“…In E. coli, mRNA degradation is a complex process that includes an initial, rate-limiting endonucleolytic cleavage (usually mediated by RNase E) followed by 3′-5′ exonucleolytic degradation of the cleavage products [28][29][30]. Endonucleolytic attacks can be blocked to some extent by increased translational efficiency because, at higher rates of translation, ribosomes occlude a greater number of endonuclease recognition sites [12,13]. Therefore, it is likely that the SD-mediated increased translational efficiency of the BTEX-SD luc mRNA led to increased stability of the mRNA.…”

“…In prokaryotes, the 5′ untranslated region (5′ UTR) of an mRNA has a significant effect on translational efficiency and mRNA stability by directing the initiation codon to the P site of the small ribosomal subunit and by masking endonuclease recognition sites in mRNAs [12][13][14]. It is clear that two major motifs in the 5′ UTRs of bacterial mRNAs, the Shine-Dalgarno (SD) sequence and translational enhancers, are directly involved in initiation of translation [15][16][17][18].…”

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin

“…pJRB510 is a high copy plasmid derived from pJRB202 (Bernstein et al, 2007). It contains the badHIaliBAbadK operon from amplified from the R. palustris chromosome, expressed from the P lacO-1 promoter.…”

“…The protein fusions of genes from the badHIaliBAbadK operon were created as described using the high copy pTB120 backbone (Bernstein et al, 2007). Protein fusions to LacZ were made to the ~20 th amino acid of each gene in the pathway (Simons et al, 1987), such that the fusion to the final gene in the operon, badK, includes the promoter and each of the 4 genes upstream of it.…”

“…Moreover, a group successfully produced the polyketides epothilone C and D in E. coli from a Sorangium cellulosum pathway using a combination of DNA synthesis, inducible promoters, chaperone co-expression, and the separation of a large protein into two polypeptides (Mutka et al, 2006). Previously, we assessed the ability of Escherichia coli to recognize unaltered foreign DNA from the high GC photosynthetic α-bacterium R. palustris (Bernstein et al, 2007). Multiple transcription signals were recognized by the host, but translation of the donor DNA was found to be less efficient which was partially attributed to the high GC content of the 5'-untranslated region (UTR) of transcripts, even in the presence of a strong RBS.…”