Enhancing functional expression of codon‐optimized heterologous enzymes in Escherichia coli BL21(DE3) by selective introduction of synonymous rare codons. Issue 5 (27th December 2016)

• Record Type: Journal Article
• Authors: Zhong, Chao; Wei, Ping; Zhang, Yi‐Heng Percival
• Is Part Of: Biotechnology and bioengineering. Volume 114:Issue 5(2017)
• Page Start: 1054
• Page End: 1064
• Publication Date: 2016-12-27
• Subjects: rare codon -- translation attenuation -- synonymous substitution -- functional protein expression -- directed evolution; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26238
• Languages: English
• ISSNs: 0006-3592

Abstract

ABSTRACT: Rare codon in a heterologous gene may cause premature termination of protein synthesis, misincorporation of amino acids, and/or slow translation of mRNA, decreasing the heterologous protein expression. However, its hypothetical function pertaining to functional protein folding has been barely reported. Here, we investigated the effects of selective introduction of synonymous rare codons (SRCs) to two codon‐optimized (i.e., rare codon‐free) genes sucrose phosphorylase (SP) gene from Thermoanaerobacterium thermosaccharolyticum and amidohydrolase gene from Streptomyces caatingaensis on their expression levels in Escherichia coli BL21(DE3). We investigated the introduction of a single SRC to the coding regions of alpha‐helix, beta‐strand, or linker in the first half of rare codon‐free sp and ah gene. The introduction of a single SRC in the beginning of the coding regions of beta‐strand greatly enhanced their soluble expression levels as compared to the other regions. Also, we applied directed evolution to test multi‐SRC‐containing sp gene mutants for enhanced soluble SP expression levels. To easily identify the soluble SP expression level of colonies growing on Petri dishes, mCherry fluorescent protein was used as a SP‐folding reporter when it was fused to the 3′ end of the sp gene mutant libraries. After three rounds of screening, the best sp gene mutant containing nine SRCs exhibited an approximately six‐fold enhancement in soluble protein expression level as compared to the wild‐type and rare codon‐free sp control. This study suggests that the selective introduction of SRCs can attenuate translation at specific points and such discontinuous attenuation can temporally separate the translation of segments of the peptide chains and actively coordinates their co‐translational folding, resulting in enhanced functional protein expression. Biotechnol. Bioeng. 2017;114: 1054–1064. © 2016 Wiley Periodicals, Inc. Abstract : The selective introduction of one or multiple synonymous rare codon (SRC) in the beginning of β‐strand coding regions within the first half of genes was found out to enhance their soluble expression levels of rare codon‐free genes. The introduction of SRC can attenuate translation at specific sites and such discontinuous attenuation can temporally separate the translation of polypeptides and coordinate their co‐translational folding.