Codon optimization prevents premature polyadenylation of heterologously-expressed cellulases from termite-gut symbionts in Aspergillus oryzae

Abstract

To achieve high expression of glycoside hydrolase family 45 endoglucanase (RsSym45EG1) from a symbiotic protist of the termite Reticulitermes speratus, synthetic sequence RsSym45eg1-co, in which the codon usage was adjusted to that of the highly-expressed tef1 gene encoding translation elongation factor 1α, was prepared and introduced into A. oryzae. The transcript level of RsSym45eg1-co was 1.8-fold higher than that of RsSym45eg1. In cells harboring RsSym45eg1, but not RsSym45eg1-co, truncated transcripts in which the coding region was prematurely terminated and followed by a poly A chain were found. The production of endoglucanase in the culture supernatant was improved by codon optimization. Truncated transcripts were also found when cellobiohydrolase and β-glucosidase from R. speratus symbionts were expressed, and the transcript level of the former was increased by codon-optimization. Our fi ndings suggest that premature polyadenylation frequently occurs in heterologous protein expression in A. oryzae, which might result in the poor yield of expressed proteins.

Heterologous Expression and Characterization of an Endoglucanase from a Symbiotic Protist of the Lower Termite, Reticulitermes speratus

“…The plasmid for transformation of A. oryzae, a molecule designed to express RsSymEG1 using α-amylase as a carrier, was generated by a set of plasmids [14] constructed with the aid of the MultiSite Gateway® three-fragment vector construction kit (MultiSite Gateway® System; Invitrogen, Carlsbad, CA, USA). Details are essentially the same as described in our recent publication [15]. Briefly, a Kex2 cleavage site followed by triglycine sequence (KRGGG) was inserted between the full-length α-amylase (AmyB) and a mature region of RsSymEG1 (residues 22-341) without the putative signal sequence.…”

Production and characterization of a recombinant beta‐1,4‐endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes

“…There have been several successful attempts to functionally express recombinant digestive enzymes from termites and their symbionts in both E. coli and Aspergillus oryzae. These include GHF9 endoglucanases from Coptotermes formosanus and C. acinaciformis in E. coli (Tokuda et al, 1999;Nakashima et al, 2002;Watanabe et al, 2002;Inoue et al, 2005;Ni et al, 2005Ni et al, , 2007Zhang et al, 2009); GHF5, GHF9 and GHF45 cellulases from Nasutitermes hindgut bacteria in E. coli (Warnecke et al, 2007); and R. speratus protist symbiont GHF7 exoglucanases in A. oryzae (Sasaguri et al, 2008;Todaka et al, 2010). With the current emphasis on biofuels and improved lignocellulose processing, there is also much interest in the development and characterization of recombinant termite and symbiont lignocellulases for biofuel production purposes (Scharf and Tartar, 2008;Matsui et al, 2009).…”

Remaining Challenges in the Metabolic Engineering of Yeasts for Biofuels

“…[50][51][52][53][54] A gene sequence is altered to improve enzyme expression. While most organisms use the same genetic code for amino acids, which are building blocks of an enzyme, the redundant nature of the code makes certain codons more frequent than others, and this frequency can change from organism to organism.…”