High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris

Abstract

Phytase is widespread in nature. It has been used as a cereal feed additive that can enhance the phosphorus and mineral absorption in monogastric animals to reduce the level of phosphorus output in manure. Phytase of Peniophora lycii is a 6'-phytase, which owns high specific activity. To achieve a high expression level of 6'-phytase in Pichia pastoris, the 1,230-bp phytase gene of P. lycii was synthesized and optimized for codon usage, G+C content, as well as mRNA secondary structures. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene (AOX1) promoter, the synthetic signal peptide (designated MF4I), which is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence, were used to transform P. pastoris. The P. pastoris strain that expressed the modified phytase gene (phy-pl-sh) with MF4I sequence produced 12.2 g phytase per liter of fluid culture, with the phytase activity of 10,540 U ml(-1). The yield of the modified phytase gene, with bias codon usage and MF4I signal, is 4.4 times higher than that of the wild type gene with MF4I signal and 13.6 times higher than that of the wild type gene with wild type S. cerevisiae signal. The recombinant phytase had one optimum pH (pH 4.5) and an optimum temperature of 50 degrees C. The P. pastoris strain expressed the modified 6-phytase gene, with the MF4I signal peptide showing great potential as a commercial phytase production system.

Enhanced delignification of lignocellulosic substrates by <i>Pichia</i> GS115 expressed recombinant laccase

“…Two laccase genes [Madhavan et al, 2014], one CDH gene, and 13 LDA genes, including four genes encoding glucose oxidases and benzoquinone reductases, are well conserved in S. commune [Ohm et al, 2012]. Because of the diversity of laccase genes from S. commune, and the ambiguity in isolating these genes using laccase specific universal primers, the expression of recombinant protein using a P.pastoris host system through codon optimization [Xiong et al, 2006] was considered for further study.…”

The construction of a short gene by a very fast, modified, and simplified gene synthesis and the analysis of various effects on this synthesis

“…One of the most critical situations which can be addressed by chemical gene synthesis is the lacking of template DNA (Chang et al, 2002). Several methods including enzymatic ligation (Smith et al, 1982;Borodina et al, 2003), ForkI method (Hayden and Mandecki, 1988) and PCRbased strategies (Ciccarelli et al, 1991;Stemmer et al, 1995;Wooddelland Burgess, 1996;Gao et al, 2003;Young and Dong, 2004;Xiong et al, 2006) have already been applied to chemical gene synthesis. In 1995, Stemmer et al described the assembly PCR technique, which was an attractive and relative low cost method in gene synthesis, as a technique in which small genes or gene fragments were synthesized.…”

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

“…These problems have often been attributed to the A/T-richness of the coding region. In several cases, expression level was improved dramatically by codon optimization (Gouka et al, 1997b;Koda et al, 2005;Xiong et al, 2006). Therefore, codon optimization is thought of as a technique to improve the production of heterologous proteins.…”