Codon Optimization ofCandida rugosa lip1 Gene for Improving Expression inPichia pastorisand Biochemical Characterization of the Purified Recombinant LIP1 Lipase

Abstract

An important industrial enzyme, Candida rugosa lipase (CRL) possesses several different isoforms encoded by the lip gene family (lip1-lip7), in which the recombinant LIP1 is the major form of the CRL multigene family. Previously, 19 of the nonuniversal serine codons (CTG) of the lip1 gene hav been successfully converted into universal serine codons (TCT) by overlap extension PCR-based multiple-site-directed mutagenesis to express an active recombinant LIP1 in the yeast Pichia pastoris. To improve the expression efficiency of recombinant LIP1 in P. pastoris, a regional synthetic gene fragment of lip1 near the 5' end of a transcript has been constructed to match P. pastoris-preferred codon usage for simple scale-up fermentation. The present results show that the production level (152 mg/L) of coLIP1 (codon-optimized LIP1) has an overall improvement of 4.6-fold relative to that (33 mg/L) of non-codon-optimized LIP1 with only half the cultivation time of P. pastoris. This finding demonstrates that the regional codon optimization the lip1 gene fragment at the 5' end can greatly increase the expression level of recombinant LIP1 in the P. pastoris system. More distinct biochemical properties of the purified recombinant LIP1 for further industrial applications are also determined and discussed in detail.

Reversible immobilization of Candida rugosa lipase on fibrous polymer grafted and sulfonated p(HEMA/EGDMA) beads

“…The enzyme lipase contains several positively charged surface exposed amino acids, i.e., histidine, lysine, and arginine amino acids through its sequence. The sequence, as well the amino acid distribution in the tertiary structure of lipase is well known [34][35][36]. The presence of these amino acid residues on the surface of lipase was considered important to plan an immobilization procedure involving these positively charged amino acid residues.…”

Effect of codon optimization on expression levels of human cystatin C in Pichia pastoris

“…It was reported that 25 of the total 61 yeast codons were optimal, whereas in Pichia, Zhao et al (2000) validated 19 optimal expression codons by analyzing the usage of 28 synonymous codons. Many researchers have managed to optimize these codons in Pichia to improve the expression of heterologous genes (Outchkourov et al, 2002;Hu et al, 2006;Chang et al, 2006;Li et al, 2008;Shumiao et al, 2010;Gao et al, 2012).…”

“…Many studies have reported significant improvements in the expression of foreign genes in yeast by codon optimization (Outchkourov et al, 2002;Chang et al, 2006;Hu et al, 2006;Li et al, 2008;Shumiao et al, 2010;Bai et al, 2011;Gao et al, 2012). The significantly higher protein yields in the native folded state, higher purity, and improved function establish the value of the gene codon optimization approach.…”

Advanced Strategies for Improving the Production of Industrial Enzymes in Heterologous Host Systems

“…Based on the codon bias of S. cerevisiae and P. pastoris, serine-encoding codons and the 5'-UTR region of the C. rugosa lipase gene were optimized to make it achieve the effective expression in the two host cells. This greatly shortens the fermentative cycle [27,28]. Wei et al [29] optimized a thermostable α-amylase gene (pfa) sequence based on the Pichia codon bias and artificially synthesized it.…”