College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, Hubei, China.
PLoS One. 2013;8(1):e53939. doi: 10.1371/journal.pone.0053939. Epub 2013 Jan 10.
Candida antarctica lipase B (CALB) is one of the most widely used and studied enzymes in the world. In order to achieve the high-level expression of CALB in Pichia, we optimized the codons of CALB gene and α-factor by using a de novo design and synthesis strategy. Through comparative analysis of a series of recombinants with different expression components, we found that the methanol-inducible expression recombinant carrying the codon-optimized α-factor and mature CALB gene (pPIC9KαM-CalBM) has the highest lipase production capacity. After fermentation parameters optimization, the lipase activity and protein content of the recombinant pPIC9KαM-CalBM reached 6,100 U/mL and 3.0 g/L, respectively, in a 5-L fermentor. We believe this strategy could be of special interest due to its capacity to improve the expression level of target gene, and the Pichia transformants carrying the codon-optimized gene had great potential for the industrial-scale production of CALB lipase.
南极假丝酵母脂肪酶 B（CALB）是世界上应用最广泛、研究最多的酶之一。为了在毕赤酵母中实现 CALB 的高效表达，我们采用从头设计和合成策略对 CALB 基因和α因子的密码子进行了优化。通过对一系列具有不同表达元件的重组体进行比较分析，我们发现携带密码子优化的α因子和成熟 CALB 基因的甲醇诱导表达重组体（pPIC9KαM-CalBM）具有最高的脂肪酶生产能力。经过发酵参数优化，重组体 pPIC9KαM-CalBM 的脂肪酶活和蛋白含量分别达到 6100 U/mL 和 3.0 g/L，在 5 L 发酵罐中实现。我们相信，由于该策略能够提高目标基因的表达水平，因此具有特殊的研究意义，而携带密码子优化基因的毕赤酵母转化体在 CALB 脂肪酶的工业规模生产中具有巨大的潜力。
