Chang Shu-Wei, Lee Guan-Chiun, Shaw Jei-Fu
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 202, Taiwan.
J Agric Food Chem. 2006 Feb 8;54(3):815-22. doi: 10.1021/jf052183k.
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.
一种重要的工业酶——皱褶假丝酵母脂肪酶（CRL）具有由lip基因家族（lip1 - lip7）编码的几种不同同工型，其中重组LIP1是CRL多基因家族的主要形式。此前，通过基于重叠延伸PCR的多位点定向诱变，已成功将lip1基因的19个非通用丝氨酸密码子（CTG）转换为通用丝氨酸密码子（TCT），以便在巴斯德毕赤酵母中表达有活性的重组LIP1。为提高重组LIP1在巴斯德毕赤酵母中的表达效率，构建了转录本5'端附近lip1的区域合成基因片段，以匹配巴斯德毕赤酵母偏好的密码子使用情况，便于简单放大发酵。目前的结果表明，共LIP1（密码子优化的LIP1）的产量水平（152 mg/L）相对于非密码子优化的LIP1（33 mg/L）总体提高了4.6倍，而巴斯德毕赤酵母的培养时间仅为一半。这一发现表明，对lip1基因片段5'端进行区域密码子优化可极大提高重组LIP1在巴斯德毕赤酵母系统中的表达水平。还详细测定并讨论了纯化的重组LIP1更独特的生化特性，以用于进一步的工业应用。
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