费希尔曲霉脂肪酶在毕赤酵母中的优化表达及高密度发酵

Optimized expression and high-density fermentation of Aspergillus fischeri lipase in Pichia pastoris

摘要:

【背景】脂肪酶广泛应用于纺织、食品、药品、皮革等工业领域，其在微生物中的异源表达研究进一步促进了脂肪酶产品的生产和应用。【目的】实现来源于费希尔曲霉的脂肪酶在毕赤酵母中的高效异源表达，探究其合适的表达及发酵条件，提高产量，降低成本。【方法】对费希尔曲霉的脂肪酶编码基因进行密码子优化后，应用pPIC9k质粒整合到毕赤酵母GS115基因组上，构建高产脂肪酶Lip605的毕赤酵母工程菌；并通过响应面发酵条件优化、筛选最适伴侣蛋白和高密度发酵相结合的方法，综合提高脂肪酶表达量。【结果】确定高产脂肪酶毕赤酵母工程菌的最优摇瓶发酵产酶条件为：甲醇3.103% (体积比)，生物素0.4 mg/L，酵母粉11.5 g/L，酵母基础氮源培养基(yeast nitrogen base，YNB) 13.4 g/L，初始pH 6.4，装液量50 mL/250 mL，转速220 r/min，温度24 °C，培养时间40 h。优化后的胞外脂肪酶酶活达到72.34 U/mL，较优化前提高了5.8倍；进一步选择12个伴侣蛋白分别与脂肪酶Lip605进行共表达，其中共表达伴侣蛋白Rpl10 (pPICZA-RPL10)效果最佳，可使Lip605表达量进一步提高46.8%；在此基础上，经过10 L发酵罐分批补料的高密度发酵，工程菌株发酵142 h，胞外脂肪酶酶活最高达到680 U/mL，蛋白浓度为15.89 g/L。【结论】应用复合策略有效提高了脂肪酶Lip605在毕赤酵母中的发酵产量，为其进一步工业化生产奠定了良好的基础。

关键词:毕赤酵母，脂肪酶Lip605，响应面，共表达，伴侣蛋白，高密度发酵

Abstract:

[Background] Lipase is widely used in textile, food, medicine, leather and other industrial fields. The study on the heterologous expression of lipase in microorganisms further promotes the production and application of lipase products. [Objective] To perform the efficient heterologous expression of lipase from Aspergillus fischeri in Pichia pastoris. Screening suitable strategies for expression and fermentation of the engineering strain to increase yield and reduce production cost of lipase. [Methods] After codon optimization of the lipase coding gene of Aspergillus fischeri, plasmid pPIC9k was used to integrate the codon optimized gene (lip605) into the genome of Pichia pastoris GS115 to construct an engineering strain for high yield of the recombinant lipase. The lipase expression was further increased by optimizing the fermentation conditions, screening the optimal chaperone protein and performing high-density fermentation, successively. [Results] The optimal fermentation conditions for the engineering strain with high lipase production in shake flask were determined as 3.103% (v/v) methanol, 0.4 mg/L biotin, 11.5 g/L yeast powder, and 13.4 g/L YNB; the initial pH was 6.4, the filling volume was 50 mL/250 mL, the speed was 220 r/min, the temperature was 24 °C, and the culture time was 40 h. After optimization, the activity of extracellular lipase reached 72.34 U/mL, which was 5.8 times higher than that of initial fermentation. Furthermore, 12 chaperones were selected for co-expression with Lip605, among which the co-expression of chaperone Rpl10 (pPICZA-RPL10) had the best effect, which further increased the expression of Lip605 by 46.8%. On this basis, after 142 h high-density fermentation with batch feeding in a 10 L fermenter, the maximum extracellular lipase activity reached 680 U/mL, with a protein concentration of 15.89 g/L. [Conclusion] In this paper, compound strategies were used to effectively improve the fermentation yield of lipase Lip605 in Pichia pastoris, which laid a good foundation for its further industrial production.

Key words:Pichia pastoris, Lipase Lip605, Response surface, Co-expression, Chaperone proteins, High-density fermentation