Zhang Baixi, Chen Haiqin, Li Min, Gu Zhennan, Song Yuanda, Ratledge Colin, Chen Yong Q, Zhang Hao, Chen Wei
Microb Cell Fact. 2013 Jul 16;12:70. doi: 10.1186/1475-2859-12-70.
Conjugated linoleic acid (CLA) has been extensively studied for decades because of its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. We previously described the production of trans-10, cis-12 CLA in Yarrowia lipolytica by expressing the gene coding for linoleic acid isomerase from Propionibacterium acnes (pai). However the stable strain produced CLA at about 0.08% of dry cell weight (DCW), a level of production which was not high enough for practical applications. The goal of the present study was to enhance production of CLA by genetic engineering of Y. lipolytica strains.
We have now co-expressed the delta 12-desaturase gene (FADS12, d12) from Mortierella alpina together with the codon-optimized linoleic acid isomerase (opai) gene in Y. lipolytica, expressed under the control of promoter hp16d modified by fusing 12 copies of UAS1B to the original promoter hp4d. A multi-copy integration plasmid was used to further enhance the expression of both genes. Using glucose as the sole carbon source, the genetically-modified Y. lipolytica produced trans-10, cis-12-CLA at a level of up to 10% of total fatty acids and 0.4% of DCW. Furthermore, when the recombinant yeast was grown with soybean oil, trans-10, cis-12-CLA now accumulated at a level of up to 44% of total fatty acids, which represented 30% of DCW after 38.5 h of cultivation. In addition, trans-10, cis-12-CLA was also detected in the growth medium up to 0.9 g/l.
We have successfully produced trans-10, cis-12-CLA with a titre of 4 g/l of culture (3.1 g/l in cells and 0.9 g/l in culture medium). Our results demonstrate the potential use of Y. lipolytica as a promising microbial cell factory for trans-10, cis-12-CLA production.
共轭亚油酸（CLA）因其对健康有益，包括预防癌症、抗动脉粥样硬化和抗肥胖作用以及调节免疫系统，已被广泛研究数十年。我们之前描述了通过在解脂耶氏酵母中表达痤疮丙酸杆菌编码亚油酸异构酶的基因（pai）来生产反式-10，顺式-12 CLA。然而，该稳定菌株产生的CLA约为干细胞重量（DCW）的0.08%，这一生产水平对于实际应用来说不够高。本研究的目标是通过对解脂耶氏酵母菌株进行基因工程来提高CLA的产量。
我们现在已在解脂耶氏酵母中共表达了来自高山被孢霉的Δ12-去饱和酶基因（FADS12，d12）以及密码子优化的亚油酸异构酶（opai）基因，它们在通过将12个UAS1B拷贝融合到原始启动子hp4d而修饰的启动子hp16d的控制下表达。使用多拷贝整合质粒进一步增强了这两个基因的表达。以葡萄糖作为唯一碳源时，基因改造后的解脂耶氏酵母产生的反式-10，顺式-12 CLA水平高达总脂肪酸的10%和DCW的0.4%。此外，当重组酵母以大豆油为培养基生长时，反式-10，顺式-12 CLA现在积累的水平高达总脂肪酸的44%，在培养38.5小时后占DCW的30%。此外，在生长培养基中也检测到反式-10，顺式-12 CLA，最高可达0.9 g/l。
我们已成功生产出反式-10，顺式-12 CLA，培养物中的产量为4 g/l（细胞中为3.1 g/l，培养基中为0.9 g/l）。我们的结果证明了解脂耶氏酵母作为生产反式-10，顺式-12 CLA的有前景的微生物细胞工厂的潜在用途。
