Biosynthesis of butenoic acid through fatty acid biosynthesis pathway in Escherichia coli
Biosynthesis of butenoic acid through fatty acid biosynthesis pathway in Escherichia coli
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DOI：
10.1007/s00253-014-6233-2
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发表时间：
2015-02-01
影响因子：
5
通讯作者：
Zhu, Kun
中科院分区：
文献类型：
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作者：
Liu, Xiping;Yu, Haiying;Zhu, Kun
丁烯酸是一种C4短链不饱和脂肪酸，主要用于制备树脂、医药、精细化学品等。然而，从石油中提取的丁烯酸价格昂贵且对环境不友好。在这里，我们报告了一种利用工程大肠杆菌中脂肪酸生物合成途径的中间体生产丁烯酸的新生物合成策略。来自 Bacteroides thetaiotaomicron 的硫酯酶基因（B.thetaiotaomicron thioesterase (bTE)）在大肠杆菌中异源表达，以特异性地将丁烯酰酰基载体蛋白（ACP）（一种脂肪酸生物合成中间体）转化为丁烯酸。在具有不同表达载体的四种不同大肠杆菌菌株中，丁烯酸的效价范围为0.07至11.4 mg/L。删除内源性fadD基因（编码酰基辅酶A合成酶）以阻断脂肪酸氧化，在一定程度上提高了所有菌株的丁烯酸产量。菌株 XP-2 (BL21-Delta fadD/pET28a-bTE) 中丁烯酸积累量最高，为 18.7 mg/L。此外，通过三氯生部分抑制菌株XP-2的烯酰基-ACP还原酶（FabI），丁烯酸产量增加了三倍，并且通过在M9培养基中提供葡萄糖和胰蛋白胨，丁烯酸滴度进一步增加至161.4mg/L。该菌株的分批补料发酵在 48 小时内将丁烯酸产量进一步提高至 4.0 g/L。丁烯酸耐受性测定表明，该菌株可耐受15-20 g/L的丁烯酸。
Butenoic acid is a C4 short-chain unsaturated fatty acid mainly used in the preparation of resins, pharmaceuticals, and fine chemicals. However, butenoic acid derived from petroleum is costly and unfriendly to the environment. Here, we report a novel biosynthetic strategy to produce butenoic acid by utilizing the intermediate of fatty acid biosynthesis pathway in engineered Escherichia coli. A thioesterase gene (B. thetaiotaomicron thioesterase (bTE)) from Bacteroides thetaiotaomicron was heterologously expressed in E. coli to specifically convert butenoyl-acyl carrier protein (ACP), a fatty acid biosynthesis intermediate, to butenoic acid. The titer of butenoic acid ranged from 0.07 to 11.4 mg/L in four different E. coli strains with varied expressing vectors. Deletion of endogenous fadD gene (encoding acyl-CoA synthetase) to block fatty acid oxidation improved the butenoic acid production in all strains to some extent. The highest butenoic acid accumulation of 18.7 mg/L was obtained in strain XP-2 (BL21-Delta fadD/pET28a-bTE). Moreover, partially inhibiting the enoyl-ACP reductase (FabI) of strain XP-2 by triclosan increased butenoic acid production by threefold, and the butenoic acid titer was further increased to 161.4 mg/L by supplying glucose and tryptone in the M9 medium. Fed-batch fermentation of this strain further enhanced butenoic acid production to 4.0 g/L within 48 h. The butenoic acid tolerance assay revealed that this strain could tolerate 15-20 g/L of butenoic acid.