Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas.
Department of Bioengineering, Rice University, Houston, Texas.
Biotechnol Bioeng. 2018 May;115(5):1311-1320. doi: 10.1002/bit.26540. Epub 2018 Feb 4.
Decanoic acid is a valuable compound used as precursor for industrial chemicals, pharmaceuticals, and biofuels. Despite efforts to produce it from renewables, only limited achievements have been reported. Here, we report an engineered cell factory able to produce decanoic acid as a major product from glycerol, and abundant and renewable feedstock. We exploit the overlapping chain-length specificity of β-oxidation reversal (r-BOX) and thioesterase enzymes to selectively generate decanoic acid. This was achieved by selecting r-BOX enzymes that support the synthesis of acyl-CoA of up to 10 carbons (thiolase BktB and enoyl-CoA reductase EgTER) and a thioesterase that exhibited high activity toward decanoyl-CoA and longer-chain acyl-CoAs (FadM). Combined chromosomal and episomal expression of r-BOX core enzymes such as enoyl-CoA reductase and thiolase (in the presence of E. coli thioesterase FadM) increased titer and yield of decanoic acid, respectively. The carbon flux toward decanoic acid was substantially increased by the use of an organic overlay, which decreased its intracellular accumulation and presumably increased its concentration gradient across cell membrane, suggesting that decanoic acid transport to the extracellular medium might be a major bottleneck. When cultivated in the presence of a n-dodecane overlay, the final engineered strain produced 2.1 g/L of decanoic acid with a yield of 0.1 g/g glycerol. Collectively, our data suggests that r-BOX can be used as a platform to selectively produce decanoic acid and its derivatives at high yield, titer and productivity.
癸酸是一种有价值的化合物，可用作工业化学品、制药和生物燃料的前体。尽管人们努力从可再生资源中生产它，但仅取得了有限的成果。在这里，我们报告了一个经过工程改造的细胞工厂，能够从甘油这种丰富且可再生的原料中生产癸酸作为主要产物。我们利用β-氧化逆转（r-BOX）和硫酯酶酶的重叠链长特异性来选择性地生成癸酸。这是通过选择支持合成长达 10 个碳原子的酰基辅酶 A 的 r-BOX 酶（硫激酶 BktB 和烯酰辅酶 A 还原酶 EgTER）和对癸酰辅酶 A 和更长链酰基辅酶 A 表现出高活性的硫酯酶（FadM）来实现的。r-BOX 核心酶如烯酰辅酶 A 还原酶和硫激酶的染色体和附加型表达（在大肠杆菌硫酯酶 FadM 的存在下）分别增加了癸酸的滴度和产量。通过使用有机覆盖物，显著增加了癸酸的碳通量，这降低了其细胞内积累，推测增加了其跨细胞膜的浓度梯度，表明癸酸向细胞外培养基的运输可能是一个主要瓶颈。当在正十二烷覆盖物的存在下培养时，最终的工程菌株产生了 2.1 g/L 的癸酸，产率为 0.1 g/g 甘油。总的来说，我们的数据表明 r-BOX 可以用作平台，以高产量、滴度和生产率选择性地生产癸酸及其衍生物。
