Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route.
Production of 3-hydroxypropionic acid in engineered Methylobacterium extorquens AM1 and its reassimilation through a reductive route.
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工程化的扭扭甲基杆菌 AM1 中 3-羟基丙酸的生产及其通过还原途径的再同化。
DOI：
10.1186/s12934-017-0798-2
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发表时间：
2017-10-30
影响因子：
6.4
通讯作者：
Yang S
中科院分区：
文献类型：
--
作者：
Yang YM;Chen WJ;Yang J;Zhou YM;Hu B;Zhang M;Zhu LP;Wang GY;Yang S
3-羟基丙酸（3-HP）是一种重要的平台化学品，用作广泛工业应用的前体，例如生产丙烯酸和1，3-丙二醇。虽然大肠杆菌或酿酒酵母是生产3-HP的主要工业微生物，但替代工程宿主具有从其他碳原料产生3-HP的潜力。扭脱甲基杆菌AM 1是一种兼性甲基营养型α-变形杆菌，是用于评估从一碳原料甲醇产生3-HP的可能性的模型系统。在这里，我们构建了一个丙二酰辅酶A途径，通过异源过表达mcr基因将甲醇转化为3-HP在M。extorquens AM1.工程菌摇瓶发酵的初始效价为6.8mg/l，通过提高启动子强度和mcr基因拷贝数，使3-HP的初始效价进一步提高到69.8mg/l。体内代谢分析显示，具有最高3-HP滴度的菌株中乙酰辅酶A池大小显著降低，表明乙酰辅酶A的供应是进一步改进的潜在瓶颈。值得注意的是，3-HP从指数期过渡到稳定期后迅速降解。代谢组学分析显示，在稳定期，向培养基中加入3-HP，细胞内3-羟基丙酰辅酶A积累，表明3-HP首先转化为其辅酶A衍生物。体外酶促测定和β-丙氨酸途径依赖性13 C-标记进一步证明，还原途径依次将3-HP-CoA转化为丙烯酰-CoA和丙酰-CoA，后者再同化到乙基丙二酰-CoA途径中。编码丙烯酰辅酶A还原酶的基因META1_4251的缺失导致3-HP在稳定期后期的降解速率降低。我们证明了在M.使AM 1脱水生成3-HP。此外，我们发现，还原途径加上乙基丙二酰辅酶A途径是主要的渠道负责降解的3-HP在生长过渡。工程师M extroquens AM 1代表了从甲醇生产3-HP的良好平台。本文的在线版本（10.1186/s12934-017-0798-2）包含补充材料，可供授权用户使用。
3-Hydroxypropionic acid (3-HP) is an important platform chemical, serving as a precursor for a wide range of industrial applications such as the production of acrylic acid and 1,3-propanediol. Although Escherichia coli or Saccharomyces cerevisiae are the primary industrial microbes for the production of 3-HP, alternative engineered hosts have the potential to generate 3-HP from other carbon feedstocks. Methylobacterium extorquens AM1, a facultative methylotrophic α-proteobacterium, is a model system for assessing the possibility of generating 3-HP from one-carbon feedstock methanol. Here we constructed a malonyl-CoA pathway by heterologously overexpressing the mcr gene to convert methanol into 3-HP in M. extorquens AM1. The engineered strains demonstrated 3-HP production with initial titer of 6.8 mg/l in shake flask cultivation, which was further improved to 69.8 mg/l by increasing the strength of promoter and mcr gene copy number. In vivo metabolic analysis showed a significant decrease of the acetyl-CoA pool size in the strain with the highest 3-HP titer, suggesting the supply of acetyl-CoA is a potential bottleneck for further improvement. Notably, 3-HP was rapidly degraded after the transition from exponential phase to stationary phase. Metabolomics analysis showed the accumulation of intracellular 3-hydroxypropionyl-CoA at stationary phase with the addition of 3-HP into the cultured medium, indicating 3-HP was first converted to its CoA derivatives. In vitro enzymatic assay and β-alanine pathway dependent 13C-labeling further demonstrated that a reductive route sequentially converted 3-HP-CoA to acrylyl-CoA and propionyl-CoA, with the latter being reassimilated into the ethylmalonyl-CoA pathway. The deletion of the gene META1_4251 encoding a putative acrylyl-CoA reductase led to reduced degradation rate of 3-HP in late stationary phase. We demonstrated the feasibility of constructing the malonyl-CoA pathway in M. extorquens AM1 to generate 3-HP. Furthermore, we showed that a reductive route coupled with the ethylmalonyl-CoA pathway was the major channel responsible for degradation of the 3-HP during the growth transition. Engineered M. extorquens AM1 represents a good platform for 3-HP production from methanol. The online version of this article (10.1186/s12934-017-0798-2) contains supplementary material, which is available to authorized users.
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影响因子：
3.6
作者：
Bjerrum, Jacob Tveiten;Wang, Yulan;Hao, Fuhua;Coskun, Mehmet;Ludwig, Christian;Guenther, Ulrich;Nielsen, Ole Haagen
通讯作者：
Nielsen, Ole Haagen
1丁醇产生的甲基甲基甲基甲基甲虫的代谢工程。
影响因子：
6.3
作者：
Hu B;Lidstrom ME
通讯作者：
Lidstrom ME
影响因子：
6.4
作者：
Kildegaard KR;Jensen NB;Schneider K;Czarnotta E;Özdemir E;Klein T;Maury J;Ebert BE;Christensen HB;Chen Y;Kim IK;Herrgård MJ;Blank LM;Forster J;Nielsen J;Borodina I
通讯作者：
Borodina I
影响因子：
3.2
作者：
Lee, Philah;Raj, Subramanian Mohan;Park, Sunghoon
通讯作者：
Park, Sunghoon
影响因子：
8.4
作者：
Liang, Wei-Fan;Cui, Lan-Yu;Xing, Xin-Hui
通讯作者：
Xing, Xin-Hui