School of Life Science, Qingdao Agricultural University, Shandong Province Key Laboratory of Applied Mycology, and Qingdao International Center on Microbes Utilizing Biogas, Qingdao, Shandong Province, China.
Central Laboratory, Qingdao Agricultural University, Qingdao, Shandong Province, China.
Microb Cell Fact. 2017 Oct 30;16(1):179. doi: 10.1186/s12934-017-0798-2.
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 C-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.
3-羟基丙酸（3-HP）是一种重要的平台化学品，可用作生产丙烯酸和 1,3-丙二醇等多种工业应用的前体。尽管大肠杆菌或酿酒酵母是生产 3-HP 的主要工业微生物，但替代工程宿主有可能利用其他碳源生产 3-HP。甲基杆菌（Methylobacterium extorquens）AM1 是一种兼性甲基营养型α变形菌，是评估从一碳甲醇源生成 3-HP 的可能性的模型系统。
本研究通过异源过表达 mcr 基因构建了丙二酰辅酶 A 途径，使 M. extorquens AM1 能够将甲醇转化为 3-HP。在摇瓶培养中，工程菌株的 3-HP 初始产量达到 6.8mg/L，通过增强启动子和 mcr 基因拷贝数，产量进一步提高到 69.8mg/L。体内代谢分析表明，在 3-HP 产量最高的菌株中，乙酰辅酶 A 池的大小显著减小，表明乙酰辅酶 A 的供应可能是进一步提高产量的瓶颈。值得注意的是，3-HP 在从指数生长期到静止期的过渡后迅速降解。代谢组学分析表明，在向培养物中添加 3-HP 后，静止期细胞内 3-羟基丙酰辅酶 A 的积累，表明 3-HP 首先转化为其辅酶 A 衍生物。体外酶促测定和依赖于β-丙氨酸途径的 C 标记进一步表明，一种还原途径依次将 3-HP-CoA 转化为丙烯酰辅酶 A 和丙酰辅酶 A，后者被重新纳入乙基丙二酰辅酶 A 途径。编码推定的丙烯酰辅酶 A 还原酶的基因 META1_4251 的缺失导致静止期后期 3-HP 降解率降低。
本研究在 M. extorquens AM1 中构建了丙二酰辅酶 A 途径来生成 3-HP，证明了该途径的可行性。此外，本研究表明，一种还原途径与乙基丙二酰辅酶 A 途径相结合是生长过渡期间 3-HP 降解的主要途径。工程化的 M. extorquens AM1 代表了从甲醇生产 3-HP 的良好平台。
