Liu Long, Guan Ningzi, Zhu Gexin, Li Jianghua, Shin Hyun-Dong, Du Guocheng, Chen Jian
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
Sci Rep. 2016 Jan 27;6:19963. doi: 10.1038/srep19963.
Propionic acid (PA) is an important chemical building block widely used in the food, pharmaceutical, and chemical industries. In our previous study, a shuttle vector was developed as a useful tool for engineering Propionibacterium jensenii, and two key enzymes-glycerol dehydrogenase and malate dehydrogenase-were overexpressed to improve PA titer. Here, we aimed to improve PA production further via the pathway engineering of P. jensenii. First, the phosphoenolpyruvate carboxylase gene (ppc) from Klebsiella pneumoniae was overexpressed to access the one-step synthesis of oxaloacetate directly from phosphoenolpyruvate without pyruvate as intermediate. Next, genes encoding lactate dehydrogenase (ldh) and pyruvate oxidase (poxB) were deleted to block the synthesis of the by-products lactic acid and acetic acid, respectively. Overexpression of ppc and deleting ldh improved PA titer from 26.95 ± 1.21 g·L(-1) to 33.21 ± 1.92 g·L(-1) and 30.50 ± 1.63 g·L(-1), whereas poxB deletion decreased it. The influence of this pathway engineering on gene transcription, enzyme expression, NADH/NAD(+) ratio, and metabolite concentration was also investigated. Finally, PA production in P. jensenii with ppc overexpression as well as ldh deletion was investigated, which resulted in further increases in PA titer to 34.93 ± 2.99 g·L(-1) in a fed-batch culture.
丙酸（PA）是一种重要的化学原料，广泛应用于食品、制药和化工行业。在我们之前的研究中，开发了一种穿梭载体作为詹氏丙酸杆菌工程改造的有用工具，并且过表达了两种关键酶——甘油脱氢酶和苹果酸脱氢酶——以提高PA产量。在此，我们旨在通过詹氏丙酸杆菌的途径工程进一步提高PA产量。首先，过表达来自肺炎克雷伯菌的磷酸烯醇式丙酮酸羧化酶基因（ppc），以直接从磷酸烯醇式丙酮酸一步合成草酰乙酸，而无需丙酮酸作为中间体。接下来，分别删除编码乳酸脱氢酶（ldh）和丙酮酸氧化酶（poxB）的基因，以阻断副产物乳酸和乙酸的合成。ppc的过表达和ldh的删除使PA产量从26.95±1.21 g·L⁻¹提高到33.21±1.92 g·L⁻¹和30.50±1.63 g·L⁻¹，而poxB的删除则降低了PA产量。还研究了这种途径工程对基因转录、酶表达、NADH/NAD⁺比率和代谢物浓度的影响。最后，研究了过表达ppc以及删除ldh的詹氏丙酸杆菌中的PA生产情况，在补料分批培养中，这使得PA产量进一步提高到34.93±2.99 g·L⁻¹。
