Tsuruno Keigo, Honjo Hiroshi, Hanai Taizo
Laboratory for Bioinformatics, Graduate School of Systems Life Sciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
Microb Cell Fact. 2015 Oct 5;14:155. doi: 10.1186/s12934-015-0342-1.
3-hydroxypropionic acid (3-HP) is an important platform for the production of C3 chemicals, including acrylic acid, methyl acrylate, and acrylamide. Microbial production of 3-HP is mainly due to glycerol metabolism. In this study, in order to improve microbial 3-HP production, we applied a metabolic toggle switch for controlling the glycerol metabolism to redirect the excess metabolic flux of central metabolic pathway toward an exogenous 3-HP producing pathway in Escherichia coli.
The metabolic toggle switch enables conditional repression of the expression of a target gene during the fermentation. We individually performed conditional repression of glpK, tpiA, and gapA, which are involved in glycerol metabolism. The conditional repression of glpK and tpiA was not effective for 3-HP production under our experimental conditions. However, gapA conditional repression contributed to improve 3-HP production (titer, 54.2 ± 1.5 mM; yield, 32.1 ± 1.3 %) compared with that for the wild type strain. Additional deletion of endogenous yqhD, which is responsible for the production of a major byproduct, 1,3-propandiol, further increased 3-HP production (titer, 67.3 ± 2.1 mM; yield, 51.5 ± 3.2 %). The titer and yield were 80 and 94 % higher than those of the wild type strain, respectively. The obtained 3-HP yield from glycerol is comparable with the highest yield ever reported for microbial 3-HP production using glycerol as a sole carbon source. The measurement of intracellular metabolites showed the metabolic toggle switch successfully controlled the metabolic flux.
The conditional repression of gapA by using the metabolic toggle switch combined with deletion of endogeneous yqhD increased 3-HP production approximately twofold from glycerol. This result indicates the metabolic toggle switch can be applied in various bio-production using diverse substrates.
3-羟基丙酸（3-HP）是生产C3化学品（包括丙烯酸、丙烯酸甲酯和丙烯酰胺）的重要平台。微生物生产3-HP主要源于甘油代谢。在本研究中，为了提高微生物生产3-HP的能力，我们应用了一种代谢切换开关来控制甘油代谢，以将中心代谢途径的过量代谢通量重定向至大肠杆菌中一条外源3-HP生产途径。
该代谢切换开关能够在发酵过程中对靶基因的表达进行条件性抑制。我们分别对参与甘油代谢的glpK、tpiA和gapA进行了条件性抑制。在我们的实验条件下，对glpK和tpiA的条件性抑制对3-HP生产无效。然而，与野生型菌株相比，gapA条件性抑制有助于提高3-HP产量（滴度，54.2±1.5 mM；产率，32.1±1.3%）。额外缺失负责产生主要副产物1,3-丙二醇的内源性yqhD，进一步提高了3-HP产量（滴度，67.3±2.1 mM；产率，51.5±3.2%）。滴度和产率分别比野生型菌株高80%和94%。从甘油获得的3-HP产率与以甘油作为唯一碳源进行微生物3-HP生产所报道的最高产率相当。细胞内代谢物的测定表明代谢切换开关成功控制了代谢通量。
利用代谢切换开关对gapA进行条件性抑制并结合缺失内源性yqhD，使甘油生产3-HP的产量提高了约两倍。该结果表明代谢切换开关可应用于使用各种底物的多种生物生产中。
