Hakvåg Sigrid, Nærdal Ingemar, Heggeset Tonje M B, Kristiansen Kåre A, Aasen Inga M, Brautaset Trygve
Department of Biotechnology and Food Sciences, Norwegian University of Science and Technology, Trondheim, Norway.
Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway.
Front Microbiol. 2020 Apr 9;11:680. doi: 10.3389/fmicb.2020.00680. eCollection 2020.
The facultative methylotroph MGA3 has previously been genetically engineered to overproduce the amino acids L-lysine and L-glutamate and their derivatives cadaverine and γ-aminobutyric acid (GABA) from methanol at 50°C. We here explored the potential of utilizing the sugar alcohol mannitol and seaweed extract (SWE) containing mannitol, as alternative feedstocks for production of chemicals by fermentation using . Extracts of the brown algae harvested in the Trondheim Fjord in Norway were prepared and found to contain 12-13 g/l of mannitol, with conductivities corresponding to a salt content of ∼2% NaCl. Initially, 12 wild type strains were tested for tolerance to various SWE concentrations, and some strains including MGA3 could grow on 50% SWE medium. Non-methylotrophic and methylotrophic growth of rely on differences in regulation of metabolic pathways, and we compared production titers of GABA and cadaverine under such growth conditions. Shake flask experiments showed that recombinant MGA3 strains could produce similar and higher titers of cadaverine during growth on 50% SWE and mannitol, compared to on methanol. GABA production levels under these conditions were however low compared to growth on methanol. We present the first fed-batch mannitol fermentation of and production of 6.3 g/l cadaverine. Finally, we constructed a recombinant MGA3 strain synthesizing the C30 terpenoids 4,4'-diaponeurosporene and 4,4'-diapolycopene, experimentally confirming that has a functional methylerythritol phosphate (MEP) pathway. Together, our results contribute to extending the range of both the feedstocks for growth and products that can be synthesized by .
兼性甲基营养菌MGA3此前已通过基因工程改造，可在50°C下从甲醇中过量生产氨基酸L-赖氨酸和L-谷氨酸及其衍生物尸胺和γ-氨基丁酸（GABA）。我们在此探索了利用糖醇甘露醇和含有甘露醇的海藻提取物（SWE）作为替代原料，通过发酵生产化学品的潜力。制备了在挪威特隆赫姆峡湾采集的褐藻提取物，发现其含有12 - 13 g/l的甘露醇，电导率相当于约2% NaCl的盐含量。最初，测试了12株野生型菌株对各种SWE浓度的耐受性，包括MGA3在内的一些菌株可以在50% SWE培养基上生长。非甲基营养菌和甲基营养菌的生长依赖于代谢途径调控的差异，我们比较了在这种生长条件下GABA和尸胺的生产效价。摇瓶实验表明，与在甲醇上生长相比，重组MGA3菌株在50% SWE和甘露醇上生长时可以产生相似且更高效价的尸胺。然而，与在甲醇上生长相比，这些条件下的GABA生产水平较低。我们展示了首次对MGA3进行的补料分批甘露醇发酵，并生产了6.3 g/l的尸胺。最后，我们构建了一种合成C30类萜4,4'-二脱辅基神经孢菌烯和4,4'-二脱辅基番茄红素的重组MGA3菌株，通过实验证实MGA3具有功能性的甲基赤藓糖醇磷酸（MEP）途径。总之，我们的结果有助于扩大MGA3生长的原料范围以及可由其合成的产品范围。
