Extension of the substrate range of the cell factory Saccharomyces cerevisiae to use C1-derived feedstocks: succinic acid as a model target product

作为上述项目的结果，我们最近已经证明了甘油（C3）和CO2（C1）的共同利用，用于通过酵母酿酒酵母的代谢工程后生产平台化学品琥珀酸（C4）。产物产率（每消耗甘油）相当于理论最大值的47%。在我们目前的项目中，已经探讨了改进这一过程的进一步可能性。在这里提出的项目中，我们计划用不来自可食用植物生物质的原料代替碳源甘油，其丰度与生物柴油生产的未来无关。甲醇（C1）和二羟基丙酮（DHA，C3）都有可能成为未来生物技术过程的碳源，因为它们可以从合成气或CO2中产生。一种有吸引力的方法是为我们的琥珀酸生产菌株配备将甲醇转化为SA（甲醇脱氢酶和甲醛酶）的线性途径，然后使用适应性实验室进化（ALE）。我们认为这是一个高风险的方法，由于热力学和动力学的限制。我们仍然认为这是有希望的。事实上，所提出的ALE策略具有优化酶福尔马林酶的动力学的潜力。此外，我们的琥珀酸途径被认为是一个强大的水槽的NADH，因此可以减弱的热力学障碍的NAD+依赖的甲醇氧化。作为甲醇作为碳源的替代品，我们提出了基于DHA（C3）作为碳源的第二种方法。在未来，DHA可以通过化学酶促过程从CO2中产生，并将DHA导入S的中心代谢。cerevisiae啤酒厂是简单的。我们以前建立的S。具有修饰的甘油催化剂的酿酒酵母菌株（特别是具有DHA途径的那些）形成了实现该目标的极好基础。ALE将解决对较高DHA浓度耐受性的改善。为了提供足够的胞质NADH用于SA生产，将进料辅助底物（例如由CO2产生的甲酸盐）。

As a result of the preceding project, we have recently demonstrated the co-utilization of glycerol (C3) and CO2 (C1) for the production of the platform chemical succinic acid (C4) by after metabolic engineering of the yeast Saccharomyces cerevisiae. The product yield (per glycerol consumed) corresponded to 47% of the theoretical maximum. Further possibilities for improvements of the process have been explored in our current project. In the project proposed here, we plan to replace the carbon source glycerol by feedstocks that do not originate from edible plant biomass, and whose abundance is independent from the future of biodiesel production. Both methanol (C1) and dihydroxyacetone (DHA, C3) have the potential to become future carbon sources for biotechnological processes since they can be generated from synthesis gas or from CO2. An attractive approach is to equip our succinic acid producing strain with a linear pathway for conversion of methanol to SA (methanol dehydrogenase and formolase) and afterwards use adaptive laboratory evolution (ALE). We consider this a high-risk approach due to both thermodynamic and kinetic constraints. We still consider it promising. In fact, the proposed ALE strategy has the potential to optimize the kinetics of the enzyme formolase. In addition, our succinic acid pathway is considered a strong sink for NADH and can therefore attenuate the thermodynamic barrier of the NAD+-dependent methanol oxidation. As an alternative to methanol as the carbon source, we propose a second approach based on DHA (C3) as a carbon source. In the future, DHA could be generated from CO2 in a chemoenzymatic process and the channeling of DHA into the central metabolism of S. cerevisiae is straightforward. Our previously constructed S. cerevisiae strains with modified glycerol catabolism (in particular those with the DHA pathway) form an excellent basis for this goal. The improvement of tolerance towards higher DHA concentrations will be addressed by ALE. In order to provide sufficient cytosolic NADH for SA production, an auxiliary substrate (such as formate generated from CO2) will be fed.