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

作为前一个项目的结果，我们最近展示了通过酿酒酵母的后代谢工程来共同利用甘油(C3)和二氧化碳(C1)来生产平台化学物质琥珀酸(C4)。产品收率(每消耗一次甘油)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进程的进一步可能性。在这里提出的项目中，我们计划用不是来自可食用植物生物质的原料来取代碳源甘油，这些原料的丰度与未来生物柴油的生产无关。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二氧化碳中产生。一个吸引人的方法是为我们的琥珀酸产生菌装备一个将甲醇转化为SA(甲醇脱氢酶和甲醇酶)的线性途径，然后使用适应性实验室进化(ALE)。我们认为，由于热力学和动力学的限制，这是一种高风险的方法。我们仍然认为这是有希望的。事实上，所提出的ALE策略有可能优化福尔马林酶的动力学。此外，我们的琥珀酸途径被认为是NADH的一个强大的汇，因此可以减弱依赖NAD+的甲醇氧化的热力学障碍。作为替代甲醇的碳源，我们提出了以DHA(C3)为碳源的第二种方法。在未来，DHA可以在化学酶过程中从二氧化碳中产生，DHA进入酿酒酵母的中心代谢是直接的。我们以前构建的具有甘油分解代谢修饰的酿酒酵母菌株(特别是那些具有DHA途径的菌株)为这一目标奠定了良好的基础。ALE将解决提高对较高DHA浓度的耐受性的问题。为了为SA的生产提供足够的胞液NADH，将补加辅助底物(例如从二氧化碳中产生的甲酸盐)。

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.