Metabolic engineering of bakers yeast for more efficient respiratory and fermentative glycerol utilization

甘油是一种丰富的碳源，并且由于其与中性糖相比相对高的还原度（DOG）而成为用于微生物发酵的有吸引力的原料。这种较高的DOG伴随着用作燃料、散装和精细化学品的还原小分子的较高理论产率。此外，它允许在用于氧化产物如有机酸的微生物生产的混合底物转化中的高得多的理论二氧化碳固定产率。酵母S.酿酒酵母是工业生物技术中流行的真菌细胞工厂，这是由于其在工业环境中的稳健性。特别地，真菌生物体在相对低的pH下生长的能力对于经济上可行的有机酸生产是非常有吸引力的，因为它有利于下游加工。在之前DFG资助的项目中，我们在改善S.以甘油为唯一碳源的酿酒酵母。特别是S.通过用异源转运蛋白（一种促进甘油转运的水甘油孔蛋白）替代酿酒酵母，以及用产生NADH的DHA途径替代内源性FAD依赖性L-G3 P途径，使流行菌株CEN.PK能够在合成甘油培养基中生长，µmax约为0.26 h-1。除了改善在甘油上的生长外，递送细胞溶质NADH的工程化甘油分解代谢途径理论上允许通过与CO2的净固定相结合的反向TCA（rTCA）途径产生氧化还原因子中性琥珀酸（SA）。我们配备了甘油利用菌株与rTCA途径，增加丙酮酸脱羧酶活性，和异源有机酸转运蛋白。最佳菌株从甘油产生SA（和小部分苹果酸），有机酸产率对应于最大理论产率的61.5%。在具有富含CO2的气相的受控生物反应器中的废气分析表明，在发酵过程中，我们的最佳菌株导致净CO2消耗（CO2-负过程）。虽然我们的结果是非常令人鼓舞的测试厌氧SA发酵，剩下的挑战是，目前的SA发酵途径消耗净ATP的事实。此外，输出SA并将其保持在细胞外是非常昂贵的能源。因此，一定部分的碳必须进入氧化TCA循环（oxTCA循环）以提供ATP用于维持和生长。目前的建议有两个主要目标。首先，必须改善一般ATP平衡，以降低细胞对oxTCA循环和呼吸的依赖性。一个特别的重点将放在ATP消耗运输过程和回补反应。第二个目标是微调进入oxTCA循环的通量，以满足细胞的能量和合成代谢需求，而不会以二氧化碳和生物质的形式损失超过必要的碳。

Glycerol is an abundant carbon source and an attractive feedstock for microbial fermentations due to its relatively high degree of reduction (DOG) compared to neutral sugars. This higher DOG is accompanied with higher theoretical yields of reduced small molecules used as fuels, bulk and fine chemicals. Moreover, it allows a much higher theoretical carbon dioxide fixation yield in mixed substrate conversions for the microbial production of oxidized products such as organic acids. The yeast S. cerevisiae is a popular fungal cell factory in industrial biotechnology due to its robustness in industrial settings. Particularly the ability of fungal organisms to grow at relatively low pH is highly attractive for an economically feasible organic acid production since it facilitates downstream processing. Within a prior DFG-funded project, we made significant progress in improving growth and fermentation of S. cerevisiae with glycerol as the sole source of carbon. Particularly, the equipment of S. cerevisiae with a heterologous transport protein (an aquaglyceroporin that facilitates glycerol transport) and the replacement of the endogenous FAD-dependent L-G3P pathway by an NADH-generating DHA pathway enabled the popular strain CEN.PK to grow in synthetic glycerol medium with a µmax of about 0.26 h-1. Apart from improving growth on glycerol, the engineered glycerol catabolic pathway delivering cytosolic NADH theoretically allows redox-factor neutral succinic acid (SA) production via the reverse TCA (rTCA) pathway which is combined with net fixation of CO2. We equipped the glycerol-utilizing strain with the rTCA pathway, an increased pyruvate decarboxylase activity, and a heterologous organic acid transporter. The best strain produced SA (and a minor portion of malate) from glycerol with an organic acid yield corresponding to 61.5 % of the maximum theoretical yield. Offgas analysis in controlled bioreactors with CO2 enriched gas-phase indicated that, over the course of the fermentation, our best strain led to net CO2 consumption (CO2-negativ process). Although our results are highly encouraging to test anaerobic SA fermentation, a remaining challenge is the fact that the current SA fermentation pathway consumes net ATP. Moreover, exporting SA and keeping it outside the cells is very energy-expensive. Thus, a certain portion of carbon has to enter the oxidative TCA cycle (oxTCA cycle) to supply ATP for maintenance and growth. The current proposal has two major goals. First, the general ATP balance has to be improved in order to decrease the dependency of the cells on the oxTCA cycle and respiration. A particular focus will be put on ATP-consuming transport processes and the anaplerotic reactions. A second goal is to fine-tune the flux into the oxTCA cycle in order to meet the energy and anabolic requirements of the cells without losing more carbon than necessary in the form of carbon dioxide and biomass.