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

甘油是一种丰富的碳源，与中性糖相比，其还原程度(DOG)相对较高，是微生物发酵的诱人原料。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更高理论产量。此外，在微生物生产有机酸等氧化产物的混合底物转化中，它允许更高的二氧化碳固定理论产率。酿酒酵母因其在工业环境中的健壮性，在工业生物技术中是一种受欢迎的真菌细胞工厂。特别是，真菌生物在相对较低的pH值下生长的能力对经济上可行的有机酸生产具有极大的吸引力，因为它促进了下游加工。在之前DFG资助的一个项目中，我们在改善以甘油为唯一碳源的酿酒酵母的生长和发酵方面取得了重大进展。特别是，酿酒酵母携带异源转运蛋白(一种促进甘油运输的水凝胶蛋白)，并用产生NADH的DHA途径取代内源FAD依赖的L-G3P途径，使广受欢迎的菌株CEN.PK能够在合成甘油培养基中生长，最大生长时间约为0.26h-1。除了促进甘油的生长外，工程甘油分解代谢途径提供胞液NADH，理论上允许氧化还原因子中性琥珀酸(SA)通过反向TCA(RTCA)途径产生，这与二氧化碳的净固定相结合。我们为利用甘油的菌株配备了RTCA途径，增加了丙酮酸脱羧酶活性，并配备了异源有机酸转运体。最好的菌株利用甘油生产SA(和少量苹果酸)，其有机酸产量相当于最大理论产量的61.5%。在富CO2气相的受控生物反应器中的尾气分析表明，在发酵过程中，我们最好的菌株导致了净CO2消耗(CO2阴性过程)。尽管我们的结果对测试厌氧SA发酵是非常令人鼓舞的，但仍然存在的挑战是，当前的SA发酵途径消耗净ATP。此外，输出SA并将其保存在细胞外是非常耗能的。因此，一定部分的碳必须进入氧化的TCA循环(oxTCA循环)，以提供维持和生长所需的ATP。目前的提议有两个主要目标。首先，必须改善总体的ATP平衡，以减少细胞对oxTCA周期和呼吸的依赖。重点将放在消耗ATP的运输过程和逆性反应上。第二个目标是微调oxTCA循环中的通量，以满足细胞的能量和合成代谢需求，而不会损失超过必要的二氧化碳和生物质形式的碳。

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.