Regmi Priti, Knesebeck Melanie, Boles Eckhard, Weuster-Botz Dirk, Oreb Mislav
Goethe University Frankfurt, Faculty of Biological Sciences, Institute of Molecular Biosciences, Max-von-Laue Straße 9, 60438, Frankfurt am Main, Germany.
Technical University of Munich, Chair of Biochemical Engineering, Boltzmannstr. 15, 85748, Garching, Germany.
Metab Eng Commun. 2024 Jul 5;19:e00245. doi: 10.1016/j.mec.2024.e00245. eCollection 2024 Dec.
Enhancing the supply of the redox cofactor NADPH in metabolically engineered cells is a critical target for optimizing the synthesis of many product classes, such as fatty acids or terpenoids. In , several successful approaches have been developed in different experimental contexts. However, their systematic comparison has not been reported. Here, we established the reduction of xylose to xylitol by an NADPH-dependent xylose reductase as a model reaction to compare the efficacy of different NADPH supply strategies in the course of a batch fermentation, in which glucose and ethanol are sequentially used as carbon sources and redox donors. We show that strains overexpressing the glucose-6-phosphate dehydrogenase Zwf1 perform best, producing up to 16.9 g L xylitol from 20 g L xylose in stirred tank bioreactors. The beneficial effect of increased Zwf1 activity is especially pronounced during the ethanol consumption phase. The same notion applies to the deletion of the aldehyde dehydrogenase gene, albeit at a quantitatively lower level. Reduced expression of the phosphoglucose isomerase Pgi1 and heterologous expression of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase Gdp1 from acted synergistically with overexpression in the presence of glucose, but had a detrimental effect after the diauxic shift. Expression of the mitochondrial NADH kinase Pos5 in the cytosol likewise improved the production of xylitol only on glucose, but not in combination with enhanced Zwf1 activity. To demonstrate the generalizability of our observations, we show that the most promising strategies - overexpression and deletion of - also improve the production of l-galactonate from d-galacturonic acid. Therefore, we expect that these findings will provide valuable guidelines for engineering not only the production of xylitol but also of diverse other pathways that require NADPH.
增强代谢工程改造细胞中氧化还原辅因子NADPH的供应，是优化许多产品类别（如脂肪酸或萜类化合物）合成的关键目标。在不同的实验背景下，已经开发出了几种成功的方法。然而，尚未有对它们进行系统比较的报道。在此，我们建立了由NADPH依赖性木糖还原酶将木糖还原为木糖醇的反应作为模型反应，以比较分批发酵过程中不同NADPH供应策略的效果，在该发酵过程中，葡萄糖和乙醇依次用作碳源和氧化还原供体。我们发现，过表达葡萄糖-6-磷酸脱氢酶Zwf1的菌株表现最佳，在搅拌罐生物反应器中，从20 g/L木糖中可产生高达16.9 g/L的木糖醇。Zwf1活性增加的有益效果在乙醇消耗阶段尤为明显。醛脱氢酶基因的缺失也有同样的效果，尽管在数量上较低。磷酸葡萄糖异构酶Pgi1的表达降低以及来自的NADP依赖性甘油醛-3-磷酸脱氢酶Gdp1的异源表达，在有葡萄糖存在的情况下与Zwf1过表达协同作用，但在双相转变后产生了有害影响。线粒体NADH激酶Pos5在胞质溶胶中的表达同样仅在葡萄糖存在时提高了木糖醇的产量，但与增强的Zwf1活性联合使用时则没有效果。为了证明我们观察结果的普遍性，我们表明最有前景的策略——Zwf1过表达和基因缺失——也提高了由D-半乳糖醛酸生产L-半乳糖酸的产量。因此，我们预计这些发现不仅将为木糖醇的生产工程，而且为其他需要NADPH的多种途径的工程提供有价值的指导。
