背景:Komagataellaphafii,一种嗜甲烷酵母,可以使用甲醇,在生态友好型生物制造中具有良好的非糖底物。K.phafii中的异化途径导致CO2形式的碳原子损失。然而,ΔFLD应变,被设计为缺乏甲醛脱氢酶-异化途径中的一种必需酶-暴露于含甲醇的培养基时显示出生长缺陷。
结果:抑制异化途径会引发甲醛的过度积累和细胞内NAD/NADH比率的下降。这里,我们设计了醇氧化酶1/二羟基丙酮合成酶1(Aox1/Das1)的双酶复合物,增强甲醛受体木酮糖-5-磷酸(Xu5P)的再生。该策略减轻了甲醛积累的有害影响和对细胞的相关毒性。同时,我们通过在TCA循环中过表达异柠檬酸脱氢酶来提高NAD+/NADH比率,促进细胞内氧化还原稳态。上述策略的优化组合的OD600,菌株DF02-1,是对照菌株DF00的4.28倍(ΔFLD,1%甲醇下的HIS4+)。随后,在菌株DF02-1中来自多形性汉逊酵母的甲醇氧化酶Mox的异源表达导致重组菌株DF02-4,其在含有3%甲醇的培养基中的OD600比对照菌株DF00高4.08倍。
结论:减少甲醛积累,NAD+/NADH比值的增加,甲醇氧化的增强有效地提高了缺乏甲醛脱氢酶的菌株ΔFLD菌株对高甲醇浓度的有效利用。本研究中实施的改性策略共同为推进K.phafii中甲醇的有效利用提供了基础框架。
BACKGROUND: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase-an essential enzyme in the dissimilation pathway-displayed growth defects when exposed to a methanol-containing medium.
RESULTS: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol.
CONCLUSIONS: The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.