重构甲醇利用途径促进毕赤酵母甲醇和CO2的共利用

Reconstruction of methanol utilization pathway enhances co-utilization of methanol and CO2 in Komagataella phaffii

摘要:

目的 工程化构建还原性甘氨酸途径(reductive glycine pathway, rGlyP)是实现甲醇与CO₂协同利用的有效策略，但该途径的高效运转受限于胞内还原型烟酰胺腺嘌呤二核苷酸(reduced nicotinamide adenine dinucleotide, NADH)供给不足。毕赤酵母(Komagataella phaffii)内源醇氧化酶(alcohol oxidase, AOX)途径氧化甲醇产生过氧化氢而非NADH，导致能量浪费并引发氧化胁迫。为解决这一瓶颈，本研究通过重构甲醇氧化途径并结合亚细胞区室化策略，优化碳通量与能量代谢。方法 以生长曲线和甲醇利用速率为指标，在敲除内源aox1和aox2的底盘菌株中对5种不同来源的NAD⁺依赖型甲醇脱氢酶(methanol dehydrogenase, MDH)进行筛选，以确定最佳MDH，并对其甲醇诱导浓度进行优化。进一步采用区室化策略，利用1型过氧化物酶体靶向信号(peroxisomal targeting signal 1, PTS1)信号肽将MDHN1T靶向定位至过氧化物酶体，实现了甲醇氧化与甲醛解毒的空间偶联。结果 筛选确定源自杀虫贪铜菌(Cupriavidus necator)的MDHN1T具有最佳催化性能，并优化出其最适甲醇诱导浓度为0.6%。重组菌株在甲醇/CO₂共利用条件下，甲醇消耗速率提升至28.98 mg/d，胞内NAD⁺和NADH总量提升至原来的1.3倍，NADH/NAD⁺提升至原来的1.2倍，生物量达到出发菌株的2.2倍。结论 本研究成功缓解了rGlyP的还原力匮乏问题，促进了毕赤酵母对甲醇和CO₂的共利用，为构建高效利用一碳资源的微生物细胞工厂提供了优质的底盘细胞及理论依据。

Abstract:

Objective The engineering of the reductive glycine pathway (rGlyP) in Komagataella phaffii (syn. Pichia pastoris) represents a promising strategy for the co-utilization of methanol and CO₂. However, the efficiency of this pathway is constrained by the insufficient supply of intracellular reduced nicotinamide adenine dinucleotide (NADH), as the native alcohol oxidase (AOX) pathway generates hydrogen peroxide rather than NADH, leading to energy loss and oxidative stress. To overcome this bottleneck, this study reconstructed the methanol oxidation pathway and employed a subcellular compartmentalization strategy to optimize the carbon flux and energy metabolism.Methods Five different sources of NAD⁺-dependent methanol dehydrogenase (MDH) were screened in an aox1/aox2-deficient strain by using the growth curve and methanol utilization rate as indicators to determine the optimal MDH, and the methanol induction concentration was optimized. Subsequently, a compartmentalization strategy was employed by fusing the peroxisomal targeting signal 1 (PTS1) to MDHN1T, which targeted the enzyme to the peroxisome to spatially couple methanol oxidation with formaldehyde detoxification.Results The MDHN1T derived from Cupriavidus necator had the best catalytic performance, and the optimum methanol induction concentration was optimized to be 0.6%. Under co-utilization conditions, the engineered strain achieved a methanol consumption rate of 28.98 mg/d, with the total intracellular NAD_total pool, NADH/NAD⁺ ratio, and biomass being 1.3, 1.2, and 2.2 folds, respectively, of those in the parental strain.Conclusion This study successfully alleviates the redox cofactor imbalance in the rGlyP and enhances co-utilization of methanol and CO₂ in K. phaffii, providing a robust chassis and a theoretical basis for the development of microbial cell factories utilizing one-carbon resources.