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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 NADtotal 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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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