利用毕赤酵母进行甲醇生物转化具有广泛的应用前景。为了深入了解该酵母甲醇代谢机理,该文以甲醇利用途径的4个过氧化物酶体同工酶为研究对象展开研究,分别为D-核酮糖-5-磷酸-3-表异构酶2(D-ribulose-5-phosphate 3-epimerase,Rpe1-2),核糖-5-磷酸异构酶2(ribose-5-phosphate isomerase,Rki1-2),转醛醇酶2(transaldolase,Tal1-2),果糖-1,6-二磷酸醛缩酶2(fructose-1,6-bisphosphate aldolase,Fba1-2)。在验证其参与甲醇代谢的基础上,分别对4个酶基因进行单敲除、四敲除,并在四敲除菌中分别进行细胞质同工酶及过氧化物酶体同工酶的回补实验。结果表明4敲除菌在甲醇培养基中受到严重的生长抑制。而单敲除菌中,Fba1-2的缺失对菌株在甲醇培养基中的生长表型影响最大;同时,回补Fba1-2也能最大程度地恢复4敲除菌株的生物量。综上,Fba1-2是甲醇同化途径中的关键酶,且同化途径的过氧化物酶体区室化对甲醇代谢途径至关重要。此外,景天庚酮糖-1,7-二磷酸酶(sedoheptulose-1,7-bisphosphatase,Shb17)基因单敲除菌和TAL1-2基因单敲除菌的表型对比结果表明,更改的卡尔文循环是甲醇代谢中木酮糖-5-磷酸(xylulose 5-phosphate,XU5P)再生的主要途径。该研究可为后续优化毕赤酵母甲醇利用途径提供理论依据。
The potential applications of Pichia pastoris for methanol bioconversion are extensive. In order to elucidate the mechanism of methanol metabolism in P. pastoris, four peroxisomal isoenzymes (i.e., Rpe1-2, Rki1-2, Tal1-2, and Fba1-2.) involved in methanol utilization (MUT) pathway were investigated. Their roles in methanol metabolism were verified by constructing single-gene knockout mutants and quadruple-gene knockout mutant. Moreover, cytoplasmic isozymes and peroxisomal isozymes were complemented in quadruple-gene knockout mutant, respectively. The results showed that the growth of quadruple-gene knockout mutant was severely inhibited in methanol medium, and the Fba1-2 deficiency had the greatest effect on the growth phenotype. At the same time, Fba1-2 complementation could rescue the biomass production of quadruple-gene knockout mutant most effectively. These results further confirm that Fba1-2 is a crucial enzyme for the methanol assimilation and highlight the significance of peroxisome localization of assimilation pathway. Additionally, by comparing the growth phenotypes of SHB17 single-gene knockout mutant and TAL1-2 single-gene knockout mutant, it is further confirmed that the altered Calvin cycle is the main pathway of regenerating XU5P in methanol metabolism. This study can provide theoretical basis for optimizing the MUT pathway in P. pastoris.
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