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更新時(shí)間：2025.06.01
代謝工程改造釀酒酵母合成葡萄糖二酸

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葡萄糖二酸是一種高附加值的有機(jī)酸,廣泛用于食品、醫(yī)藥和化工領(lǐng)域.為獲得生產(chǎn)葡萄糖二酸的微生物細(xì)胞工廠,通過(guò)共表達(dá)小鼠來(lái)源的肌醇加氧酶(MIOX)及惡臭假單胞菌來(lái)源的醛酸脫氫酶(Udh),在釀酒酵母Saccharomyces cerevisiae CEN.PK2-1C中構(gòu)建了葡萄糖二酸合成途徑,產(chǎn)量為(28.28±3.15) mg/L.在此基礎(chǔ)上,通過(guò)調(diào)控前體肌醇的合成途徑,發(fā)現(xiàn)肌醇-1-磷酸合成酶(INO1)是葡萄糖二酸合成途徑的限速酶,過(guò)量表達(dá)INO1,葡萄糖二酸產(chǎn)量達(dá)到(107.51±10.87) mg/L,提高了2.8倍.進(jìn)一步弱化競(jìng)爭(zhēng)支路中磷酸果糖激酶(PFKl)的表達(dá),最終葡萄糖二酸的產(chǎn)量達(dá)到(230.22±10.75) mg/L,為進(jìn)一步獲得高產(chǎn)葡萄糖二酸細(xì)胞工廠提供基礎(chǔ).

葡萄糖異構(gòu)酶基因工程菌的改造初探

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In order to realize stable overexpression of mutant glucose isomerase(GI)gene from Streptomyces diastaticus No.7 M1033 in E.coli, a 1.2 kb fragment containing the intact coding sequence of the protein was amplified specifically from plasmid pTKD GI1 by PCR.At the same time,45 bp unnecessary sequence at GI gene upstream was deleted.The amplified fragment was inserted into the expression vector pBV220 to obtain the recombinant plasmid pBZGI1,which was introduced into E.coli DH5α.Data gathered from passage of the generations of the strains showed that pBZGI1 in DH5α was much more stable than pTKD GI1 in K38/pGP1 2.Induced at 42℃,pBZGI1 overexpressed the mutant GI,which accounted for about 55% of total soluble proteins and was purified through heat treatment,DEAE Sepharose FF column and Sephacrcyl S 300 column.It also showed that the thermostability of the purified GI didn't decline though the undesired 15 amino acids present in N terminal was deleted.

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