过表达全局调控因子LaeA对橘青霉美伐他汀的合成及产孢的影响

Overexpression of LaeA enhances mevastatin production and reduces sporulation of Penicillium citrinum

摘要:

摘要:【目的】研究橘青霉中全局性调控因子LaeA过表达对美伐他汀生物合成过程及产孢的影响。【方法】同源克隆法从橘青霉中克隆获得laeA基因，构建pGiHTGi-laeA载体，经农杆菌介导转化转入橘青霉，利用hygromycin基因进行阳性克隆子的PCR筛选。使用HPLC方法比较PGA发酵液中橘青霉野生型菌株WT和LaeA过表达菌株OE::laeA的美伐他汀产量差异。血球计数法进行孢子产量比较。荧光定量PCR方法分析WT和OE::laeA中美伐他汀合成基因簇表达量。【结果】成功构建pGiHTGi-laeA载体，并获得LaeA过表达橘青霉菌株(OE:: laeA)。OE:: laeA 中，laeA基因表达量较WT增加29%，mlcB表达量较WT增加72%，mlcR表达量增加153%，孢子产量由(2.38±0.24)×107/cm2减少到(1. 40±0.11)×107/cm2，美伐他汀产量从(35.77±4.63)mg/L提高到(201.46±9.98)mg/L。【结论】在橘青霉中过表达LaeA可能通过提高mlcR和mlcB基因的转录来增加美伐他汀的产量，同时过表达laeA基因不利于孢子的形成。这些结果为美伐他汀生物合成的全局性调控和高产菌株的开发提供基础。

Abstract:

Abstract:［Objective］ To study the regulation of laeA overexpression on mevastatin production and sporulation in Penicillium citrinum． ［Methods］We cloned the laeA gene from Penicillium citrinum and constructed the vector pGiHTGilaeA．The plasmid pGiHTGi-laeA was transformed in Penicillium citrinum by agrobacterium tumefaciens-mediated transformation．Positive transformants were detected by cloning the hygromycin gene．The mevastatin production of the wild type and OE::laeA was compared by HPLC．The conidia number was counted by blood counting chamber．The biosynthetic gene cluster expression quantity of mevastatin in the wild type and OE::laeA were analyzed by qRT-PCR.［Results］We constructed the plasmid pGiHTGi-laeA，and screened the positive transformants that overexpress the laeA in Penicillium citrinum．With the overexpression of laeA，the mevastatin production was increased from (0.69±0.12)mg/g to (4.02±0.50) mg/g dry cell weight．Compared to the wild type strain，the laeA expression quantity in the OE::laeA strain increased 29%，and the mlcB expression increased 72%，the mlcＲ expression increased 153%． Moreover，the overexpression of laeA would decrease the conidia number．［Conclusion］Overexpression of LaeA enhances mevastatin production and reduces sporulation of Penicillium citrinum，with increases expression of pathway-regulator mlcR，and biosynthetic gene MlcR．These results could guide global regulatory mechanism of mevastatin biosynthesis and the exploitation of high-production strain.