Zheng Yueliang, Cao Shuang, Huang Yuqi, Liao Guojian, Hu Changhua
Wei Sheng Wu Xue Bao. 2014 Dec 4;54(12):1438-45.
To study the regulation of laeA overexpression on mevastatin production and sporulation in Penicillium citrinum.
We cloned the laeA gene from Penicillium citrinum and constructed the vector pGiHTGi-laeA. 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.
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.
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.
研究过表达laeA对桔青霉中洛伐他汀产量及孢子形成的调控作用。
从桔青霉中克隆laeA基因，构建载体pGiHTGi-laeA。通过根癌农杆菌介导的转化法将质粒pGiHTGi-laeA转入桔青霉。通过克隆潮霉素基因检测阳性转化子。采用高效液相色谱法比较野生型和OE::laeA的洛伐他汀产量。用血细胞计数板计数分生孢子数。通过qRT-PCR分析野生型和OE::laeA中洛伐他汀生物合成基因簇的表达量。
构建了质粒pGiHTGi-laeA，并筛选出在桔青霉中过表达laeA的阳性转化子。随着laeA的过表达，洛伐他汀产量从（0.69±0.12）mg/g增加到（4.02±0.50）mg/g干细胞重量。与野生型菌株相比，OE::laeA菌株中laeA表达量增加29%，mlcB表达量增加72%，mlcR表达量增加153%。此外，laeA的过表达会减少分生孢子数。
LaeA的过表达增强了桔青霉中洛伐他汀的产量并减少了孢子形成，同时增加了途径调节因子mlcR和生物合成基因MlcR的表达。这些结果可为洛伐他汀生物合成的全局调控机制及高产菌株的开发提供指导。
