黑曲霉柠檬酸工业菌株原生质体制备与转化

摘要

Aspergillus niger is the major industrial strain for citric acid production. In spite of many successes of modern molecular biology approaches in engineering laboratory or protein-producing strains ofA. niger, there is few positive report on its application for citric acid industrial strains mainly due to the hard-to-transform nature of these strains. In this study, the protoplast-PEG mediated genetic transformation system for citric acid industrial strain was extensively studied, suggesting an optimized protocol for protoplast preparation, regeneration and DNA transformation. The concentration of protoplasts reached up to 106 /mL by lysing younger mycelia for 2.5 h after 48 h incubation of a proper amount of conidia spores in enrichment medium. The optimal lysing enzyme mixtures comprised of 1.5% lysing enzyme, 0.5% snail enzyme and 0.2% lysozyme. Concentration of protoplast influenced the protoplast-PEG mediated transformation efficiency, which reached the maximal when the concentration of protoplast was higher than 106/mL. The genetic transformation system established in this study should pave the way to molecular biology study of the citric acid hyper-producing strains, for further understanding its acid-tolerant physiology and for rational design of the industrial strain for further improvement of the citric acid production process as well as creation of new organic acid-producing cell factories.%黑曲霉是柠檬酸工业化生产的主要发酵菌株。尽管现代遗传操作技术在黑曲霉的实验室菌株、蛋白质生产菌株等的应用中获得了成功，但是柠檬酸工业菌株遗传转化异常困难，成为柠檬酸工业持续升级的重要限制因素。以柠檬酸工业生产实际应用的黑曲霉菌株为研究对象，对其原生质体的制备与再生以及PEG介导的转化等条件进行了细致优化。结果表明，柠檬酸高产工业菌株原生质体的制备需选取丰富培养基中培养48 h的年轻菌丝体，在1.5%裂解酶-0.5%蜗牛酶-0.2%溶菌酶的复合酶解体系下裂解2.5 h，原生质体的制备浓度可达106个/mL以上，原生质体再生效率可达90%以上。原生质体的浓度是原生质体-PEG介导转化方法的关键，当原生质体浓度达到106个/mL以上时，高产柠檬酸菌株的转化效率大幅提高。成功建立了由原生质体-PEG所介导的高产柠檬酸黑曲霉菌株的遗传转化体系。