Helbig Florian, Steighardt Jörg, Roos Werner
Department of Cellular Physiology, Institute of Pharmaceutical Biology, University of Halle, Halle, Germany.
Appl Environ Microbiol. 2002 Apr;68(4):1524-33. doi: 10.1128/AEM.68.4.1524-1533.2002.
On searching for endogenous, low-molecular-weight effectors of benzodiazepine alkaloid biosynthesis in Penicillium cyclopium uric acid was isolated from ethanolic or autoclaved mycelial extracts of this fungus. The isolation was based on a three-step high-pressure liquid chromatography procedure guided by a microplate bioassay, and uric acid was identified by mass spectrometry and the uricase reaction. Conidiospore suspensions that were treated with this compound during the early phase of outgrowth developed emerged cultures with an enhanced rate of alkaloid production. Uric acid treatment did not increase the in vitro measurable activity of the rate-limiting biosynthetic enzyme, cyclopeptine synthetase. However, these cultures displayed a reduced rate of uptake of the alkaloid precursor L-phenylalanine into the vacuoles of the hyphal cells as assayed in situ. It is suggested that the depressed capacity of vacuolar uptake caused by the contact of outgrowing spores with uric acid liberated from hyphal cells results in an enhanced availability of the precursor L-phenylalanine in the cytoplasm and thus accounts at least in part for the increase in alkaloid production.
在寻找环孢青霉中苯二氮卓生物碱生物合成的内源性低分子量效应物时，从该真菌的乙醇提取物或高压灭菌菌丝体提取物中分离出了尿酸。分离基于微板生物测定指导的三步高压液相色谱程序，通过质谱和尿酸酶反应鉴定出尿酸。在生长早期用该化合物处理的分生孢子悬浮液培养出的培养物中生物碱产量增加。尿酸处理并未增加限速生物合成酶环肽合成酶的体外可测量活性。然而，如原位测定所示，这些培养物中生物碱前体L-苯丙氨酸进入菌丝细胞液泡的摄取速率降低。有人提出，生长中的孢子与从菌丝细胞释放的尿酸接触导致液泡摄取能力下降，从而使细胞质中前体L-苯丙氨酸的可用性增加，因此至少部分解释了生物碱产量的增加。
