Sim K L, Perry D
School of Biological and Health Sciences, University of Westminster, London, UK.
Glycoconj J. 1997 Aug;14(5):661-8. doi: 10.1023/a:1018505130422.
The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 microg ml(-1). Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition.
α-甘露糖苷酶抑制剂苦马豆素由丝状真菌绿僵菌产生。已知其衍生的主要代谢途径是通向赖氨酸的途径。为了提高苦马豆素的产量，研究赖氨酸及其生物合成中间体以及苦马豆素本身在细胞内水平的变化是很有意义的，这些变化伴随着培养条件或微生物遗传学的改变。察氏定义培养基已用于这些研究。开发了一种反相高效液相色谱法，用于分析菌丝体提取物中的赖氨酸、酵母氨酸、α-氨基己二酸和哌啶酸。该方法基于用9-芴甲基氯甲酸酯（FMOC）进行柱前衍生，FMOC是一种已知可用于含氨基化合物衍生的试剂。用醋酸盐缓冲液/乙腈梯度洗脱实现了四种代谢物的分离，通过在1至20μg ml(-1)浓度下的紫外吸收对其进行定量。苦马豆素浓度使用先前描述的基于酶的方法测定，但现在应用于细胞内和细胞外样品。对添加L-赖氨酸的培养基中苦马豆素积累末期的菌丝体提取物进行分析，结果显示与对照菌丝体相比，哌啶酸积累，赖氨酸积累程度较小。将培养基pH控制在9.0导致苦马豆素产量下降，同时细胞内哌啶酸水平升高。为了产生可能预期会产生更多苦马豆素的赖氨酸过量生产者，分离了对有毒赖氨酸类似物2-氨基乙基半胱氨酸（AEC）具有耐受性的自发突变体。令人惊讶的是，四个独立分离的突变体产生的苦马豆素产量较低，但酵母氨酸积累水平较高。因此，对AEC的耐受性似乎是由于酵母氨酸向苦马豆素生物合成的转移减少，从而使足够的赖氨酸生物合成以克服AEC竞争。
