Nagy Viviana, Seidl Verena, Szakacs George, Komoń-Zelazowska Monika, Kubicek Christian P, Druzhinina Irina S
Department of Agricultural Chemical Technology, Technical University of Budapest, Gellert ter 4, 1111 Budapest, Hungary.
Appl Environ Microbiol. 2007 Nov;73(21):7048-58. doi: 10.1128/AEM.00995-07. Epub 2007 Sep 7.
Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed. The geographic origin of the strains was irrelevant for chitinase production. The improved chitinase production by strains containing this haplotype was not due to better growth on N-acetyl-beta-D-glucosamine or glucosamine. Isoenzyme electrophoresis showed that neither the isoenzyme profile of N-acetyl-beta-glucosaminidases or the endochitinases nor the intensity of staining of individual chitinase bands correlated with total chitinase in the culture filtrate. The superior chitinase producers did not exhibit similarly increased cellulase formation. Biolog Phenotype MicroArray analysis identified lack of N-acetyl-beta-D-mannosamine utilization as a specific trait of strains with the chitinase-overproducing haplotype. This observation was used to develop a plate screening assay for rapid microbiological identification of the strains. The data illustrate that desired industrial properties may be an attribute of certain populations within a species, and screening procedures should thus include a balanced mixture of all genotypes of a given species.
为生物技术目的选择合适的菌株通常是一个由高通量方法支持的随机过程。以哈茨木霉/里氏木霉产生几丁质酶为模型，我们测试了能否通过DNA条形码诊断出具有优异酶形成能力的真菌菌株。我们分析了50株哈茨木霉/里氏木霉分离株的两个系统发育标记位点的序列，即编码rRNA的基因簇的内部转录间隔区1（ITS1）和ITS2以及延伸因子1-α基因（tef1）的大内含子，这些分离株还经过测试以确定它们在固态发酵（SSF）中产生几丁质酶的能力。对于携带与哈茨木霉模式菌株CBS 226.95的一个等位基因相对应的观察到的ITS1、ITS2和tef1等位基因之一的菌株，在统计学上获得了支持的优异几丁质酶产量。开发了一种基于tef1的DNA条形码工具TrichoCHIT，用于快速鉴定这些菌株。菌株的地理来源与几丁质酶的产生无关。含有这种单倍型的菌株几丁质酶产量的提高并非由于在N-乙酰-β-D-葡萄糖胺或葡萄糖胺上生长更好。同工酶电泳表明，N-乙酰-β-葡萄糖苷酶或内切几丁质酶的同工酶谱以及单个几丁质酶条带的染色强度均与培养滤液中的总几丁质酶无关。优异的几丁质酶生产者并未表现出类似增加的纤维素酶形成。Biolog表型微阵列分析确定缺乏对N-乙酰-β-D-甘露糖胺的利用是具有几丁质酶高产单倍型菌株的一个特定特征。这一观察结果被用于开发一种平板筛选试验，用于快速微生物鉴定这些菌株。数据表明，所需的工业特性可能是一个物种内某些群体的属性，因此筛选程序应包括给定物种所有基因型的平衡混合物。
