Wong Valerie L, Ellison Christopher E, Eisen Michael B, Pachter Lior, Brem Rachel B
Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California, United States of America.
Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States of America.
PLoS One. 2014 May 13;9(5):e96784. doi: 10.1371/journal.pone.0096784. eCollection 2014.
Strain selection and strain improvement are the first, and arguably most important, steps in the industrial production of biological compounds by microorganisms. While traditional methods of mutagenesis and selection have been effective in improving production of compounds at a commercial scale, the genetic changes underpinning the altered phenotypes have remained largely unclear. We utilized high-throughput Illumina short read sequencing of a wild Penicillium chrysogenum strain in order to make whole genome comparisons to a sequenced improved strain (WIS 54-1255). We developed an assembly-free method of identifying chromosomal rearrangements and validated the in silico predictions with a PCR-based assay and Sanger sequencing. Despite many rounds of mutagen treatment and artificial selection, WIS 54-1255 differs from its wild progenitor at only one of the identified rearrangements. We suggest that natural variants predisposed for high penicillin production were instrumental in the success of WIS 54-1255 as an industrial strain. In addition to finding a previously published inversion in the penicillin biosynthesis cluster, we located several genes related to penicillin production associated with these rearrangements. By comparing the configuration of rearrangement events among several historically important strains known to be high penicillin producers to a collection of recently isolated wild strains, we suggest that wild strains with rearrangements similar to those in known high penicillin producers may be viable candidates for further improvement efforts.
菌株选择和菌株改良是微生物工业化生产生物化合物的首要步骤,也可以说是最重要的步骤。虽然传统的诱变和选择方法在商业规模上提高化合物产量方面很有效,但支撑表型改变的基因变化在很大程度上仍不清楚。我们利用高通量Illumina短读长测序技术对一株野生产黄青霉进行测序,以便与一株已测序的改良菌株(WIS 54-1255)进行全基因组比较。我们开发了一种无需组装的方法来识别染色体重排,并通过基于PCR的检测和桑格测序验证了计算机预测结果。尽管经过了多轮诱变处理和人工选择,但在已识别的重排中,WIS 54-1255与其野生祖先仅在一处存在差异。我们认为,倾向于高产青霉素的自然变体对WIS 54-1255作为工业菌株的成功起到了重要作用。除了在青霉素生物合成簇中发现一个先前已发表的倒位外,我们还定位了几个与这些重排相关的青霉素生产相关基因。通过比较几个已知高产青霉素的历史重要菌株与一组最近分离的野生菌株之间重排事件的构型,我们认为,具有与已知高产青霉素菌株相似重排的野生菌株可能是进一步改良的可行候选菌株。