Efficient Reconstitution of Basidiomycota Diterpene Erinacine Gene Cluster in Ascomycota Host Aspergillus oryzae Based on Genomic DNA Sequences
Efficient Reconstitution of Basidiomycota Diterpene Erinacine Gene Cluster in Ascomycota Host Aspergillus oryzae Based on Genomic DNA Sequences
复制标题
基于基因组 DNA 序列在子囊菌科宿主黑曲霉中高效重建基生真菌二萜 Erinacine 基因簇
DOI：
10.1021/jacs.9b08935
复制
发表时间：
2019-10-02
影响因子：
15
通讯作者：
Oikawa, Hideaki
中科院分区：
文献类型：
--
作者：
Liu, Chengwei;Minami, Atsushi;Oikawa, Hideaki
为了开发蘑菇代谢物基因组挖掘的通用方法，我们研究了使用基因组DNA序列的生物活性二萜类化合物erinacines的生产。在这份报告中，我们首先确定了高表达位点（热点）曲霉菌通过测序的基因组DNA从高产转化，这是在我们以前的生物合成研究中获得的。基因组编辑敲入所有的erinacine生物合成基因直接到热点表明，A。蘑菇基因组DNA基因序列中超过90%的内含子被正确剪接。然后，我们重建了erinacine生物合成基因簇使用两轮敲入的cDNA和新开发的可重复的基因工程质粒回收。在100%转化率下，我们获得了成功生产erinacine Q及其中间体的酶。在这项研究中，我们阐明了erinacines的生物合成途径，包括功能独特的羟基化支持脱氢-genate EriH和木糖特异性糖基化，通过引入植物基因提供UDP-木糖。我们新开发的热点敲入和质粒回收使我们能够避免耗时的筛选过程，并通过无标记基因组编辑无限制地引入生物合成基因。
To develop the versatile methodology for genome mining of mushroom metabolites, we examined the production of bioactive diterpenes erinacines using genomic DNA sequences. In this report, we initially identified high expression loci (hot spots) in Aspergillus oryzae by sequencing the genomic DNAs from highly yielding transformants which were obtained in our previous biosynthetic studies. Genome editing knock-in of all erinacine biosynthetic genes directly to the hot spot showed that A. oryzae correctly spliced more than 90% of the introns in the mushroom genomic DNA gene sequences. Then, we reconstituted the erinacine biosynthetic gene cluster using two rounds of knock-in of the cDNAs and newly developed repeatable genetic engineering by plasmid recycling. At 100% transformation rate, we obtained a transformant that successfully produced erinacine Q and its intermediates. In this study, we elucidated a biosynthetic pathway of erinacines involving functionally unique hydroxylation supported by dehydro-genate EriH and xylose-specific glycosylation by introducing plant genes for supplying UDP-xylose. Our newly developed hot spot knock-in and plasmid recycling allowed us to avoid a time-consuming screening process and to use unlimited introduction of biosynthetic genes due to marker-free genome editing.