Wiemann Philipp, Soukup Alexandra A, Folz Jacob S, Wang Pin-Mei, Noack Andreas, Keller Nancy P
1Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI 53706 USA.
Present Address: Hexagon Bio, Menlo Park, CA 94025 USA.
Fungal Biol Biotechnol. 2018 Mar 13;5:6. doi: 10.1186/s40694-018-0049-2. eCollection 2018.
Sequencing of fungal species has demonstrated the existence of thousands of putative secondary metabolite gene clusters, the majority of them harboring a unique set of genes thought to participate in production of distinct small molecules. Despite the ready identification of key enzymes and potential cluster genes by bioinformatics techniques in sequenced genomes, the expression and identification of fungal secondary metabolites in the native host is often hampered as the genes might not be expressed under laboratory conditions and the species might not be amenable to genetic manipulation. To overcome these restrictions, we developed an inducible expression system in the genetic model .
We genetically engineered a strain of devoid of producing eight of the most abundant endogenous secondary metabolites to express the sterigmatocystin Zn(II)Cys transcription factor-encoding gene and its cofactor under control of the nitrate inducible / promoter. Furthermore, we identified a subset of promoters from the sterigmatocystin gene cluster that are under nitrate-inducible AflR/S control in our production strain in order to yield coordinated expression without the risks from reusing a single inducible promoter. As proof of concept, we used this system to produce β-carotene from the carotenoid gene cluster of .
Utilizing one-step yeast recombinational cloning, we developed an inducible expression system in the genetic model and show that it can be successfully used to produce commercially valuable metabolites.
真菌物种测序已证明存在数千个假定的次生代谢物基因簇，其中大多数含有一组独特的基因，这些基因被认为参与不同小分子的产生。尽管通过生物信息学技术在已测序的基因组中能够轻易鉴定关键酶和潜在的基因簇基因，但真菌次生代谢物在天然宿主中的表达和鉴定常常受到阻碍，因为这些基因在实验室条件下可能不表达，而且该物种可能不适合进行基因操作。为了克服这些限制，我们在遗传模型中开发了一种诱导表达系统。
我们对一种不产生八种最丰富的内源性次生代谢物的菌株进行基因工程改造，使其在硝酸盐诱导型/nit promoter的控制下表达编码柄曲霉素锌（II）半胱氨酸转录因子的基因及其辅因子。此外，我们从柄曲霉素基因簇中鉴定出一组启动子，它们在我们的生产菌株中受硝酸盐诱导型AflR/S控制，以便实现协调表达，而不会因重复使用单个诱导型启动子而产生风险。作为概念验证，我们使用该系统从的类胡萝卜素基因簇中生产β-胡萝卜素。
利用一步酵母重组克隆技术，我们在遗传模型中开发了一种诱导表达系统，并表明它可成功用于生产具有商业价值的代谢物。
