背景:真菌物种的测序表明存在数千个推定的次生代谢物基因簇,他们中的大多数拥有一组独特的基因,这些基因被认为参与不同小分子的生产。尽管通过生物信息学技术在测序的基因组中容易鉴定关键酶和潜在的簇基因,真菌次生代谢产物在天然宿主中的表达和鉴定通常受到阻碍,因为这些基因在实验室条件下可能无法表达,并且该物种可能不适合进行遗传操作。为了克服这些限制,我们在构巢曲霉的遗传模型中开发了可诱导的表达系统。
结果:我们通过基因工程改造了一个不产生8种最丰富的内源性次级代谢产物的念珠芽孢杆菌菌株,在硝酸盐诱导型niiA/niaD启动子的控制下表达杂色霉素Zn(II)2Cys6转录因子编码基因aflR及其辅因子aflS。此外,我们在我们的生产菌株中鉴定了在硝酸盐诱导型AflR/S控制下的杂色细胞素基因簇中的启动子子集,以便在没有重复使用单个诱导型启动子的风险的情况下产生协调表达.作为概念的证明,我们使用该系统从镰刀菌的类胡萝卜素基因簇中生产β-胡萝卜素。
结论:利用一步酵母重组克隆,我们在遗传模型A.nidulans中开发了一种诱导型表达系统,并表明它可以成功地用于生产具有商业价值的代谢产物。
BACKGROUND: 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 Aspergillus nidulans.
RESULTS: We genetically engineered a strain of A. nidulans devoid of producing eight of the most abundant endogenous secondary metabolites to express the sterigmatocystin Zn(II)2Cys6 transcription factor-encoding gene aflR and its cofactor aflS under control of the nitrate inducible niiA/niaD 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 Fusarium fujikuroi.
CONCLUSIONS: Utilizing one-step yeast recombinational cloning, we developed an inducible expression system in the genetic model A. nidulans and show that it can be successfully used to produce commercially valuable metabolites.