Kwak Suryang, Crook Nathan, Yoneda Aki, Ahn Naomi, Ning Jie, Cheng Jiye, Dantas Gautam
The Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine in St. Louis, 4515 McKinley Avenue, Room 5121, Campus Box 8510, Saint Louis, MO, 63110, USA.
Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, Saint Louis, MO, 63110, USA.
Biotechnol Biofuels Bioprod. 2022 Oct 8;15(1):104. doi: 10.1186/s13068-022-02189-9.
Terpenes are one of the most diverse and abundant classes of natural biomolecules, collectively enabling a variety of therapeutic, energy, and cosmetic applications. Recent genomics investigations have predicted a large untapped reservoir of bacterial terpene synthases residing in the genomes of uncultivated organisms living in the soil, indicating a vast array of putative terpenoids waiting to be discovered.
We aimed to develop a high-throughput functional metagenomic screening system for identifying novel terpene synthases from bacterial metagenomes by relieving the toxicity of terpene biosynthesis precursors to the Escherichia coli host. The precursor toxicity was achieved using an inducible operon encoding the prenyl pyrophosphate synthetic pathway and supplementation of the mevalonate precursor. Host strain and screening procedures were finely optimized to minimize false positives arising from spontaneous mutations, which avoid the precursor toxicity. Our functional metagenomic screening of human fecal metagenomes yielded a novel β-farnesene synthase, which does not show amino acid sequence similarity to known β-farnesene synthases. Engineered S. cerevisiae expressing the screened β-farnesene synthase produced 120 mg/L β-farnesene from glucose (2.86 mg/g glucose) with a productivity of 0.721 g/L∙h.
A unique functional metagenomic screening procedure was established for screening terpene synthases from metagenomic libraries. This research proves the potential of functional metagenomics as a sequence-independent avenue for isolating targeted enzymes from uncultivated organisms in various environmental habitats.
萜类化合物是种类最多、含量最丰富的天然生物分子类别之一，广泛应用于多种治疗、能源和化妆品领域。最近的基因组学研究预测，生活在土壤中的未培养生物基因组中存在大量未开发的细菌萜类合酶库，这表明有大量假定的萜类化合物有待发现。
我们旨在开发一种高通量功能宏基因组筛选系统，通过减轻萜类生物合成前体对大肠杆菌宿主的毒性，从细菌宏基因组中鉴定新型萜类合酶。通过使用编码异戊烯基焦磷酸合成途径的诱导型操纵子和添加甲羟戊酸前体来实现前体毒性。对宿主菌株和筛选程序进行了精细优化，以尽量减少自发突变产生的假阳性，避免前体毒性。我们对人类粪便宏基因组进行的功能宏基因组筛选产生了一种新型β-法尼烯合酶，它与已知的β-法尼烯合酶没有氨基酸序列相似性。表达筛选出的β-法尼烯合酶的工程酿酒酵母从葡萄糖中产生了120mg/L的β-法尼烯（2.86mg/g葡萄糖），生产力为0.721g/L∙h。
建立了一种独特的功能宏基因组筛选程序，用于从宏基因组文库中筛选萜类合酶。本研究证明了功能宏基因组学作为一种不依赖序列的途径，从各种环境栖息地的未培养生物中分离目标酶的潜力。
