Department of Energy, Agile BioFoundry, Emeryville, CA, 94608, USA.
Department of Biomass Science and Conversion Technology, Sandia National Laboratories, 7011 East Ave, Livermore, CA, 94550, USA.
Microb Cell Fact. 2020 Feb 5;19(1):24. doi: 10.1186/s12934-020-1293-8.
Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials.
The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L.
This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.
红酵母（Rhodosporidium toruloides）已成为从木质纤维素生产生物制品的有前途的宿主，部分原因是它能够利用木质纤维素原料生长，耐受生长抑制剂，并共同利用糖和木质素衍生的单体。贝壳杉烯衍生物具有从治疗剂到新型树脂基材料等多种潜在应用。
采用设计、构建、测试和学习（DBTL）方法来工程化生产非天然二萜贝壳杉烯在红酵母（R. toruloides）中。在第一个 DBTL 循环中，在红酵母（R. toruloides）中表达贝壳杉烯合酶（KS）后，似乎关键限制因素是二萜前体香叶基香叶基二磷酸（GGPP）的可用性。进一步进行了 DBTL 循环，以选择最佳的 GGPP 合酶，并平衡其与 KS 的表达，这需要红酵母（R. toruloides）中两个最强的启动子，腺嘌呤核苷酸转运酶（ANT）和翻译延伸因子 1（TEF1）来驱动来自藤仓镰孢菌的 KS 和来自鸡的 FPP 合酶的突变体的表达，该突变体产生 GGPP。使用玉米秸秆水解物在 2 L 生物反应器中扩大培养，导致贝壳杉烯产量达到 1.4 g/L。
本研究建立在先前证明红酵母（R. toruloides）作为生产单萜和倍半萜的强大而多功能宿主的潜力的工作基础上，并且是该生物体中首次生产非天然二萜的证明。
