BACKGROUND 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. RESULTS 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. CONCLUSION 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.

中文翻译：

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在球形假单胞菌中由木质纤维素水解产物生产ENT-天竺葵。

背景技术环孢假单胞菌已成为从木质纤维素生产生物产品的有希望的宿主，部分是由于其能够在木质纤维素原料上生长，耐受生长抑制剂并共同利用糖和木质素衍生的单体。Ent-Kaurene衍生物具有广泛的潜在应用范围，从治疗到新型的基于树脂的材料。结果采用了设计，构建，测试和学习（DBTL）方法，以工程化了拟南芥中非天然二萜烯-丁烯的生产。在第一个DBTL周期中，在贝壳杉中表达了贝壳杉烯合酶（KS）之后，一个关键的限制似乎是二萜前体香叶基香叶基二磷酸香叶酯（GGPP）的可用性。进行了进一步的DBTL循环以选择最佳的GGPP合酶，并使其与KS平衡表达，要求R.toruloides中最强的两个启动子ANT（腺嘌呤核苷酸转位酶）和TEF1（翻译延伸因子1）来驱动藤本赤霉菌中KS的表达以及生产GGPP的鸡盖氏FPP合酶的突变形式。使用玉米秸秆水解产物在2 L生物反应器中按比例扩大培养量，导致对丁苯脲的滴定度为1.4 g / L。结论这项研究建立在先前的研究基础之上，证明R. toruloides作为生产单-倍半萜和倍半萜的健壮和多功能宿主的潜力，并且是该生物中非天然二萜生产的首次证明。ANT（腺嘌呤核苷酸转位酶）和TEF1（翻译延伸因子1）可驱动富士赤霉菌（Kibberella fujikuroi）的KS表达以及来自鸡胆虫（Galus gallus）的FPP合酶的突变形式，该突变体可产生GGPP。使用玉米秸秆水解产物在2 L生物反应器中按比例扩大培养量，导致对丁苯脲的滴定度为1.4 g / L。结论这项研究建立在先前的研究基础之上，证明R. toruloides作为生产单-倍半萜和倍半萜的健壮和多功能宿主的潜力，并且是该生物中非天然二萜生产的首次证明。ANT（腺嘌呤核苷酸转位酶）和TEF1（翻译延伸因子1）可驱动富士赤霉菌（Kibberella fujikuroi）的KS表达以及来自鸡胆虫（Galus gallus）的FPP合酶的突变形式，该突变体可产生GGPP。使用玉米秸秆水解产物在2 L生物反应器中按比例扩大培养量，导致对丁苯脲的滴定度为1.4 g / L。结论这项研究建立在先前的研究基础之上，证明R. toruloides作为生产单-倍半萜和倍半萜的强大和多功能宿主的潜力，并且是该生物中非天然二萜生产的首次证明。