Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, 300072, China.
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Microb Cell Fact. 2019 May 3;18(1):77. doi: 10.1186/s12934-019-1127-8.
Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory.
In this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH- or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the β-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures.
Here, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.
白桦脂酸是一种五环三萜类化合物，具有抗病毒和抗肿瘤的潜力，但植物中白桦脂酸的含量较低，无法满足对该化合物的需求。假丝酵母脂肪酶作为一种油脂酵母，由于其能够积累大量的油脂，这些油脂可以储存疏水性产物并为萜烯合成提供足够的前体，因此是生产高度疏水性化合物的有前途的微生物细胞工厂。然而，与其他萜类化合物的生产相比，白桦脂酸和相关三萜类化合物的异源生产工程尚未得到系统发展，因此微生物中白桦脂酸的生产仍不尽如人意。
在本研究中，我们通过工程化四个功能模块，即异源 CYP/CPR、MVA、乙酰辅酶 A 生成和氧化还原辅因子供应模块，应用多模块策略系统地提高了假丝酵母脂肪酶中白桦脂酸和相关三萜类化合物的生物合成。首先，通过筛选来自 5 个不同来源的 25 种细胞色素 P450 单加氧酶 (CYPs) 和 NADPH-细胞色素 P450 还原酶 (CPRs) 的组合，我们选择了两种最佳的白桦脂酸生产菌株。然后，在这两种菌株中过表达 MVA 模块中的 ERG1、ERG9 和 HMG1，这极大地提高了白桦脂酸的产量，使一株（YLJCC56）的白桦脂酸产量达到 51.87±2.77mg/L。然后，我们通过引入 NADPH 或 NADH 生成酶来工程化氧化还原辅因子供应模块，并通过直接过表达乙酰辅酶 A 合酶或强化β-氧化途径来工程化乙酰辅酶 A 生成模块，这进一步提高了总三萜类化合物的产量（白桦脂、白桦脂酸和白桦脂醛的产量总和）。最后，我们将这些模块进行组合工程化，在摇瓶培养中，总三萜类化合物的产量达到 204.89±11.56mg/L（由 65.44%的白桦脂、23.71%的白桦脂酸和 10.85%的白桦脂醛组成）。
在这里，我们系统地工程化了假丝酵母脂肪酶，并获得了迄今为止微生物中报道的最高白桦脂酸和总三萜类化合物产量。我们的研究为 P450 酶的异源利用以及假丝酵母脂肪酶中三萜类化合物生产的操纵提供了合适的参考。
