紫杉二烯是高效抗癌药物紫杉醇生物合成的重要前体，但其微生物生物合产量非常低。华东理工大学许建和教授团队以解脂耶氏酵母为底盘细胞，首次在该酵母中生物合成了紫杉二烯。首先，作者采用“推-拉”策略，使紫杉二烯产量提高234%。随后将紫杉二烯合酶分别与5种可溶性标签融合表达，增加紫杉二烯合酶在该酵母中的可溶性表达，当与SUMO融合表达后紫杉二烯的产量提高了62.3%。随后，采用多拷贝迭代整合的方法进一步提高紫杉二烯产量，经过三轮整合后，紫杉二烯的产量达到了23.7 mg/L。最后，通过优化补料分批发酵条件，将紫杉二烯产量提高到了101.4 mg/L。这个是首次在解脂耶氏酵母中完成紫杉二烯生物合成的报道，该文章为解脂耶氏酵母可持续生产紫杉二烯和其他二萜化合物提供了良好的范例。相关文章以 “Improving solubility and copy number of taxadiene synthase to enhance the titer of taxadiene in Yarrowia lipolytica” 发表在Synthetic and Systems Biotechnology期刊上。

Fig. 1. Production pathways of taxadiene from glucose in Y. lipolytica. In Y. lipolytica, isoprenoids are synthesized from acetyl-CoA through the MVA pathway. Pink box represents the endogenous MVA pathway. Dashed boxes represent alternative metabolic pathways for the biosynthesis of taxadiene. Red arrows indicate overexpression and thin black arrows indicate reduced expression.

Fig. 2. Engineered taxadiene biosynthesis pathways in Y. lipolytica. Taxadiene production in engineered strains overexpressing TASY alone and simultaneously overexpressing MVA pathway rate-limiting enzymes tHMG1, GGSP1, and TASY. Experiments were conducted in triplicate, with error bars representing the standard deviation from the mean value.

Fig. 3. “Push–pull” strategy analysis of taxadiene biosynthesis. (a) Taxadiene production in engineered strains obtained from increasing the gene copy number, decreasing SQS expression by replacing the strong promotor, and fusion by expressing ERG20 with GGSP1. (b) Concentration of GGOH in strains WT, YP12, YP23, YP24, and YP27 after 140 h of cultivation. Experiments were conducted in triplicate, with error bars representing the standard deviation from the mean value.

Fig. 4. Test of fusion tag universality. (a) Scheme of chromosomally integrated fusion tags with tasy genetic constructs. SUMO, GST, MBP, NusA, and TrxA were fused with TASY before being integrated into the chromosome XPR2 site. (b) Taxadiene production in the engineered strains by overexpressing TASY with different fusion tags. (c) Concentration of GGOH in the engineered strains detected at 140 h. Experiments were conducted in triplicate, with error bars representing the standard deviation from the mean value.

Fig. 5. Multicopy integration of 26s rDNA into genome to improve taxadiene production. (a) Multicopy integration scheme of (SUMO-TASY)*2 in Y. lipolytica. After two loxP sequences were recognized by Cre, the ORF-URA between the loxP sites was excised for the next round of genes integration. (b) Effect of increasing rounds of integration of (SUMO-TASY)*2 into the genome on the formation of taxadiene in engineered Y. lipolytica. (c) The copy number of tasy and GGOH concentration in the highest titer strain per integration. Experiments were conducted in triplicate, with error bars representing the standard deviation from the mean value.

Fig. 6. Fed-batch fermentation of metabolically engineered strain M4. Taxadiene production of recombinant strain M6 in batch fermentation. Fermentation was performed in a 5-L jar fermenter containing 2 L of medium.

Improving solubility and copy number of taxadiene synthase to enhance the titer of taxadiene in Yarrowia lipolytica