Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae.
Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae.
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酿酒酵母中藏红花酸的异源生物合成和操作
DOI：
10.1186/s12934-017-0665-1
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发表时间：
2017-03-29
影响因子：
6.4
通讯作者：
Yuan Y
中科院分区：
文献类型：
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作者：
Chai F;Wang Y;Mei X;Yao M;Chen Y;Liu H;Xiao W;Yuan Y
背景：藏红花素在抗肿瘤、抗氧化、降压、抗动脉粥样硬化和抗抑郁等方面具有优异的活性，天然存在于藏红花中，在医疗和食品领域具有巨大的应用潜力。微生物生产番石榴素近年来受到越来越多的关注。然而，只有EVOVA公司的一项专利和Lou等人的一份报告说明了微生物合成西红花苷的可行性，但目前尚无具体的滴度数据报道。酿酒酵母通常被认为是食品安全和多产的宿主，对关键酶的操纵对于平衡代谢通量，从而提高产量至关重要。因此，为了促进酿酒葡萄球菌的crocetin生产，必须对CrtZ、CCD和ALD等关键酶进行组合工程。结果：通过在已有的β-胡萝卜素生产菌株中引入外源的CrtZ和CCD，酿酒葡萄球菌成功实现了胡萝卜素的生物合成。与30°C培养相比，20°C培养的西红花苷产量提高到223 μg/L。通过组合筛选藏红花9个品种的CrtZ和4个CCD，获得了最佳的CrtZ/CCD组合和351 μg/L的crocetin滴度。然后通过筛选Bixa orellana的异源ald （Bix_ALD）和Synechocystis sp. PCC6803的Syn_ALD （Syn_ALD）以及内源性ALD6，加入Syn_ALD后，crocetin的效价进一步提高了1.8倍。最后，CCD2和Syn_ALD的过表达在摇瓶水平上达到了最高滴度1219 μg/L。最终，通过补料分批发酵，在5-L生物反应器中，西红花苷的产量达到6278 μg/L，这是目前报道的真核细胞中西红花苷的最高滴度。结论：本研究通过对酿酒酵母菌进行工程改造，实现了西红花苷的生产。通过对CrtZ、CCD和ALD三个关键酶的组合操作，筛选酶源和调节蛋白表达水平（反应温度和拷贝数），红花素滴度较起始菌株逐步提高129.4倍（从9.42 μg/L提高到1219 μg/L）。在5-L的生物反应器中，微生物中报道的红花素滴度最高（6278 μg/L）。这项研究为微生物过量生产具有复杂结构的所需化合物的一系列反应中涉及的关键酶操作提供了很好的见解。
Background:Due to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.Results:By introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.Conclusions:Saccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.