The sesquiterpenoid abscisic acid (ABA) is mostly known for regulating developmental processes and abiotic stress responses in higher plants. Recent studies show that ABA also exhibits a variety of pharmacological activities. Affordable and sustainable production will be required to utilize the compound in agriculture and as a potential pharmaceutical. Saccharomyces cerevisiae is an established workhorse for the biotechnological production of chemicals. In this study, we constructed and characterised an ABA-producing S. cerevisiae strain using the ABA biosynthetic pathway from Botrytis cinerea. Expression of the B. cinerea genes bcaba1, bcaba2, bcaba3 and bcaba4 was sufficient to establish ABA production in the heterologous host. We characterised the ABA-producing strain further by monitoring ABA production over time and, since the pathway contains two cytochrome P450 enzymes, by investigating the effects of overexpressing the native S. cerevisiae or the B. cinerea cytochrome P450 reductase. Both, overexpression of the native or heterologous cytochrome P450 reductase, led to increased ABA titres. We were able to show that ABA production was not affected by precursor or NADPH supply, which suggested that the heterologous enzymes were limiting the flux towards the product. The B. cinerea cytochrome P450 monooxygenases BcABA1 and BcABA2 were identified as pathway bottlenecks and balancing the expression levels of the pathway enzymes resulted in 4.1-fold increased ABA titres while reducing by-product formation. This work represents the first step towards a heterologous ABA cell factory for the commercially relevant sesquiterpenoid.

中文翻译：

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酿酒酵母中多步骤异源途径的整合用于生产脱落酸

倍半萜类脱落酸 (ABA) 主要用于调节高等植物的发育过程和非生物胁迫反应。最近的研究表明 ABA 还表现出多种药理活性。为了在农业中利用该化合物并作为潜在的药物，需要经济且可持续的生产。酿酒酵母是化学品生物技术生产的成熟主力。在本研究中，我们利用灰霉病菌的 ABA 生物合成途径构建并鉴定了产生 ABA 的酿酒酵母菌株。灰霉病菌基因 bcaba1、bcaba2、bcaba3 和 bcaba4 的表达足以在异源宿主中建立 ABA 生产。我们通过监测 ABA 随时间的产生情况，进一步表征了 ABA 产生菌株，并且由于该途径包含两种细胞色素 P450 酶，因此通过研究过度表达天然酿酒酵母或灰葡萄孢细胞色素 P450 还原酶的影响，进一步表征了 ABA 产生菌株。天然或异源细胞色素 P450 还原酶的过度表达都会导致 ABA 滴度增加。我们能够证明 ABA 的产生不受前体或 NADPH 供应的影响，这表明异源酶限制了产品的通量。B. cinerea 细胞色素 P450 单加氧酶 BcABA1 和 BcABA2 被确定为途径瓶颈，平衡途径酶的表达水平导致 ABA 滴度增加 4.1 倍，同时减少副产物形成。这项工作代表了迈向商业相关倍半萜类异源 ABA 细胞工厂的第一步。