Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

蓖麻油酸 (C18:1-OH, RA) 是一种有价值的羟基脂肪酸，具有多种用途。目前RA的工业来源依赖于蓖麻子油的水解。然而，有毒化合物蓖麻毒素的共存以及这种植物的供应不稳定，促使人们探索有希望的替代方案：在异源植物或微生物中产生 RA。在这项研究中，我们对产油酵母解脂耶氏酵母进行了改造，以产生游离脂肪酸 (FFA) 形式的 RA。首先，我们过表达来自麦角菌的真菌Δ12油酸羟化酶基因（CpFAH12），同时删除与脂肪酸降解（MEF1和PEX10）和油酸去饱和（FAD2）相关的基因。由于 Δ12 油酸羟化酶可转化位于磷脂酰胆碱 (PC) sn -2 位点的油酸 (C18:1)，因此我们接下来重点关注增加含有油酸的 PC 池。这一目标是通过实施旨在增强 PC 和 C18 脂肪酸生物合成的代谢工程策略来实现的。为了增加PC池，我们通过删除分别参与二酰基甘油和三酰基甘油生产的磷脂酸磷酸酶基因（ PAH1和APP1）和二酰基甘油酰基转移酶基因（DGA1 ）来将流量转向磷脂生物合成。此外，通过CDS1、PSD1、CHO2和OPI3基因的过表达增强了通过 CDP-DAG 途径的 PC 生物合成。随后，为了增加 PC 内的油酸含量，我们过表达参与 C16 至 C18 脂肪酸转化的异源脂肪酸延伸酶基因 ( MaC16E )。随着RA产生滴度的升高，产生的RA主要以FFA形式存在，导致细胞生长受到抑制。通过 Triton X-100 处理诱导 RA 分泌，可以减轻生长抑制，该过程通过将流量重新定向到 RA 合成，同时放大 RA 的产生。最终的工程菌株 JHYL-R146 在用 5% Triton X-100 处理的培养基中产生了 2.061 g/L 的游离 RA，占产生的总 FFA 的 74%。生成游离 RA 具有绕过使用蓖麻子油作为 RA 来源时所需的水解阶段的额外好处。这一成就代表了迄今为止报道的从葡萄糖合成 RA 的最高水平，强调了解脂耶氏酵母作为可持续 RA 生产宿主的潜力。