Faculty of Biology, Center for Biotechnology (CeBiTec), Bielefeld University, Universitätsstrasse 27, 33615, Bielefeld, Germany.
Department of Molecular Sciences, Macquarie University, Sydney, NSW, Australia.
Microb Cell Fact. 2022 Sep 14;21(1):190. doi: 10.1186/s12934-022-01910-5.
Eukaryotic algae have recently emerged as hosts for metabolic engineering efforts to generate heterologous isoprenoids. Isoprenoid metabolic architectures, flux, subcellular localization, and transport dynamics have not yet been fully elucidated in algal hosts.
In this study, we investigated the accessibility of different isoprenoid precursor pools for C sesquiterpenoid generation in the cytoplasm and chloroplast of Chlamydomonas reinhardtii using the Abies grandis bisabolene synthase (AgBS) as a reporter. The abundance of the C sesquiterpene precursor farnesyl pyrophosphate (FPP) was not increased in the cytosol by co-expression and fusion of AgBS with different FPP synthases (FPPSs), indicating limited C precursor availability in the cytoplasm. However, FPP was shown to be available in the plastid stroma, where bisabolene titers could be improved several-fold by FPPSs. Sesquiterpene production was greatest when AgBS-FPPS fusions were directed to the plastid and could further be improved by increasing the gene dosage. During scale-up cultivation with different carbon sources and light regimes, specific sesquiterpene productivities from the plastid were highest with CO as the only carbon source and light:dark illumination cycles. Potential prenyl unit transporters are proposed based on bioinformatic analyses, which may be in part responsible for our observations.
Our findings indicate that the algal chloroplast can be harnessed in addition to the cytosol to exploit the full potential of algae as green cell factories for non-native sesquiterpenoid generation. Identification of a prenyl transporter may be leveraged for further extending this capacity.
真核藻类最近成为代谢工程的宿主，以产生异源类异戊二烯。在藻类宿主中，类异戊二烯代谢结构、通量、亚细胞定位和运输动力学尚未完全阐明。
在这项研究中，我们使用白冷杉双萜合酶（AgBS）作为报告基因，研究了不同类异戊二烯前体池在衣藻细胞质和叶绿体中生成 C 倍半萜烯的可及性。AgBS 与不同的 FPP 合酶（FPPSs）共表达和融合，并未增加细胞质中 C 倍半萜前体法呢基焦磷酸（FPP）的丰度，表明细胞质中 C 前体的可用性有限。然而，FPP 被证明在质体基质中是可用的，在那里，FPPSs 可以将双萜烯产量提高几倍。当 AgBS-FPPS 融合物被导向质体时，倍半萜烯的产量最大，并且可以通过增加基因剂量进一步提高。在不同碳源和光照条件下进行放大培养时，质体中特定倍半萜烯的产物得率在 CO 作为唯一碳源和光暗光照循环时最高。根据生物信息学分析提出了潜在的prenyl 单元转运蛋白，它们可能部分负责我们的观察结果。
我们的发现表明，除了细胞质外，藻类叶绿体还可以被利用，以充分发挥藻类作为非天然倍半萜烯生成的绿色细胞工厂的潜力。prenyl 转运蛋白的鉴定可能会进一步扩展这种能力。
