Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India.
School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi, India.
Appl Environ Microbiol. 2020 Aug 18;86(17). doi: 10.1128/AEM.00414-20.
and have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. , a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes ( and ) of encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) Therefore, we cloned codon-optimized copies of the genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase () gene from also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of can be effectively utilized for the heterologous production of high-value phytochemicals. To date, the major host organisms used for the heterologous production of terpenoids, i.e., and , do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.
和 已被广泛用于各种次生代谢产物的异源生产。两者都没有内源性的高通量异戊二烯途径，这是萜类化合物生产所必需的。 ，一种非光合 GRAS（一般认为是安全的）细菌，在光照下产生类胡萝卜素。当一个编码抗σ因子（ChrR1）的基因失活时，类胡萝卜素的产量增加了几倍。我们利用这个 突变体（Car-1）作为一个宿主，用于两种高价值植物化学物质的异源生产，香叶醇和 amorpha-1,4-二烯。当过量表达和纯化时，编码天然香叶基香叶基二磷酸合酶（GGPPS）的 基因（ 和 ）没有产生香叶基焦磷酸（GPP）。因此，我们克隆了 编码 GPP 合酶（GPPS）和香叶醇合酶（GES）的基因的密码子优化拷贝，以显示 Car-1 菌株中类胡萝卜素生物合成途径的内源性中间产物被用于异源生产香叶醇。同样，从 克隆和表达一个密码子优化的 amorpha-1,4-二烯合酶（）基因也导致了 Car-1 中 amorpha-1,4-二烯的异源生产。使用气相色谱-质谱联用（GC-MS）和 GC 来估计香叶醇或 amorpha-1,4-二烯的含量。这些结果表明，Car-1 是代谢工程的一个有前途的宿主，因为 中类胡萝卜素生物合成途径的天然可用的中间产物内源池可以有效地用于异源生产高价值的植物化学物质。迄今为止，用于萜类化合物异源生产的主要宿主生物，即 和 ，没有高通量异戊二烯途径，并且需要繁琐的代谢工程来增加前体池。由于产类胡萝卜素的细菌携带内源性的高通量异戊二烯途径，我们使用产类胡萝卜素的 突变体作为宿主，以证明其适合作为 geraniol 和 amorpha-1,4-二烯异源生产的概念验证。使用 作为模型系统的优点包括：（i）类胡萝卜素的非必需性，以及（ii）通过单一突变过度生产类胡萝卜素以利用高碳通量生产萜类化合物的可能性。
