Escherichia coli and Saccharomyces cerevisiae 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. Azospirillum brasilense, 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 A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro. Therefore, we cloned codon-optimized copies of the Catharanthus roseus 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 A. brasilense. Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua 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 A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.

中文翻译：

---

工程化类胡萝卜素的类胡萝卜素高产菌株，用于异源生产香叶醇和紫穗槐二烯。

大肠杆菌和酿酒酵母已被广泛用于多种次级代谢产物的异源生产。两者都没有产生萜类化合物所需的内源性高通量类异戊二烯途径。一种非光合作用的GRAS（通常被认为是安全的）细菌巴西细螺旋藻，在有光的情况下会产生类胡萝卜素。灭活编码抗西格玛因子（ChrR1）的基因后，类胡萝卜素的产量会增加很多倍。我们使用此巴西乳杆菌突变体（Car-1）作为宿主，以异源生产两种高价值的植物化学成分香叶醇和吗啡二烯。巴西拟南芥的克隆基因（crtE1和crtE2）当过度表达和纯化时，编码天然香叶基香叶基香叶基焦磷酸合酶（GGPPS）的体外不产生香叶基香叶基磷酸（GPP）。因此，我们克隆了编码GPP合酶（GPPS）和香叶醇合酶（GES）的长春花玫瑰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体被用于异源生产香叶醇。巴西。同样，从青蒿中克隆并表达了经密码子优化的非晶质合成酶（ads）基因也导致了Car-1中异源二氢吗啡的生产。使用气相色谱-质谱（GC-MS）和GC估算香叶醇或紫苏二烯的含量。这些结果表明，Car-1是代谢工程的有前途的宿主，因为巴西拟南芥类胡萝卜素生物合成途径的中间体的天然可用内源库可有效地用于高价值植物化学物质的异源生产。