Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, University of Bielefeld, P,O, Box 100131, D-33501, Bielefeld, Germany.
BMC Microbiol. 2012 Sep 10;12:198. doi: 10.1186/1471-2180-12-198.
Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin.
Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ε-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained.
C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.
谷氨酸棒杆菌含有糖基化的 C50 类胡萝卜素脱植基叶黄素作为黄色色素。从异戊烯焦磷酸开始，它在非甲羟戊酸途径中产生，脱植基叶黄素通过中间体法呢基焦磷酸、香叶基香叶基焦磷酸、番茄红素和叶黄素合成。
在这里，我们表明，类胡萝卜素基因簇 crtE-cg0722-crtBIYeYfEb 的基因是共转录的，并对定义的基因缺失突变体进行了特征描述。基因缺失分析表明，crtI、crtEb 和 crtYeYf 分别编码唯一的类胡萝卜素脱氢酶、番茄红素延伸酶和类胡萝卜素 C45/C50 ε-环化酶。然而，谷氨酸棒杆菌的基因组还编码第二个类胡萝卜素基因簇，包括 crtB2I2-1/2，该基因簇被证明是共转录的。外源表达 crtB2 可以弥补谷氨酸棒杆菌ΔcrtB 中类胡萝卜素合酶 CrtB 的缺乏，因此，谷氨酸棒杆菌拥有两种功能性的类胡萝卜素合酶，即 CrtB 和 CrtB2。由于不能从质粒上表达 crtI2-1/2 来弥补谷氨酸棒杆菌ΔcrtI 中类胡萝卜素脱氢酶 CrtI 的缺乏，因此无法获得 crtI2-1/2 编码的类胡萝卜素脱氢酶的遗传证据。以番茄红素为例，估计了谷氨酸棒杆菌过量生产类胡萝卜素的潜力。crtEb 基因的缺失阻止了番茄红素转化为脱植基叶黄素，并导致番茄红素积累到 0.03±0.01mg/g 细胞干重（CDW）。当在谷氨酸棒杆菌ΔcrtEb 中过表达 crtE、crtB 和 crtI 基因以将香叶基香叶基焦磷酸转化为番茄红素时，获得了强烈红色着色的细胞和 80 倍增加的番茄红素含量 2.4±0.3mg/g CDW。
谷氨酸棒杆菌在 C50 类胡萝卜素脱植基叶黄素的生物合成中具有一定程度的冗余性，因为它拥有两种功能性的类胡萝卜素合酶基因。仅对导致番茄红素的末端反应进行代谢工程改造就导致了相当大的番茄红素产量，表明谷氨酸棒杆菌可能成为类胡萝卜素生产的潜在宿主。
