Functional characterization of the Xanthophyllomyces dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors.
Functional characterization of the Xanthophyllomyces dendrorhous farnesyl pyrophosphate synthase and geranylgeranyl pyrophosphate synthase encoding genes that are involved in the synthesis of isoprenoid precursors.
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DOI：
10.1371/journal.pone.0096626
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Cifuentes V
中科院分区：
文献类型：
--
作者：
Alcaíno J;Romero I;Niklitschek M;Sepúlveda D;Rojas MC;Baeza M;Cifuentes V
树状叶黄酵母可合成类胡萝卜素虾青素，该虾青素因其抗氧化和色素沉着特性而在生物技术中得到应用。然而，野生型菌株产生的类胡萝卜素含量太低，不具有工业竞争力。考虑到这一背景，了解虾青素的合成如何在这种酵母中被控制和调节是必不可少的。在这项工作中，表征了在 X. dendrorhous 中从异戊烯基焦磷酸 (IPP, C5) 和二甲基烯丙基焦磷酸 (DMAPP, C5) 合成类胡萝卜素前体香叶基香叶基焦磷酸 (GGPP, C20) 的步骤。两个异戊二烯基转移酶编码基因 FPS 和 crtE 在大肠杆菌中表达。使用重组大肠杆菌蛋白提取物进行的酶测定表明，FPS 和 crtE 分别编码法尼基焦磷酸 (FPP, C15) 合酶和 GGPP 合酶。 X. dendrorhous FPP-合酶从IPP和DMAPP产生香叶基焦磷酸(GPP，C10)，并从IPP和GPP产生FPP，而X. dendrorhous GGPP-合酶仅利用FPP和IPP作为底物来产生GGPP。此外，FPS 和 crtE 基因在 X. dendrorhous 中过度表达，导致类胡萝卜素总产量增加。由于亲本菌株是二倍体，这些基因的等位基因之一的缺失并不影响类胡萝卜素的总产量，但其组成发生了显着改变。这些结果表明，这些基因的过度表达可能会引起更高的胡萝卜素生成碳通量，很可能涉及胡萝卜素酶复合物的早期形成。相反，缺失突变体中胡萝卜素生成的碳通量较低，可能会延迟或导致胡萝卜素酶复合物的部分形成，这可以解释虾青素类胡萝卜素前体在这些突变体中的积累。总之，FPS 和 crtE 基因代表了良好的候选基因，可以进行操纵以促进 X. dendrorhous 中的类胡萝卜素生物合成。
The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.
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影响因子：
2.7
作者：
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