As an ideal carotenoids producer, Blakeslea trispora has gained lots of attentions due to its large biomass and high production of β-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified since few investigations have been conducted at molecular level in B. trispora. In this study, the homologous gene of carotenogenesis regulatory gene (crgA) was cloned from the mating type (-) of B. trispora and the deduced CrgA protein was analyzed for its primary structure and domains. In order to clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora. In contrast to the wild-type strain, the crgA null mutant was defective in sporulation, but accumulated much more β-carotene (31.2% improvement at the end) accompanied by the enhanced transcription levels of three structural genes (hmgR, carB and carRA) for carotenoids over whole culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription level of structural gene and carotenoids production were restored as present in wild-type strain. Gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway, thus the increase of precursors and fat content contributes to the accumulation of carotenoids.

IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids in large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the current study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis and other anabolic phenotypes. This will lead to a better understanding of global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.

作为理想的类胡萝卜素生产者，Blakeslea trispora因其大量的生物质以及高产的β-胡萝卜素和番茄红素而备受关注。然而，由于在三孢散孢菌中在分子水平上进行了很少的研究，所以仍然需要弄清三孢散孢子中的胡萝卜素生成调控。在这项研究中，胡萝卜素调节基因的同源基因（crgA）从交配型克隆（ - ）的B. trispora并分析其一级结构和结构域推导CrgA蛋白。为了澄清crgA在介导调控B. trispora，我们使用基因敲除和互补的策略研究crgA表达对三孢菌的表型的影响。与野生型菌株相反，crgA null突变体的孢子形成缺陷，但积累了更多的β-胡萝卜素（末尾提高了31.2％），并伴随着三个结构基因（hmgR，carB和carRA）的转录水平提高。用于整个培养时间内的类胡萝卜素。当crgA的野生型拷贝被补充到crgA中时如野生型菌株中所示，恢复了无效突变体，孢子形成，结构基因的转录水平和类胡萝卜素的产生。进行了基于气相色谱-质谱（GC-MS）的代谢组学方法和多元统计分析，以研究细胞内代谢物谱。三羧酸（TCA）循环成分和某些氨基酸的含量降低，糖酵解中间体和脂肪酸的含量提高，表明更多的代谢通量被驱使进入甲羟戊酸（MVA）途径，因此前体和脂肪含量的增加有助于类胡萝卜素的积累。

重要合子Blakeslea trispora是大规模生产类胡萝卜素的重要菌株。然而，在这种丝状真菌中，类胡萝卜素生物合成的调节机制仍未得到很好的理解。在当前的研究中，我们试图研究crgA如何影响类胡萝卜素，类胡萝卜素生物合成和其他合成代谢表型的结构基因的表达。这将使人们更好地了解类胡萝卜素生物合成的整体调节机制，并在将来促进对该菌株的工程改造，以提高类胡萝卜素的产量。