β-胡萝卜素广泛用于食品、饲料、医药和化妆品等领域,利用微生物细胞合成高附加值的天然β-胡萝卜素,具有重要的研究意义。如何调控代谢途径中相关基因的表达强度,是在酿酒酵母体内合成β-胡萝卜素的关键因素。该文利用来源于三孢布拉氏霉Blakeslea trispora的β-胡萝卜素合成酶转化出发菌株酿酒酵母LFD18,酿酒酵母能够从头合成β-胡萝卜素,并在细胞中积累。在研究过程中,平板上发现一株菌落颜色明显较周围菌落更深地突变株命名为ZS19 reddish,经实验验证突变株的β-胡萝卜素产量显著提高。针对上述两种不同颜色菌落,进行转录组分析,主要是发现一些与产物合成有关的基因,通过GO与PATHWAY分析发现与碳代谢和脂质体合成有关的6个上调基因,hxk1、dpp1、lpp1、fdh1、hmg2和gre2表达水平上调。通过定量PCR和单个基因过表达验证,最终通过同时过量表达hxk1、dpp1、lpp1和fdh1基因构建命名为ZS20菌株,β-胡萝卜素摇瓶发酵产量提高到194.3 mg/L,是ZS19 reddish菌株的1.3倍,是出发菌株ZS19的2.1倍,最终ZS20菌株经发酵罐发酵后β-胡萝卜素产量为314.3 mg/L,因此,通过进一步PATHWAY分析构建重组菌或许可以提高β-胡萝卜素的产量。
β-Carotene belonging to the carotenoid family, is a class of important functional pigments with commercial value, exists in plants, fruits and microorganisms as functional cosmetics, food colorants and feed supplements are widely used in pharmaceuticals, health products, cosmetics, and other fields. Epidemiological studies have consistently shown that people who eat more carotenoid-rich fruits and vegetables, as well as those with higher serum β-carotene levels, have a lower risk of cancer, especially lung cancer, so it is of great significance to synthesize high value-added natural carotene from microbial cells. To regulate the expression intensity of related genes in metabolic pathways is a key factor in the synthesis of β-carotene in Saccharomyces cerevisiae. Metabolic engineering is a promising strategy for β-carotene biosynthesis because there are currently quite a number of compounds produced on an industrial scale by engineered microbial cells, including 1,3-propanediol, 3-eicosapentaenoic acid, and 2,3-butanediol. It is mainly synthesized through the mevalonate (MVA) pathway in S. cerevisiae β-carotene, from acetyl coenzyme A to β-carotene, in these 12 steps, each step uses a strong promoter and multiple gene copies to generate an efficient biosynthetic pathway that can produce 100 times more β-carotenes, such as thmg1 overexpression and MVA pathway related genes, have been studied to improve the synthesis of β-carotene. Construction of engineered S. cerevisiae strains to improve production β-carotene is mainly overexpressed β-carotene. How to select the overexpression gene target is a common problem in the supply of precursors for the β-carotene synthase and the optimization of culture conditions. In this manuscript, β-carotene synthases derived from Blakeslea trispora was heterologous expressed in LFD18, and S. cerevisiae was able to synthesize and accumulate β-carotene de novo. In this research, the color of a mutant colony ZS19 reddish was found significantly deeper than that of the surrounding colony. As a result, the content of β-carotene in the mutant strain was significantly increased. Transcriptome analysis was then performed, and it was found that the expression levels of 6 genes related to carbon metabolism and liposome synthesis through GO and PATHWAY, hxk1, dpp1, lpp1, fdh1, hmg2, and gre2 were up-regulated. Through quantitative PCR verification and single gene overexpression and simultaneous overexpression verification, the ZS20 strain was finally named by simultaneous overexpression of hxk1, dpp1, lpp1, and fdh1 genes, and the fermentation yield of β-carotene shaker was increased to 194.3 mg/L, which was 1.3 times that of ZS19 reddish strain and 2.1 times that of the starting strain ZS19 The yield of β-carotene of ZS20 strain was 314.3 mg/L after fermentation in fermenter. Subsequently, the expression of hxk1, dpp1, lpp1, fdh1, hmg2, and gre2 genes can be precisely regulated by promoter regulation, antisense RNA regulation and precise regulation of CRISPR-Cas9-based metabolic pathway, and the yield of β-carotene can be further improved by further optimizing the medium and culture methods. At the same time, the metabolic pathways related to product synthesis and their regulation can be further improved by further PATHWAY analysis, and the yield of recombinant bacteria β-carotene can be further improved.
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