Heme has attracted considerable attention due to its indispensable biological roles and applications in healthcare and artificial foods. The development and utilization of edible microorganisms instead of animals to produce heme is the most promising method to promote the large-scale industrial production and safe application of heme. However, the cytotoxicity of heme severely restricts its efficient synthesis by microorganisms, and the cytotoxic mechanism is not fully understood. In this study, the effect of heme toxicity on Saccharomyces cerevisiae was evaluated by enhancing its synthesis using metabolic engineering. The results showed that the accumulation of heme after the disruption of heme homeostasis caused serious impairments in cell growth and metabolism, as demonstrated by significantly poor growth, mitochondrial damage, cell deformations, and chapped cell surfaces, and these features which were further associated with substantially elevated reactive oxygen species (ROS) levels within the cell (mainly H2O2 and superoxide anion radicals). To improve cellular tolerance to heme, 5 rounds of laboratory evolution were performed, increasing heme production by 7.3-fold and 4.2-fold in terms of the titer (38.9 mg/L) and specific production capacity (1.4 mg/L/OD600), respectively. Based on comparative transcriptomic analyses, 32 genes were identified as candidates that can be modified to enhance heme production by more than 20% in S. cerevisiae. The combined overexpression of 5 genes (SPS22, REE1, PHO84, HEM4 and CLB2) was shown to be an optimal method to enhance heme production. Therefore, a strain with enhanced heme tolerance and ROS quenching ability (R5-M) was developed that could generate 380.5 mg/L heme with a productivity of 4.2 mg/L/h in fed-batch fermentation, with S. cerevisiae strains being the highest producers reported to date. These findings highlight the importance of improving heme tolerance for the microbial production of heme and provide a solution for efficient heme production by engineered yeasts.

中文翻译：

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酿酒酵母的多维工程设计通过探索血红素的细胞毒性和耐受性来高效生产血红素


血红素因其在医疗保健和人工食品中不可或缺的生物学作用和应用而引起了相当大的关注。开发利用可食用微生物代替动物生产血红素是促进血红素大规模工业化生产和安全应用的最有前途的方法。然而，血红素的细胞毒性严重限制了微生物对其有效合成，细胞毒机制尚不完全清楚。在本研究中，通过使用代谢工程增强其合成来评价血红素毒性对酿酒酵母的影响。结果表明，血红素稳态破坏后血红素的积累导致细胞生长和代谢严重受损，表现为生长明显不良、线粒体损伤、细胞变形和细胞表面皲裂，这些特征进一步与细胞内活性氧 （ROS） 水平显着升高相关（主要是 H 2 O 2 和超氧阴离子自由基）。为了提高细胞对血红素的耐受性，进行了 5 轮实验室进化，血红素产量分别在滴度 （38.9 mg/L） 和比生产能力 （1.4 mg/L/OD600） 方面增加了 7.3 倍和 4.2 倍。基于比较转录组学分析，32 个基因被确定为候选基因，可以对其进行修饰以将酿酒酵母中的血红素产生提高 20% 以上。5 个基因 （SPS22 、 REE1 、 PHO84 、 HEM4 和 CLB2） 的联合过表达被证明是增强血红素产生的最佳方法。因此，开发了一种具有增强血红素耐受性和 ROS 淬灭能力 （R5-M） 的菌株，该菌株可以产生 380.5 mg/L 的血红素，生产率为 4。补料分批发酵中为 2 mg/L/h，酿酒酵母菌株是迄今为止报道的最高生产菌株。这些发现强调了提高血红素耐受性对微生物产生血红素的重要性，并为通过工程酵母高效生产血红素提供了解决方案。