Multidimensional engineering of Saccharomyces cerevisiae for the efficient production of heme by exploring the cytotoxicity and tolerance of heme

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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引用次数: 0

Abstract

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.

通过探索血红素的细胞毒性和耐受性,对酿酒酵母进行多维工程改造,以高效生产血红素。
血红素因其不可或缺的生物学作用以及在医疗保健和人工食品中的应用而备受关注。开发和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制约了其在微生物中的高效合成,而且其细胞毒性机理尚未完全清楚。本研究利用代谢工程技术提高了血红素的合成能力,从而评估了血红素毒性对酿酒酵母的影响。结果表明,血红素平衡被破坏后,血红素的积累会严重影响细胞的生长和新陈代谢,表现为明显的生长不良、线粒体损伤、细胞变形和细胞表面皲裂,而这些特征又与细胞内活性氧(ROS)水平(主要是 H2O2 和超氧阴离子自由基)的大幅升高有关。为了提高细胞对血红素的耐受性,进行了 5 轮实验室进化,使血红素产量在滴度(38.9 毫克/升)和特定生产能力(1.4 毫克/升/OD600)方面分别提高了 7.3 倍和 4.2 倍。根据转录组学比较分析,确定了 32 个候选基因,这些基因经改造后可使 S. cerevisiae 的血红素产量提高 20% 以上。结果表明,联合过表达 5 个基因(SPS22、REE1、PHO84、HEM4 和 CLB2)是提高血红素产量的最佳方法。因此,我们培育出了一株具有更强血红素耐受性和ROS淬灭能力的菌株(R5-M),该菌株在饲料批量发酵中可产生380.5毫克/升血红素,生产率为4.2毫克/升/小时,是迄今为止报道的血红素生产率最高的S. cerevisiae菌株。这些发现强调了提高血红素耐受性对微生物生产血红素的重要性,并为工程酵母高效生产血红素提供了解决方案。
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来源期刊
Metabolic engineering
Metabolic engineering 工程技术-生物工程与应用微生物
CiteScore
15.60
自引率
6.00%
发文量
140
审稿时长
44 days
期刊介绍: Metabolic Engineering (MBE) is a journal that focuses on publishing original research papers on the directed modulation of metabolic pathways for metabolite overproduction or the enhancement of cellular properties. It welcomes papers that describe the engineering of native pathways and the synthesis of heterologous pathways to convert microorganisms into microbial cell factories. The journal covers experimental, computational, and modeling approaches for understanding metabolic pathways and manipulating them through genetic, media, or environmental means. Effective exploration of metabolic pathways necessitates the use of molecular biology and biochemistry methods, as well as engineering techniques for modeling and data analysis. MBE serves as a platform for interdisciplinary research in fields such as biochemistry, molecular biology, applied microbiology, cellular physiology, cellular nutrition in health and disease, and biochemical engineering. The journal publishes various types of papers, including original research papers and review papers. It is indexed and abstracted in databases such as Scopus, Embase, EMBiology, Current Contents - Life Sciences and Clinical Medicine, Science Citation Index, PubMed/Medline, CAS and Biotechnology Citation Index.
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