谷氨酸和脯氨酸促进 Cereibacter sphaeroides DT.1 番茄红素积累的作用与机制

The role of glutamate and proline in enhancing lycopene accumulation in Cereibacter sphaeroides DT.1 and its mechanism

摘要

[目的]本文旨在分离鉴定具有积累番茄红素能力的紫色非硫细菌,分析番茄红素的合成途径,研究谷氨酸和脯氨酸促进番茄红素积累的机制。[方法]采用双层板法从底泥中富集分离紫色非硫细菌,通过基因组测序和代谢组分析,揭示番茄红素合成的代谢途径;比较分离菌株在不同条件及不同培养基中生长及积累番茄红素的性能,研究不同氨基酸对分离菌株积累番茄红素的影响,通过差异转录组结合差异代谢组学分析谷氨酸或脯氨酸促进番茄红素积累的机制。[结果]分离鉴定到1株具有番茄红素合成能力的紫色非硫细菌,经16S rRNA基因及全基因组比对,将该菌株鉴定并命名为Cereibacter sphaeroides DT.1。基因组测序和代谢组结果表明该菌株通过4-磷酸甲基赤藓糖醇(MEP)途径合成番茄红素,且该途径的八氢番茄红素去饱和酶(CrtI)具有4步脱氢功能,可以催化八氢番茄红素经四步脱氢形成番茄红素。在好氧黑暗条件下,8种氨基酸可以提高番茄红素的产量,其中谷氨酸和脯氨酸促进效果最佳,在酵母粉培养基中添加400 mg·L^-1的谷氨酸或脯氨酸,番茄红素积累量分别较对照提高99.4%和101.7%。添加谷氨酸或脯氨酸后非环化类胡萝卜素1,2-水合酶基因crtC的表达水平显著下降,丙酮酸、乙酰辅酶A等番茄红素合成前体物质的积累明显增加。[结论]在酵母粉培养基中添加谷氨酸或脯氨酸可以显著提高C. sphaeroides DT.1中番茄红素的产量;谷氨酸或脯氨酸提高番茄红素产量的机制主要为下调非环化类胡萝卜素1,2-水合酶基因的表达,促进番茄红素合成前体物质的积累。

Abstract

[Objectives] The paper aimed to isolate and identify purple non-sulfur bacteria capable of accumulating lycopene, analyze the biosynthetic pathway of lycopene, and investigate the mechanisms by which glutamate and proline promote the accumulation of lycopene. [Methods] The double-layer plate method was used to enrich and isolate purple non-sulfur bacteria from fish pond sediments. Genome sequencing and metabolomics analysis were used to explore the metabolic pathways of lycopene synthesis. The growth of and lycopene accumulation in the isolated strains were compared under different conditions and in different culture media. The effects of the addition of different amino acids on the lycopene accumulation in the isolated strains were studied. The mechanisms by which glutamate or proline promote lycopene accumulation were analyzed through differential transcriptomics combined with differential metabolomics. [Results] A purple non-sulfur bacterium capable of synthesizing lycopene was isolated and identified. Through comparison of the 16S rRNA gene and whole-genome sequences, the strain was identified and named asCereibacter sphaeroides DT.1. Genome sequencing and metabolomic results indicated that this strain synthesized lycopene via the methylerythritol 4-phosphate (MEP) pathway. Moreover, the phytoene desaturase (CrtI) in this pathway possessed a 4-step desaturation activity. Under aerobic dark conditions, increased lycopene yield was observed due to the addition of eight amino acids, among which glutamic acid and proline showed the most significant promoting effects. Compared to the control, accumulation of lycopene increased by 99.4% and 101.7%, respectively by adding 400 mg·L^-1 of glutamic acid or proline to the yeast extract medium. Addition of glutamic acid or proline led to a significant decrease in the expression level of the acyclic carotenoid 1,2-hydratase gene crtC, and a significant increase in the accumulation of lycopene synthesis precursors such as pyruvate and acetyl-CoA. [Conclusions] Lycopene yield ofC. sphaeroides DT.1 could be significantly enhanced by the addition of either glutamate or proline addition. The mechanisms for enhanced lycopene production by glutamate or proline addition included downregulation of the expression levels of acyclic carotenoid 1,2-hydratase genes and promotion of the accumulation of precursor for lycopene synthesis.

关键词

Cereibacter sphaeroides / 番茄红素 / 氨基酸 / crtC基因

Key words

Cereibacter sphaeroides / lycopene / amino acids / crtC gene

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基金资助

国家重点研发计划项目(2019YFC1906404)

江苏省碳达峰碳中和科技创新专项(BE2022421)