通过将前体5-氨基乙酰丙酸(5-aminolevulinic acid, 5-ALA)合成模块和5-ALA合成血红素模块分别设计在解淀粉芽孢杆菌与大肠杆菌中,构建了血红素合成共培养体系,实现了血红素的积累。首先,重构解淀粉芽孢杆菌的高谷氨酸合成代谢流方向,改善谷氨酸流向血红素前体5-ALA的效率,并在大肠杆菌中表达5-ALA合成血红素的7个途径基因。通过对上述2株工程菌共培养发酵实现了血红素积累。此外,通过适配性优化关键途径基因、阻遏竞争途径代谢流、载体工程以及发酵过程优化等策略来进一步改善共培养体系合成血红素的能力,获得了65.38 mg/L的血红素积累。该研究首次采用共培养体系从葡萄糖生产血红素,为其他天然产物的生物合成提供了崭新的思路。
Heme has a variety of physiological functions in vivo and has been widely used in food, medicine, and other fields. Compared with traditional extraction methods, microbial synthesis of heme has the advantages of low cost and environmental friendliness. In this study, we constructed a co-culture system for heme biosynthesis by engineering the 5-aminolevulinic acid (5-ALA) synthesis module and the heme synthesis from the 5-ALA module into Bacillus amyloliquefaciens NX-2S154 with high glutamate anabolic flux and Escherichia coli BL21 with high expression ability of heterologous enzymes, respectively. Firstly, the polyglutamic acid synthase gene pgsBCA in B. amyloliquefaciens was knocked out by CRISPR-cas9n, and the fermentation results showed that B. amyloliquefaciens NX-2S154 had high intracellular glutamate synthesis flux, which indicated that it could provide sufficient glutamate precursors for the co-culture system of heme synthesis. Secondly, we divided the heme synthesis pathway into the precursor 5-ALA synthesis module and the module for heme synthesis from 5-ALA, which were constructed in B. amyloliquefaciens NX-2S154 and E. coli BL21, respectively, to obtain engineered strains B. amy-1 and E. coli-a. Co-cultivation of engineered strains B.amy-1 and E.coli-a resulted in a heme accumulation of 7.84 mg/L. Thirdly, the precursor 5-ALA synthesis module in B. amyloliquefaciens (B. amy-1) was further optimized to improve the efficiency of central carbon flux to heme through adaptively optimizing key pathway genes gltX, hemA ,and hemL, and knocking out ldh, pta, nas and prob genes to block the biosynthesis of by-products lactate, acetate, N-acetylaminoglutamate and proline. The above strategies resulted in the titer of heme in the co-culture system reaching 18.16 mg/L, which was 131.62% higher than that of the initial co-culture system. In addition, the 5-ALA extracellular transport pathway and the heme extracellular transport pathway were constructed to improve the synthesis efficiency of heme in the co-culture system and obtained 28.36 mg/L of heme. Finally, combining these strategies and further optimizing the fermentation process, we obtained a heme titer of 65.38 mg/L and a substrate molar conversion rate of 0.000382 mol/mol (heme/glucose) in a 7.5 L fermenter. The substrate conversion rate of the heme co-culture system constructed in this study was significantly higher than that of traditional single bacterial fermentation. These results also indicated that the co-culture strategy used in this study has great potential in constructing efficient cell factories to biosynthesize complex natural products.
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