枯草芽孢杆菌(Bacillus subtilis)作为公认安全(Generally Recognized as Safe,GRAS)菌株,是生产食品级血红素的理想底盘,但其粪卟啉依赖性(coproporphyrin-dependent,CPD)合成途径中关键酶的调控机制尚不明确。为解析并突破该途径的限速步骤,该研究系统评价了强化尿卟啉原脱羧酶(UroD,hemE编码)和粪血红素脱羧酶(ChdC,hemQ编码)的表达对血红素合成的影响。采用无标记基因修饰技术,将强组成型启动子驱动的hemE和hemQ基因分别或组合整合到B.subtilis BSH11基因组的中性位点ydaP和yvkC,构建基因过表达菌株,并通过摇瓶发酵和实时荧光定量PCR分析其血红素产量和基因转录水平。结果表明,单独强化hemE表达效果最为显著,其转录水平最高提升303倍,发酵48 h后胞外血红素产量达到(23.90±1.49) mg/L,较对照菌株提高826%;单独强化hemQ表达对产量无明显促进作用;而两基因共表达时,胞外血红素产量较hemE单过表达菌株反而下降35%。研究证实,UroD是B.subtilis血红素CPD合成途径的关键限速步骤,而ChdC的催化能力在当前条件下并非主要限制因素。该发现为后续在B.subtilis这一安全底盘中,通过精准代谢工程策略高效合成食品级血红素提供了核心靶点和理论指导。
Bacillus subtilis, a Generally Recognized as Safe (GRAS) strain, serves as an ideal chassis for producing food-grade heme.However, the regulatory mechanisms of key enzymes within its coproporphyrin-dependent (CPD) synthesis pathway remain unclear.To elucidate and overcome the rate-limiting steps in this pathway, this study systematically evaluated the impact of enhancing the expression of uroporphyrinogen decarboxylase (UroD, encoded by hemE) and coproheme decarboxylase (ChdC, encoded by hemQ) on heme synthesis.The genes hemE and hemQ, controlled by a strong constitutive promoter, were integrated into the neutral genomic loci ydaP and yvkC of B.subtilis BSH11, both individually and in combination, using a markerless gene modification technique.The resulting engineered strains were then evaluated for heme production in shake-flask fermentation and for gene transcription levels by Real-time quantitative PCR (RT-qPCR).The results showed that overexpressing hemE alone yielded the most significant improvement.Its transcription level increased by up to 303-fold, leading to an extracellular heme production of 23.90±1.49 mg/L after 48 h of fermentation, an 826% increase compared to the control strain.In contrast, overexpression of hemQ alone did not significantly promote heme production.Furthermore, the co-expression of both genes resulted in a 35% decrease in extracellular heme yield compared to the strain overexpressing only hemE.These findings demonstrate that UroD is a key rate-limiting step in the CPD pathway of B.subtilis, whereas the catalytic ability of ChdC is not the primary limiting factor under the current conditions.This discovery identifies a core target and provides theoretical guidance for efficiently synthesizing food-grade heme in the safe B.subtilis chassis via precise metabolic engineering.
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