Zhang Jian, Li Qingbin, Wang Qi, Zhao Jingyu, Zhu Yuan, Su Tianyuan, Qi Qingsheng, Wang Qian
National Glycoengineering Research Center, State Key Laboratory of Microbial Technology, Shandong University, Qingdao, 266237, People's Republic of China.
CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101, People's Republic of China.
Biotechnol Biofuels Bioprod. 2023 Mar 1;16(1):33. doi: 10.1186/s13068-023-02285-4.
Heme has attracted much attention because of its wide applications in medicine and food. The products of genes hemBCDEFY convert 5-aminolevulinic acid to protoporphyrin IX (PPIX; the immediate precursor of heme); protoporphyrin ferrochelatase (FECH) inserts Fe into PPIX to generate heme. Biosynthesis of heme is limited by the need for optimized expression levels of multiple genes, complex regulatory mechanisms, and low enzymatic activity; these problems need to be overcome in metabolic engineering to improve heme synthesis.
We report a heme biosensor-guided screening strategy using the heme-responsive protein HrtR to regulate tcR expression in Escherichia coli, providing a quantifiable link between the intracellular heme concentration and cell survival in selective conditions (i.e., the presence of tetracycline). This system was used for rapid enrichment screening of heme-producing strains from a library with random ribosome binding site (RBS) variants and from a FECH mutant library. Through up to four rounds of iterative evolution, strains with optimal RBS intensities for the combination of hemBCDEFY were screened; we obtained a PPIX titer of 160.8 mg/L, the highest yield yet reported in shaken-flask fermentation. A high-activity FECH variant was obtained from the saturation mutagenesis library. Fed-batch fermentation of strain SH20C, harboring the optimized hemBCDEFY and the FECH mutant, produced 127.6 mg/L of heme.
We sequentially improved the multigene biosynthesis pathway of PPIX and performed in vivo directed evolution of FECH, based on a heme biosensor, which demonstrated the effectiveness of the heme biosensor-based pathway optimization strategy and broadens our understanding of the mechanism of heme synthesis.
血红素因其在医学和食品中的广泛应用而备受关注。hemBCDEFY基因的产物将5-氨基乙酰丙酸转化为原卟啉IX（PPIX；血红素的直接前体）；原卟啉亚铁螯合酶（FECH）将铁插入PPIX以生成血红素。血红素的生物合成受到多个基因优化表达水平需求、复杂调控机制和低酶活性的限制；在代谢工程中需要克服这些问题以改善血红素合成。
我们报道了一种利用血红素响应蛋白HrtR调节大肠杆菌中tcR表达的血红素生物传感器引导的筛选策略，在选择性条件下（即存在四环素）提供了细胞内血红素浓度与细胞存活之间的可量化联系。该系统用于从具有随机核糖体结合位点（RBS）变体的文库和FECH突变体文库中快速富集筛选产血红素菌株。通过多达四轮的迭代进化，筛选出了hemBCDEFY组合具有最佳RBS强度的菌株；我们获得了160.8mg/L的PPIX滴度，这是摇瓶发酵中报道的最高产量。从饱和诱变文库中获得了高活性FECH变体。对含有优化的hemBCDEFY和FECH突变体的SH20C菌株进行补料分批发酵，产生了127.6mg/L的血红素。
我们基于血红素生物传感器依次改进了PPIX的多基因生物合成途径，并对FECH进行了体内定向进化，这证明了基于血红素生物传感器的途径优化策略的有效性，并拓宽了我们对血红素合成机制的理解。
