State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No.2 Yuanmingyuan West Road, Haidian District, Beijing, 100193, China.
Microb Cell Fact. 2023 Mar 28;22(1):59. doi: 10.1186/s12934-023-02067-5.
Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme.
Here, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3-107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold.
High intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.
血红素蛋白，如血红蛋白、辣根过氧化物酶和细胞色素 P450（CYP）酶，具有广泛的用途，在食品、医疗保健、医学和生物分析等领域有广泛的应用。作为辅因子，血红素的可用性对血红素蛋白的正确折叠和功能起着关键作用。然而，血红素蛋白的功能生产通常具有挑战性，主要是由于细胞内血红素的供应不足。
本文构建了一种多功能的高产血红素大肠杆菌底盘，用于高效生产各种高价值的血红素蛋白。首先，通过强化基于 C4 途径的血红素合成途径，开发了一种高产血红素的 Komagataella phaffii 菌株。然而，分析结果表明，工程化 K. phaffii 菌株产生的大多数红色化合物都是血红素合成的中间产物，无法激活血红素蛋白。随后，选择大肠杆菌菌株作为宿主开发产血红素底盘。为了精细调整大肠杆菌中基于 C5 途径的血红素合成途径，构建了 52 株携带不同血红素合成基因组合的重组菌株。获得了一株产高血红素的突变株 Ec-M13，其中间产物积累可忽略不计。然后，在 Ec-M13 中评估了三种血红素蛋白（一种染料脱色过氧化物酶（Dyp）、六种氧转运蛋白（血红蛋白、肌红蛋白和豆血红蛋白）和三种 CYP153A 亚家族 CYP 酶）的功能表达。正如预期的那样，与在野生型菌株中表达相比，表达在 Ec-M13 中的血红素结合 Dyp 和氧转运蛋白的组装效率提高了 42.3-107.0%。Dyp 和 CYP 酶的活性在 Ec-M13 中表达时也显著提高。最后，使用携带三种 CYP 酶的全细胞生物催化剂用于壬二酸的生产。细胞内血红素的高供应可以将壬二酸的产量提高 1.8-6.5 倍。
在没有明显积累血红素合成中间体的情况下，在工程大肠杆菌中实现了高细胞内血红素的产生。证实了 Dyp、血红蛋白、肌红蛋白、豆血红蛋白和 CYP 酶的功能表达。观察到这些血红素蛋白的组装效率和活性增强。这项工作为构建高产血红素的细胞工厂提供了有价值的指导。开发的突变株 Ec-M13 可用作功能生产难以表达的血红素蛋白的通用平台。
