Engineering strategies for microbial synthesis, customized modification, and application of hemoglobin

Hemoglobin is a functional protein with heme as a cofactor, playing a crucial role in transporting oxygen and maintaining nitric oxide metabolic balance. Besides its physiological functions, hemoglobin has broad potential applications in medicine and biotechnology. However, the widespread use of hemoglobin is constrained by limited natural sources, challenges in heterologous synthesis, and functional restrictions that hinder efficient application. In this review, we discuss the key challenges and solutions associated with microbial synthesis of hemoglobin. We systematically elucidate the engineering strategies to improve the stability, autoxidation rate, heme-binding capacity, oxygen transport efficiency, and nitric oxide scavenging rate of hemoglobin, with particular emphasis on the use of artificial intelligence algorithms to customize the function modification of hemoglobin. Also, we provide a comprehensive overview of the various applications of hemoglobin, including artificial oxygen carriers, medical treatments requiring enhanced oxygen supply, synthesis of high-value products, biocatalysis, artificial foods, agriculture, functional substance testing, and bioactive peptide production, with a special focus on the potential of hemoglobin mutants and derivatives in expanding its use across various fields. Finally, we explore the prospects for accelerating the resolution of hemoglobin synthesis and overcoming the application challenges by integrating Pareto-optimal and iterative bioengineering frameworks, deep learning, synthetic biology, and other advanced technologies.