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Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P 21 , P 43 , and P veg ) and three inducible promoters (P grac100 , P xylA , and P tet * ) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 μg/L, was achieved when regulated by the P tet * promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SP yycP resulted in increased HLF expression to 1 099.87 μg/L, with 498.68 μg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 μg/L. This study successfully demonstrated the secretory expression of HLF in B . subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B . subtilis cell factories for lactoprotein synthesis.

Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.