5-Aminolevulinic acid(5-ALA)serves as a key precursor for tetrapyrrole compounds biosynthesis.Conventional high-producing strains often rely on multi-copy plasmids,causing instability and elevated costs.In this study,...5-Aminolevulinic acid(5-ALA)serves as a key precursor for tetrapyrrole compounds biosynthesis.Conventional high-producing strains often rely on multi-copy plasmids,causing instability and elevated costs.In this study,a plasmid-free,inducer-free,and antibiotic-free 5-ALA cell factory was constructed via chromosomal integration to improve strain robustness and scalability.Using DLKcat-based computational screening,an efficient 5-aminolevu-linate synthase(ALAS)derived from Methylocystis sp.was identified,achieving a 5-ALA titer of 3.60 g/L in shake flasks.Subsequently,promoter library-based tuning of the downstream gene hemB increased the titer by 15.83%.Further rational engineering of key metabolic targets increased the titer to 7.14 g/L.Transcriptomic analysis enabled the identification and introduction of transcription factor PspC,yielding strain ALA33 with 8.04 g/L 5-ALA.Without the addition of exogenous antibiotics or inducers,ALA33 achieved a 5-ALA titer of 40.88 g/L through fed-batch fermentation,showing a practical strategy for stable and efficient industrial production of 5-ALA and tetrapyrroles.展开更多
基金supported by the National Key Research and Development Program of China(2023YFA0914500)the National Natural Science Foundation of China(32470067,32370040)+2 种基金the Jiangsu Provincial Frontier Technology Research and Development Program(BF2024012)the Basic Research Program of Jiangsu(BK20233003)the Fundamental Research Funds for the Central Universities(JUSRP124018).
文摘5-Aminolevulinic acid(5-ALA)serves as a key precursor for tetrapyrrole compounds biosynthesis.Conventional high-producing strains often rely on multi-copy plasmids,causing instability and elevated costs.In this study,a plasmid-free,inducer-free,and antibiotic-free 5-ALA cell factory was constructed via chromosomal integration to improve strain robustness and scalability.Using DLKcat-based computational screening,an efficient 5-aminolevu-linate synthase(ALAS)derived from Methylocystis sp.was identified,achieving a 5-ALA titer of 3.60 g/L in shake flasks.Subsequently,promoter library-based tuning of the downstream gene hemB increased the titer by 15.83%.Further rational engineering of key metabolic targets increased the titer to 7.14 g/L.Transcriptomic analysis enabled the identification and introduction of transcription factor PspC,yielding strain ALA33 with 8.04 g/L 5-ALA.Without the addition of exogenous antibiotics or inducers,ALA33 achieved a 5-ALA titer of 40.88 g/L through fed-batch fermentation,showing a practical strategy for stable and efficient industrial production of 5-ALA and tetrapyrroles.
