摘要
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.
基金
supported by the National Key Research and Development Program of China(2023YFA0914500)
the National Natural Science Foundation of China(32470067,32370040)
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).
