Abstract
BackgroundSalvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases. Recently, production of SAA in engineered Escherichia coli was established via the artificial biosynthetic pathway of SAA on the multiple plasmids in our previous work. However, the plasmid-mediated system required to supplement expensive inducers and antibiotics during the fermentation process, restricting scale-up production of SAA. Microbial cell factory would be an attractive approach for constitutive production of SAA by chromosome engineering.ResultsThe limited enzymatic reactions in SAA biosynthetic pathway from glucose were grouped into three modules, which were sequentially integrated into chromosome of engineered E. coli by λ Red homologous recombination method. With starting strain E. coli BAK5, in which the ptsG, pykF, pykA, pheA and tyrR genes were previously deleted, chassis strain BAK11 was constructed for constitutive production of precursor l-tyrosine by replacing the 17.7-kb mao-paa cluster with module 1 (PlacUV5-aroGfbr-tyrAfbr-aroE) and the lacI gene with module 2 (Ptrc-glk-tktA-ppsA). The synthetic 5tacs promoter demonstrated the optimal strength to drive the expression of hpaBC-d-ldhY52A in module 3, which then was inserted at the position between nupG and speC on the chromosome of strain BAK11. The final strain BKD13 produced 5.6 g/L of SAA by fed-batch fermentation in 60 h from glucose without any antibiotics and inducers supplemented.ConclusionsThe plasmid-free and inducer-free strain for SAA production was developed by targeted integration of the constitutive expression of SAA biosynthetic genes into E. coli chromosome. Our work provides the industrial potential for constitutive production of SAA by the indel microbial cell factory and also sets an example of further producing other valuable natural and unnatural products.
Highlights
Salvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases
Sufficient supplement of l-tyrosine facilitates the production of SAA
Plasmid-mediated l-tyrosine producer strains needed the addition of corresponding antibiotics and IPTG to control gene overexpression of interest [37,38,39]
Summary
Salvianic acid A (SAA), a valuable natural product from herbal plant Salvia miltiorrhiza, exhibits excellent antioxidant activities on food industries and efficacious therapeutic potential on cardiovascular diseases. Microbial cell factory would be an attractive approach for constitutive production of SAA by chromosome engineering. Salvianic acid A (SAA, 3-(3′,4′-dihydroxyphenyl)-2-hydroxypropanoic acid), called danshensu, is the major bioactive ingredient of traditional Chinese herb plant Salvia miltiorrhiza (danshen) which is widely used for the prevention and treatment of vascular diseases in clinic [1, 2]. Salvianolic acid B has already been used to alleviate angina pectoris and treat coronary heart diseases in clinic [7]. Salvianolic acid A has been approved by China Food and Drug Administration (CFDA) into phase I clinical trial. Conjugates of SAA with cysteine show better vascular-protective effect than SAA [9]
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