ABSTRACT
This study engineered ß-carotene ketolase CrtW and ß-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from ß-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L-1 without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of ß-carotene ketolase and ß-carotene hydroxylase were improved for conversion of ß-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.
Subject(s)
Escherichia coli/metabolism , Metabolic Engineering/methods , Mixed Function Oxygenases/genetics , Oxygenases/genetics , Biosynthetic Pathways , Carotenoids/chemistry , Carotenoids/metabolism , Mixed Function Oxygenases/chemistry , Oxygenases/chemistry , Xanthophylls/chemistry , Xanthophylls/metabolismABSTRACT
Momordica charantia L. is a vegetable widely cultivated around the world. Its fruits have been used in Asian countries as a folk medicine for the treatment of non-insulin-dependent diabetes mellitus. Here we report eight compounds isolated from the fruits of M. charantia. On the basis of NMR and MS spectroscopic analyses, these compounds were identified as momordicolide ((10E)-3-hydroxyl-dodeca-10-en-9-olide, 1), monordicophenoide A (4-hydroxyl-benzoic acid 4-O-beta-D-apiofuranosyl (1 --> 2)-O-beta-D-glucopyranoside, 2), dihydrophaseic acid 3-O-beta-D-glucopyranoside (3), 6,9-dihydroxy-megastigman-4,7-dien-3-one (blumenol, 4), guanosine (5), adenosine (6), uracil (7) and cytosine (8). Among them, 1 and 2 are new compounds. Compounds 3-5 were isolated from this plant for the first time.
Subject(s)
Momordica charantia/chemistry , Fruit/chemistry , Glycosides/analysis , Glycosides/chemistry , Triterpenes/analysis , Triterpenes/chemistryABSTRACT
OBJECTIVE: To study the effect of Supplemented Taoren Chengqi decoction (STCD) on the secretion of insulin and proliferation of NIT-1. METHOD: The effect of STCD and the serum of rat after orally administrating of STCD on the secretion of insulin and proliferation of NIT-1 were studied. The proliferation of NIT-1 was measured by 3H-TdR incorporation and cell counting methods, while the secretion of insulin was measured from the cultured medium by the ultra sensitive rat insulin ELISIA kit. RESULT: Both the STCD and the serum of rat after orally administrating of STCD significantly could increased the secretion of insulin and proliferation of NIT-1. CONCLUSION: The treatment of the diabetic patients by STCD might be through with its improvement of secretion of insulin and proliferation on pancreatic beta-cell.