ABSTRACT
This study engineered β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from β-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 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 β-carotene ketolase and β-carotene hydroxylase were improved for conversion of β-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.
ABSTRACT
The growth changes of glutathione (GSH) and ergosterol in Saccharomyces cerevisiae (CICC1447 and CICC1339) were detected under 0.5Mpa pressure with compressed high-pure air (O-2∶N-2=21∶79). The results showed that logarithmic phases of the two strains were delayed; their biomass and special growth rate were lower than those of control sample (0.1MPa) and the double time were prolonged under 0.5MPa. High-pressure could increase the content of GSH obviously, compared to ambient atmosphere control samples. When the holding time was 3h, the content of GSH and ergosterol in CICC1447 increased 42.6% and 20.1%, respectively. However, the content of GSH in CICC1339 increased 58.7% when the holding time was 6h, while ergosterol content reduced. The results indicated that different yeast strains have different stress-response mechanism to copy with high-pressure shock.