Screening Of Key Genes In Astaxanthin Biosynthesis Pathway And Construction Of Efficient Cell Factory

Carotenoids are a group of high value-added terpenoids with isoprene as a structural unit.As its strong anti-oxidation and elimination of active singlet oxygen,it has a wide range of applications in food,cosmetics and health products.Beta carotene ketone enzyme(CrtW)and beta carotene hydroxylase(CrtZ)are two key enzymes in astaxanthin biosynthesis pathway.Using Escherichia coil as a cell factory,the key gene cluster crtW-crtZ in the synthetic pathway of astaxanthin was systematically optimized,in this study.The main purpose of the project was to obtain the high-level synthesis of astaxanthin.The crtW and crtZ genes from eukaryotic or prokaryotic microorganisms,were screened and cloned in the expression vector pCA12 separately.They were heterologously expressed in GY02 by vector pCA12 and the appropriate auto-induction temperature was optimized to 30 ?.Through screening,we obtain three sets of relatively better crtW and crtZ combination:(1)crtW-derived from Brevundimonas sp.SD212and Pantoea ananatis crtZ genes combination(BreW-PanZ)(2)Agrobacteritum aurantiacum crtW and Agrobacterium aurantiacum crtZ genes combination(AauW-AauZ)(3)Haematococcus pluvialis crtW and Agrobacterium aurantiacum crtZ genes combination(W148-AauZ).These three combinations can increase astaxanthin yields to 11.83 mg/g DCW,9.24 mg/g DCW,and 11.79 mg/g DCW.And the corresponding strain was named LY01;LY03;LY05,respectively.Due to the problems of plasmid instability,measures were taken as follows:the two key gene of crtW-crtZ combinations were ligated into four different copy number plasmids(only the brew-panz combination was explored in this study),then co-transformed into GY02 with pCA 12 respectively.The results indicate that the brew-panz gene cluster is compatible with the plasmid copy number of A(high-copy)and Y(high-copy),and the corresponding yields up to 10.6 mg/g DCW and 10.3 mg/g DCW,respectively.IPP and DMAPP are two important intermediates in terpenoids synthesis pathway,which are also the influx of MEP and exogenous MVA pathways in E.coli.Isopentenyl diphosphate isomerase(IDI)is a regulates the synthesis of IPP and DMAPP of terpenes,has different meanings in the metabolic pathway,and it is also one of the main targets of metabolic engineering.Therefore,a copy of the idi was integrated into on E.coli chromosome using the means of CRISPR in this study.Subsequently,recombining report plasmid with crtW and crtZ of different sources were transported into the cells,respectively.Through screening the production of astaxanthin,it was found that the over expression of IDI was helpful for the expression of AauW-PanZ.The yield was increased from the original 4.45 mg/g DCW up to 12.25 mg/g DCW,and the yield increased by 2.75 times.The LY01 strain was cultured for 45.5h in high density.The cell density OD600 could reach 183 and the dry weight of astaxanthin per unit weight was 8.43 mg/g,which was reduced by 28.7%compared with the yield of shake flasks fermentation(dry weight 11.83 mg/g).However,high density fermentation has its own advantages.The yield per unit volume is increased from 35.92 mg/L of shake flasks fermentation to 455.68 mg/L of the production of fermentor,and the output per unit volume has been increased by 11.69 times.