Optimization Of Metabolic Pathway For Synthesis Of Astaxanthin From Escherichia Coli

Astaxanthin(astaxanthin)is 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 feed additives.The demand for astaxanthin is increasing,but the way of astaxanthin’s production is restricted.At present,using Escherichia coil as a cell factory for microbial fermentation to realise astaxanthin’s production has good prospects.Zeaxanthin is an important intermediate in the production process of astaxanthin,and it is also a research hotspot of terpenoids,Astaxanthin can be obtained through the action of beta carotene hydroxylase(CrtZ).Therefore,in this study,we use a engineering strain of βcarotene named PCA09 which stored in our laboratory as starting strain.Then we select twoβ-hydroxylase gene crtZ from different sources(Pantoea ananatis crtZ genes named PanZ and Agrobacterium aurantiacum crtZ gene named AauZ)and introduced into PCA09 to obtained the engineering production strain of zeaxanthin named HZ01 and HZ02.Then we fused the plasmids of HZ01 and HZ02 and obtained two new strains named HZ11 and HZ12.The yields of the new strain was increased and it could be reached in 7.4 mg/g DCW and 13.2 mg/g DCW respectively.We select HZ 12 and continue to introduce the crtW gene which from two different sources(Agrobacterium aurantiacum crtW named AauW and Brevundimonas sp.SD212 crtW named BreW)into it.And then we obtained two engineering strains of astaxanthin named HAOland HA02.We fused the two plasmid of HA01 and HA02 respectively and then obtained two new strains named HA03 and HA04.The yields of astaxanthin was 8.4 mg/g DCW and 10.7 mg/g DCW,respectively.Identifying the key genes and removing the bottleneck of the related metabolic pathway in the engineered strain is an important method to increase yield.Therefore,in order to increase the yield of HA04,measures were taken as follows:First,construct the plasmid about the downstream pathway gene for astaxanthin production and transferred into the HA04 respectively.We found crtW and crtZ are two critical factors and it is more conducive to accumulat astaxanthinIt that enhance the expression crtZ.Finally,we changed the expression intensity of crtZ through replaced promoter to increase the level of astaxanthin synthesis.The results show that the genes of crtZ and crtW from different sources have an important effect on the production of astaxanthin.And for this experiment,the crtZ gene derived from Pantoea ananatis(PanZ)and the crtW gene derived from Brevundimonas sp.SD212(BreW)compared with the crtZ gene derived from Agrobacterium aurantiacum(AauZ)and crtW gene derived from Agrobacterium aurantiacum(AauW),the accumulation of astaxanthin is more favorable.As for HA04,crtZ gene is the main limiting factor for astaxanthin accumulation and tac promoter and Pantoea ananatis crtZ gene is the best combination(tac-PanZ)for astaxanthin’s production which yield can be reach in 13.3 mg/g DCW.