| Natural astaxanthin(3,3’-dihydroxy-4,4’-diketo-β,β’-carotene,C40H52O4)is the strongest antioxidant found in nature,and its antioxidant activity is 38 times ofβ-carotene,500 times of vitamin E and 6000 times of vitamin C.Haematococcus pluvialis is the most ideal synthetic tool for natural astaxanthin,but current industrial production is less efficient.It cannot be synthesized in animals,but it is an essential substance for human and animal life activities.Because of its important biological functions and good coloring effects,it is widely used in aquaculture,medicine,cosmetics and advanced nutrition and health care products.H.pluvialis is the preferred synthetic tool for natural astaxanthin,but for industrial production,its growth cycle is too long and the synthesis efficiency is not high enough,resulting in high production costs and abnormally high end product prices.It has become a bottleneck for the commercial production of natural astaxanthin by humans using Haematococcus.Dunaliella salina is a green microalgae that can produce a large amount of natural astaxanthin precursorβ-carotene under adverse conditions,but lack two enzymes,β-carotene hydroxylase（CRTR-B）andβ-carotene ketolase（BKT）,in the D.salina is convertedβ-carotene to astaxanthin.In order to convert a large amount ofβ-carotene into natural astaxanthin in D.salina,this study uses the D.salina cells as the chassis to express BKT and CRTR-B from H.pluvialis by synthetic biology.The two genes are homologously recombined into the chloroplast genome,prolonging the carotenoid synthesis pathway in D.salina,and forming a cell factory that efficiently produces natural astaxanthin.First,16S-trn A/trn I-23S was amplified as a homologous arm at both ends of the insertion site,and the atp A 5’UTR and psb A 5’UTR were amplified as an endogenous promoter to regulate the expression of the foreign gene.Using the chloroplast genome to have the characteristics of polycistronics,the bkt and crt R-B genes were ligated using the Rbs sequence to form a simple polycistronic gene cluster.The chloroplast homologous recombination vector p MD-bkt-crtr（16S-trn A-atp A-bkt-crt R-B-rbc L-psb A-bar-trn I-23S）was constructed using a homologous recombination kit in which the bkt and crt R-B genes were used as one gene clusters were all regulated by the atp A promoter,while the psb A promoter was used to regulate expression of the herbicide resistance gene bar.After introduction of the foreign gene into the recipient cells by the gene gun and screening with glufosinate,we obtained a number of algae with glufosinate resistance.After PCR,southern blot and western blot screening,we obtained a strain of D.salina-bkt-crt.The expression of exogenous genes in D.salina-bkt-crt was increased by 5-7 times under high light and nitrogen deficiency induction by RT-PCR.The presence of（3S,3’S）-all-trans astaxanthin and canthaxanthin in D.salina-bkt-crt containing BKT and CRTR-B enzymes was verified by HPLC under light induction.The maximum contents of total astaxanthin and canthaxanthin were 77.5±7.7 and 50.1±0.8μg g-1,respectively.This study preliminarily verified the feasibility of D.salina as a synthetic biological chassis cell and also showed that D.salina has the potential of a cell factory for efficient astaxanthin production. |
