Metabolic Engineering Of Synechocystis Sp.PCC6803 For Producing Astaxanthin

Astaxanthin(3,3'-dihydroxy-4,4'-dione-?,?'-carotene,C₄₀H₅₂O₄)is a secondary carotenoid that has been found in nature.Astaxanthin is the strongest antioxidant,its antioxidant activity is 1000 times that of vitamin E.Astaxanthin is mainly extracted from algae sources?Haematococcus pluvialis?,Phaffia rhodozyma,and synthetic methods by various methods.The source of algae containing astaxanthin is mainly Haematococcus pluvialis.The structure of algae astaxanthin is 3S,3'S,its structure is stable.After human or animal ingestion,it can exert some important protective functions and maximize its biological effects.The astaxanthin in Phaffia rhodozyma is 3R,3'R,while the synthetic astaxanthin contains 3S,3'S;3S,3'R;3R,3'R.H.pluvialis is the preferred source for natural astaxanthin because of its high content.In various stress conditions,H.pluvialis rapidly synthesizes and accumulates astaxanthin to 5%of the cell dry weight?DW?.This process is affected by environmental factors such as light,temperature,pH,and nutrient availability.High light,C/N ratio,and salinity all contribute to the accumulation of astaxanthin.However,the production of astaxanthin by H.pluvialis on a large scale is hindered by the long growth cycle of this species;it is affected by the seasons and the weather;it is susceptible to bacterial and algal contamination;and it cannot take into account both cells growth and accumulation of astaxanthin.These factors severely limit astaxanthin production using H.pluvialis on an industrial scale.Microbial production of astaxanthin via metabolic engineering has emerged as an attractive alternative.Synechocystis sp.PCC6803 is a prokaryote capable of photosynthesis.It is the phototrophic organism to have its genome completely sequenced and has the efficient and mature genetic transformation systems.Synechocystis sp.PCC6803 is a model organism for the study of metabolic engineering and also an excellent chassis for constructing cell factories by using synthetic biology.It is both autotrophic and heterotrophic,grows rapidly,and is suitable for large-scale cultivation;thus,it has various potential applications.Synechocystis sp.PCC6803 can synthesize echinenone and zeaxanthin,which are precursors of astaxanthin.Based on the above characteristics,we hypothesized that Synechocystis sp.PCC6803 would be an excellent chassis for astaxanthin production.However,compared with H.pluvialis,Synechocystis sp.PCC6803 lacks the enzymes?-carotene ketolase?BKT?and carotenoid hydroxylase?CRTR-B?,encoded by the genes bkt and crtR-B respectively.Here,we extended the carotenoid synthesis pathway in Synechocystis sp.PCC6803 to explore an alternative to H.pluvialis for astaxanthin production.First,the accumulation of astaxanthin was increased by choosing efficient promoters.Through codon optimization,the bkt and crtR-B coding sequences from H.pluvialis were adapted for expression in Synechocystis sp.PCC6803.Then,culture conditions were optimized to increase astaxanthin production.Consequently,the highest amount of astaxanthin produced was 4.81±0.06 mg/g DW.