Construction Of Astaxanthin Cell Factory And Optimization Of Fermentation Process

Astaxanthin is a kind of carotenoid with strong antioxidant ability.It also has a variety of biological activities,including anti-inflammatory,anti-cancer and immune system protection properties.At present,it has been widely used in aquaculture,food,animal feed,health products,cosmetics and the same industry.With the continuous development of metabolic engineering,de novo biosynthesis of astaxanthin using host bacteria such as Escherichia coli has attracted much attention.In this study,the E.coli strain CAR026 with high production ofβ-carotene was selected as the starting strain for the production of astaxanthin.The astaxanthin biosynthesis pathway was constructed by introducingβ-carotene hydroxylase（Crt Z）andβ-carotene ketolase（Crt W）genes.The yield of astxanthin was improved by balancing the mounts of crt Z and crt W*and the expression of molecular chaperone genes（gro EL-gro ES）.Furthermore,the high production strain was product 1.52g/L astaxanthin with optimized of fermentation process in a 5 L fermenter.The experiment got the following research results:1、Using the CRISPR/Cas9 method,the crt YZW*gene was integrated into the mgs A site of the CAR026 strain chromosome to eliminate the crt Y gene bottleneck.Obtained stable genetic strain,named Ast005,produced 15.45 mg/L and 3.03 mg/g DCW of astaxanthin in shake flasks.To reduce the accumulation of intermediate products and improve the production of astaxanthin,the mounts of crt Z and crt W*in Ast005 was coordinated by using one-step homologous recombination and two-step homologous recombination methods.In addition,the molecular chaperone genes gro ES-gro EL were regulated to further improve the astaxanthin yield.The astaxanthin production of strain Gro-46 reached 26 mg/L and 6.17mg/g DCW in the shaking flask,which was 68.3% and 103.6% higher than that of Ast005strain,respectively.2、The beat stain Gro-46 produced 1.18 g/L astaxanthin with a yield of 7.8 mg/g DCW after 60 h of fermentation under fed-batch conditions,which were 34.4%and 32%higher than Ast005 strain,respectively.The fed-batch fermentation conditions（fermentation temperature,IPTG induction time,neutralizer and DO）were optimized for improving astaxanthin production.The astaxanthin production under the optimized fermentation conditions(30℃,when the OD₆₀₀ reached 65,use the final concentration of 0.5 m M IPTG to induce,the ammonia as a neutralizer to maintain p H 7.0 and the DO control at 20%)reached 1.52 g/L and 10.52 mg/g,which were 28.8%and 34.9%higher than before optimization.To the best of our knowledge,this is the highest astaxanthin obtained using engineered E.coli to date.