Impact Study On Carotenoids Production Of CrtYB And Asy Knocking-out In Phaffia Rhodozyma Strains

Phaffia rhodozyma is highly commercial value,for its carotenoids particularly astaxanthin is widely used in aquaculture,medical products,health products,cosmetics and so on.The wild strain of P.rhodozyma is with a low astaxanthin production and how to enhance its astaxanthin production is a research hotspot.Exploring the astaxanthin synthesis approach and the regulation mechanism of P.rhodozyma has the vital significance on development of natural astaxanthin resource.In order to learn more about the metabolic rule of astaxanthin,find the relationship between the carotenoids synthesis and the expression of related genes,and construct commercially valuable engineering strain from P.rhodozyma,the following works have been done:Firstly,we cloned the genes of phytoene synthase/lycopene cyclase(crtYB)and astaxanthin synthase(asy)in astaxanthin-hyperproducing strain JMU-MVP14 using PCR technique,and studied the homology of crtYB gens or asy genes in different astaxanthin-producing level strains.The results showed that the homologies of new cloned crtYB gene and reported crtYB gene(Accession No.DQ016503)and their amino acid sequences were 99.3% and 100%,respectively.The homologies of new cloned asy gene and reported asy genes(Accession No.s DQ201828 and EU713462)were 99.9% and 99.7%.The homologies of their amino acid sequences were 99.8% and 100%.There was no significant difference among crtYB genes or asy genes in different astaxanthin-producing level strains.Then,we cloned homologous fragments of crtYB gene or asy gene to construct knocking-out vectors for crtYB gene and asy gene in P.rhodozyma,respectively.The vector for crtYB gene knocking-out was named pMD-?crtYB and was about 6600 bp.The vector for asy gene knocking-out was named pMD-?asy and was almost 7700 bp.After that,we knocked crtYB gene out in astaxanthin-hyperproducing strain JMU-MVP14 and astaxanthin-lowproducing strain JMU-VDL668.When crtYB gene was knocked out,all the mutant colonies were white-colored phenotype and no carotenoids were detected.Compared with original strain JMU-MVP14,there was no noticeable influence on growth of JMU-MVP?crtYB within a decrease in the amount of acid fatty by 43.15% and an increase in the amount of ergosterol by 53.62%.While,the biomass of JMU-VDL?crtYB was improved about 35% than original strain JMU-VDL668 with a decrease in the amount of acid fatty by 55.43% and an increase in the amount of ergosterol by 15.80%.Finally,we knocked asy gene out in P.rhodozyma strains JMU-MVP14 and JMU-VDL668 and obtained JMU-MVP?asy and JMU-VDL?asy accordingly to investigate the influence on growth and carotenoids production of P.rhodozyma strains with lacking in asy gene.Both the two mutants were with unconspicuous changes in phenotype and biomasses.Compared with orignal strain,the amounts of carotenoids and ?-carotene of JMU-MVP?asy were decreased.And the amount of astaxanthin was equal with orignal strain before 24 h,much less than orignal strain in 24-56 h,over orignal strain after 56 h and then eventually slightly improved.The amount of carotenoids of JMU-VDL?asy was equal with orignal strain before 24 h,little more than orignal strain in 24-48 h and over orignal strain after 48 h.In 0-8 h,there was no astaxanthin detected in JMU-VDL?asy or JMU-VDL668.And the amount of astaxanthin of JMU-VDL?asy was less than orignal strain in 8-40 h,over orignal strain after 48 h and eventually increased by almost 25%.During the whole fermentation process,there was no ?-carotene detected in either JMU-VDL668 or JMU-VDL?asy.