Molecular Cloning Of The Key Enzyme Genes Of Carotenogenesis In Dunaliella And The Construction Of Their Heterologous Expression System

Natural carotenoids are high in demand in the global market owing to its effectivefunctional characteristics and the growing natural and healthy preference of consumers today.Currently, carotenoids have been widely applied in fields of nutrition, medicine, food,cosmetic, and feed. The halotolerant eukaryotic green alga Dunaliella has been the people’sfavor for its massive accumulation of natural-carotene. This valuable alga has beenexploited commercially for natural-carotene and-carotene-rich Dunaliella powder in anumber of countries and regions since1980s. However, the traditional process optimization of-carotene production with Dunaliella can not meet the increasing market demand nowadays.It is an effective method to alleviate the current tense market demand by means of moleculargenetic and breeding techniques to improve the desired carotenoids yield on theunderstanding and mastery of the mechanism of highly-carotene accumulation of Dunaliellafrom the perspective of molecular biology. In addition, it has an important social significanceto ensure and improve food production by enhancing the saline and alkali tolerance of cropthat benefits from genetic breeding study of carotenogenic pathway.A multiple and efficient carotenogenic genes pool is the prerequisite in genetic breeding forenhancing the production of desired carotenoids. Since the green alga Dunaliella accumulatemassive-carotene under stress conditions, it is implied that the carotenogenic enzymes ofDunaliella have high catalytic activity in the conversion of carotenoids. Considering theprogress of carotenogenic pathway and the results of our groups these years, genes ofcarotenogenic pathway of Dunaliella have been cloned in this work. Based on the molecularcloning techniques, including degenerate PCR, rapid amplification of cDNA end (RACE),and gene splicing by overlap extension PCR (SOE PCR), nucleotide sequences respectivelyfor the phytoene desaturase (PDS),-carotene desaturase (ZDS), and lycopene-cyclase(LCY-B) of Dunaliella bardawil, as well as for the ZDS of Dunaliella salina were firstcloned presently. Subsequently, a series of bioinformatics tools have been used for theanalysis of nucleotide and protein sequences of carotenogenic enzymes of D. salina and D.bardawil to display the coding scheme of carotenogenic genes, amino acid composition andits physicochemical property of carotenogenic enzymes, the subcellular localization of theseenzymes, and so on. A potential mechanism of the massive-carotene accumulation ofDunaliella was defined at the level of gene expression and enzyme catalytic reactionattributing to codon preference analysis and protein domain specificity respectively.According to the results of protein interactions, subcellular localization, and prediction for the transmembrane domain, it was hypothesized that in Dunaliella carotenoids biosynthesis wasperformed by the multisubunit aggregate of carotenogenic enzymes, and LCY-B acts as theconnection point between this multisubunit aggregate and thylakoid membrane. Finally,homology modeling was employed to predict the advanced structures of carotenogenicenzymes of Dunaliella, which would promote study of enzyme function and engineering.A series of carotenoids-generated vectors, including pACCRT-EIB-DbL, pPaEIDbS-I,pPaEBDbZ-I, pPaEBDbDZ-I, pPaEDbSDZ-I, pPaEIBY-I, pPaEIBDbL-I, pPaEIDbSL-I,pPaEDbSDZL-I, pPaEIYDbS-I, pPaEYDbSDZ-I, pPaEBYDbDZ-I, and pPaEBDbDZL-I,were constructed with different combinations of nucleotide sequences of carotenogenic genesof Dunaliella and Pantoea ananatis to expressed in host cell Escherichia coli DH5for theanalysis of catalytic efficacy and efficiency of carotenogenic enzymes of Dunaliella. It wasproposed that two isomerases, Z-ISO and CRTISO, were required during the carotenogenesisof Dunaliella, because the substitution for the P. ananatis crtI with Dunaliella DbPDS orDbZDS could not achieve the conversion from phytoene to lycopene, but the intermediate-carotene. Interestingly, results also displayed that the substitution for the P. ananatis crtY(pACCAR16crtX) with Dunaliella DbLCY-B (pACCRT-EIB-DbL) leads to a remarkableincreased-carotene accumulation of transformant, by48.78%. Subsequently, univariateanalysis method and response surface methodology have been used to optimize the culturalcondition and extraction process for the-carotene yield of transformant. Results showed thatthe optimal medium composition contained16.17g/L tryptone,10g/L yeast extract,5g/LNaCl,21.15g/L glycerol,78.95g/L NH4Cl, and12.68g/L Na2HPO4·7H2O, and the optimalcultural temperature was29°C. Actually, in this optimal cultural condition the-caroteneyield of pACCRT-EIB-DbL transformant was2.82±0.07mg/L. The experimental data offermentation displayed that the max-carotene yield of pACCRT-EIB-DbL transformant was6.30mg/L at56h under the optimal cultural condition.