A Novel Producing Approach Of Lycopene

Lycopene is a kind of red-colored carotenoid, which widely distribute in animals and plants. Molecular formula is C40H56 and molecular weight is 536.88. It named lycopene because of first discovery in tomato. In recent years, the investigation on lycopene at home and abroad showed that lycopene have some superior functions, so it has been widely used in the fields such as food, cosmetic and drug. Now the study on the ways of lycopene production is becoming a hot spot by many companies at home and abroad, and we can obtain lycopene by microorganism fermentation, especially with carotenoid synthesis genes cloning in different species.The gene crtI relating to lycopene synthesis, encodes the phytoene desaturase, which has different functions in diverse species. In the Erwinia herbicola, the dasaturase produces lycopene though 4-steps dehydrogenation, but in the photosynthetic bacteria Rhodobacter sphaeroides, the dasaturase produces neurosporene instead of lycopene by 3-steps dehydrogenation. In this research, the crtI gene of E.herbicola was heterologously expressed in the R.sphaeroides mutant bacteria TC72, and making TC72 to produce lycopene after transformation, which provides a new way to produce lycopene. The main results obtained are listed bellow.1. The expression vector pRKR5-crtI was successfully introduced into photosynthetic bacteria R.sphaeroides by conjugative transfer. By the multiply alignment of amino acid sequences and nucleotide acid sequences of several E.herbicola family crtI gene in Genbank, a pair of degenerate primers was designed against the conserved regions that was used for PCR to amplify a 629bp DNA segment from the E. herbicola069. The nucleotide acid sequence compared by Blast showed that the similarity to E. herbicola pv. Milletiae (GenBank accession number: AB076662) was the highest with 95.8%. Based on the DNA sequence of E. herbicola pv. Milletiae, we designed a pair of primers and the full length of crtI gene was acquired (GenBank accession number: DQ408590). Then the crtI gene was cloned into pRKR5 vector controlled by puc promoter from R. sphaeroides, to construct pRKR5-crtI expression cassette. Then the expression construct was introduced into photosynthetic mutant bacteria TC72 by conjugative transfer.2. The engineering bacteria can accumulate a red pigment induced by oxygen concentration regulating. The puc promoter can high express by anaerobic conditions, so we could regulate oxygen concentration of the engineering bacteria and make it to produce red pigment.3. The red pigment was testified as lycopene by absorbance spectra analysis and HPLC analysis. The engineering bacteria could grow up to 2.36gDCW/L and accumulate lycopene to 1.52mg/gDCW. Compared with other engineering bacteria producing lycopene , such as Escherichia coli and Candida utilis, the output of lycopene in the transformant had some increase.4. Study on the growth curve and fermentation curve of engineering bacteria and comparison strains, the results as follows. Their growth curves are quite similar, both through 3 periods: delayed phase, growth index phase and stationary phase. Their fermentation curves were obviously different: the lycopene output of comparison strains is zero; but for engineering bacteria, the output of lycopene significantly improved with the length of time, it reached maximum in initial stage of stationary phase and the accumulated lycopene can be degraded after stationary phase, so the suitable culture time of engineering bacteria is initial stage of stationary phase.