Sll0147 IN SYNECHOCYSTIS SP. PCC 6803: THE CruA LYCOPENE CYCLASE
Open Access
- Author:
- Xiong, Wei
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- April 07, 2011
- Committee Members:
- Donald Bryant, Thesis Advisor/Co-Advisor
Donald Ashley Bryant, Thesis Advisor/Co-Advisor - Keywords:
- cyanobacteria
lycopene cyclase
CruA - Abstract:
- The sll0147 gene in the cyanobacterium Synechocystis sp. PCC 6803 was studied to determine its role in carotenoid biosynthesis. Through complementation of a cruA mutant of Synechococcus sp. PCC 7002, and by in vitro assays with purified protein, the sll0147 product was shown to be a lycopene cyclase, converting lycopene, 1-hydroxy-lycopene and γ-carotene into β-carotene. Sll0147 belongs to the newly identified family of lycopene cyclases, the CruA family, so Sll0147 has been re-designated as CruA of Synechocystis sp. PCC 6803. The CruA of Synechococcus sp. PCC 7002 was previously and indirectly shown to be a lycopene cyclase in the carotenoid biosynthetic pathway of that organism. Sequence comparisons show that the sll0147 product of Synechocystis sp. PCC 6803 shares 65% amino acid sequence identity with the CruA of Synechococcus sp. PCC 7002. A cruA deletion mutant of Synechococcus sp. PCC 7002, designated as Synechococcus sp. PCC 7002 ΔcruA::aacC1, segregated fully. Unlike the wild-type strain, the ΔcruA::aacC1 mutant accumulated lycopene, 1-hydroxy-lycopene and γ-carotene. By using the heterologous gene expression system in Synechococcus sp. PCC 7002 based on its endogenous plasmid pAQ1, two complementation strains were obtained based on the Synechococcus sp. PCC 7002 ΔcruA::aacC1 strain, named 7002_pAQ1::6803cruA-N(His)10 and 7002_pAQ1::6803cruA-C(His)6 respectively. Pigment analysis of these two strains showed the same pigments profile as that of the wild-type, demonstrating the sll0147 product had lycopene cyclase activity in vivo. In contrast, expression of these same proteins in Escherichia coli strains producing lycopene showed no activity. These data strongly suggested that E. coli strains were unable to produce an essential cofactor for activity of the sll0147 gene product. To obtain additional information about the Sll0147/CruA protein, CruA was purified from Synechococcus sp. PCC 7002 expression strains by metal chelation chromatography of detergent solubilized membranes. The as-purified Sll0147 product was yellow-green in color due to the presence of non-covalently bound chlorophyll a and pheophytin a. Small amounts of lycopene and β-carotene were also detected in the as-purified protein fractions. Gel electrophoresis, immunoblotting, and mass spectrometry identified the yellow-green protein as the product of sll0147/cruA. The as-purified CruA protein was able to convert lycopene and γ-carotene to β-carotene in vitro. These enzymatic data provide the first direct evidence that Sll0147 and by extension other CruA proteins are lycopene cyclases capable of synthesizing β-carotene from lycopene. The findings reported in this thesis fill a major gap in the biosynthetic pathway of carotenoids in Synechocystis sp. PCC 6803 specifically and cyanobacteria in general.