Identification of a lycopene beta-cyclase required for bacteriorhodopsin biogenesis in the archaeon Halobacterium salinarum

Biogenesis of the light-driven proton pump bacteriorhodopsin in the archaeon Halobacterium salinarum requires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor. As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified an H. salinarum gene required for conversion of lycopene to beta-carotene, a retinal precursor. The gene, designated crtY, is predicted to encode an integral membrane protein homologous to lycopene beta-cyclases identified in bacteria and fungi. To test crtY function, we constructed H. salinarum strains with in-frame deletions in the gene. In the deletion strains, bacteriorhodopsin, retinal, and beta-carotene were undetectable, whereas lycopene accumulated to high levels ( approximately 1.3 nmol/mg of total cell protein). Heterologous expression of H. salinarum crtY in a lycopene-producing Escherichia coli strain resulted in beta-carotene production. These results indicate that H. salinarum crtY encodes a functional lycopene beta-cyclase required for bacteriorhodopsin biogenesis. Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.     

Effects of light and low oxygen tension on pigment biosynthesis in Halobacterium salinarum, revealed by a novel method to quantify both retinal and carotenoids

A novel method for analyzing halobacterial pigments was developed, in which retinal was liberated from halobacterial rhodopsins as retinal oxime by hydroxylamine, ethyl beta-apo-8'-carotenoate was introduced as an internal standard, and the pigments including bacterioruberin and beta-carotene were analyzed by HPLC at the same time. With this method, we revealed that light enhances the biosynthesis of bacterioruberin and the conversion of beta-carotene to retinal, but does not affect beta-carotene biosynthesis in Halobacterium salinarum strain Oyon Moussa-16. Low oxygen tension given in the light brought a slight increase in retinal accumulation, although its biosynthesis from beta-carotene is an oxygenation reaction. This paradox could be explained by the increase in beta-carotene biosynthesis.     

Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker

To facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine-5'-monophosphate decarboxylase. H. salinarum was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can select for mutations in ura3. A spontaneous 5-FOA-resistant mutant was found to contain an insertion in ura3 and was a uracil auxotroph. Integration of ura3 at the bacterioopsin locus (bop ) of this mutant restored 5-FOA sensitivity and uracil prototrophy. Parallel results were obtained with a Deltaura3 strain constructed by gene replacement and with derivatives of this strain in which ura3 replaced bop. These results show that H. salinarum ura3 encodes functional orotidine-5'-monophosphate decarboxylase. To demonstrate ura3-based gene replacement, a Deltabop strain was constructed by transforming a Deltaura3 host with a bop deletion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 was included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5-FOA, yielding Deltabop recombinants at a frequency of > 10-2 in both approaches. These studies establish an efficient new genetic strategy towards the systematic knockout of genes in an archaeon.     

Genetic transfer of the pigment bacteriorhodopsin into the eukaryote Schizosaccharomyces pombe

The gene encoding for bacterio-opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector. After transformation in Schizosaccharomyces pombe, bacterio-opsin (BO) is expressed and was detected by antisera. The precursor protein of BO (pre-BO) is processed by cleavage of amino acids at the N-terminal end as in H. halobium. Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes. Therefore, in contrast to the expression in E. coli, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis. The kinetics of the ground state signal of the photocycle BR in protoplasts is demonstrated by flash spectroscopy and is comparable to that of the natural system. The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques. This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.     

Induced chirality of the light-harvesting carotenoid salinixanthin and its interaction with the retinal of xanthorhodopsin

In xanthorhodopsin, a retinal protein-carotenoid complex of Salinibacter ruber, the carotenoid salinixanthin functions as a light-harvesting antenna in supplying additional excitation energy for retinal isomerization and proton transport. Another retinal protein, archaerhodopsin, has been shown to contain a carotenoid, bacterioruberin, but without an antenna function. We report here that the binding site confers a chiral geometry on salinixanthin in xanthorhodopsin and confirm that the same is true for bacterioruberin in archaerhodopsin. Cell membranes containing these rhodopsins exhibit CD spectra with sharp positive bands in the visible region where the carotenoids absorb, and in the case of xanthorhodopsin a negative band at 536 nm, as well as bands in the UV region. The carotenoid in ethanol has very weak optical activity in the visible region of the spectrum. Denaturation of the opsin upon deprotonation of the Schiff base at pH 12.5 eliminates the induced CD bands in both proteins. In one of these proteins, but not in the other, the carotenoid binding site depends entirely on the retinal. Hydrolysis of the retinal Schiff base of xanthorhodopsin with hydroxylamine eliminates the induced CD bands of salinixanthin. In contrast, hydrolysis of the Schiff base in archaerhodopsin does not abolish the CD bands of bacterioruberin. Thus, consistent with its antenna function, the carotenoid binding site interacts closely with the retinal only in xanthorhodopsin, and this interaction is the major source of the CD bands. In this protein, protonation of the counterion with a decrease in pH from 8 to 5 causes significant changes in the CD spectrum. The observed spectral features suggest that binding of salinixanthin in xanthorhodopsin involves the cyclohexenone ring of the carotenoid and its conformational heterogeneity is restricted.     

Expression, purification, and structural characterization of the bacteriorhodopsin-aspartyl transcarbamylase fusion protein.

We are testing a strategy for creating three-dimensional crystals of integral membrane proteins which involves the addition of a large soluble domain to the membrane protein to provide crystallization contacts. As a test of this strategy we designed a fusion between the membrane protein bacteriorhodopsin (BR) and the catalytic subunit of aspartyl transcarbamylase from Escherichia coli. The fusion protein (designated BRAT) was initially expressed in E. coli at 51 mg/liter of culture, to yield active aspartyl transcarbamylase and an unfolded bacterio-opsin (BO) component. In Halobacterium salinarum, BRAT was expressed at a yield of 7 mg/liter of culture and formed a high-density purple membrane. The visible absorption properties of BRAT were indistinguishable from those of BR, demonstrating that the fusion with aspartyl transcarbamylase had no effect on BR structure. Electron microscopy of BRAT membrane sheets showed that the fusion protein was trimeric and organized in a two-dimensional crystalline lattice similar to that in the BR purple membrane. Following solubilization and size-exclusion purification in sodium dodecyl sulfate, the BO portion of the fusion was quantitatively refolded in tetradecyl maltoside (TDM). Ultracentrifugation demonstrated that BR and BRAT-TDM mixed micelles had molecular masses of 138 and 162 kDa, respectively, with a stoichiometry of one protein per micelle. High TDM concentrations (>20 mM) were required to maintain BRAT solubility, hindering three-dimensional crystallization trials. We have demonstrated that BR can functionally accommodate massive C-terminal fusions and that these fusions may be expressed in quantities required for structural investigation in H. salinarum.     

Characterization of a bacterioruberin‐producing Haloarchaea isolated from the marshlands of the Odiel river in the southwest of Spain

In this work, we describe the isolation, identification, pigment characterization, and optimization of the culture conditions for a haloarchaea strain isolated from salt evaporation ponds in the Odiel river, at Southwest of Spain. The haloarchaea belongs to the genus Halorobrum, as deduced from the analysis of its 16S rRNA encoding gene and has been designated as Halorubrum sp. SH1. The growth conditions for the new strain were optimized studying temperature, NaCl concentration, agitation rate and light intensity. The C50‐carotenoids, bacterioruberin, and its derivatives bisanhydrobacterioruberin and trisanhydrobacterioruberin, were found to be the predominant pigments produced by this strain of Halorubrum, as determined using HPLC‐DAD and UHPLC‐ESI‐MS/MS techniques. This extremely halophilic archaeon could be a good candidate for the production of bacterioruberins of high added‐value due to their coloring, antioxidant, and possible anticancer properties. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:592–600, 2016     

表面活性剂TritonX－100对菌紫质蛋白紫外荧光性质的影响

用荧光光谱方法研究了ＴｒｉｔｏｎＸ－１００（以下缩写为ＴＸ－１００）对菌紫质蛋白（Ｂａｃｔｅｒｉｏｒｈｏｄｏｐｓｉｎ,ＢＲ）及视黄醛生色团漂白后的紫膜（Ｂａｔｅｒｉｏｐｓｉｎ,ＢＯ）紫外荧光性质的影响．结果表明：表面活性剂ＴＸ－１００使ＢＲ中色氨酸在３２６ｎｍ处的荧光发射强度增加。随着ＴＸ－１００对ＢＲ的增溶,改变了生色团的构象环境,破坏了ＢＲ中色氨酸与生色团之间的能量转移。增溶后的ＢＲ中,Ｔｒｐ趋向于更疏水性的环境。     

Das rote Haloarchaeon Halobacterium salinarum

Halobacterium salinarum – microbe of the year 2017 The microbe of the year 2017 thrives in harsh and salty environments, but offers some traits for applications in biotechnology or biomedical research. Among these are the carotinoide bacterioruberin, salt‐adapted proteins and enzymes, but also the light‐driven proton pump bacteriorhodopsin and gas vesicles. Halobacterium salinarum lives planktonic and is motile, or adheres to solid surfaces and forms biofilms. Biofilms protect the cells in stress conditions and support the exchange of DNA. Haloarchaea are extremely resistant and survive hundreds of years enclosed in salt crystals. Hbt. salinarum offers a lot to explore and is enjoyable because of its red color in nature.     

Dynamics of primary events in the photocycle of excited bacteriorhodopsin

Transient dynamic behavior of the excited bacteriorhodopsin (BR), which was isolated from the strain H. salinarum, was studied at excitation wavelength from 585 to 639 nm. With the one-color femtosecond (fs) pump-probe technique, we revealed the primary events in BR's photocycle that took place in an ultrafast time scale. From the analysis of the decay components of the dynamical traces, it was evident that the isomerization of the retinal     

Carotenoid biotechnology in plants for nutritionally improved foods

Carotenoids participate in light harvesting and are essential for photoprotection in photosynthetic plant tissues. They also furnish non-photosynthetic flowers and fruits with yellow to red colors to attract animals for pollination and dispersal of seeds. Although animals can not synthesize carotenoids de novo , carotenoid-derived products such as retinoids (including vitamin A) are required as visual pigments and signaling molecules. Dietary carotenoids also provide health benefits based on their antioxidant properties. The main pathway for carotenoid biosynthesis in plants and microorganisms has been virtually elucidated in recent years, and some of the identified biosynthetic genes have been successfully used in metabolic engineering approaches to overproduce carotenoids of interest in plants. Alternative approaches that enhance the metabolic flux to carotenoids by upregulating the production of their isoprenoid precursors or interfere with light-mediated regulation of carotenogenesis have been recently shown to result in increased carotenoid levels. Despite spectacular achievements in the metabolic engineering of plant carotenogenesis, much work is still ahead to better understand the regulation of carotenoid biosynthesis and accumulation in plant cells. New genetic and genomic approaches are now in progress to identify regulatory factors that might significantly contribute to improve the nutritional value of plant-derived foods by increasing their carotenoid levels.     

Pathway engineering strategies for production of beneficial carotenoids in microbial hosts

Carotenoids, such as lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin have many benefits for human health. In addition to the functional role of carotenoids as vitamin A precursors, adequate consumption of carotenoids prevents the development of a variety of serious diseases. Biosynthesis of carotenoids is a complex process and it starts with the common isoprene precursors. Condensation of these precursors and subsequent modifications, by introducing hydroxyl- and keto-groups, leads to the generation of diversified carotenoid structures. To improve carotenoid production, metabolic engineering has been explored in bacteria, yeast, and algae. The success of the pathway engineering effort depends on the host metabolism, specific enzymes used, the enzyme expression levels, and the strategies employed. Despite the difficulty of pathway engineering for carotenoid production, great progress has been made over the past decade. We review metabolic engineering approaches used in a variety of microbial hosts for carotenoid biosynthesis. These advances will greatly expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet.     

Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara

Cara Cara is a spontaneous bud mutation of Navel orange (Citrus. sinensis L. Osbeck) characterized by developing fruits with a pulp of bright red coloration due to the presence of lycopene. Peel of mutant fruits is however orange and indistinguishable from its parental. To elucidate the basis of lycopene accumulation in Cara Cara, we analyzed carotenoid profile and expression of three isoprenoid and nine carotenoid genes in flavedo and pulp of Cara Cara and Navel fruits throughout development and maturation. The pulp of the mutant accumulated high amounts of lycopene, but also phytoene and phytofluene, from early developmental stages. The peel of Cara Cara also accumulated phytoene and phytofluene. The expression of isoprenoid genes and of carotenoid biosynthetic genes downstream PDS (phytoene desaturase) was higher in the pulp of Cara Cara than in Navel. Not important differences in the expression of these genes were observed between the peel of both oranges. Moreover, the content of the plant hormone ABA (abscisic acid) was lower in the pulp of Cara Cara, but the expression of two genes involved in its biosynthesis was higher. The results suggest that an altered carotenoid composition may conduct to a positive feedback regulatory mechanism of carotenoid biosynthesis in citrus fruits. Increased levels of isoprenoid precursors in the mutant that could be channeled to carotenoid biosynthesis may be related to the red-fleshed phenotype of Cara Cara.