Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus: Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C50 Carotenoid Cyclases

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C₅₀ carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C₄₀ lycopene, C₄₅ nonaflavuxanthin, C₅₀ flavuxanthin, and C₅₀ sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C₅₀ carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ɛ-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C₅₀ carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ɛ-cyclic C₅₀ carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ɛ- and γ-cyclic C₅₀ carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C₅₀ carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.Carotenoids are natural pigments synthesized by bacteria, fungi, algae, and plants, and more than 750 different carotenoids have been isolated from natural sources (17). They possess important biological functions as protectants against light and oxygen excess in photosynthetic processes (32, 38), and they have been proposed to reduce the risk of certain cancers, cardiovascular disease, and Alzheimer disease due to their antioxidative properties (20, 46). The global market for carotenoids used as food colorants and nutritional supplements was estimated at approximately $935 million in 2005 (11). More than 95% of all natural carotenoids are based on a symmetric C₄₀ phytoene backbone, and only a small number of C₃₀ and even fewer C₅₀ carotenoids have been discovered (42).C₅₀ carotenoids have multiple conjugated double bonds, and they contain at least one hydroxyl group; both these features contribute to strong antioxidative properties (17, 30, 32, 38). In nature, C₅₀ carotenoids are synthesized by bacteria of the order Actinomycetales, and to date, only two different C₅₀ carotenoid biosynthetic pathways have been described in the literature. The biosynthetic pathways of the ɛ-cyclic C₅₀ carotenoid decaprenoxanthin [2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-ɛ,ɛ-carotene] and the β-cyclic C₅₀ carotenoid C.p.450 [2,2′-bis-(4-hydroxy-3-methybut-2-enyl)-β,β-carotene] have been elucidated in Corynebacterium glutamicum (22, 23) and in Dietzia sp. CQ4 (41), respectively. For both pathways, the common precursor, C₄₀ lycopene, is synthesized from C₁₅ farnesyl pyrophosphate (FPP) via the methylerythritol 4-phosphate (MEP) pathway, which is present in most eubacteria (33). Effective lycopene production has been achieved in genetically engineered noncarotenogenic hosts, such as Escherichia coli and Saccharomyces cerevisiae (9). Accordingly, the potential of using such biotechnologically relevant hosts for heterologous production of any lycopene-derived carotenoids has generated high interest.The biosynthesis of cyclic C₅₀ carotenoids from lycopene is catalyzed by lycopene elongase and carotenoid cyclases. Even though most carotenoids in plants and microorganisms exhibit cyclic structures, cyclization reactions were predominantly known for C₄₀ pathways (45) catalyzed by monomeric enzymes that have been isolated from plants and bacteria (5, 16, 27, 29, 31, 36). In C. glutamicum, the genes crtYe, crtYf, and crtEb were identified as being involved in the conversion of lycopene to the ɛ-cyclic C₅₀ carotenoid decaprenoxanthin (22, 44). Sequential elongation of lycopene into the acyclic C₅₀ carotenoid flavuxanthin was catalyzed by the crtEb gene product lycopene elongase. Subsequent cyclization to decaprenoxanthin was catalyzed by a heterodimeric C₅₀ carotenoid, ɛ-cyclase, encoded by crtYe and crtYf (22). C. glutamicum can synthesize both mono- and diglucosylated decaprenoxanthin; however, the genetic and enzymatic bases for glucosylation of decaprenoxanthin are unknown. Analogous to decaprenoxanthin, biosynthesis of the β-cyclic C₅₀ carotenoid C.p.450 in Dietzia sp. CQ4 from lycopene involves lycopene elongase and C₅₀ carotenoid β-cyclase activities (41).While most cyclic carotenoids exhibit β-rings, ɛ-ring-containing pigments are common in higher plants (7), and carotenoids substituted only with γ-rings are rarely observed in plants and algae (14). To date, no biosynthetic pathway for γ-cyclic C₅₀ carotenoids has been reported in the literature.Micrococcus luteus NCTC2665 (the “Fleming strain”) is a Gram-positive bacterium belonging to the family Micrococcaceae within the order Actinomycetales. The carotenoids, including the γ-cyclic C₅₀ sarcinaxanthin [(2R,6R,2′R,6′R)-(2,2′-bis(4-hydroxy-3-methyl-2-butenyl)-γ,γ-carotene)], synthesized by this bacterium have been identified and structurally elucidated (26). We recently isolated and characterized several wild-type M. luteus strains from the sea surface microlayer of the middle part of the Norwegian coast (39). Here, we report one additional such marine M. luteus isolate, designated Otnes7, forming color-intensive colonies indicating high sarcinaxanthin production levels. Both Otnes7 and NCTC2665 were used as M. luteus model strains, and the sarcinaxanthin biosynthetic gene clusters were cloned from both strains. The complete sarcinaxanthin biosynthetic pathway from lycopene was elucidated, including glucosylation, and we also explored the potential of using Otnes7-derived genes to achieve effective heterologous production of sarcinaxanthin in E. coli. The results add important new knowledge of the biosynthesis of C₅₀ carotenoids, and in particular, they highlight the diverse functions of C₅₀ carotenoid cyclases leading to synthesis of structurally different carotenoids.     

Cold adapted features of Vibrio salmonicida catalase: characterisation and comparison to the mesophilic counterpart from Proteus mirabilis

The gene encoding catalase from the psychrophilic marine bacterium Vibrio salmonicida LFI1238 was identified, cloned and expressed in the catalase-deficient Escherichia coli UM2. Recombinant catalase from V. salmonicida (VSC) was purified to apparent homogeneity as a tetramer with a molecular mass of 235 kDa. VSC contained 67% heme b and 25% protoporphyrin IX. VSC was able to bind NADPH, react with cyanide and form compounds I and II as other monofunctional small subunit heme catalases. Amino acid sequence alignment of VSC and catalase from the mesophilic Proteus mirabilis (PMC) revealed 71% identity. As for cold adapted enzymes in general, VSC possessed a lower temperature optimum and higher catalytic efficiency (k _cat/K _m) compared to PMC. VSC have higher affinity for hydrogen peroxide (apparent K _m) at all temperatures. For VSC the turnover rate (k _cat) is slightly lower while the catalytic efficiency is slightly higher compared to PMC over the temperature range measured, except at 4°C. Moreover, the catalytic efficiency of VSC and PMC is almost temperature independent, except at 4°C where PMC has a twofold lower efficiency compared to VSC. This may indicate that VSC has evolved to maintain a high efficiency at low temperatures.     

Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose Biosynthesis

dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.     

In vivo tumor growth inhibition and biodistribution studies of locked nucleic acid (LNA) antisense oligonucleotides

Locked nucleic acids (LNA) are novel high-affinity DNA analogs that can be used as genotype-specific drugs. The LNA oligonucleotides (LNA PO ODNs) are very stable in vitro and in vivo without the need for a phosphorothiolated backbone. In this study we tested the biological fate and the efficacy in tumor growth inhibition of antisense oligonucleotides directed against the gene of the large subunit of RNA polymerase II (POLR2A) that are completely synthesized as LNA containing diester backbones. These full LNA oligonucleotides strongly reduce POLR2A protein levels. Full LNA PO ODNs appeared to be very stable compounds when injected into the circulation of mice. Full LNA PO ODNs were continuously administered for 14 days to tumor-bearing nude mice. Tumor growth was inhibited sequence specifically at dosages from 1 mg/kg/day. LNA PO ODNs appeared to be non-toxic at dosages <5 mg/kg/day. Biodistribution studies showed the kidneys to have the highest uptake of LNA PO ODNs and urinary secretion as the major route of clearance. This report shows that LNA PO ODNs are potent genotype-specific drugs that can inhibit tumor growth in vivo.     

WRN interacts physically and functionally with the recombination mediator protein RAD52

Werner syndrome (WS) is a premature aging disorder that predisposes affected individuals to cancer development. The affected gene, WRN, encodes an RecQ homologue whose precise biological function remains elusive. Altered DNA recombination is a hallmark of WS cells suggesting that WRN plays an important role in these pathways. Here we report a novel physical and functional interaction between WRN and the homologous recombination mediator protein RAD52. Fluorescence resonance energy transfer (FRET) analyses show that WRN and RAD52 form a complex in vivo that co-localizes in foci associated with arrested replication forks. Biochemical studies demonstrate that RAD52 both inhibits and enhances WRN helicase activity in a DNA structure-dependent manner, whereas WRN increases the efficiency of RAD52-mediated strand annealing between non-duplex DNA and homologous sequences contained within a double-stranded plasmid. These results suggest that coordinated WRN and RAD52 activities are involved in replication fork rescue after DNA damage.     

Identification of cofactor discrimination sites in NAD-isocitrate dehydrogenase from Pyrococcus furiosus

The role of Asp-328 and Ile-329 as a cofactor discrimination site of the NAD-dependent isocitrate dehydrognase (NAD-IDH) from Pyrococcus furiosus has been verified by replacing these residues with Lys and Tyr, respectively, which are the corresponding residues in NADP-IDH from Escherichia coli. The Asp-328-Lys mutant showed dual coenzyme specificity, whereas introduction of the double mutation, Asp-328-Lys/Ile-329-Tyr shifted the cofactor preference from NAD to NADP. NADP-dependent P. furiosus IDH retained thermostability and thermoactivity compared with NAD-IDH.     

Hypomorphic phenotype in mice with pituitary‐specific knockout of steroidogenic factor 1

Summary: The bacteriophage Cre recombinase provides a powerful approach for tissue‐specific gene inactivation. Using a Cre transgene driven by the common alpha subunit of glycoprotein hormones (αGSU‐Cre), we have previously inactivated steroidogenic factor 1 (SF‐1) in the anterior pituitary, causing hypogonadotropic hypogonadism with sexual infantilism, sterility, and severe gonadal hypoplasia. We now explore the molecular mechanisms underlying a hypomorphic gonadal phenotype in mice carrying two floxed SF‐1 alleles (F/F) relative to mice carrying one recombined and one floxed allele (F/R). Because their Cre‐mediated disruption of the locus encoding SF‐1 was less efficient, αGSU‐Cre, F/F mice retained some gonadotropin‐expressing cells in the anterior pituitary, thereby stimulating some gonadal function. This novel in vivo model for exploring the effects of differing levels of gonadotropins on gonadal development highlights the need for careful genotype‐phenotype comparisons in studies using Cre recombinase to produce tissue‐specific knockouts. genesis 30:65–69, 2001. © 2001 Wiley‐Liss, Inc.     

Anaerobic Benzene Biodegradation: A Microcosm Survey

Benzene-amended microcosms prepared with saturated soil or sediment from five hydrocarbon-contaminated sites and one pristine site were monitored for a year and a half to determine the rate of benzene biodegradation under a variety of electron-accepting conditions. Sustainable benzene degradation was observed under specific conditions in microcosms from four of the six sites. Significant differences were observed between sites with respect to lag times before the onset of degradation, rates of degradation, sustainability of the activity, and environmental conditions supporting degradation. Benzene degradation was observed under sulfate-reducing, nitrate-reducing, and iron(III)-reducing conditions, but not under methanogenic conditions. The presence of competing substrates such as toluene, xylenes, and ethylbenzene was found to inhibit anaerobic benzene degradation in microcosms where sulfate or possibly nitrate was the electron acceptor for benzene degradation, but not in microcosms from where iron(III) was the electron acceptor. The presence of organic matter, indicated by a high fraction organic carbon (f_oc), also appeared to inhibit the biodegradation of benzene; microcosms constructed with soils with the highest f_oc exhibited the longest lag times before the onset of benzene degradation. The initial extent of hydrocarbon contamination did not appear to correlate with anaerobic benzene-degrading activity.     

Analytical procedures for the quantification of isotopic amino acid incorporation into photosynthetic proteins of Synechocystis PCC 6803

The mechanism of oxygen evolution has been an enigma for nearly two centuries. Pioneering work by Bessel Kok, Pierre Joliot, and many others during the last quarter century has provided valuable insight into this most unique and important chemical reaction. The late 1970s and early 1980s saw the introduction of biochemical techniques for the purification of photosynthetic complexes that have, in turn, stimulated the biophysical chemists and spectroscopists to apply high resolution techniques in order to resolve the structure/function relationships in these protein complexes. Valuable information about events at the atomic level can be gained through isotopic substitution of particular amino acids thought to be important in the catalytic process. The ability to generate functional auxotrophs in the photosynthetic cyanobacterium Synechocystis 6803 has been used successfully to identify the redox active components Z and D as tyrosine residues in the reaction center of Photosystem II. In this report, we present results of the application of specific isotopic labeling for high resolution spectroscopy of purified PS II particles. We have developed analytical procedures for monitoring the incorporation of both ²H and ¹⁷O labeled amino acids by gas chromatography-mass spectroscopic analysis. We also show that the growth curve of cells subjected to obligate auxotrophy displays two distinct stationary phases; one that corresponds to depletion of exogenous amino acids, and a second that corresponds to the normal cell density at stationary phase. Cells harvested at the second stationary phase show little or no retention of the labeled amino acid.Abbreviations D1 D2 reaction center core proteins of Photosystem II encoded by the psbA and psbD genes, respectively - FTIR Fourier transform infrared - ENDOR electron-nuclear double resonance - ESEEM electron spin-echo envelope modulation - EXAFS extended X-ray absorption fine structure - iBuCF isobutylchloroformate - OD optical density - XANES X-ray absorption near-edge structure - Y_D Y_Z redox active tyrosines of PS II