Identification of a cytochrome bc1-aa3 supercomplex in Rhodobacter sphaeroides.

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc1 and aa3-type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O2 reduction activity of the cyt. bc1-aa3 supercomplex. The potential role of the membrane-anchored cyt. cy as a component in supercomplexes was also investigated.

Plasmid-Free System and Modular Design for Efficient 5-Aminolevulinic Acid Production by Engineered Escherichia coli.

5-Aminolevulinic acid (ALA) is an essential intermediate for many organisms and has been considered for the applications of medical especially in photodynamic therapy of cancer recently. However, ALA production via chemical approach is complicated; hence, microbial manufacturing has received more attentions. In this study, a modular design to simultaneously express ALA synthase from Rhodobacter sphaeroides (RshemA), a non-specific ALA exporter (RhtA), and chaperones was first developed and discussed. The ALA production was significantly increased by coexpressing RhtA and RshemA. Besides, ALA was enhanced by the cofactor pyridoxal phosphate (PLP) which was supplied by expressing genes of pdxK and pdxY or direct addition. However, inclusion bodies of RshemA served as an obstacle; thus, chaperones DnaK and GroELS were introduced to reform the conformation of proteins and successfully improved ALA production. Finally, a plasmid-free strain RrGI, as the robust chassis, was established and a 6.23-fold enhancement on ALA biosynthesis and led to 7.47 g/L titer and 0.588 g/L/h productivity under the optimal cultural condition.

In Situ, Protein-Mediated Generation of a Photochemically Active Chlorophyll Analogue in a Mutant Bacterial Photosynthetic Reaction Center.

All possible natural amino acids have been substituted for the native LeuL185 positioned near the B-side bacteriopheophytin (HB) in the bacterial reaction center (RC) from Rhodobacter sphaeroides. Additional mutations that enhance electron transfer to the normally inactive B-side cofactors are present. Approximately half of the isolated RCs with Glu at L185 contain a magnesium chlorin (CB) in place of HB. The chlorin is not the common BChl a oxidation product 3-desvinyl-3-acetyl chlorophyll a with a C-C bond in ring D and a C═C bond in ring B but has properties consistent with reversal of these bond orders, giving 17,18-didehydro BChl a. In such RCs, charge-separated state P+CB- forms in ∼5% yield. The other half of the GluL185-containing RCs have a bacteriochlorophyll a (BChl a) denoted ßB in place of HB. Residues His, Asp, Asn, and Gln at L185 yield RCs with ≥85% ßB in the HB site, while most other amino acids result in RCs that retain HB (≥95%). To the best of our knowledge, neither bacterial RCs that harbor five BChl a molecules and one chlorophyll analogue nor those with six BChl a molecules have been reported previously. The finding that altering the local environment within a cofactor binding site of a transmembrane complex leads to in situ generation of a photoactive chlorin with an unusual ring oxidation pattern suggests new strategies for amino acid control over pigment type at specific sites in photosynthetic proteins.

Dynamic band-shift signal in two-dimensional electronic spectroscopy: A case of bacterial reaction center.

Optical nonlinear spectroscopies carry a high amount of information about the systems under investigation; however, as they report polarization signals, the resulting spectra are often congested and difficult to interpret. To recover the landscape of energy states and physical processes such as energy and electron transfer, a clear interpretation of the nonlinear signals is prerequisite. Here, we focus on the interpretation of the electrochromic band-shift signal, which is generated when an internal electric field is established in the system following optical excitation. Whereas the derivative shape of the band-shift signal is well understood in transient absorption spectroscopy, its emergence in two-dimensional electronic spectroscopy (2DES) has not been discussed. In this work, we employed 2DES to follow the dynamic band-shift signal in reaction centers of purple bacteria Rhodobacter sphaeroides at 77 K. The prominent two-dimensional derivative-shape signal appears with the characteristic formation time of the charge separated state. To explain and characterize the band-shift signal, we use expanded double-sided Feynman diagram formalism. We propose to distinguish two types of Feynman diagrams that lead to signals with negative amplitude: excited state absorption and re-excitation. The presented signal decomposition and modeling analysis allows us to recover precise electrochromic shifts of accessory bacteriochlorophylls, identify additional signals in the B band range, and gain a further insight into the electron transfer mechanism. In a broader perspective, expanded Feynman diagram formalism will allow for interpretation of all 2D signals in a clearer and more intuitive way and therefore facilitate studying the underlying photophysics.

Time-Resolved Electrometric Study of the F→O Transition in Cytochrome c Oxidase. The Effect of Zn2+ Ions on the Positive Side of the Membrane.

The effect of Zn2+ on the P-side of proteoliposomes containing membrane-incorporated Rhodobacter sphaeroides cytochrome c oxidase was investigated by the time-resolved electrometrics following a single electron injection into the enzyme prepared in the F state. The wild-type enzyme was examined along with the two mutants, N139D and D132N. All obtained data indicate that the primary effect of Zn2+ added from the P-side of the membrane is slowing of the pumped proton release from the proton loading site (PLS) to the bulk aqueous phase on the P-side of the membrane. The results strongly suggest the presence of two pathways by which the pumped proton can exit the protein from the PLS and of two separate binding sites for Zn2+. A model is presented to explain the influence of Zn2+ on the kinetics of membrane-potential generation by the wild-type COX, as well as by the N139D and D132N mutants.

Introducing a delivery system for melanogenesis inhibition in melanoma B16F10 cells mediated by the conjugation of tyrosine ammonia-lyase and a TAT-penetrating peptide.

Hyperpigmentation disorders negatively influence an individual's quality of life and may cause emotional distress. Over the years, various melanogenesis inhibitors (mainly tyrosinase inhibitors) have been developed, most of which with low efficacy or high toxicity. Although metabolic engineering by deviation in the flux of substrate is of considerable interest, trials to develop a melanogenesis inhibitor based on L-tyrosine (L-Tyr) restriction are missing. We propose a novel proteinaceous melanogenesis inhibitor called tyrosine ammonia-lyase (TAL), an enzyme that catalyzes the conversion of L-Tyr to p-coumaric acid and ammonia. Since the cell membrane can act as a barrier for intracellular protein delivery, we have covalently conjugated a recombinant TAL enzyme from Rhodobacter sphaeroides (RsTAL) to a trans-activator of transcription (TAT) cell-penetrating peptide (CPP) to afford the intracellular delivery. The heterologously expressed TAT-RsTAL fusion protein was delivered successfully into B16F10 melanocytes as confirmed by the direct fluorescence microscopy with increased intensity from 30 to 180 min. TAT-RsTAL showed sufficient intracellular activity of about 0.83 ± 0.04 and 0.34 ± 0.03 nmol•mg-1 •s-1 for the native and inclusion body-extracted conjugates, respectively. The conjugate inhibited melanin biosynthesis in B16F10 cells in a time-dependent manner. Melanin accumulation was inhibited by 12.7 ± 6.2%, 28.2 ± 5.7%, and 33.9 ± 2.9% compared to the nontreated control groups after 24, 48, and 72 hr of incubation, respectively. L-Tyr restriction had no significant effect on the cell viability up to a concentration of 100 µgml-1 even after 72 hr. According to the observed hypopigmentary effect of the conjugate in this study, TAT-RsTAL can be suggested as a melanogenesis inhibitor for further investigations.

Characterization of a Rhodobacter sphaeroides primary fatty acid kinase.

Widely distributed among prokaryotes, short chain fatty acid kinases provide a path for fatty acid entry into central metabolic pathways. These enzymes catalyze the reversible, ATP-dependent synthesis of acyl-phosphates, which leads to the production of acyl-CoA derivatives by a coordinate acyltransferase. To date, characterized representatives of short chain fatty acid kinases exhibit relatively narrow substrate specificity. In this work, biochemical characterization of a predicted acetate kinase from Rhodobacter sphaeroides reveals a novel enzyme with broad substrate specificity for primary fatty acids of varying lengths (C2--C8).

The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth.

Plant photosynthesis and growth are often limited by the activity of the CO2-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants. Here, we show this impediment does not extend to Rubisco from Rhodobacter sphaeroides (RsRubisco)-a red-type Rubisco able to assemble in plant chloroplasts. In transplastomic tobRsLS lines expressing a codon optimized Rs-rbcLS operon, the messenger RNA (mRNA) abundance was ∼25% of rbcL transcript and RsRubisco ∼40% the Rubisco content in WT tobacco. To mitigate the low activation status of RsRubisco in tobRsLS (∼23% sites active under ambient CO2), the metabolic repair protein RsRca (Rs-activase) was introduced via nuclear transformation. RsRca production in the tobRsLS::X progeny matched endogenous tobacco Rca levels (∼1 µmol protomer·m2) and enhanced RsRubisco activation to 75% under elevated CO2 (1%, vol/vol) growth. Accordingly, the rate of photosynthesis and growth in the tobRsLS::X lines were improved >twofold relative to tobRsLS. Other tobacco lines producing RsRubisco containing alternate diatom and red algae S-subunits were nonviable as CO2-fixation rates (kcatc) were reduced >95% and CO2/O2 specificity impaired 30-50%. We show differences in hybrid and WT RsRubisco biogenesis in tobacco correlated with assembly in Escherichia coli advocating use of this bacterium to preevaluate the kinetic and chloroplast compatibility of engineered RsRubisco, an isoform amenable to directed evolution.

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase.

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

Substrate recognition induces sequential electron transfer across subunits in the nitrogenase-like DPOR complex.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR is made of electron donor (BchL) and acceptor (BchNB) component proteins. BchNB is further composed of two subunits each of BchN and BchB arranged as an α2ß2 heterotetramer with two active sites for substrate reduction. Such oligomeric architectures are found in several other electron transfer (ET) complexes, but how this architecture influences activity is unclear. Here, we describe allosteric communication between the two identical active sites in Rhodobacter sphaeroides BchNB that drives sequential and asymmetric ET. Pchlide binding to one BchNB active site initiates ET from the pre-reduced [4Fe-4S] cluster of BchNB, a process similar to the deficit spending mechanism observed in the structurally related nitrogenase complex. Pchlide binding in one active site is recognized in trans by an Asp-274 from the opposing half, which is positioned to serve as the initial proton donor. A D274A variant DPOR binds to two Pchlide molecules in the BchNB complex, but only one is bound productively, stalling Pchlide reduction in both active sites. A half-active complex combining one WT and one D274A monomer also stalled after one electron was transferred in the WT half. We propose that such sequential electron transfer in oligomeric enzymes serves as a regulatory mechanism to ensure binding and recognition of the correct substrate. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.

Cytochrome c oxidase oxygen reduction reaction induced by cytochrome c on nickel-coordination surfaces based on graphene oxide in suspension.

BACKGROUND: The electrochemical and spectroscopic investigation of bacterial electron-transfer proteins stabilized on solid state electrodes has provided an effective approach for functional respiratory enzyme studies. METHODS: We assess the biocompatibility of carboxylated graphene oxide (CGO) functionalized with Nickel nitrilotriacetic groups (CGO-NiNTA) ccordinating His-tagged cytochrome c oxidase (CcO) from Rhodobacter sphaeroides. RESULTS: Kinetic studies employing UV-visible absorption spectroscopy confirmed that the immobilized CcO oxidized horse-heart cytochrome c (Cyt c) albeit at a slower rate than isolated CcO. The oxygen reduction reaction as catalyzed by immobilized CcO could be clearly distinguished from that arising from CGO-NiNTA in the presence of Cyt c and dithiothreitol (DTT) as a sacrificial reducing agent. Our findings indicate that while the protein content is about 3.7‰ by mass with respect to the support, the contribution to the oxygen consumption activity averaged at 56.3%. CONCLUSIONS: The CGO-based support stabilizes the free enzyme which, while capable of Cyt c oxidation, is unable to carry out oxygen consumption in solution on its own under our conditions. The turnover rate for the immobilized CcO was as high as 240 O2 molecules per second per CcO unit. GENERAL SIGNIFICANCE: In vitro investigations of electron flow on isolated components of bacterial electron-transfer enzymes immobilized on the surface of CGO in suspension are expected to shed new light on microbial bioenergetic functions, that could ultimately contribute toward the improvement of performance in living organisms.

Characterisation of the Cyanate Inhibited State of Cytochrome c Oxidase.

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane. Oxygen consumption is inhibited by various substances. Here we tested the relatively unknown inhibition of cytochrome c oxidase (CcO) with isocyanate. In contrast to other more common inhibitors like cyanide, inhibition with cyanate was accompanied with the rise of a metal to ligand charge transfer (MLCT) band around 638 nm. Increasing the cyanate concentration furthermore caused selective reduction of heme a. The presence of the CT band allowed for the first time to directly monitor the nature of the ligand via surface-enhanced resonance Raman (SERR) spectroscopy. Analysis of isotope sensitive SERR spectra in comparison with Density Functional Theory (DFT) calculations identified not only the cyanate monomer as an inhibiting ligand but suggested also presence of an uretdion ligand formed upon dimerization of two cyanate ions. It is therefore proposed that under high cyanate concentrations the catalytic site of CcO promotes cyanate dimerization. The two excess electrons that are supplied from the uretdion ligand lead to the observed physiologically inverse electron transfer from heme a3 to heme a.

Dynamics of the Excited State in Photosynthetic Bacterial Reaction Centers.

In the initial charge-separation reaction of photosynthetic bacterial reaction centers, a dimer of strongly interacting bacteriochlorophylls (P) transfers an electron to a third bacteriochlorophyll (BL). It has been suggested that light first generates an exciton state of the dimer and that an electron then moves from one bacteriochlorophyll to the other within P to form a charge-transfer state (PL+PM-), which passes an electron to BL. This scheme, however, is at odds with the most economical analysis of the spectroscopic properties of the reaction center and particularly with the unusual temperature dependence of the long-wavelength absorption band. The present paper explores this conflict with the aid of a simple model in which exciton and charge-transfer states are coupled to three vibrational modes. It then uses a similar model to show that the main experimental evidence suggesting the formation of PL+PM- as an intermediate could reflect pure dephasing of vibrational modes that modulate stimulated emission.

High-Pressure Modulation of Primary Photosynthetic Reactions.

Photochemical charge separation is key to biological solar energy conversion. Although many features of this highly quantum-efficient process have been described, others remain poorly understood. Herein, ultrafast fluorescence barospectroscopy is used for the first time to obtain insights into the mechanism of primary charge separation in a YM210W mutant bacterial reaction center under novel surrounding modulating conditions. Over a range of applied hydrostatic pressures reaching 10 kbar, the rate of primary charge separation monotonously increased and that of the electron transfer to secondary acceptor decreased. While the inferred free energy gap for charge separation generally narrowed with increasing pressure, a pressure-induced break of a protein-cofactor hydrogen bond observed at ∼2 kbar significantly (by 219 cm-1 or 27 meV) increased this gap, resulting in a drop in fluorescence. The findings strongly favor a model for primary charge separation that incorporates charge recombination and restoration of the excited primary pair state, over a purely sequential model. We show that the main reason for the almost threefold acceleration of the primary electron transfer rate is the pressure-induced increase of the electronic coupling energy, rather than a change of activation energy. We also conclude that across all applied pressures, the primary electron transfer in the mutant reaction center studied can be considered nonadiabatic, normal region, and thermally activated.

Quantification, regulation and production of 5-aminolevulinic acid by green fluorescent protein in recombinant Escherichia coli.

5-Aminolevulinic acid (5-ALA) is an unnatural amino acid and has been approved as a biodegradable, non-toxic pesticide and herbicide with applications in sustainable agriculture. 5-ALA can also be applied for cancer targeting via tumor localization and photodynamic therapy. Herein, we developed a feasible quantification, regulation and production method of 5-ALA in Escherichia coli is based on the chimera of 5-ALA synthetase from Rhodobacter sphaeroides (RshemA) and super-fold green fluorescent protein (sfGFP) under the control of dual promoters/double plasmids. 5-ALA production based on quantification with the reporter sfGFP was unsuccessfully for the RshemA-sfGFP fusion protein owing to a steric hindrance effect, but was effective using dual constitutive promoters (i.e., J23100 and PLacI) for RshemA and sfGFP independently. Moreover, a simple quantification method based on the linear relationship between 5-ALA concentration and the change in sfGFP intensity was calculated with the Hill equation according to the results of dual plasmids which composed of RshemA-threonine/homoserine exporter (RhtA) and the sensing plasmid pSU-T7-sfGFP. Compared with the conventional detection method for 5-ALA using Ehrlich's reagent, our proposed method is advantages in effectiveness, real-time detection, and outstanding sensitivity. Finally, the highest yield of 5-ALA was obtained in E. coli D2TT strain, reaching 2.46 g/L of 5-ALA produced in a 2.5-L baffle flask fermentation. Hence, this approach shows strong potential for improving 5-ALA production with appropriate regulation and detection based on the fluorescent signal.

Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry.

Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme. Replacement of the protonatable residue Glu552, part of the same loop, by an Ala, resulted in a similar decrease in the proton-pumping stoichiometry without loss of the O2-reduction activity or changes in the proton-uptake kinetics. The data show that minor structural changes at the orifice of the D pathway, at a distance of ~40 Šfrom the proton gate of cytochrome c oxidase, may alter the proton-pumping stoichiometry of the enzyme.

Identification and Characterization of a New Class of (6-4) Photolyase from Vibrio cholerae.

Light is crucial for many biological activities of most organisms, including vision, resetting of circadian rhythm, photosynthesis, and DNA repair. The cryptochrome/photolyase family (CPF) represents an ancient group of UV-A/blue light sensitive proteins that perform different functions such as DNA repair, circadian photoreception, and transcriptional regulation. The CPF is widely distributed throughout all organisms, including marine prokaryotes. The bacterium Vibrio cholerae was previously shown to have a CPD photolyase that repairs UV-induced thymine dimers and two CRY-DASHs that repair UV-induced single-stranded DNA damage. Here, we characterize a hypothetical gene Vca0809 encoding a new member of CPF in this organism. The spectroscopic analysis of the purified protein indicated that this enzyme possessed a catalytic cofactor, FAD, and photoantenna chromophore 6,7-dimethyl 8-ribityl-lumazin. With a slot blot-based DNA repair assay, we showed that it possessed (6-4) photolyase activity. Further phylogenetic and computational analyses enabled us to classify this gene as a member of the family of iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP). Therefore, we named this gene Vc(6-4) FeS-BCP.

Dissecting the cytochrome c2-reaction centre interaction in bacterial photosynthesis using single molecule force spectroscopy.

The reversible docking of small, diffusible redox proteins onto a membrane protein complex is a common feature of bacterial, mitochondrial and photosynthetic electron transfer (ET) chains. Spectroscopic studies of ensembles of such redox partners have been used to determine ET rates and dissociation constants. Here, we report a single-molecule analysis of the forces that stabilise transient ET complexes. We examined the interaction of two components of bacterial photosynthesis, cytochrome c2 and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC-LH1-PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome c2 molecules. Microscale patterns of cytochrome c2 and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome c2 Following the transient association of photo-oxidised RC and reduced cytochrome c2 molecules, retraction of the RC-functionalised probe met with resistance, and forces between 112 and 887 pN were required to disrupt the post-ET RC-c2 complex, depending on the retraction velocities used. If tip-attached RCs were reduced instead, the probability of interaction with reduced cytochrome c2 molecules decreased 5-fold. Thus, the redox states of the cytochrome c2 haem cofactor and RC 'special pair' bacteriochlorophyll dimer are important for establishing a productive ET complex. The millisecond persistence of the post-ET cytochrome c2[oxidised]-RC[reduced] 'product' state is compatible with rates of cyclic photosynthetic ET, at physiologically relevant light intensities.

Economical synthesis of 14C-labeled aminolevulinic acid for specific in situ labeling of plant tetrapyrroles.

The application of metabolic radiolabeling techniques to plant tetrapyrroles, i.e., chlorophyll and hemes, is complicated by the difficulty of obtaining sufficient quantities of radiolabeled aminolevulinic acid (ALA). ALA, the first committed intermediate in the tetrapyrrole biosynthetic pathway, is inconvenient to synthesize chemically and is generally not produced in significant quantities in biological systems. Radiolabeled ALA is therefore usually quite expensive and available only in limited quantities. Here, we describe bulk biosynthesis and purification of 14C-labeled ALA from 14C glycine. We first cloned ALA synthase (ALAS) from Rhodobacter sphaeroides into an expression vector for expression and purification as a fusion with maltose-binding protein. We then used the purified ALAS to synthesize ALA in vitro from 14C-labeled glycine and succinyl-coenzyme A. Finally, we used ion exchange chromatography to separate the ALA product from the crude reaction. We achieved conversion and recovery efficiencies of 80-90%, and chlorophyll radiolabeling experiments with the 14C ALA product revealed no detectable non-specific incorporation into proteins. The ability to economically produce robust quantities of 14C ALA using common methodologies provides a new tool for working with tetrapyrroles, which includes both hemes and chlorophylls and their respective binding proteins. This tool allows the specific detection and quantification of the tetrapyrrole of interest from standard acrylamide gels or hybridization transfer membranes via radiographic imaging, which enables a wide array of experiments involving spatial and temporal resolution of the movement of pigments as they are synthesized, incorporated into their target binding proteins, and eventually degraded.

Co-production of farnesol and coenzyme Q10 from metabolically engineered Rhodobacter sphaeroides.

BACKGROUND: Farnesol is an acyclic sesquiterpene alcohol present in the essential oils of various plants in nature. It has been reported to be valuable in medical applications, such as alleviation of allergic asthma, gliosis, and edema as well as anti-cancerous and anti-inflammatory effects. Coenzyme Q10 (CoQ10), an essential cofactor in the aerobic respiratory electron transport chain, has attracted growing interest owing to its clinical benefits and important applications in the pharmaceutical, food, and health industries. In this work, co-production of (E,E)-farnesol (FOH) and CoQ10 was achieved by combining 3 different exogenous terpenes or sesquiterpene synthase with the RNA interference of psy (responsible for phytoene synthesis in Rhodobacter sphaeroides GY-2). RESULTS: FOH production was significantly increased by overexpressing exogenous terpene synthase (TPS), phosphatidylglycerophosphatase B (PgpB), and sesquiterpene synthase (ATPS), as well as RNAi-mediated silencing of psy coding phytoene synthase (PSY) in R. sphaeroides strains. Rs-TPS, Rs-ATPS, and Rs-PgpB respectively produced 68.2%, 43.4%, and 21.9% higher FOH titers than that of the control strain. Interestingly, the CoQ10 production of these 3 recombinant R. sphaeroides strains was exactly opposite to that of FOH. However, CoQ10 production was almost unaffected in R. sphaeroides strains modified by psy RNA interference. The highest FOH production of 40.45 mg/L, which was twice as high as that of the control, was obtained from the TPS-PSYi strain, where the exogenous TPS was combined with the weakening of the phytoene synthesis pathway via psy RNA interference. CoQ10 production in TPS-PSYi, ATPS-PSYi, and PgpB-PSYi was decreased and lower than that of the control strain. CONCLUSIONS: The original flux that contributed to phytoene synthesis was effectively redirected to provide precursors toward FOH or CoQ10 synthesis via psy RNA interference, which led to weakened carotenoid synthesis. The improved flux that was originally involved in CoQ10 production and phytoene synthesis was redirected toward FOH synthesis via metabolic modification. This is the first reported instance of FOH and CoQ10 co-production in R. sphaeroides using a metabolic engineering strategy.