Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum

Heider S, Peters-Wendisch P, Wendisch VF. Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum. BMC Microbiology. 2012;12(1): 198.Background Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. Results Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 epsilon-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum DeltacrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum DeltacrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 +/- 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum DeltacrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 +/- 0.3 mg/g CDW were obtained. Conclusion C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production

Characterization of 3-phosphoglycerate kinase from Corynebacterium glutamicum and its impact on amino acid production

Komati Reddy G, Wendisch VF. Characterization of 3-phosphoglycerate kinase from Corynebacterium glutamicum and its impact on amino acid production. BMC Microbiology. 2014;14(1): 54.Background Corynebacterium glutamicum cg1790/pgk encodes an enzyme active as a 3-phosphoglycerate kinase (PGK) (EC 2.7.2.3) catalyzing phosphoryl transfer from 1,3-biphosphoglycerate (bPG) to ADP to yield 3-phosphoglycerate (3-PG) and ATP in substrate chain phosphorylation. Results C. glutamicum 3-phosphoglycerate kinase was purified to homogeneity from the soluble fraction of recombinant E. coli. PGKHis was found to be active as a homodimer with molecular weight of 104 kDa. The enzyme preferred conditions of pH 7.0 to 7.4 and required Mg2+ for its activity. PGKHis is thermo labile and it has shown maximal activity at 50–65°C. The maximal activity of PGKHis was estimated to be 220 and 150 U mg-1 with KM values of 0.26 and 0.11 mM for 3-phosphoglycerate and ATP, respectively. A 3-phosphoglycerate kinase negative C. glutamicum strain ∆pgk was constructed and shown to lack the ability to grow under glycolytic or gluconeogenic conditions unless PGK was expressed from a plasmid to restore growth. When pgk was overexpressed in L-arginine and L-ornithine production strains the production increased by 8% and by 17.5%, respectively. Conclusion Unlike many bacterial PGKs, C. glutamicum PGK is active as a homodimer. PGK is essential for growth of C. glutamicum with carbon sources requiring glycolysis and gluconeogenesis. Competitive inhibition by ADP reveals the critical role of PGK in gluconeogenesis by energy charge. Pgk overexpression improved the productivity in L-arginine and L-ornithine production strains

Influence of local surface albedo variability and ice crystal shape on passive remote sensing of thin cirrus

Airborne measurements of solar spectral radiance reflected by cirrus are performed with the HALO-Solar Radiation (HALO-SR) instrument onboard the High Altitude and Long Range Research Aircraft (HALO) in November 2010. The data are used to quantify the influence of surface albedo variability on the retrieval of cirrus optical thickness and crystal effective radius. The applied retrieval of cirrus optical properties is based on a standard two-wavelength approach utilizing measured and simulated reflected radiance in the visible and near-infrared spectral region. Frequency distributions of the surface albedos from Moderate resolution Imaging Spectroradiometer (MODIS) satellite observations are used to compile surface-albedo-dependent lookup tables of reflected radiance. For each assumed surface albedo the cirrus optical thickness and effective crystal radius are retrieved as a function of the assumed surface albedo. The results for the cirrus optical thickness are compared to measurements from the High Spectral Resolution Lidar (HSRL). The uncertainty in cirrus optical thickness due to local variability of surface albedo in the specific case study investigated here is below 0.1 and thus less than that caused by the measurement uncertainty of both instruments. It is concluded that for the retrieval of cirrus optical thickness the surface albedo variability is negligible. However, for the retrieval of crystal effective radius, the surface albedo variability is of major importance, introducing uncertainties up to 50%. Furthermore, the influence of the bidirectional reflectance distribution function (BRDF) on the retrieval of crystal effective radius was investigated and quantified with uncertainties below 10%, which ranges below the uncertainty caused by the surface albedo variability. The comparison with the independent lidar data allowed for investigation of the role of the crystal shape in the retrieval. It is found that if assuming aggregate ice crystals, the HSRL observations fit best with the retrieved optical thickness from HALO-SR

Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus

Markert B, Stolzenberger J, Brautaset T, Wendisch VF. Characterization of two transketolases encoded on the chromosome and the plasmid pBM19 of the facultative ribulose monophosphate cycle methylotroph Bacillus methanolicus. BMC Microbiology. 2014;14(1): 7.Background Transketolase (TKT) is a key enzyme of the pentose phosphate pathway (PPP), the Calvin cycle and the ribulose monophosphate (RuMP) cycle. Bacillus methanolicus is a facultative RuMP pathway methylotroph. B. methanolicus MGA3 harbors two genes putatively coding for TKTs; one located on the chromosome (tktC) and one located on the natural occurring plasmid pBM19 (tktP). Results Both enzymes were produced in recombinant Escherichia coli, purified and shown to share similar biochemical parameters in vitro. They were found to be active as homotetramers and require thiamine pyrophosphate for catalytic activity. The inactive apoform of the TKTs, yielded by dialysis against buffer containing 10 mM EDTA, could be reconstituted most efficiently with Mn2+ and Mg2+. Both TKTs were thermo stable at physiological temperature (up to 65°C) with the highest activity at neutral pH. Ni2+, ATP and ADP significantly inhibited activity of both TKTs. Unlike the recently characterized RuMP pathway enzymes fructose 1,6-bisphosphate aldolase (FBA) and fructose 1,6-bisphosphatase/sedoheptulose 1,7-bisphosphatase (FBPase/SBPase) from B. methanolicus MGA3, both TKTs exhibited similar kinetic parameters although they only share 76% identical amino acids. The kinetic parameters were determined for the reaction with the substrates xylulose 5-phosphate (TKTC: kcat/KM: 264 s-1 mM-1; TKTP: kcat/KM: 231 s-1 mM) and ribulose 5-phosphate (TKTC: kcat/KM: 109 s-1 mM; TKTP: kcat/KM: 84 s-1 mM) as well as for the reaction with the substrates glyceraldehyde 3-phosphate (TKTC: kcat/KM: 108 s-1 mM; TKTP: kcat/KM: 71 s-1 mM) and fructose 6-phosphate (TKTC kcat/KM: 115 s-1 mM; TKTP: kcat/KM: 448 s-1 mM). Conclusions Based on the kinetic parameters no major TKT of B. methanolicus could be determined. Increased expression of tktP, but not of tktC during growth with methanol [J Bacteriol 188:3063–3072, 2006] argues for TKTP being the major TKT relevant in the RuMP pathway. Neither TKT exhibited activity as dihydroxyacetone synthase, as found in methylotrophic yeast, or as the evolutionary related 1-deoxyxylulose-5-phosphate synthase. The biological significance of the two TKTs for B. methanolicus methylotrophy is discussed

Atmospheric radiative effects of an in situ measured Saharan dust plume and the role of large particles

This work will present aerosol size distributions measured in a Saharan dust plume between 0.9 and 12 km altitude during the ACE-2 campaign 1997. The distributions contain a significant fraction of large particles of diameters from 4 to 30 μm. Radiative transfer calculations have been performed using these data as input. Shortwave, longwave as well as total atmospheric radiative effects (AREs) of the dust plume are investigated over ocean and desert within the scope of sensitivity studies considering varied input parameters like solar zenith angle, scaled total dust optical depth, tropospheric standard aerosol profiles and particle complex refractive index. The results indicate that the large particle fraction has a predominant impact on the optical properties of the dust. A single scattering albedo of ω<sub><I>o</I></sub>=0.75–0.96 at 550 nm was simulated in the entire dust column as well as 0.76 within the Saharan dust layer at ~4 km altitude indicating enhanced absorption. The measured dust leads to cooling over the ocean but warming over the desert due to differences in their spectral surface albedo and surface temperature. The large particles absorb strongly and they contribute at least 20% to the ARE in the dusty atmosphere. <br><br> From the measured size distributions modal parameters of a bimodal lognormal column volume size distribution were deduced, resulting in a coarse median diameter of ~9 μm and a column single scattering albedo of 0.78 at 550 nm. A sensitivity study demonstrates that variabilities in the modal parameters can cause completely different AREs and emphasises the warming effect of the large mineral dust particles

Optical thickness and effective radius of Arctic boundary-layer clouds retrieved from airborne nadir and imaging spectrometry

Arctic boundary-layer clouds in the vicinity of Svalbard (78° N, 15° E) were observed with airborne remote sensing and in situ methods. The cloud optical thickness and the droplet effective radius are retrieved from spectral radiance data from the nadir spot (1.5°, 350–2100 nm) and from a nadir-centred image (40°, 400–1000 nm). Two approaches are used for the nadir retrieval, combining the signal from either two or five wavelengths. Two wavelengths are found to be sufficient for an accurate retrieval of the cloud optical thickness, while the retrieval of droplet effective radius is more sensitive to the number of wavelengths. Even with the comparison to in-situ data, it is not possible to definitely answer the question which method is better. This is due to unavoidable time delays between the in-situ measurements and the remote-sensing observations, and to the scarcity of vertical in-situ profiles within the cloud

Desert Dust Air Mass Mapping in the Western Sahara, using Particle Properties Derived from Space-based Multi-angle Imaging

Coincident observations made over the Moroccan desert during the SAhara Mineral dUst experiMent (SAMUM) 2006 field campaign are used both to validate aerosol amount and type retrieved from Multi-angle Imaging SpectroRadiometer (MISR) observations, and to place the sub-orbital aerosol measurements into the satellite's larger regional context. On three moderately dusty days for which coincident observations were made, MISR mid-visible aerosol optical thickness (AOT) agrees with field measurements point-by-point to within 0.05 to 0.1. This is about as well as can be expected given spatial sampling differences; the space-based observations capture AOT trends and variability over an extended region. The field data also validate MISR's ability to distinguish and to map aerosol air masses, from the combination of retrieved constraints on particle size, shape, and single-scattering albedo. For the three study days, the satellite observations (a) highlight regional gradients in the mix of dust and background spherical particles, (b) identify a dust plume most likely part of a density flow, and (c) show an air mass containing a higher proportion of small, spherical particles than the surroundings, that appears to be aerosol pollution transported from several thousand kilometers away

Computing the shortest elementary flux modes in genome-scale metabolic networks

This article is available open access through the publisher’s website through the link below. Copyright @ The Author 2009.Motivation: Elementary flux modes (EFMs) represent a key concept to analyze metabolic networks from a pathway-oriented perspective. In spite of considerable work in this field, the computation of the full set of elementary flux modes in large-scale metabolic networks still constitutes a challenging issue due to its underlying combinatorial complexity. Results: In this article, we illustrate that the full set of EFMs can be enumerated in increasing order of number of reactions via integer linear programming. In this light, we present a novel procedure to efficiently determine the K-shortest EFMs in large-scale metabolic networks. Our method was applied to find the K-shortest EFMs that produce lysine in the genome-scale metabolic networks of Escherichia coli and Corynebacterium glutamicum. A detailed analysis of the biological significance of the K-shortest EFMs was conducted, finding that glucose catabolism, ammonium assimilation, lysine anabolism and cofactor balancing were correctly predicted. The work presented here represents an important step forward in the analysis and computation of EFMs for large-scale metabolic networks, where traditional methods fail for networks of even moderate size. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online (http://bioinformatics.oxfordjournals.org/cgi/content/full/btp564/DC1).Fundação Calouste Gulbenkian, Fundação para a Ciência e a Tecnologia (FCT) and Siemens SA Portugal