Increased Product Formation Research Articles

Biosensoren für die mikrobielle Stammentwicklung im Hochdurchsatzformat

Microbial strain development for the biotechnological production of small molecules is time-consuming and laborious. This can be attributed to the fact that increased product formation usually does not confer a phenotype, which would allow interfacing with high-throughput screening technologies. Recently, advances were made in the construction of biosensors for detecting small molecules at the single cell level and first examples for their application in combination with FACS demonstrate the potential of such biosensors for microbial strain development.

Solar Spectrum Photocatalytic Conversion of CO2 and Water Vapor Into Hydrocarbons Using TiO2 Nanoparticle Membranes

A viable option for recycling carbon dioxide is through the sunlight-powered photocatalytic conversion of CO2 and water vapor into hydrocarbon fuels over highly active nanocatalysts. With photocatalytic CO2 reduction sunlight, a renewable energy source as durable as the sun, is used to drive the catalytic reaction with the resultant fuel products compatible with the current hydrocarbon-based energy infrastructure. The use of co-catalyst (Cu, Pt)-sensitized TiO2 nanoparticle wafers in the photocatalytic conversion of CO2 and water vapor to hydrocarbon fuels, with optimal humidity levels and exposure times established. We also attempted to increase product formation by sputtering both co-catalysts on the nanoparticle wafer's surface, with the resulting product rates significantly higher than that of either the Cu or Pt coated samples. When the TiO2 nanoparticle wafers are used in a flow-through membrane implementation we find a significant increase in product rates of formation, including methane, hydrogen, and carbon monoxide. We believe that nanocatalyst-based flow-through membranes are a viable route for achieving large-scale and low cost photocatalytic solar fuel production.

Proline addition increases the efficiency of l‐lysine production by Corynebacterium glutamicum

The wild type of Corynebacterium glutamicum does not excrete l‐lysine, but mutants are developed to use C. glutamicum for the large‐scale production of this amino acid. l‐lysine is derived from oxaloacetate and pyruvate. A reduced entry of these precursors into the citric acid cycle triggers l‐lysine synthesis, but there is simultaneously an unfavorable reduction in growth rate. Here, we further investigated strains with reduced citrate synthase activity and observed that proline supplementation partly reversed the growth rate reduction, and simultaneously increased l‐lysine accumulation by 10%. The combined effect of faster growth and higher l‐lysine formation lead to an increase in l‐lysine productivity of about 14%. Small scale bioreactor cultivations showed an increase of the glucose uptake rate from 3.96 to 5.12 mmol gCDW−1 h−1 as the effect of proline addition. Deletion of the proline‐utilization gene putA demonstrated that proline did not serve to refill the citric acid cycle. The combined data show that proline addition has a favorable effect on l‐lysine formation and sugar uptake, which indicates that engineering sugar uptake is a future option for increased product formation.

ANALYZING THE GENOMIC VARIATION OF MICROBIAL CELL FACTORIES IN THE ERA OF “NEW BIOTECHNOLOGY”

The application of genome-scale technologies, both experimental and in silico, to industrial biotechnology has allowed improving the conversion of biomass-derived feedstocks to chemicals, materials and fuels through microbial fermentation. In particular, due to rapidly decreasing costs and its suitability for identifying the genetic determinants of a phenotypic trait of interest, whole genome sequencing is expected to be one of the major driving forces in industrial biotechnology in the coming years. We present some of the recent studies that have successfully applied high-throughput sequencing technologies for finding the underlying molecular mechanisms for (a) improved carbon source utilization, (b) increased product formation, and (c) stress tolerance. We also discuss the strengths and weaknesses of different strategies for mapping industrially relevant genotype-to-phenotype links including exploiting natural diversity in natural isolates or crosses between isolates, classical mutagenesis and evolutionary engineering.

Regulation of dolichol-linked glycosylation

In the majority of congenital disorders of glycosylation, the assembly of the glycan precursor GlcNAc(2)Man(9)Glc(3) on the polyprenol carrier dolichyl-pyrophosphate is compromised. Because N-linked glycosylation is essential to life, most types of congenital disorders of glycosylation represent partial losses of enzymatic activity. Consequently, increased availability of substrates along the glycosylation pathway can be beneficial to increase product formation by the compromised enzymes. Recently, we showed that increased dolichol availability and improved N-linked glycosylation can be achieved by inhibition of squalene biosynthesis. This review summarizes the current knowledge on the biosynthesis of dolichol-linked glycans with respect to deficiencies in N-linked glycosylation. Additionally, perspectives on therapeutic treatments targeting dolichol and dolichol-linked glycan biosynthesis are examined.

Kinetics of tRNA Folding Monitored by Aminoacylation

We describe a strategy for tracking Mg2+‐initiated folding of 32P‐labeled tRNA molecules to their native structures based on capacity for aminoacylation by the cognate aminoacyl‐tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by addition of tRNA synthetase and further incubation, yields a rapid burst of aminoacyl‐tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. Application of the approach to study the influence of post‐transcriptional modifications in folding of E. coli tRNA1 Gln reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the apparent folding pathway. This assay allows the use of 32P‐labeled tRNA in integrated studies combining folding, post‐transcriptional processing and aminoacylation reactions. The assay also allows tracking folding of mitochondrial tRNAs, the native structures of many of which are poorly characterized.

Kinetics of tRNA folding monitored by aminoacylation

We describe a strategy for tracking Mg²⁺-initiated folding of ³²P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by the addition of aminoacyl-tRNA synthetase and further incubation, yields a rapid burst of aminoacyl-tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of the enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA₁(Gln) reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of ³²P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions.

Pore scale analysis of the impact of mixing-induced reaction dependent viscosity variations

Expanding interest in enhanced subsurface natural resource recovery and carbon sequestration motivates study of reacting flows in porous media. In this work, we examine the case of reaction products that increase or decrease the viscosity of the fluid. Parallel reactant streams flow through porous media and react transversely along the centerline. We utilize a pore scale, finite element numerical method that couples the reaction with fluid flow through two arrangements of porous media at three Damkohler (Da) numbers and two viscosity conditions. When the product increases the fluid viscosity, the flow velocity is reduced and higher amounts of product are formed due to increased diffusion time. Conversely, reduced fluid viscosity leads to greater fluid velocity and lower amount of product formation. An exception is the viscous thinning case of high Peclet (Pe) number and high Da where an instability develops (in low Reynolds (Re) number flow) that enhances mixing between the reactants, resulting in increasing product formation.

Traces of certain drug molecules can enhance heme-enzyme catalytic outcomes

We report that trace amounts (nano- to picomolar concentrations) of dapsone and amiodarone in reaction mixtures of structurally and functionally distinct heme enzymes gave increased product formation rates from diverse substrates. These enhancements are found to be lowered by mild radical quenchers; a lowering well differentiated from heme active site inhibitions. Based on the non-specific nature of the activations in the diverse heme enzyme reactions studied, a generic mechanism is proposed to explain additive-based activity enhancements in heme enzymology.

Significant Increase in Phenacetin Oxidation on L382V Substitution in Human Cytochrome P450 1A2

Human CYP1A2 is an important drug-metabolizing enzyme, similar in sequence to CYP1A1 but with distinct substrate specificity. We have previously shown that residue 382 affected CYP1A1 and CYP1A2 specificities with alkoxyresorufins. To determine whether this residue is also important for the metabolism of other substrates, we have investigated phenacetin oxidation by single (T124S, T223N, V227G, N312L, and L382V) and multiple (L382V/T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/N312L) mutants of CYP1A2. The enzymes were expressed in Escherichia coli and purified. All the CYP1A2 mutants that contained the L382V substitution displayed much higher activities than the wild-type enzyme, with k(cat) values 3-fold higher, in contrast to other mutants, for which k(cat) decreased. Likewise, a significant increase in specificity, expressed as the k(cat)/K(m) ratio, was observed for the mutants containing the L382V substitution. The efficiency of coupling of reducing equivalents to acetaminophen formation was decreased for all the single mutants except L382V, for which the coupling increased. This effect was also observed with multiple CYP1A2 mutants containing the L382V substitution. Low activities of the four other single mutants were likely caused by dramatically increased uncoupling to water. In contrast, the increase in activity of the L382V-containing mutants resulted from decreased water formation. This finding is consistent with molecular dynamics results, which showed decreased phenacetin mobility leading to increased product formation. The results of these studies confirm the importance of residue 382 in CYP1A2-catalyzed oxidations and show that a single residue substitution can dramatically affect enzymatic activity.

Increased product formation induced by a directed secondary substrate limitation in a batch Hansenula polymorpha culture

By the use of directed limitations of secondary substrates, the metabolic flux should be deflected from biomass production to product formation. In order to study the impact of directed limitations caused by various secondary substrates on the growth and product formation of the methylotrophic yeast Hansenula polymorpha, the cultivation systems respiration activity monitoring system (RAMOS) and BioLector were used in parallel. While the RAMOS device allows the online monitoring of the oxygen transfer rate in shake flasks, the BioLector enables in microtiter plates the monitoring of scattered light and the fluorescence intensity of the green fluorescent protein (GFP). Secondary substrate limitations of phosphate, potassium, and magnesium were analyzed in batch fermentations. The sole carbon source was either 10 g/L glucose or 10 g/L glycerol. The expression of the GFP gene is controlled by the FMD promoter (formate dehydrogenase). In batch cultures with glucose as carbon source, a directed limitation of phosphate increased the GFP production 1.87-fold, compared to phosphate unlimited conditions. Under potassium-limited conditions with glycerol as sole carbon source, the GFP production was 1.41-fold higher compared to unlimited conditions. A limitation of the substrate magnesium resulted in a 1.22-fold increase GFP formation in the case of glycerol as carbon source.

The effect of syngas composition on the growth and product formation of Butyribacterium methylotrophicum

A carbon monoxide-adapted strain of Butyribacterium methylotrophicum was grown submerged on different head space gas mixtures of carbon monoxide, hydrogen and carbon dioxide. Carbon monoxide was utilized by the B. methylotrophicum strain with acetic and butyric acid as main products. Supplemental carbon dioxide increased initial growth rate and increased product formation especially acetic acid production. Acetic acid was produced in concentrations between 0.6 and 1.3 g/l. Butyric acid, lactic acid and ethanol were produced in lesser concentrations. The total carbon yield to products was between 90% and 110%. Carbon monoxide was almost completely utilized before hydrogen utilization started. Supplemental hydrogen increased the formation of butyric acid and made it possible to convert also carbon dioxide to organic products. Separation of growth and product formation may offer interesting process design options.

Influence of process conditions on end product formation from Clostridium thermocellum 27405 in solid substrate cultivation on paper pulp sludge

Solid substrate cultivation of thermophilic, anaerobic bacteria offers an alternative production method for many bio-based chemicals; however the process must be optimized for each substrate-organism fermentation. The effects of initial substrate moisture content (SMC, 30%, 50% and 70% wet-basis), supplemental nutrient concentration (SNC, 12%, 50% and 100%) and duration of cultivation time (6, 10 and 14 days), on product formation (lactate, ethanol and acetate) by Clostridium thermocellum 27405 were examined during growth on paper pulp sludge. Water activities at moisture contents above 30% wet-basis were essentially identical (∼0.99), yet the water contents differed significantly, and affected the metabolic activity of C. thermocellum. Increases in initial substrate moisture content from 50% to 70% for cultures supplemented with 50% or 100% nutrients resulted in a 75–145 mM increase in total end products. At 70% SMC, the addition of 100% SNC generated a 56% increase in product formation above the addition of 50% nutrient supplementation. Increases in the quantity of free water present in the solid substrate cultivation system up to the water holding capacity of the paper pulp sludge led to improved performance of this anaerobic bacterium. While nutrient supplementation is common in the form of salts for many aerobic microorganisms, efficient metabolism for anaerobic C. thermocellum grown in SSC was highly dependent on added salts, vitamins and reducing agents. Further studies are needed to determine if this is a general effect for other anaerobes grown in solid substrate cultures.

Phosphorylation of Bovine Adrenodoxin by Protein Kinase CK2 Affects the Interaction with Its Redox Partner Cytochrome P450scc (CYP11A1)

Adrenodoxin (Adx), a [2Fe-2S] vertebrate-type ferredoxin, transfers electrons from the NADPH-dependent flavoprotein Adx reductase (AdR) to mitochondrial cytochrome P450 enzymes of the CYP11A and CYP11B families, which catalyze key reactions in steroid hormone biosynthesis. Adx is a known phosphoprotein, but the kinases that phosphorylate Adx have remained mostly obscure. The aim of this study was to identify previously unknown Adx phosphorylating kinases and to acquire a deeper insight into the functional consequences of such a modification. Here, we show for the first time that bovine Adx is a substrate of protein kinase CK2, whereas bovine CYP11A1, CYP11B1, and AdR are not phosphorylated by this kinase. CK2 phosphorylation of mature Adx requires the presence of both the catalytic alpha-subunit and the regulatory beta-subunit of CK2 and takes place exclusively at residue Thr-71, which is located within the redox partner interaction domain of the protein. We created two Adx mutants, Adx-T71E (imitating a phosphorylation) and Adx-T71V (which cannot be phosphorylated at this site), respectively, and investigated how these mutations affected the interaction of Adx with its redox partners. These data were supplemented with detailed spectroscopic and functional assays using the phosphorylated protein. All Adx species behaved like wild type (Adx-WT) with respect to their redox potential, iron-sulfur cluster symmetry, and overall backbone structure. Substrate conversion assays catalyzed by CYP11A1 showed an increase in product formation when Adx-T71E or CK2-phosphorylated Adx were used as electron carrier instead of Adx-WT, whereas the activity toward CYP11B1 was not altered using these Adx species. Additionally, Adx-T71E represents the only full-length Adx mutant which leads to an increase in CYP11A1 product formation. Therefore, characterizing this full-length mutant helps to improve our knowledge on the functional effects of phosphorylations on complex redox systems.

A perspective of metabolic engineering strategies: moving up the systems hierarchy.

Metabolic engineering has been established as an important field in biotechnology. It involves the analysis, design, and alteration of the stoichiometric network using sophisticated mathematical and molecular biology techniques. It allows for improvement of pathway kinetics by removing flux bottlenecks, balancing precursors, and recycling cofactors used to increase product formation. The next step in the systems hierarchy is the constructive manipulation of regulatory networks. As our understanding of regulation continues to expand rapidly, engineering of intracellular regulation will become an integral aspect of metabolic engineering.

Lipoxygenases in human and bovine corneal epithelia

Oxidatively-modified low density lipoprotein (LDL) is thought to play a significant role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic fatty lesions. Recently, lipoxygenases have been implicated as a major enzymatic pathway involved in rabbit endothelial cell-mediated LDL modification. We investigated the effect of LDL on porcine aortic endothelial cell (PAEC) and human umbilical vein (HUVEC) and aortic endothelial cell (HAEC) lipoxygenase activity. By thin layer chromatography, we observed that human LDL stimulated the metabolism of radiolabeled arachidonic acid to 12+15-hydroxyeicosatetraenoic acid (HETE) in indomethacin-treated PAEC. Furthermore, radiolabeled linoleic acid, a specific substrate for the 15-lipoxygenase, was metabolized to its respective product 13-hydroxyoctadecadienoic acid (13-HODE) in the presence of LDL. Increased product formation in both studies was inhibited by the lipoxygenase blockers nordihydroguaiaretic acid (NDGA) and RG 6866. 15-HETE was confirmed as the predominant HETE product in LDL-treated cells by high performance liquid chromatography. Both porcine- and human-derived LDL stimulated the CL release of 15-HETE from cells as determined by radioimmunoassay. Release of immunoreactive 15-HETE was inhibited by NDGA, RG 6866, and 5,8,11,14-eicosatetraynoic acid (ETYA) but not by the selective 5-lipoxygenase inhibitor RG 5901. These lipoxygenase inhibitors had similar effects on the modification of LDL. Our results suggest that the oxidative modification of LDL by endothelial cells may be mediated in part through activation of 15-lipoxygenase.

Glycosylated α-chymotrypsin as a catalyst for kyotorphin synthesis in water-organic media

α-Chymotrypsin was covalently modified with cellobiose by chemical means. After adsorption on to a porous polyamide support, both the native and the glycosylated immobilized derivatives were used to synthesize a kyotorphin derivative (N-benzoyl-l-tyrosyl-l-argininamide) in acetonitrile/water. Glycosylated chymotrypsin gave a 125% increase in product formation (750 nmol mg−1 catalyst in 3 h) at 60% (v/v) acetonitrile/water. Maximal protective effect of this glycosylation process was at 70% (v/v) acetonitrile/water, at which concentration the half-life of the glycosylated enzyme was 20-times longer than that of the native form (52 min and 2.8 min, respectively).

Expression and Coupling of Human Cytochrome P450 2E1 and NADPH–Cytochrome P450 Oxidoreductase in Dual Expression and Co-infection Systems with Baculovirus in Insect Cells

In order to study the interaction between human cytochrome P450 2E1 (h2E1) and NADPH–P450 oxidoreductase (hOR) in a native membrane environment, we used two approaches to express both h2E1 and hOR in a baculovirus expression system. For a dual-expression system, h2E1 and hOR were coexpressed inSpodoptera frugiperda(Sf9) insect cells using a recombinant baculovirus carrying both h2E1 and hOR cDNAs (v-h2E1-hOR). The h2E1 cDNA was expressed under the control of the polyhedrin promoter PPolh, whereas hOR cDNA was expressed under the control of the P10promoter. The expressed enzymes were catalytically active in the cell membrane preparations. The estimated molar expression ratio of h2E1 to hOR in the membranes was 1:5. The apparentKmandkcatforN-nitrosodimethylamine (NDMA) demethylase activity were 145 μMand 2.4 min−1, respectively. When Sf9 cells were co-infected with the dual-expression virus (v-h2E1-hOR) and human cytochrome b5recombinant virus (v-hb5), a 9-fold decrease in theKmof NDMA demethylase activity (16 μM) was observed in the membrane preparations, whereas thekcatwas increased to 4 min−1. In the second approach, recombinant viruses of h2E1 and hOR (v-h2E1 and v-hOR) were used to co-infect the Sf9 cells. In this double-expression system, with a fixed amount of v-h2E1, the expression of h2E1 in the Sf9 cells decreased as the amount of v-hOR increased. Western blot analysis of the membrane preparations showed that the level of hOR increased, but the level of h2E1 decreased with increasing amounts of v-hOR. A corresponding decrease in h2E1 mRNA, however, was not observed. In the presence of human cytochrome b5(hb5), the optimal h2E1:hOR molar ratio for h2E1 catalytic activity was 1:1. In order to further investigate the hb5effect on h2E1-catalyzed reactions in the native membranes, we co-infected Sf9 cells with v-h2E1, v-hOR, and v-hb5and obtained a membrane preparation containing h2E1, hOR, and hb5. Stoichiometric analysis with membrane preparations from double-infection and triple-infection systems revealed that the presence of hb5decreased NADPH oxidation and H2O2formation by 72 and 80%, respectively, but increased product formation from NDMA 13-fold. These results suggest that hb5enhances the coupling between h2E1 and hOR for product formation. These studies also demonstrate that the baculovirus–insect cell system can produce high levels of expression of functional h2E1, hOR, and hb5for mechanistic studies.

Contribution of excited states in the heavy ion radiolysis of cyclooctane

The yields of the major end products produced in cyclooctane have been used to probe the effects of the track structure on radiation chemical processes induced by γ-rays, 1–15 MeV protons (LET 50–10 eV/nm), 5–20 MeV helium ions (LET 150–70 eV/nm), 7–29 MeV carbon ions (LET 800–660 eV/nm) and 10 MeV oxygen ions (LET 1150 eV/nm). There is a gradual decrease in the yields of cyclooctene, bicyclooctyl, and bicyclo[3.3.0]octane (pentalane) with increasing LET and there appears to be a considerable change in the yields of individual primary decomposition channels with varying LET. Shifting from γ-radiolysis to proton radiolysis results in an increase in product formation through the triplet state with a concomitant decrease in the singlet excited state, probably due to the enhanced probability of cross combination reactions of electrons and cations in the high density region of heavy particle tracks. It appears that the yield of both the triplet and singlet excited states of cyclooctane slowly decrease with increasing LET.

Effect of ilvBN-encoded α-acetolactate synthase expression on diacetyl production in Lactococcus lactis

Conversion of pyruvate to α-acetolactate, which is broken down to diacetyl and acetoin, can be catalysed by two α-acetolactate synthases in Lactococcus lactis. The enzyme encoded by the als gene (Als) has previously been shown to have a low affinity for pyruvate, which limits the formation of diacetyl. In this study we have expressed from a plasmid the ilvBN genes, which encode the other α-acetolactate synthase (IlvBN). This plasmid-directed enzyme expression provided up to 3.6-fold increased product formation in the L. lactis MG1363 and IL1403 backgrounds. Plasmid-based expression of the ilvBN genes, in an IL1403 derivative from which the leu.ilv.ald and flanking genes had been deleted, yielded up to 0.1 mM diacetyl where-as the host strain produced none. In addition, IlvBN, with a K m value of 8.3 mM, was shown to have a greater affinity for pyruvate than does Als.