Mol Glucose Research Articles

Kinetic analysis of cellulose synthase of Gluconacetobacter hansenii in whole cells and in purified form

The Gram-negative bacterium, Gluconacetobacter hansenii, has been long studied and is a model for cellulose synthesis. It produces cellulose, using the enzyme AcsA-AcsB, of exceptionally high crystallinity in comparison to the cellulose of higher plants. We determined the rate of cellulose synthesis in whole cells measured as moles of glucose incorporated into cellulose per second per mole of enzyme. This was determined by quantifying the rate of cellulose synthesis (over a short time span, such that the enzyme concentration is not changing due to cell growth) and the amount of enzyme in the whole cell by quantitative western blotting. We found that the whole cell rate of 24 s−1 is much faster than the kcat, measured from steady-state kinetic analysis, of 1.7 s−1. Our whole cell rates are consistent with previous studies using microscopy. We postulate that the rationale for this difference is the presence of an alternative in vivo priming mechanism. This in turn can increase the rate of initiation, which we previously postulated to be the rate-limiting step in catalysis.

Genome shuffling and high-throughput screening of Brevibacterium flavum MDV1 for enhanced l-valine production

L-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for L-valine grows rapidly, there is an increasing interest to develop an efficient L-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield L-valine strains to replace the traditional L-valine assay. L-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six L-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an L-valine yield of 30.1g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30L automated fermentor, MDVR2-21 accumulated 70.1g/L of L-valine (0.598mol L-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120m3 fermentor, this strain produced > 66.8g/L L-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of L-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.

High yield production of four-carbon dicarboxylic acids by metabolically engineered Escherichia coli.

Several metabolic engineered Escherichia coli strains were constructed and evaluated for four-carbon dicarboxylic acid production. Fumarase A, fumarase B and fumarase C single, double and triple mutants were constructed in a ldhA adhE mutant background overexpressing the pyruvate carboxylase from Lactococcus lactis. All the mutants produced succinate as the main four-carbon (C4) dicarboxylic acid product when glucose was used as carbon source with the exception of the fumAC and the triple fumB fumAC deletion strains, where malate was the main C4-product with a yield of 0.61-0.67mol (mole glucose)-1. Additionally, a mdh mutant strain and a previously engineered high-succinate-producing strain (SBS550MG-Cms pHL413-Km) were investigated for aerobic malate production from succinate. These strains produced 40.38mM (5.41g/L) and 50.34mM (6.75g/L) malate with a molar yield of 0.53 and 0.55mol (mole succinate)-1, respectively. Finally, by exploiting the high-succinate production capability, the strain SBS550MG-Cms243 pHL413-Km showed significant malate production in a two-stage process from glucose. This strain produced 133mM (17.83g/L) malate in 47h, with a high yield of 1.3mol (mole glucose)-1 and productivity of 0.38gL-1h-1.

Impairment of NADH dehydrogenase and regulation of anaerobic metabolism by the small RNA RyhB and NadE for improved biohydrogen production in Enterobacter aerogenes

BackgroundEnterobacter aerogenes is a facultative anaerobe and is one of the most widely studied bacterial strains because of its ability to use a variety of substrates, to produce hydrogen at a high rate, and its high growth rate during dark fermentation. However, the rate of hydrogen production has not been optimized. In this present study, three strategies to improve hydrogen production in E. aerogenes, namely the disruption of nuoCDE, overexpression of the small RNA RyhB and of NadE to regulate global anaerobic metabolism, and the redistribution of metabolic flux. The goal of this study was to clarify the effect of nuoCDE, RyhB, and NadE on hydrogen production and how the perturbation of NADH influences the yield of hydrogen gas from E. aerogenes.ResultsNADH dehydrogenase activity was impaired by knocking out nuoCD or nuoCDE in E. aerogenes IAM1183 using the CRISPR-Cas9 system to explore the consequent effect on hydrogen production. The hydrogen yields from IAM1183-CD(∆nuoC/∆nuoD) and IAM1183-CDE (∆nuoC/∆nuoD/∆nuoE) increased, respectively, by 24.5 and 45.6% in batch culture (100 mL serum bottles). The hydrogen produced via the NADH pathway increased significantly in IAM1183-CDE, suggesting that nuoE plays an important role in regulating NADH concentration in E. aerogenes. Batch-cultivating experiments showed that by the overexpression of NadE (N), the hydrogen yields of IAM1183/N, IAM1183-CD/N, and IAM1183-CDE/N increased 1.06-, 1.35-, and 1.55-folds, respectively, compared with IAM1183. Particularly worth mentioning is that the strain IAM118-CDE/N reached 2.28 mol in H2 yield, per mole of glucose consumed. IAN1183/R, IAM1183-CD/R, and IAM1183-CDE/R showed increasing H2 yields in batch culture. Metabolic flux analysis indicated that increased expression of RyhB led to a significant shift in metabolic patterns. We further investigated IAM1183-CDE/N, which had the best hydrogen-producing traits, as a potential candidate for industry applications using a 5-L fermenter; hydrogen production reached up to 1.95 times greater than that measured for IAM1183.ConclusionsKnockout of nuoCD or nuoCDE and the overexpression of nadE in E. aerogenes resulted in a redistribution of metabolic flux and improved the hydrogen yield. Overexpression of RyhB had an significant change on the hydrogen production via NADH pathway. A combination of strategies would be a novel approach for developing a more economic and efficient bioprocess for hydrogen production in E. aerogenes. Finally, the latest CRISPR-Cas9 technology was successful for editing genes in E. aerogenes to develop our engineered strain for hydrogen production.

Producción de Hidrógeno a partir de la fermentación de residuos agroindustriales de la piña

<p>Se estudió la generación de hidrógeno a partir de la fermentación anaeróbica de residuos agroindustriales de corazón de piña utilizando como inóculo lodos pretratados provenientes de una planta de tratamiento de agua residual del tipo UASB. Mediante un diseño factorial 23, se determinaron los efectos de la concentración inicial de sustrato, del pH inicial del medio de fermentación y de la formulación de nutrientes, sobre el rendimiento en la producción de hidrógeno. Los mejores resultados se obtuvieron utilizando un pH inicial de 5,5, con una concentración inicial de sustrato de 5 (g de glucosa)/L y usando una formulación de nutrientes basada en magnesio, hierro, zinc y sodio. Posteriormente, utilizando las condiciones de operación anteriores, se realizó un escalamiento de la fermentación en un bioreactor de 5 L y se alcanzó un rendimiento máximo en la producción de hidrógeno de 1,541 (mol H2)/(mol glucosa). Este rendimiento es comparable con datos reportados en la literatura cuando se ha usado glucosa como sustrato. Los resultados obtenidos sugieren que los residuos de corazón de piña podrían ser utilizados como una fuente renovable para la producción de hidrógeno.</p>

Biological synthesis of 2,5-bis(hydroxymethyl)furan from biomass-derived 5-hydroxymethylfurfural by E. coli CCZU-K14 whole cells

Biocatalytic upgrading of bio-based platform chemical 5-hydroxymethylfurfural (5-HMF) to 2,5-bis(hydroxymethyl)furan (BHMF) is currently of great interest due to the product specificity, mild reaction and high efficiency. In this work, 200mM 5-HMF could be effectively biotransformed to BHMF at 90.6% with highly 5-HMF-tolerant recombinant E. coli CCZU-K14 whole cells at pH 6.5 and 30°C under the optimum reaction conditions (cosubstrate glucose 1.0mol glucose/(mol 5-HMF), D-xylose 400mM, l-glutamic acid 250mM, Mg2+ 1.5mM, 0.2mol β-cyclodextrin/(mol 5-HMF), CTAB (cetyltrimethyl ammonium bromide) 12.5mM, and 0.1g wet cells/mL). It was found that E. coli CCZU-K14 was highly tolerant to 5-HMF (up to 400mM). Effective bioreduction of biomass-derived 5-HMF (≤200) to BHMF was successfully demonstrated in this study. In conclusion, this strategy showed high potential application for the synthesis of BHMF.

Can artificial neural network and response surface methodology reliably predict hydrogen production and COD removal in an UASB bioreactor?

Artificial Neural Network (ANN) and Response Surface Methodology (RSM) modeling was employed in an Upflow Anaerobic Sludge Blanket (UASB) bioreactor for optimization of hydrogen yield and COD (Chemical Oxygen Demand) removal efficiency. Experimental data were generated by running seventeen fermentation experiments at varying hydraulic retention times, immobilized cell volumes and temperatures. RSM and ANN models predicted similar optimum conditions for these process parameters. Upon validation, the prediction error for ANN and RSM was observed to be 2.22 and 9.64% on hydrogen yield and 1.01 and 6.34% on COD removal. These results suggested a greater accuracy and higher reliability of ANN in modeling and optimizing the bioprocess parameter interactions associated to the fermentation process. In addition, the study demonstrated a higher molar biohydrogen yield (0.90 mol-H2/mol glucose) and COD removal efficiency (84.81%) in the UASB system optimized by ANN modeling.

Metabolism and kinetic study of bioH 2 production by anaerobic sludge under different acid pretreatments

Abstract This study presents results pertaining to bioH 2 production carried out in batch experiments under different inorganic acid pretreatments of sludge. The sludge was collected from an anaerobic digester and was subjected to different acid (H 2 SO 4 , H 3 PO 4 , HNO 3 , and HCl) pretreatments at pH 3 for 24 h. The results showed that electron equivalent balance closure was within −12 to +10% for all experiments; the portion of electrons as bioH 2 fluctuated from 16% to 25%. H 2 SO 4 pretreatments significantly increased H 2 yields to 3.3 mol H 2 /mol glucose followed by HNO 3 , HCl, and H 3 PO 4 pretreatments which averaged 2.5 ± 0.5. Enthalpy (ΔH°) and Gibb’s free energies (ΔG°) fluctuated from 120.5 to 192.7 (kJ/mol) and from −2.2 to −5.9 (kJ/mol). The modified Gompertz was assessed and determined that the maximum bioH 2 production potential, 1551 mL and maximum rate of bioH 2 production, 303 mL/h occurred using the H 2 SO 4 pretreatment.

English

This work aimed to develop an efficient L-leucine industrial production strain of Corynebacterium glutamicum by using metabolic engineering. A recombinant C. glutamicum strain was constructed by expressing a feedback-resistant leuA-encoded 2-isopropylmalate synthase (IPMS) that carries three amino acid exchanges (R529H, G532D and L535V) from the mutant strain C. glutamicum ML1-9 which was obtained by screening for structural analogues. In order to improve the expression of IPMS, a strong promoter (tac promoter) was used to ensure efficient expression of the rate-limiting enzyme. In addition, reasonable metabolic modifications on the central carbon metabolic pathway and competitive metabolic pathways to optimize the L-leucine biosynthesis pathway by redistribution of various types of precursors and repression of negative regulation were used aimed for increased L-leucine production. The modifications involved (1) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (2) deletion of the gene encoding the AlaT to decrease the concentration of extracellular L-alanine, and increased availability of pyruvate for L-leucine formation, (3) deletion of the gene encoding the threonine dehydratase to abolish L-isoleucine synthesis and to eliminate the intermediate precursor of L-isoleucine biosynthesis competing with L-leucine biosynthesis, (4) inactivation of the pantothenate synthetase to increase α- ketoisovalerate formation, and to enable its further conversion to L-leucine, and (5) inactivation of lactate dehydrogenase to decrease lactate production and its pyruvate consumption, concomitant to decreased glucose consumption rates and prevention of lactic acid to restrict cell growth. The production performance of the engineered strain MDLeu-19/pZ8-1leuAr was characterized with cultivations in a bioreactor. Under fed-batch conditions in a 50-L automated fermentor, the best producer strain accumulated 38.1 g L-1 of L-leucine; the molar product yield being 0.42 mol L-leucine per mole of glucose (glucose conversion rate attained 26.4%). Moreover, during large-scale fermentation using a 150-m3 fermentor, this strain produced more than 37.5 g L-1 L-leucine and the glucose conversion rate was 25.8%, making this process potentially viable for industrial production. Key words: Corynebacterium glutamicum, L-leucine, metabolic engineering, fermentation, industrial production.

The cancer Warburg effect may be a testable example of the minimum entropy production rate principle

Cancer cells consume more glucose by glycolytic fermentation to lactate than by respiration, a characteristic known as the Warburg effect. In contrast with the 36 moles of ATP produced by respiration, fermentation produces two moles of ATP per mole of glucose consumed, which poses a puzzle with regard to the function of the Warburg effect. The production of free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) per mole linearly varies with the fraction (x) of glucose consumed by fermentation that is frequently estimated around 0.9. Hence, calculation shows that, in respect to pure respiration, the predominant fermentative metabolism decreases around 10% the production of entropy per mole of glucose consumed in cancer cells. We hypothesize that increased fermentation could allow cancer cells to accomplish the Prigogine theorem of the trend to minimize the rate of production of entropy. According to the theorem, open cellular systems near the steady state could evolve to minimize the rates of entropy production that may be reached by modified replicating cells producing entropy at a low rate. Remarkably, at CO2 concentrations above 930 ppm, glucose respiration produces less entropy than fermentation, which suggests experimental tests to validate the hypothesis of minimization of the rate of entropy production through the Warburg effect.

Electrode-assisted acetoin production in a metabolically engineered Escherichia coli strain

BackgroundThis paper describes the metabolic engineering of Escherichia coli for the anaerobic fermentation of glucose to acetoin. Acetoin has well-established applications in industrial food production and was suggested to be a platform chemical for a bio-based economy. However, the biotechnological production is often hampered by the simultaneous formation of several end products in the absence of an electron acceptor. Moreover, typical production strains are often potentially pathogenic. The goal of this study was to overcome these limitations by establishing an electrode-assisted fermentation process in E. coli. Here, the surplus of electrons released in the production process is transferred to an electrode as anoxic and non-depletable electron acceptor.ResultsIn a first step, the central metabolism was steered towards the production of pyruvate from glucose by deletion of genes encoding for enzymes of central reactions of the anaerobic carbon metabolism (ΔfrdA-D ΔadhE ΔldhA Δpta–ack). Thereafter, the genes for the acetolactate synthase (alsS) and the acetolactate decarboxylase (alsD) were expressed in this strain from a plasmid. Addition of nitrate as electron acceptor led to an anaerobic acetoin production with a yield of up to 0.9 mol acetoin per mol of glucose consumed (90% of the theoretical maximum). In a second step, the electron acceptor nitrate was replaced by a carbon electrode. This interaction necessitated the further expression of c-type cytochromes from Shewanella oneidensis and the addition of the soluble redox shuttle methylene blue. The interaction with the non-depletable electron acceptor led to an acetoin formation with a yield of 79% of the theoretical maximum (0.79 mol acetoin per mol glucose).ConclusionElectrode-assisted fermentations are a new strategy to produce substances of biotechnological value that are more oxidized than the substrates. Here, we show for the first time a process in which the commonly used chassis strain E. coli was tailored for an electrode-assisted fermentation approach branching off from the central metabolite pyruvate. At this early stage, we see promising results regarding carbon and electron recovery and will use further strain development to increase the anaerobic metabolic turnover rate.

Applying thermodynamics to digestion/gasification processes: the Attainable Region approach

The research shows theoretical calculations on the thermodynamics of digestion/gasification processes where glucose is used as a surrogate for biomass. The change in Enthalpy (∆H) and Gibbs Free Energy (∆G) is used to obtain the Attainable Region (AR) that shows the overall thermodynamic limits for digestion/gasification from 1 mol of glucose. Gibbs Free Energy and Enthalpy (G–H) plots were calculated for the temperature range 25–1500 °C. The results show the effect of temperature on the AR for the processes when water is in both liquid and gas states using 25 °C, 1 bar as the reference state. The AR results show that the production of CO, H2, CH4 and CO2 are feasible at all temperatures studied. The minimum Gibbs Free Energy becomes more negative from −418.68 kJ mol−1 at 25 °C to −3024.34 kJ mol−1 at 1500 °C while the process shifts from exothermic (−141.90 kJ mol−1) to endothermic (1161.80 kJ mol−1) for the respective temperatures. Methane and carbon dioxide are favoured products (minimum Gibbs Free Energy) for temperatures up to about 600 °C, and this therefore includes Anaerobic Digestion. The process is exothermic below 500 °C, and thus Anaerobic Digestion requires heat removal. As the temperature continues to increase, hydrogen production becomes more favourable than methane production. The production of gas is endothermic above 500 °C, and it needs a supply of heat that could be done, either by combustion or by electricity (plasma gasification). The calculations show that glucose conversion at temperatures around 700 °C favours the production of carbon dioxide and hydrogen at minimum G. Generally, the results show that the gas from high-temperature gasification (>~800 °C) typically carries the energy mainly in syngas components CO and H2, whereas at low-temperature gasification (<500 °C) the energy is carried in CH4. The overall analysis for the temperature range (25–1500 °C) also suggests a close relationship between biogas production/digestion and gasification as biogas production can be referred to as a form of low-temperature gasification.

ADVANCES IN BIOLOGICAL HYDROGEN PRODUCTION PROCESSES

Biological hydrogen production processes offer a technique through which renewable energy sources like biomass can be utilized for the generation of the cleanest energy carrier for the use of mankind. Hydrogen intensive research work has already been carried out on the advancement of these processes, such as the development of genetically modified microorganism, metabolic engineering, improvement of the reactor designs, use of different solid matrices for the immobilization of whole cells, biochemical assisted bioreactor, development of two-stage processes, etc. for higher H 2 -production rates. Maximum H 2 yield is found to be 7.1 mol H 2 /mol glucose. However, major bottlenecks for the commercialization of these processes are lower H 2 yield and rate of H 2 production. Suitable microbial cultures are required to handle waste materials efficiently, which are usually complex in nature. This will serve dual purposes: clean energy generation and bioremediation. Scale-up studies on fermentative H 2 -production processes have been done successfully. Pilot plant trials of the photo-fermentation processes require more attention. Use of cheaper raw materials and efficient biological hydrogen production processes will surely make them more competitive with the conventional H 2 generation processes in near future.

Produção de hidrogênio a partir da manipueira em reator anaeróbio de leito fixo

The use of agro-industrial waste for the production of hydrogen has shown a very promising trend, because its improper disposal creates environmental problems. Thus, the objective of the research was to evaluate the production of hydrogen from cassava processing residue in anaerobic fixed bed reactor operated under progressively increasing organic loading rate (OLR) of 12 kg.m-3.d-1 a 96 kg.m-3.d-1. The support material for the adhesion of biomass was expanded clay with a diameter between 2.80 - 3.35 mm, and the reactor was inoculated with anaerobic sludge pre heat-treated. The reactor was operated for 250 days and the progressive increase of ORL was carried out keeping the COD affluent around 4000 mg. L-1, throughout the operation of the reactor and varying the hydraulic retention time (HRT) of 8 hours to 1 hour. The maximum yield of hydrogen was obtained in HRT of 2h (1.66 mol H2 / mol glucose). The soluble metabolites present during operation of the reactor were acetic acid (30.72% to 84.9%), butyric acid (2.89% to 29.13%), propionic acid (3.98 to 13.09%), caproic acid (0.55% and 22.79%) and ethanol (3.64% to 10.46%). Methane production was observed along with hydrogen in all operating phases.

Aerobiosis–anaerobiosis transition has a significant impact on organic acid production by Corynebacterium glutamicum

Abstract Corynebacterium glutamicum is known to produce organic acids under anaerobic culture conditions, in particular, lactic, succinic, and acetic acids. Our study is focused on acetic and succinic acid production using a lactate dehydrogenase-deficient strain of C. glutamicum . Usually, with this bacterium, the organic acid production process is based on an initial aerobic growth phase, followed by a rapid deoxygenation and an anaerobic production phase. In our study, we demonstrated that this strategy was unfavorable for the production of organic acids. Conversely, we showed that applying the best transition strategy based on progressive deoxygenation significantly increased the concentration of organic acids up to 640%. This was observed either by applying controlled dissolved oxygen concentrations or by decreasing the steps of gas flow rates. Our results also showed that applying constant oxygen transfer flux throughout the culture, and thus in the absence of the anaerobic phase, promoted constant production yields (approximately 0.5 mol of succinate or acetate per mole of glucose). In this case, acetate production (120 mM) was favored over succinate production (132 mM), resulting in a decrease in the molar ratio of products (succinate/acetate) from 4.8 to 1.1 between progressive deoxygenation and constant OTR cultures.

Genome sequence and gene expression studies reveal novel hydrogenases mediated hydrogen production by Clostridium biohydrogenum sp. nov., MCM B-509T

The present study describes Clostridium biohydrogenum sp. nov., MCM B-509T as one of the most efficient hydrogen producing dark fermentative bacteria. MCM B-509 produced 3.35 mol hydrogen per mole of glucose consumed. The draft genome sequence of MCM B-509 provided insights into gene repertoire involved in hydrogen metabolism. Genome analysis revealed the presence of key enzymes such as Fe hydrogenase, NiFe Hydrogenases, NAD dependent hydroxy-butyrate dehydrogenase and D-3 Phosphoglycerate dehydrogenase. Fe-hydrogenases were identified as novel hydrogenases on the basis of low sequence homology with the reference sequences in GenBank database as well as predicted 3D structure. Gene expression studies revealed genes encoding hydrogenases were upregulated and solventogenic pathway down regulated during exponential growth phase. Reverse was observed during stationary growth phase explaining high hydrogen production and decline during respective growth phases. This paper illustrates genetic investigation to explain high hydrogen production potential of C. biohydrogenum sp. nov., MCM B-509T.

Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli

Itaconic acid is a high potential platform chemical which is currently industrially produced by Aspergillus terreus. Heterologous production of itaconic acid with Escherichia coli could help to overcome limitations of A. terreus regarding slow growth and high sensitivity to oxygen supply. However, the performance achieved so far with E. coli strains is still low.We introduced a plasmid (pCadCS) carrying genes for itaconic acid production into E. coli and applied a model-based approach to construct a high yield production strain. Based on the concept of minimal cut sets, we identified intervention strategies that guarantee high itaconic acid yield while still allowing growth. One cut set was selected and the corresponding genes were iteratively knocked-out. As a conceptual novelty, we pursued an adaptive approach allowing changes in the model and initially calculated intervention strategy if a genetic modification induces changes in byproduct formation. Using this approach, we iteratively implemented five interventions leading to high yield itaconic acid production in minimal medium with glucose as substrate supplemented with small amounts of glutamic acid. The derived E. coli strain (ita23: MG1655 ∆aceA ∆sucCD ∆pykA ∆pykF ∆pta ∆Picd::cam_BBa_J23115 pCadCS) synthesized 2.27g/l itaconic acid with an excellent yield of 0.77mol/(mol glucose). In a fed-batch cultivation, this strain produced 32g/l itaconic acid with an overall yield of 0.68mol/(mol glucose) and a peak productivity of 0.45g/l/h. These values are by far the highest that have ever been achieved for heterologous itaconic acid production and indicate that realistic applications come into reach.

Metabolic engineering of Corynebacterium glutamicum for methionine production by removing feedback inhibition and increasing NADPH level.

Relieving the feedback inhibition of key enzymes in a metabolic pathway is frequently the first step of producer-strain construction by genetic engineering. However, the strict feedback regulation exercised by microorganisms in methionine biosynthesis often makes it difficult to produce methionine at a high level. In this study, Corynebacterium glutamicum ATCC 13032 was metabolically engineered for methionine production. First, the metD gene encoding the methionine uptake system was deleted to achieve extracellular accumulation of methionine. Then, random mutagenesis was performed to remove feedback inhibition by metabolic end-products. The resulting strain C. glutamicum ENM-16 was further engineered to block or decrease competitive branch pathways by deleting the thrB gene and changing the start codon of the dapA gene, followed by point mutations of lysC (C932T) and pyc (G1A, C1372T) to increase methionine precursor supply. To enrich the NADPH pool, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the pentose phosphate pathway were mutated to reduce their sensitivity to inhibition by intracellular metabolites. The resultant strain C. glutamicum LY-5 produced 6.85±0.23gmethioninel(-1) with substrate-specific yield (Y P/S) of 0.08mol per mol of glucose after 72h fed-batch fermentation. The strategies described here will be useful for construction of methionine engineering strains.

Co-production of hydrogen and ethanol by pfkA-deficient Escherichia coli with activated pentose-phosphate pathway: reduction of pyruvate accumulation.

BackgroundFermentative hydrogen (H2) production suffers from low carbon-to-H2 yield, to which problem, co-production of ethanol and H2 has been proposed as a solution. For improved co-production of H2 and ethanol, we developed Escherichia coli BW25113 ΔhycA ΔhyaAB ΔhybBC ΔldhA ΔfrdAB Δpta-ackA ΔpfkA (SH8*) and overexpressed Zwf and Gnd, the key enzymes in the pentose-phosphate (PP) pathway (SH8*_ZG). However, the amount of accumulated pyruvate, which was significant (typically 0.20 mol mol−1 glucose), reduced the co-production yield.ResultsIn this study, as a means of reducing pyruvate accumulation and improving co-production of H2 and ethanol, we developed and studied E. coli SH9*_ZG with functional acetate production pathway for conversion of acetyl-CoA to acetate (pta-ackA+). Our results indicated that the presence of the acetate pathway completely eliminated pyruvate accumulation and substantially improved the co-production of H2 and ethanol, enabling yields of 1.88 and 1.40 mol, respectively, from 1 mol glucose. These yields, significantly, are close to the theoretical maximums of 1.67 mol H2 and 1.67 mol ethanol. To better understand the glycolytic flux distribution, glycolytic flux prediction and RT-PCR analyses were performed.ConclusionThe presence of the acetate pathway along with activation of the PP pathway eliminated pyruvate accumulation, thereby significantly improving co-production of H2 and ethanol. Our strategy is applicable to anaerobic production of biofuels and biochemicals, both of which processes demand high NAD(P)H.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0510-5) contains supplementary material, which is available to authorized users.

Biohydrogen Production from Simple Carbohydrates with Optimization of Operating Parameters

Hydrogen could be alternative energy carrier in the future as well as source for chemical and fuel synthesis due to its high energy content, environmentally friendly technology and zero carbon emissions. In particular, conversion of organic substrates to hydrogen via dark fermentation process is of great interest. The aim of this study was fermentative hydrogen production using anaerobic mixed culture using different carbon sources (mono and disaccharides) and further optimization by varying a number of operating parameters (pH value, temperature, organic loading, mixing intensity). Among all tested mono- and disaccharides, glucose was shown as the preferred carbon source exhibiting hydrogen yield of 1.44 mol H(2)/mol glucose. Further evaluation of selected operating parameters showed that the highest hydrogen yield (1.55 mol H(2)/mol glucose) was obtained at the initial pH value of 6.4, T=37 °C and organic loading of 5 g/L. The obtained results demonstrate that lower hydrogen yield at all other conditions was associated with redirection of metabolic pathways from butyric and acetic (accompanied by H(2) production) to lactic (simultaneous H(2) production is not mandatory) acid production. These results therefore represent an important foundation for the optimization and industrial-scale production of hydrogen from organic substrates.