Engineering <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for the production of pyruvate

A Corynebacterium glutamicum strain with inactivated pyruvate dehydrogenase complex and a deletion of the gene encoding the pyruvate:quinone oxidoreductase produces about 19 mM l-valine, 28 mM l-alanine and about 55 mM pyruvate from 150 mM glucose. Based on this double mutant C. glutamicum △aceE △pqo, we engineered C. glutamicum for efficient production of pyruvate from glucose by additional deletion of the ldhA gene encoding NAD⁺-dependent l-lactate dehydrogenase (LdhA) and introduction of a attenuated variant of the acetohydroxyacid synthase (△C–T IlvN). The latter modification abolished overflow metabolism towards l-valine and shifted the product spectrum to pyruvate production. In shake flasks, the resulting strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN produced about 190 mM pyruvate with a Y _P/S of 1.36 mol per mol of glucose; however, it still secreted significant amounts of l-alanine. Additional deletion of genes encoding the transaminases AlaT and AvtA reduced l-alanine formation by about 50%. In fed-batch fermentations at high cell densities with adjusted oxygen supply during growth and production (0–5% dissolved oxygen), the newly constructed strain C. glutamicum △aceE △pqo △ldhA △C–T ilvN △alaT △avtA produced more than 500 mM pyruvate with a maximum yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol g_(CDW)⁻¹ h⁻¹ (i.e., 0.08 g g_(CDW) ⁻¹ h⁻¹) in the production phase.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

Direct production of cadaverine from soluble starch using <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> coexpressing α-amylase and lysine decarboxylase

Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 α-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli–C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5’-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation of l-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources.     

Microbial production of L -glutamate and L -glutamine by recombinant Corynebacterium glutamicum harboring Vitreoscilla hemoglobin gene vgb

Vitreoscilla hemoglobin (VHb) gene vgb equipped with a native promoter Pvgb or a tac promoter Ptac was introduced into Corynebacterium glutamicum ATCC14067, respectively. Ptac was proven to be more suitable for expressing VHb protein in higher concentration in both Escherichia coli and C. glutamicum strains compared with the native vgb promoter Pvgb. VHb-expressing C. glutamicum exhibited higher oxygen uptake rate and enhanced cell growth. Recombinant C. glutamicum harboring vgb gene equipped with Ptac promoter produced 23% more l-glutamate in shake-flask culture and grew to 30% more cell density and formed 22% more l-glutamate in fermentor studies compared with the wild-type strain. When a site-directed mutagenesis in which Tyr405 was replaced by a phenylalanine residue (Y405F) was performed on glutamine synthesis gene, recombinant C. glutamicum overexpressing the mutated gene glnA′ was able to produce l-glutamine effectively. Co-expression of vgb and glnA′ genes in C. glutamicum produced 17 g/l l-glutamine in shake flask culture, approximately 30% more than that produced by the recombinant harboring only glnA′ gene. In fermentor cultivation, the recombinant yielded 25% more cells and produced 40.5 g/l l-glutamine. In this study, it was clearly demonstrated that VHb significantly enhanced cell growth, l-glutamate, and l-glutamine production by recombinant C. glutamicum.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.     

Carbohydrate metabolism in Corynebacterium glutamicum and applications for the metabolic engineering of l-lysine production strains

Carbohydrates exclusively serve as feedstock for industrial amino acid production with Corynebacterium glutamicum. Due to the industrial interest, knowledge about the pathways for carbohydrate metabolization in C. glutamicum steadily increases, enabling the rational design of optimized strains and production processes. In this review, we provide an overview of the metabolic pathways for utilization of hexoses (glucose, fructose), disaccharides (sucrose, maltose), pentoses (d-ribose, l-arabinose, d-xylose), gluconate, and β-glucosides present in C. glutamicum. Recent approaches of metabolic engineering of l-lysine production strains based on the known pathways are described and evaluated with respect to l-lysine yields.     

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD⁺-dependent l-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator d-fructose-1,6-bisphosphate (Fru-1,6-P₂). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with V _max for the reduction of pyruvate being approximately tenfold that of the value for l-lactate oxidation at pH 7.5. No deviation from Michaelis–Menten kinetics was observed in the presence of Fru-1,6-P₂, while a sigmoidal response (indicative of positive cooperativity) was seen towards l-lactate without Fru-1,6-P₂. Strikingly, when introduced into an lldD ⁻ strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on l-lactate as the sole carbon source.     

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a K_m of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside.     

A flow injection analysis system with encapsulated high-density <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells for rapid determination of biochemical oxygen demand

The biochemical oxygen demand (BOD) determination was studied using a novel flow injection analysis (FIA) system with encapsulated Saccharomyces cerevisiae cells and an oxygen electrode and was compared with conventional 5-day BOD tests. S. cerevisiae cells were packed in a calcium alginate capsule at a dry cell weight of 250 g/l of capsule core. The level of dissolved oxygen (DO) was reduced due to the enhanced respiratory activity of the microbial cells when the injected nutrient passed through the bioreactor. The decrease in DO (ΔDO) was intensified with the amount of microbial cells packed in the bioreactor. However, the specific ΔDO decreased as the amount of cells loaded in the bioreactor increased. The ΔDO value was dependent on the pH and temperature of the mobile phase and reached its maximum value at 35°C and pH 7–8. Also, ΔDO became larger at longer response times as the flow rate of the mobile phase decreased. The measurement of ΔDO was repeated more than six times consecutively using a 20-ppm standard glucose and glutamic acid solution, which confirmed the reproducibility with a standard deviation of 0.95%. A strong linear correlation between ΔDO and BOD was also observed. The 5-day BOD values of actual water and wastewater samples were in accordance with the BOD values obtained by this FIA method using encapsulated S. cerevisiae cells. Unlike the cell-immobilized bead system, there was no contamination of the bioreactor resulting from any leak of yeast cells from the sensor capsules during BOD measurements.     

Effect of tween 80 and dimethyl sulfoxide on biosynthesis ofl-lysine in regulatory mutants ofCorynebacterium glutamicum

By using dimethyl sulfoxide or Tween 80 (1 or 0.2 %), the production ofl-lysine was increased by 20–28 and 23–25%, respectively, in regulatory mutant strains ofCorynebacterium glutamicum. The stimulation observed is supposed to be caused by influencing cellular surface structures. Translated by Č. Novotny     

Metabolic activity of Corynebacterium glutamicum grown on l-lactic acid under stress

Respiration measurement in shake flasks is introduced as a new method to characterize the metabolic activity of microorganisms during and after stress exposure. The major advantage of the new method is the possibility to determine the metabolic activity independent of manual sampling without the necessity to change the culture vessel or the cultivation medium. This excludes stress factors, which may be induced by transferring the microorganisms to plates or respirometers. The negative influence, which interruptions of the shaker during sampling times may have on the growth of microorganisms was demonstrated. The applicability of the method was verified by characterizing the behavior of Corynebacterium glutamicum grown on the carbon source l-lactic acid under stress factors such as carbon starvation, anaerobic conditions, lactic acid, osmolarity, and pH. The following conditions had no effect on the metabolic activity of C. glutamicum: a carbon starvation of up to 19 h, anaerobic conditions, lactic acid concentrations up to 10 g/l, 3-(N-morpholino) propanesulfonic acid buffer concentrations up to 42 g/l, or pH from 6.4 to 7.4. Lactic-acid concentrations from 10 to 30 g/l lead to a decrease of the growth rate and the biomass substrate yield without effecting the oxygen substrate conversion. Without adaptation, the organism did not grow at pH≤5 or ≥9.     

Factors affecting the production of <Emphasis Type="SmallCaps">l</Emphasis>-xylulose by resting cells of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l⁻¹. A specific productivity of 1.09 ± 0.10 g g⁻¹ h⁻¹ was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.     

Purification and characterization of an extracellular alpha-L-arabinosidase from a novel isolate Bacillus pumilus ARA and its over-expression in Escherichia coli

The α-l-arabinosidase, AraB, was induced when Bacillus pumilus ARA was grown at 50°C in a minimal medium containing xylan. A 56-kDa protein with α-l-arabinosidase activity was purified from culture supernatant to gel electrophoretic homogeneity. The optimal activity was at pH 6.4 and 60°C over a 10-min assay. The purified enzyme was stable over a pH range of 5.2–7.6 and had a 1-h half life at 70°C. The enzyme released arabinose from oat spelt xylan. Kinetic experiments at 60°C with p-nitrophenyl α-l-arabinofuranoside as substrate gave a K _m, and V _max of 1.05 mM and 240 U per mg of protein. The NH₂-terminal amino acid sequence of the enzyme was determined, and its gene araB was subsequently cloned, sequenced, and over-expressed in Escherichia coli. The open reading frame of araB consists of a 1,479-bp fragment encoding a protein of 472 amino acids, which belonged to family 51 of the glycoside hydrolases with an identity of 67% to the protein encoded by abfB of Bacillus subtilis 168.     

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.     

Expression of glf Z.m. increases D-mannitol formation in whole cell biotransformation with resting cells of Corynebacterium glutamicum

A recombinant oxidation/reduction cycle for the conversion of D-fructose to D-mannitol was established in resting cells of Corynebacterium glutamicum. Whole cells were used as biocatalysts, supplied with 250 mM sodium formate and 500 mM D-fructose at pH 6.5. The mannitol dehydrogenase gene (mdh) from Leuconostoc pseudomesenteroides was overexpressed in strain C. glutamicum ATCC 13032. To ensure sufficient cofactor [nicotinamide adenine dinucleotide (reduced form, NADH)] supply, the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae N10 was coexpressed. The recombinant C. glutamicum cells produced D-mannitol at a constant production rate of 0.22 g (g cdw)⁻¹ h⁻¹. Expression of the glucose/fructose facilitator gene glf from Zymomonas mobilis in C. glutamicum led to a 5.5-fold increased productivity of 1.25 g (g cdw)⁻¹ h⁻¹, yielding 87 g l⁻¹ D-mannitol from 93.7 g l⁻¹ D-fructose. Determination of intracellular NAD(H) concentration during biotransformation showed a constant NAD(H) pool size and a NADH/NAD⁺ ratio of approximately 1. In repetitive fed-batch biotransformation, 285 g l⁻¹ D-mannitol over a time period of 96 h with an average productivity of 1.0 g (g cdw)⁻¹ h⁻¹ was formed. These results show that C. glutamicum is a favorable biocatalyst for long-term biotransformation with resting cells. Dedicated to Prof. Hermann Sahm on the occasion of his 65th birthday.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Cloning,expression, and characterization of an insoluble glucan-producing glucansucrase from <Emphasis Type="BoldItalic">Leuconostoc mesenteroides</Emphasis> NRRL B-1118

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K _M 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0–6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-d-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked d-glucopyranosyl units and a low degree of branching.     

Cloning, purification, and characterization of a thermostable alpha-L-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari

The gene, AbfAC26Sari, encoding an α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a V _max and K _m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its activity was strongly inhibited by 1 mM Cu²⁺ and Hg²⁺. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose. Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme.     

Zhihengliuella salsuginis sp. nov., a moderately halophilic actinobacterium from a subterranean brine

A novel moderately halophilic, alkaliphilic, non-motile, non-sporulating, catalase-positive, oxidase-negative, aerobic, coccus-shaped, Gram-positive bacterium, designated strain JSM 071043^T, was isolated from a subterranean brine sample collected from a salt mine in Hunan Province, China. Growth occurred with 0.5–20% (w/v) NaCl (optimum 5–10%) at pH 6.5–10.5 (optimum pH 8.5) and at 10–40°C (optimum 25–30°C). Good growth also occurred in the presence of 0.5–20% (w/v) KCl (optimum 5–8%) or 0.5–25% (w/v) MgCl₂·6H₂O (optimum 5–10%). The peptidoglycan type was A4α (l-Lys–l-Ala–l-Glu) and major cell-wall sugars were tyvelose and mannose. The major cellular fatty acids were anteiso-C_15:0, iso-C_16:0 and anteiso-C_17:0. Strain JSM 071043^T contained MK-9 and MK-8 as the predominant menaquinones and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the major polar lipids. The DNA G + C content was 67.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JSM 071043^T was a member of the suborder Micrococcineae, and was most closely related to Zhihengliuella halotolerans YIM 70185^T (sequence similarity 98.9%) and Zhihengliuella alba YIM 90734^T (98.2%), and the three strains formed a distinct branch in the phylogenetic tree. The combination of phylogenetic analysis, DNA–DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the proposal that strain JSM 071043^T represents a novel species of the genus Zhihengliuella, for which the name Z. salsuginis sp. nov. is proposed. The type strain is JSM 071043^T (= DSM 21149^T = KCTC 19466^T).