Enhanced glutamic acid production by a H+-ATPase-defective mutant of Corynebacterium glutamicum

Previously we reported that a mutant of Corynebacterium glutamicum ATCC14067 with reduced H+-ATPase activity, F172-8, showed an approximately two times higher specific rate of glucose consumption than the parent, but no glutamic acid productivity under the standard biotin-limited culture conditions, where biotin concentration was set at 5.5 microg/l in the production medium (Sekine et al., Appl. Microbiol. Biotechnol., 57, 534-540 (2001)). In this study, various culture conditions were tested to check the glutamic acid productivity of strain F172-8. The mutant was found to produce glutamic acid under exhaustive biotin limitation, where the biotin concentration of the medium was set at 2.5 microg/l with much smaller inoculum size. When strain F172-8 was cultured under the same biotin-limited conditions using a jar fermentor, 53.7 g/l of glutamic acid was produced from 100 g/l glucose, while the parent produced 34.9 g/l of glutamic acid in a medium with 5.5 microg/l biotin. The glutamic acid yield of strain F172-8 also increased under Tween 40-triggered production conditions (1.2-fold higher than the parent strain). The amounts of biotin-binding enzymes were investigated by Western blot analysis. As compared to the parent, the amount of pyruvate carboxylase was lower in the mutant; however, the amount of acetyl-CoA carboxylase did not significantly change under the glutamic acid production conditions. To the best of our knowledge, this is the first report showing that the H+-ATPase-defective mutant of C. glutamicum is useful in glutamic acid production.     

光滑球拟酵母新霉素抗性株加速葡萄糖代谢

为进一步提高光滑球拟酵母发酵生产丙酮酸的生产强度,在能量代谢分析的基础上提出了降低ATP合成酶活性、但不影响NADH氧化的育种策略。通过亚硝基胍诱变,获得一株新霉素抗性突变株N07,该菌株F1ATPase活性降低65%、丙酮酸产量高于48gL且单位细胞消耗葡萄糖能力提高38%。添加双环己基碳二亚胺(DCCD)、叠氮钠(NaN3)、新霉素显著降低出发株F1ATPase活性但不影响突变株F1ATPase活性。突变菌株胞内ATP含量下降23.7%导致生长速率和最终菌体浓度(为出发菌株的76%)均低于出发菌株,但葡萄糖消耗速度和丙酮酸生产速度分别提高34%和42.9%,发酵周期缩短12h。进一步研究发现,突变株糖酵解途径中关键酶磷酸果糖激酶、丙酮酸激酶和磷酸甘油醛激酶的活性提高了63.7%、28.8%和14.4%,电子传递链关键酶活性提高10%。结果表明降低真核微生物F1ATPase活性有效地提高了糖酵解关键酶活性而加速葡萄糖代谢。     

Carboxylic acid consumption and production by Corynebacterium glutamicum

Corynebacterium glutamicum is well-known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Previous studies of the effect of gradients in scale-down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation. This study, therefore, addresses the impact of carboxylic acids, for example, acetate and l -lactate, on the cultivation of the cadaverine producing strain C. glutamicum DM1945Δact3:P_tuf-ldcC^opt and their potential role in scale up related performance losses. A fluctuating power input in shake flask and stirred tank cultivations with mineral salt was applied to mimic discontinuous oxygen availability. Results demonstrate, whenever sufficient oxygen was available, C. glutamicum recovered from previously occurring stressful conditions like an oxygen limiting phase. Reassimilation of acids was detected simultaneously. In cultures, which were supplemented with either acetate or l -lactate, a rapid cometabolization of both acids in presence of glucose was observed, showing conversion rates of 7.8 and 3.8 mmol g_{cell dry weight}⁻¹ hr⁻¹, respectively. Uptake of these acids was accompanied by increased oxygen consumption. Proteins related to oxidative stress response, glycogen synthesis, and the main carbon metabolism were found in altered concentrations under oscillatory cultivation conditions. (Proteomics data are available via ProteomeXchange with identifier PXD012760). Virtually no impact on growth or product formation was observed. We conclude that the reduced growth and product formation in scale-down cultivations when complex media was used is not caused by the accumulation of carboxylic acids.     

Enhanced valine production in Corynebacterium glutamicum with defective H+-ATPase and C-terminal truncated acetohydroxyacid synthase

We have reported increased glutamate production by a mutant of Corynebacterium glutamicum ATCC14067 (strain F172-8) with reduced H(+)-ATPase activity under biotin-limiting culture conditions (Aoki et al. Biosci. Biotechnol. Biochem., 69, 1466-1472 (2005)). In the present study, we examined valine production by an H(+)-ATPase-defective mutant of C. glutamicum. Using the double-crossover chromosome replacement technique, we constructed a newly defined H(+)-ATPase-defective mutant from ATCC13032. After transforming the new strain (A-1) with a C-terminal truncation of acetohydroxyacid synthase gene (ilvBN), valine production increased from 21.7 mM for the wild-type strain to 46.7 mM for the A-1 in shaking flask cultures with 555 mM glucose. Increased production of the valine intermediate acetoin was also observed in A-1, and was reduced by inserting acetohydroxyacid isomeroreductase gene (ilvC) into the ilvBN plasmid. After transformation with this new construct, valine production increased from 38.3 mM for the wild-type strain to 95.7 mM for A-1 strain. To the best of our knowledge, this is the first report indicating that an H(+)-ATPase-defective mutant of C. glutamicum is capable of valine production. Our combined results with glutamate and valine suggest that the H(+)-ATPase defect is also effective in the fermentative production of other practical compounds.     

Glutamic acid production with gel-entrapped Corynebacterium glutamicum

A glutamic acid producing microorganism (Corynebacterium glutamicum) is entrapped in a polyacrylamide gel. These immobilized microorganisms were used to produce glutamic acid in successive batches of fresh medium. Free microorganisms similarly used produced much less glutamic acid under similar conditions.     

Sequential optimization approach for enhanced production of glutamic acid from Corynebacterium glutamicum 2262 using date juice

To improve glutamic acid production from Corynebacterium glutamicum 2262 using date juice, a culture medium was screened and optimized using the statistical experimental designs of Plackett-Burman and response-surface methodology. In the first step, a two-level Plackett-Burman design was adopted to select the most important nutrients influencing the glutamic acid production, which showed that the date juice sugars, urea, peptone, and glycine betaine were the most significant ingredients (P < 0.05). Finally, response surface Box-Behnken design was employed to develop a mathematical model to identify the optimum concentrations of key components for higher glutamic acid production, which revealed the following: date juice (45 g/L), urea (16.9 g/L), peptone (15 g/L), and glycine betaine (12 g/L). The high correlation between the predicted and observed values indicated the validity of the model. Glutamic acid concentration increased significantly with optimized medium (33.2 g/L) when compared with non-optimized medium (12 g/L).     

Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum

Corynebacterium glutamicum, an established industrial amino acid producer, has been genetically modified for efficient succinate production from the renewable carbon source glucose under fully aerobic conditions in minimal medium. The initial deletion of the succinate dehydrogenase genes (sdhCAB) led to an accumulation of 4.7 g l^?1 (40 mM) succinate as well as high amounts of acetate (125 mM) as by‐product. By deleting genes for all known acetate‐producing pathways (pta‐ackA, pqo and cat) acetate production could be strongly reduced by 83% and succinate production increased up to 7.8 g l^?1 (66 mM). Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l^?1 (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)^?1 h^?1. This value represents the highest productivity among currently described aerobic bacterial succinate producers. Optimization of the production conditions by decoupling succinate production from cell growth using the most advanced producer strain (C. glutamicumΔpqoΔpta‐ackAΔsdhCABΔcat/pAN6‐pyc^P458Sppc) led to an additional increase of the product yield to 0.45 mol succinate mol^?1 glucose and a titre of 10.6 g l^?1 (90 mM) succinate.     

Modelling L-glutamic acid production with Corynebacterium glutamicum under biotin limitation

The production of L-glutamic acid with Corynebacterium glutamicum under biotin limitation was studied. Assuming a formal type of cell maturation, an adequate formal kinetic model was developed. This model includes growth, dependent on biotin, and uses the same retention term for describing the lag phase and cell maturation. Special attention was paid to the graphical interpretation of the performance between the variables, which is relevant for kinetics. Comparison between experiments and the model resulted in different degrees of agreement. However, the main trend of the experimental patterns of the complex bioprocess can clearly be mirrored in this model.     

谷氨酸棒杆菌H+-ATPase基因失活提高谷氨酸产生量

为了证实在谷氨酸棒杆菌中,利用H⁺-ATPase基因失活构建高产谷氨酸基因工程菌的应用可行性,通过重组PCR技术部分缺失H⁺-ATPaseγ亚基基因序列,采用插入失活方法构建H⁺-ATPase失活的谷氨酸棒杆菌。考察了其谷氨酸产生能力及对生长速率的影响。实验结果表明,H⁺-ATPase失活的谷氨酸棒杆菌在含有100g/L的葡萄糖培养基中摇瓶发酵,其谷氨酸最大累积量为51.6g/L, 比野生菌株提高了42.9%。生长速率研究结果表明,H⁺-ATPase失活的谷氨酸棒杆菌生长速率略低于野生谷氨酸棒杆菌。证实了H⁺-ATPase基因失活对提高谷氨酸产量的作用,为利用H⁺-ATPase基因构建高产谷氨酸基因工程菌株提供了科学依据。     

Glutamate excretion as a major kinetic bottleneck for the thermally triggered production of glutamic acid by Corynebacterium glutamicum

The study was aimed at evaluating the extent of flux control exercised by the amino acid excretion step on the glutamate production flux in C. glutamicum 2262 strain that is induced for glutamate excretion by an upward temperature shift. Cells initially induced to excrete glutamate were cultivated at different controlled temperatures between 33 and 40 degrees C, and changes in glutamate excretion flux and intracellular concentration were determined in response to increased culture temperature. The fastest growth rate of 0.45 h(-1) and the lowest glutamate excretion rate of 1 mmole/g dw x h were observed at 33 degrees C, together with a high intracellular 0.5 mmole/g dw glutamate accumulation. On the contrary, the fastest glutamate excretion rate of 6 mmole/g dw x h was obtained at 40 degrees C, when cell growth was arrested and the internal glutamate level reduced to 0.25 mmol/g dw. The observed sixfold increase in excretion flux as a result of the temperature increase clearly suggests a specific effect of temperature on the glutamate export system which appears as the major kinetic bottleneck for the glutamate production flux. This conclusion is corroborated by the high internal accumulation of glutamate which, even under the fastest excretion conditions, severely inhibits the activity of the glutamate biosynthesis pathway.     

Feedback-resistant acetohydroxy acid synthase increases valine production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km(r)). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum DeltailvA DeltapanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.     

Modelling of the L-glutamic acid production with Corynebacterium glutamicum under biotin limitation

Production of L-glutamic acid with Corynebacterium glutamicum, under biotin limitation was studied. On the base of formal kinetic approach, a mathematical model was developed, which included formal growth inhibition with product, and production repression with substrate. For the testing of computer simulations experiments were carried out and the results have been compared.     

Importance of phosphoenolpyruvate carboxylase of Corynebacterium glutamicum during the temperature triggered glutamic acid fermentation

To give clues about the respective importance of phosphoenol-pyruvate carboxylase (PEPc) and pyruvate carboxylase (Pc) in Corynebacterium glutamicum metabolism during a temperature triggered glutamic acid fermentation, PEPc activity was genetically amplified and Pc activity was suppressed by biotin limitation in the culture medium. In absence of Pc activity, glutamate production was dramatically reduced whereas lactate excretion was strongly increased. Whereas PEPc amplification in excess of biotin (4 mg/L) only slightly modified the cell kinetics, under biotin limiting conditions this amplification strongly improved the glutamate production (4 microg/L). When Pc was absent, PEPc activity was sufficient to allow up to 70% of the maximal glutamate production rate and seemed to have an important anaplerotic role, especially at the beginning of the production phase. In contrast, Pc was predominant during the remainder of the glutamate fermentation.     

Bio-based production of organic acids with Corynebacterium glutamicum

The shortage of oil resources, the steadily rising oil prices and the impact of its use on the environment evokes an increasing political, industrial and technical interest for development of safe and efficient processes for the production of chemicals from renewable biomass. Thus, microbial fermentation of renewable feedstocks found its way in white biotechnology, complementing more and more traditional crude oil-based chemical processes. Rational strain design of appropriate microorganisms has become possible due to steadily increasing knowledge on metabolism and pathway regulation of industrially relevant organisms and, aside from process engineering and optimization, has an outstanding impact on improving the performance of such hosts. Corynebacterium glutamicum is well known as workhorse for the industrial production of numerous amino acids. However, recent studies also explored the usefulness of this organism for the production of several organic acids and great efforts have been made for improvement of the performance. This review summarizes the current knowledge and recent achievements on metabolic engineering approaches to tailor C. glutamicum for the bio-based production of organic acids. We focus here on the fermentative production of pyruvate, l-and d-lactate, 2-ketoisovalerate, 2-ketoglutarate, and succinate. These organic acids represent a class of compounds with manifold application ranges, e.g. in pharmaceutical and cosmetics industry, as food additives, and economically very interesting, as precursors for a variety of bulk chemicals and commercially important polymers.Funding Information Work in the laboratories of the authors was supported by the Fachagentur Nachwachsende Rohstoffe (FNR) of the Bundesministerium für Ernährung, Landwirtschaft und Verbraucherschutz (BMELV; FNR Grants 220-095-08A and 220-095-08D; Bio-ProChemBB project, ERA-IB programme), by the Deutsche Bundesstiftung Umwelt (DBU Grant AZ13040/05) and the Evonik Degussa AG.     

Effect of glutamic acid oversynthesis on the development of cyanide-resistant respiration in the bacterium Corynebacterium glutamicum

The composition of the respiratory chains of the wild stain Corynebacterium glutamicum and of its mutant differing in their ability for the glutamic acid oversynthesis in a medium with melassa was studied. Under excess of biotine and the parent strain is incapable of acid oversynthesis, while the mutant forms and excretes the acid. Both bacterial strains contain menaquinone and equal sets of cytochromes C550, b556, b563, and a600. The membrane-bound dehydrogenases of the parent strain are represented by NADH-, NADPH- and succinate dehydrogenases. Unlike the parent strain, the mutant membrane preparation does not oxidize NADPH. Both strains do not practically differ in their menaquinone content. The cyanide-resistant oxidase of a non-cytochrome nature appears in the wild strain during its transfer to the stationary growth phase. Induction of glutamic acid oversnythesis by addition of penicilline prevents the formation of the cyanide-resistant oxidase. On the contrary, the mutant transfer to the stationary growth phase is not accompanied by a formation of cyanide-resistant oxidase, which appears only after cessation of glutamic acid oversynthesis. Induction of the cyanide-resistant respiration by addition of cyanide inhibits the acid oversynthesis. Oxidation of substrates by membrane preparations of both bacterial strains in the absence and presence of cyanide is not followed by the hydrogen peroxide formation. It is assumed that there exist competitive interactions between the supersynthesis of glutamic acid and the cyanide-resistant respiration. The possible structure of the respiratory chain of Cor. glutamicum is discussed.     

A method for the determination of pyruvate carboxylase activity during the glutamic acid fermentation with Corynebacterium glutamicum

A discontinuous lactate dehydrogenase coupled assay is described for the evaluation of the pyruvate carboxylase activity (Pc, EC 6.4.1.1) in a glutamate overproducing strain of Corynebacterium glutamicum. After an initial permeabilisation period of the cells, the method consisted of the fluorometric determination of the remaining pyruvate level after transformation into oxaloacetate by the endogenous Pc. The assay was demonstrated to be powerful and enabled the determination of the C. glutamicum Pc activity grown on different carbon sources. Besides, this method was used to assay Pc activity in C. glutamicum 2262 during a temperature triggered glutamate producing process with biotin excess or limitation.