Mechanism of Transfection with Deoxyribonucleic Acid from the Temperate Bacillus Bacteriophage φ105

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.     

l-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum

Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for l-valine production by over-expressing ilvEBN ^r C genes at 31?°C in 72?h fermentation. Different strategies were carried out to reduce the by-products’ accumulation in l-valine fermentation and also to increase the availability of precursor for l-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of l-isoleucine. Effect of different relative dissolved oxygen on l-valine production and by-products’ formation was recorded, indicating that 15?% saturation may be the most appropriate relative dissolved oxygen for l-valine fermentation with almost no l-lactic acid and l-glutamate formed. To minimize l-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of l-alanine accumulated by alaT inactivated strains harboring ilvEBN ^r C genes, l-alanine concentration was reduced to 0.18?g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, and 0.22?g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. Meanwhile, l-valine production and conversion efficiency were enhanced to 31.15?g/L and 0.173?g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, 38.82?g/L and 0.252?g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. This study provides combined strategies to improve l-valine yield by minimization of by-products’ production.     

Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s⁻¹), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s⁻¹), and high tolerance to product inhibition (K_i for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.     

N-Acetylornithine-δ-aminotransferase-deficient and N-Acetylglutamokinase-deficient Arginine Auxotrophs of Corynebacterium glutamicum

An N-acetylglutamokinase-deficient arginine-requiring mutant, KY9390 and an N-acetylornithine aminotransferase-deficient arginine-requiring mutant, AA-7 were derived from a wild-type strain and an l-arginine-producing mutant of Corynebacterium glutamicum, respectively. KY 9390 accumulated 7.5 mg per ml of N-acetylglutamic acid and AA-7 accumulated 2 mg per ml of N-acetylglutamate-γ-semialdehyde, intermediates of arginine biosynthesis, in a culture medium.

The production of N-acetylglutamate-γ-semialdehyde by AA-7 was not affected by the concentration of l-arginine in the medium, whereas that of N-acetylglutamic acid by KY 9390 was inhibited by the addition of l-arginine in the medium.     

Corynebacterium glutamicum,a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro‐aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild‐type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L^?1) corresponded to the lower k_La value of 5 h^?1. Above 5 h^?1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L^?1. Taking into account this threshold, a fed‐batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild‐type strain was able to produce 93.6 g L^?1 of succinate, which is one of the highest concentration reported in the literature.     

Microbial production of l-leucine from α-ketoisocaproate by Corynebacterium glutamicum

Summary A process for l-leucine production was studied using Corynebacterium glutamicum for the conversion of -ketoisocaproate. When this precursor was added to the culture medium in a concentration of 20 g/l about 16 g/l l-leucine were formed after a fermentation time of 57 h and the molar yield was 91%. Using a fed-batch culture it was possible to produce 24 g/l of l-leucine from 32 g/l of -ketoisocaproate within 23 h. Enzymatic studies indicate that in this glutamate-producing bacterium -ketoisocaproate is converted into l-leucine via the transaminase B reaction and l-glutamate is regenerated by the glutamate dehydrogenase. By the addition of -ketoisocaproate to the culture medium the specific activity of transaminase B was increased threefold.     

The amrG1 gene is involved in the activation of acetate in Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic, Gram-positive microorganism, well known as a pro-ducer of several amino acids. Amino acid products are used on a large scale for food industry flavouring, feed additive, pharmaceutical and cosmetic purpose[1,2]. The organism is able to grow not only on glucose, fructose and lactose, but also on acetate, lactate as its sole carbon source. The growth on acetate requires its activation to acetyl-CoA. In C. glutamicum, acetate is activated in a two-step …     

Enhanced production of neoagarobiose from agar with Corynebacterium glutamicum producing exo-type and endo-type β-agarases

Neoagarobiose (NA2) derived from agar marine biomass is a rare reagent that acts as an anti-melanogenesis reagent and moisturizer. Here, for the economical manufacturing of NA2, we developed the co-secretory production system of endo-type β-agarases (DagA) and exo-type β-agarases (EXB3) in Corynebacterium glutamicum. For this purpose, we first developed a secretory system of DagA via Tat pathway. To improve the secretion efficiency, we coexpressed two Tat pathway components (TatA and TatC), and to improve the purity of secreted DagA in the culture supernatant, two endogenous protein genes (Cg2052 and Cg1514) were removed. Using the engineered strain (C. glutamicum SP002), we confirmed that DagA as high as 1.53 g l^-1 was successfully produced in the culture media with high purity (72.7% in the supernatant protein fraction). Next, we constructed the expression system (pHCP-CgR-DagA-EXB3) for the simultaneous secretion of EXB3 via Sec-pathway together with DagA, and it was clearly confirmed that DagA and EXB3 were successfully secreted as high as 54% and 24.5%, respectively. Finally, using culture medium containing DagA and EXB3, we successfully demonstrated the conversion of high-concentration agar (40 g l^-1) into NA2 via a two-stage hydrolysis process.     

Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.     

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of β-alanine

β-Alanine is an important β-amino acid with a growing demand in a wide range of applications in chemical and food industries. However, current industrial production of β-alanine relies on chemical synthesis, which usually involves harmful raw materials and harsh production conditions. Thus, there has been increasing demand for more sustainable, yet efficient production process of β-alanine. In this study, we constructed Corynebacterium glutamicum strains for the highly efficient production of β-alanine through systems metabolic engineering. First, aspartate 1-decarboxylases (ADCs) from seven different bacteria were screened, and the Bacillus subtilis ADC showing the most efficient β-alanine biosynthesis was used to construct a β-alanine-producing base strain. Next, genome-scale metabolic simulations were conducted to optimize multiple metabolic pathways in the base strain, including phosphotransferase system (PTS)-independent glucose uptake system and the biosynthesis of key precursors, including oxaloacetate and L-aspartate. TCA cycle was further engineered for the streamlined supply of key precursors. Finally, a putative β-alanine exporter was newly identified, and its overexpression further improved the β-alanine production. Fed-batch fermentation of the final engineered strain BAL10 (pBA2_tr18) produced 166.6 g/L of β-alanine with the yield and productivity of 0.28 g/g glucose and 1.74 g/L/h, respectively. To our knowledge, this production performance corresponds to the highest titer, yield and productivity reported to date for the microbial fermentation.     

Involvement of meso-α,∊-Diamino-pimelate D-Dehydrogenase in Lysine Biosynthesis in Corynebacterium glutamicum

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

Improved d,l-α-hydroxybutyrate conversion to l-isoleucine with Corynebacterium glutamicum mutants with increased d-lactate utilization

Summary Corynebacterium glutamicum possesses NAD-independent lactate dehydrogenases. The d-lactate dehydrogenase is consitutive, the l-lactate dehydrogenase is inducible. Enzyme measurements, gel electrophoretic studies and mutant studies suggest that both enzymes are responsible for the oxidation of the chemically synthesized precursor dl--hydroxybutyrate. Mutants with increased d-lactate utilization were selected. In mutant dl-4 the specific activity of the d-lactate dehydrogenase is increased 3 fold. This mutant utilizes the d-isomer of hydroxybutyrate to completion, which does not occur in the wild type. This results in the formation of 103 mmol/l l-isoleucine by mutant dl-4 as compared to 71 mmol/l in its ancestor.     

Synthesis of β-alanine from l-aspartate using l-aspartate-α-decarboxylase from Corynebacterium glutamicum

β-Alanine is mainly produced by chemical methods in current industrial processes. Here, panD from Corynebacterium glutamicum encoding l-aspartate-α-decarboxylase (ADC) was cloned and expressed in Escherichia coli BL21(DE3). ADC _C.g catalyzes the α-decarboxylation of l-aspartate to β-alanine. The purified ADC _C.g was optimal at 55 °C and pH 6 with excellent stability at 16–37 °C and pH 4–7. A pH–stat directed, fed-batch feeding strategy was developed for enzymatic synthesis of β-alanine to keep the pH value within 6–7.2 and thus attenuate substrate inhibition. A maximum conversion of 97.2 % was obtained with an initial 5 g l-aspartate/l and another three feedings of 0.5 % (w/v) l-aspartate at 8 h intervals. The final β-alanine concentration was 12.85 g/l after 36 h. This is the first study concerning the enzymatic production of β-alanine by using ADC.     

Improving the secretion of cadaverine in Corynebacterium glutamicum by cadaverine–lysine antiporter

Cadaverine (1,5-pentanediamine, diaminopentane), the desired raw material of bio-polyamides, is an important industrial chemical with a wide range of applications. Biosynthesis of cadaverine in Corynebacterium glutamicum has been a competitive way in place of petroleum-based chemical synthesis method. To date, the cadaverine exporter has not been found in C. glutamicum. In order to improve cadaverine secretion, the cadaverine–lysine antiporter CadB from Escherichia coli was studied in C. glutamicum. Fusion expression of cadB and green fluorescent protein (GFP) gene confirmed that CadB could express in the cell membrane of C. glutamicum. Co-expression of cadB and ldc from Hafnia alvei in C. glutamicum showed that the cadaverine secretion rate increased by 22 % and the yield of total cadaverine and extracellular cadaverine increased by 30 and 73 %, respectively. Moreover, the recombinant strain cultured at acid and neutral pH separately hardly had any difference in cadaverine concentrations. These results suggested that CadB could be expressed in the cell membrane of C. glutamicum and that recombinant CadB could improve cadaverine secretion and the yield of cadaverine. Moreover, the pH value did not affect the function of recombinant CadB. These results may be a promising metabolic engineering strategy for improving the yield of the desired product by enhancing its export out of the cell.     

Enhanced production of α-ketoglutarate by fed-batch culture in the metabolically engineered strains of Corynebacterium glutamicum

The fed-batch culture system was employed to enhance production of α-ketoglutarate (α-KG) by the strainsof Corynebacterium glutamicum, whose genes encoding the key enzymes responsible for the biosynthesis of L-glutamate from α-KG were deleted. In a shake flask fermentation, C. glutamicum JH110 in which the 3 genes, gdh (encoding glutamate dehydrogenase), gltB (encoding glutamate synthase), and aceA (encoding isocitrate lyase) were disrupted showed the highest production of α-KG (12.4 g/L) compared to the strains JH102 (gdh mutant), JH103 (gltB mutant), and JH107 (gdh gltB double mutant). In the fed-batch cultures using a 5 L-jar fermenter, the strain JH107 produced more α-KG (19.5 g/L), but less glutamic acid (23.3 g/L) than those produced by the parent strain HH109, as well as JH102. The production of α-KG was significantly enhanced and the accumulation of glutamicacid was minimized by the ammonium-limited fed-batch cultures employing C. glutamicum JH107. Further improvement of α-KG production by the strain JH107 was achieved through the ammonium-limited fed-batch culture with the feeding of molasses, and the levels of α-KG and glutamic acid produced were 51.1 and 0.01 g/L, respectively.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation in Corynebacterium glutamicum

The β-ketoadipate pathway, a primarily chromo-somally encoded catabolic route that plays a signifi-cant role in the degradation of aromatic compounds, is widely distributed in soil bacteria and fungi[1]. This pathway consists of two parallel branches of which the aromatic rings are cleaved by either protocatechuate 3,4-dioxygenase or catechol 1,2-dioxygenase. The two branches converge at β-ketoadipate enol-lactone in bacteria, and three additional steps complete the con-version of the latter…