Acetate Kinase Activity Research Articles

Ethanol Catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h^–1 and biomass yields up to 0.47 g dry weight (g ethanol)^–1. Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.

Citrate metabolism by Enterococcus faecium and Enterococcus durans isolated from goat’s and ewe’s milk: influence of glucose and lactose

Citrate metabolism by Enterococcus faecium ET C9 and Enterococcus durans Ov 421 was studied as sole energy source and in presence of glucose or lactose. Both strains utilized citrate as the sole energy source. Enterococcus faecium ET C9 showed diauxic growth in the presence of a limiting concentration of glucose. Neither strain used citrate until glucose was fully metabolized. The strains showed co-metabolism of citrate and lactose. Lactate, acetate, formate, and flavour compounds (diacetyl, acetoin, and 2,3-butanediol) were detected in both strains. The highest production of flavour compounds was detected during growth of E. durans Ov 421 in media supplemented with citrate-glucose and citrate-lactose. Citrate lyase was inducible in both strains. Acetate kinase activities presented the highest values in LAPTc medium, with E. faecium ET C9 displaying a specific activity 2.4-fold higher than E. durans. The highest levels of alpha-acetolactate synthase specific activity were detected in E. durans grown in LAPTc+g, in accordance with the maximum production of flavour compounds detected in this medium. Diacetyl and acetoinreductases displayed lower specific activity values in the presence of citrate. Enterococcus faecium and E. durans displayed citrate lyase, acetate kinase, alpha-acetolactate synthase, and diacetyl and acetoin reductase activities. These enzymes are necessary for conversion of citrate to flavour compounds that are important in fermented dairy products.

Identification of RamA, a Novel LuxR-Type Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

In Corynebacterium glutamicum, the acetate-activating enzymes phosphotransacetylase and acetate kinase and the glyoxylate cycle enzymes isocitrate lyase and malate synthase are coordinately up-regulated in the presence of acetate in the growth medium. This regulation is due to transcriptional control of the respective pta-ack operon and the aceA and aceB genes, brought about at least partly by the action of the negative transcriptional regulator RamB. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, mass spectrometry, and peptide mass fingerprinting, we identified a LuxR-type transcriptional regulator, designated RamA, which binds to the pta-ack and aceA/aceB promoter regions. Inactivation of the ramA gene in the genome of C. glutamicum resulted in mutant RG2. This mutant was unable to grow on acetate as the sole carbon and energy source and, in comparison to the wild type of C. glutamicum, showed very low specific activities of phosphotransacetylase, acetate kinase, isocitrate lyase, and malate synthase, irrespective of the presence of acetate in the medium. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. By electrophoretic mobility shift analysis, purified His-tagged RamA protein was shown to bind specifically to the pta-ack and the aceA/aceB promoter regions, and deletion and mutation studies revealed in both regions two binding motifs each consisting of tandem A/C/TG4-6T/C or AC4-5A/G/T stretches separated by four or five arbitrary nucleotides. Our data indicate that RamA represents a novel LuxR-type transcriptional activator of genes involved in acetate metabolism of C. glutamicum.

Construction and characterization of ack knock‐out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation

Propionibacterium acidipropionici produces propionic acid from glucose with acetic acid, succinic acid, and CO2 as byproducts. In this work, inactivation of ack gene, encoding acetate kinase (AK), by gene disruption and integrational mutagenesis was studied as a method to reduce acetate formation in propionic acid fermentation. The partial ack gene of approximately 750 bp in P. acidipropionici was cloned using a PCR-based method with degenerate primers and sequenced. The deduced amino acid sequence had 88% similarity and 76% identity with the amino acid sequence of AK from Bacillus subtilis. The partial ack gene was used to construct a linear DNA fragment with an inserted tetracycline resistance cassette and a nonreplicative integrational plasmid containing a tetracycline resistance gene cassette. These DNA constructs were then introduced into P. acidipropionici by electroporation, resulting in two mutants, ACK-Tet and TAT-ACK-Tet, respectively. Southern hybridization confirmed that the ack gene in the mutant ACK-Tet was disrupted by the inserted tetracycline resistance gene. As compared to the wild-type, the activities of AK were reduced by 26% and 43% in ACK-Tet and TAT-ACK-Tet mutants, respectively. The specific growth rate of these mutants was reduced by approximately 25% to 0.10/h (0.13/h for the wild-type), probably because of reduced acetate and ATP production. Both mutants produced approximately 14% less acetate from glucose. Although ack disruption alone did not completely eliminate acetate production, the propionate yield was increased by approximately 13%.

Construction and Characterization of ack Deleted Mutant of Clostridium tyrobutyricum for Enhanced Butyric Acid and Hydrogen Production

Clostridium tyrobutyricum produces butyrate, acetate, H(2), and CO(2) as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by approximately 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by approximately 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with approximately 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 degrees C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h(-1) for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized-cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.

TheamrG1 gene is involved in the activation of acetate inCorynebacterium glutamicum

During growth of Corynebacterium glutamicum on acetate as its carbon and energy source, the expression of the pta-ack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genes amrG1 and amrG2 found in the deregulated transposon mutant C. glutamicum G25. The amrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C. glutamicum. We constructed an in-frame deletion mutant and an over-expressing strain of amrG1 in the C. glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, the amrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of the amrG1 gene in C. glutamicum 13032 had the adverse regulatory effect. These results indicate that the amrG1 gene encodes a repressor or co-repressor of the pta-ack operon.

Butyric acid and hydrogen production by Clostridium tyrobutyricum ATCC 25755 and mutants

Clostridium tyrobutyricum produces butyric acid, acetic acid, hydrogen and carbon dioxide as its main fermentation products. In this work, mutants with inactivated pta gene, encoding phosphotransacetylase (PTA) and ack gene, encoding acetate kinase (AK), were studied for their potential to improve butyric acid production in the fermentation. PTA and AK are two key enzymes in the acetate-producing pathway. PTA and AK activities in the pta-deleted mutant (PPTA-Em) were reduced by 44% and 91%, respectively, whereas AK activity in the ack-deleted mutant (PAK-Em) decreased by 50%. Meanwhile, the activity of butyrate kinase (BK) in PPTA-Em increased by 44% and hydrogenase activity in PAK-Em increased by 40%. As compared with the wild type, the specific growth rate of the mutants decreased by 32% (from 0.28 to 0.19 h −1) because of the impaired PTA-AK pathway. Meanwhile, butyric acid production by these mutants was improved greatly, with higher butyric acid yield (>0.4 g/g versus 0.34 g/g) and final concentration (43 g/L versus 29 g/L), which also indicated that the mutants had better tolerance to butyric acid inhibition. However, acetate production in the mutants was not significantly reduced even though more butyrate was produced from glucose, suggesting the existence of additional acetate forming pathway in C. tyrobutyricum. Also, hydrogen production by PAK-Em mutant increased significantly, with higher hydrogen yield (2.61 mol/mol glucose versus 1.35 mol/mol glucose) and H 2/CO 2 ratio (1.43 versus 1.08). The SDS-PAGE also showed significantly different expression levels of proteins with molecular mass around 32 and 70 kDa. These results suggested that integrational mutagenesis resulted in global metabolic shift and phenotypic changes, which also improved production of butyric acid and hydrogen from glucose in the fermentation.

Construction and characterization of pta gene‐deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation

Clostridium tyrobutyricum ATCC 25755 is an acidogenic bacterium, producing butyrate and acetate as its main fermentation products. In order to decrease acetate and increase butyrate production, integrational mutagenesis was used to disrupt the gene associated with the acetate formation pathway in C. tyrobutyricum. A nonreplicative integrational plasmid containing the phosphotransacetylase gene (pta) fragment cloned from C. tyrobutyricum by using degenerate primers and an erythromycin resistance cassette were constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome inactivated the target pta gene and produced the pta-deleted mutant (PTA-Em), which was confirmed by Southern hybridization. SDS-PAGE and two-dimensional protein electrophoresis results indicated that protein expression was changed in the mutant. Enzyme activity assays using the cell lysate showed that the activities of PTA and acetate kinase (AK) in the mutant were reduced by more than 60% for PTA and 80% for AK. The mutant grew more slowly in batch fermentation with glucose as the substrate but produced 15% more butyrate and 14% less acetate as compared to the wild-type strain. Its butyrate productivity was approximately 2-fold higher than the wild-type strain. Moreover, the mutant showed much higher tolerance to butyrate inhibition, and the final butyrate concentration was improved by 68%. However, inactivation of pta gene did not completely eliminate acetate production in the fermentation, suggesting the existence of other enzymes (or pathways) also leading to acetate formation. This is the first-reported genetic engineering study demonstrating the feasibility of using a gene-inactivation technique to manipulate the acetic acid formation pathway in C. tyrobutyricum in order to improve butyric acid production from glucose.

RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum.

The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions. Directed deletion of the ramB gene in the genome of C. glutamicum resulted in mutant strain RG1. Whereas the wild type of C. glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum.

Metabolic analysis of acetate accumulation during xylose consumption by Paenibacillus polymyxa.

Paenibacillus polymyxa ATCC 12321 produced more acetic acid and less butanediol from xylose than from glucose. The product yields from xylose were ethanol (0.72 mol/mol sugar), (R,R)-2,3-butanediol (0.31 mol/mol sugar), and acetate (0.38 mol/mol sugar) while those from glucose were ethanol (0.74 mol/mol sugar), (R,R)-2,3-butanediol (0.46 mol/mol sugar), and acetate (0.05 mol/mol sugar). Higher acetate kinase activity and lower acetate uptake ability were found in xylose-grown cells than in glucose-grown cells. Furthermore, phosphoketolase activity was higher in xylose-grown cells than in glucose-grown cells. In fed-batch culture on xylose, glucose feeding raised the butanediol yield to 0.56 mol/mol sugar and reduced acetate accumulation to 0.04 mol/mol sugar.

Glucose metabolism in Lactococcus lactis MG1363 under different aeration conditions: requirement of acetate to sustain growth under microaerobic conditions.

Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h(-1)) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO(2), and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) needed for fatty acid synthesis since acetyl-CoA can be synthesized from acetate by means of acetate kinase and phosphotransacetylase activities.

Highly sensitive simultaneous bioluminescent measurement of acetate kinase and pyruvate phosphate dikinase activities using a firefly luciferase-luciferin reaction and its application to a tandem bioluminescent enzyme immunoassay.

We have developed a simultaneous bioluminescent measurement of acetate kinase (AK) and pyruvate phosphate dikinase (PPDK) activities and its application to a tandem enzyme immunoassay. The principle of the proposed assay is as follows. In the first step, AK generates ATP from ADP and acetylphosphate, and the ATP is determined by the firefly luciferase-luciferin reaction. In the second step, the bioluminescent intensity from AK is eliminated by adding glucose and ADP-dependent hexokinase, which forms AMP from ADP. At the same time, the PPDK catalyzes the interconversion of AMP, diphosphate, and phosphoenolpyruvate to ATP, phosphate and pyruvate. The ATP formed by PPDK is also determined by the firefly luciferase-luciferin reaction. The detection limits (at blank + 3SD) of AK and PPDK were 1.03 x 10(-20) and 2.05 x 10(-20) mol per assay, respectively. The method was applicable to a bioluminescent enzyme immunoassay for the assay of insulin and C-peptide in the same sample.

Acetate utilization and butyryl coenzyme A (CoA):acetate-CoA transferase in butyrate-producing bacteria from the human large intestine.

Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine.

Evidence for a Transition State Analog, MgADP-Aluminum Fluoride-Acetate, in Acetate Kinase from Methanosarcina thermophila

Aluminum fluoride has become an important tool for investigating the mechanism of phosphoryl transfer, an essential reaction that controls a host of vital cell functions. Planar AlF(3) or AlF(4)(-) molecules are proposed to mimic the phosphoryl group in the catalytic transition state. Acetate kinase catalyzes phosphoryl transfer of the ATP gamma-phosphate to acetate. Here we describe the inhibition of acetate kinase from Methanosarcina thermophila by preincubation with MgCl(2), ADP, AlCl(3), NaF, and acetate. Preincubation with butyrate in place of acetate did not significantly inhibit the enzyme. Several NTPs can substitute for ATP in the reaction, and the corresponding NDPs, in conjunction with MgCl(2), AlCl(3), NaF, and acetate, inhibit acetate kinase activity. Fluorescence quenching experiments indicated an increase in binding affinity of acetate kinase for MgADP in the presence of AlCl(3), NaF, and acetate. These and other characteristics of the inhibition indicate that the transition state analog, MgADP-aluminum fluoride-acetate, forms an abortive complex in the active site. The protection from inhibition by a non-hydrolyzable ATP analog or acetylphosphate, in conjunction with the strict dependence of inhibition on the presence of both ADP and acetate, supports a direct in-line mechanism for acetate kinase.

An NMR and enzyme study of the carbon metabolism of Neisseria meningitidis.

The pathogenic neisseriae are fastidious bacteria that are only able to grow on a restricted range of carbon sources. The genome sequence of Neisseria meningitidis strain MC58 predicts the presence of a complete citric acid cycle (CAC), but there have been no detailed biochemical studies of carbon metabolism in this important pathogen. In this study, both NMR and conventional enzyme assays were used to investigate the central metabolic pathways of a serogroup B strain (K454). (13)C-NMR labelling patterns of amino acids from hydrolysed cell proteins after growth with either 2- or 3-[(13)C]pyruvate were consistent with the operation of a complete oxidative CAC. Enzyme assays showed that cell-free extracts contained all the CAC enzymes predicted from the genome sequence, including a membrane-bound malate:quinone oxidoreductase which is present in place of the conventional NAD-linked cytoplasmic malate dehydrogenase. (1)H-NMR studies showed that growth on glucose, lactate and, especially, pyruvate, resulted in the excretion of significant amounts of acetate into the culture supernatant. This occurred via the phosphotransacetylase (PTA)-acetate kinase (ACK) pathway. Extremely high specific activities of PTA (7-14 micromol min(-1) mg(-1)) were detected in cell-free extracts, although ACK activities were much lower (46-298 nmol min(-1) mg(-1)). Expression of PTA and ACK activities was not co-ordinately regulated during growth on combinations of carbon sources. This may be related to the presence of two ackA paralogues in N. meningitidis which are, unusually, unlinked to the pta gene.

Adaptation of Lactobacillus sakei to meat: a new regulatory mechanism of ribose utilization?

Lactobacillus sakei is a lactic acid bacterium commonly found on meat and meat products. Meat is a rich substrate but contains small amounts of sugars, mainly glucose and ribose. The phosphotransferase system (PTS) is a multienzymatic complex responsible for sugar uptake. It is also involved in the regulation of metabolism through various mechanisms (catabolite activation and repression, inducer expulsion and exclusion). The ptsHI operon of L. sakei, encoding the general enzymes of the PTS, was studied and mutants were constructed. On ribose, these mutants grow twice as fast as the wild-type strain. This phenotype was never described in other bacteria and suggests that the PTS regulates ribose utilization. When compared to what is known from the regulations involving the PTS in other bacteria, this mechanism might be new. In Bacillus subtilis and Escherichia coli, in which ribose catabolism was investigated, ribose is transported by an ABC transporter, encoded by rbsABCD genes and then phosphorylated by the rbsK encoded ribose kinase. Ribose-5P is then metabolized through the pentose-P pathway involving xylulose-5P phosphoketolase and acetate kinase. Whereas phosphoketolase and acetate kinase activities remained unchanged in L. sakei ptsI mutants, ribose kinase activity and uptake were increased by a factor of 2.5 and 1.5, respectively. The target of the PTS regulation would thus be transport and/or phosphorylation of ribose. The gene cluster encoding a ribose transporter, ribose kinase and a regulator was cloned and sequenced. In L. sakei no gene encoding RbsA, RbsB or RbsC could be found. However, rbsD was present as well as a new gene rbsU, encoding a protein homologous to a glucose transporter responsible for facilitated diffusion of glucose. The rbsUDK operon is induced by ribose via the regulator RbsR encoded by rbsR located downstream of rbsUDK. In ptsI mutants, this operon was not overexpressed on ribose. This shows that the regulation of ribose utilization is not a transcriptional regulation. Upstream from the rbs operon, a gene encoding acetate kinase (ackA) was found. In other bacteria in which these genes were identified, ackA and the rbs operon are not linked. Moreover, in B. subtilis, ackA is regulated by catabolite activation whereas the rbs operon is repressed by catabolite repression, two mechanisms involving the PTS. In L. sakei, ackA and the rbs operon are adjacent on the

Production of luciferase‐magnetic particle complex by recombinant Magnetospirillum sp. AMB‐1

Luciferase-bacterial magnetic particle (BMP) complexes were produced by recombinant Magnetospirillum sp. AMB-1. We constructed plasmids pKML and pNELM, respectively, by fusing luc to the 5′ and 3′ terminal of magA, encoding an integral iron translocating protein situated in the BMP membrane, of AMB-1. In addition, we produced bifunctional active-fusion proteins on BMPs by using a plasmid pAcML. In this plasmid, acetate kinase and luciferase genes were fused to the N-terminus and the C-terminus of MagA, respectively. Bacterial magnetic particles isolated from transconjugants for pKML, pNELM and pAcML exhibited luciferase activity. Bacterial magnetic particles isolated from transconjugants for pAcML also exhibited acetate kinase activity. Fed-batch culture of pKML transconjugant yielded 2.6 mg BMPs per liter of culture, and 95% conversion of iron into magnetite was obtained, at a nitrate concentration of 1.4 mM. Continuous feeding of iron as ferric quinate significantly enhanced growth and total magnetic production. Final cell concentration of 1.8 × 109 cells/mL and 6 mg per liter of culture was obtained. Magnetite production by fed-batch culture of AMB-1 was about 3 times that obtained by batch culture. There were no significant differences in BMPs yield between recombinant AMB-1 cultivated by fed-batch culture and wild type of AMB-1. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 704–709, 2000.

Relationships between cellobiose catabolism, enzyme levels, and metabolic intermediates in Clostridium cellulolyticum grown in a synthetic medium.

Continuous cultures, under cellobiose sufficient concentrations (14. 62 mM) using a chemically defined medium, were examined to determine the carbon regulation selected by Clostridium cellulolyticum. Using a synthetic medium, a q(cellobiose) of 2.57 mmol g cells(-1) h(-1) was attained whereas the highest value obtained on complex media was 0.68 mmol g cells(-1) h(-1) (Payot et al. 1998. Microbiology 144:375-384). On a synthetic medium at D = 0.035 h(-1) under cellobiose excess, lactate and ethanol biosynthesis were able to use the reducing equivalents supplied by acetic acid formation and the H(2)/CO(2) ratio was found equal to 1. At a higher dilution rate (D = 0.115 h(-1)), there was no lactate production and the pathways toward ethanol and NADH-ferredoxin-hydrogenase contributed to balance the reducing equivalents; in this case a H(2)/CO(2) ratio of 1.54 was found. With increasing D, there was a progressive increase (i) in the steady-state concentration of NADH and NAD(+) pools from 11.8 to 22.1 micromol (g cells) (-1), (ii) in the intracellular NADH/NAD(+) ratios from 0.43 to 1.51. On synthetic media, under cellobiose excess the carbon flow was also equilibrated by three overflows: exopolysaccharide, extracellular protein, and amino acid excretions. At D = 0.115 h(-1), 34% of the cellobiose consumed was converted into exopolysaccharides; this deviation of the carbon flow and the increase of the phosphoroclastic activity decreased dramatically the pyruvate excretion and explained the break in lactate production. Whatever the dilution rate, C. cellulolyticum, using ammonium and cellobiose excess, always spilled usual amino acids accompanied by other amino compounds. In vitro, GAPDH, phosphoroclastic reaction, alcohol dehydrogenase, and acetate kinase activities were high under conditions giving high in vivo specific production rates. There were also correlations between the in vitro lactate dehydrogenase activity and in vivo lactate production, but in contrast with the preceding activities, these two parameters decreased with D. All the results demonstrate that C. cellulolyticum was able to optimize carbon catabolism from cellulosic substrates in a synthetic medium.

Elucidation of enzymes in fermentation pathways used by Clostridium thermosuccinogenes growing on inulin.

Based on the presence and absence of enzyme activities, the biochemical pathways for the fermentation of inulin by Clostridium thermosuccinogenes DSM 5809 are proposed. Activities of nine enzymes (lactate dehydrogenase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, phosphotransacetylase, acetate kinase, pyruvate kinase, and alcohol dehydrogenase) were measured at four temperatures (37, 47, 58, and 70 degrees C). Each of the enzymes increased 1.5 to 2.0-fold in activity between 37 and 58 degrees C, but only lactate dehydrogenase, fumarate reductase, malate dehydrogenase, and fumarase increased at a similar rate between 58 and 70 degrees C. No acetate kinase activity was observed at 70 degrees C. Arrhenius energies were calculated for each of these nine enzymes and were in the range of 9.8 to 25.6 kcal/mol. To determine if a relationship existed between product formation and enzyme activity, serum bottle fermentations were completed at the four temperatures. Maximum yields (in moles per mole hexose unit) for succinate (0.23) and acetate (0.79) and for biomass (29.5 g/mol hexose unit) occurred at 58 degrees C, whereas the maximum yields for lactate (0.19) and hydrogen (0.25) and the lowest yields for acetate (0.03) and biomass (19.2 g/mol hexose unit) were observed at 70 degrees C. The ratio of oxidized products to reduced products changed significantly, from 0.52 to 0.65, with an increase in temperature from 58 to 70 degrees C, and there was an unexplained detection of increased reduced products (ethanol, lactate, and hydrogen) with a concomitant decrease in oxidized-product formation at the higher temperature.

Production of luciferase-magnetic particle complex by recombinantMagnetospirillum sp. AMB-1

Luciferase-bacterial magnetic particle (BMP) complexes were produced by recombinant Magnetospirillum sp. AMB-1. We constructed plasmids pKML and pNELM, respectively, by fusing luc to the 5' and 3' terminal of magA, encoding an integral iron translocating protein situated in the BMP membrane, of AMB-1. In addition, we produced bifunctional active-fusion proteins on BMPs by using a plasmid pAcML. In this plasmid, acetate kinase and luciferase genes were fused to the N-terminus and the C-terminus of MagA, respectively. Bacterial magnetic particles isolated from transconjugants for pKML, pNELM and pAcML exhibited luciferase activity. Bacterial magnetic particles isolated from transconjugants for pAcML also exhibited acetate kinase activity. Fed-batch culture of pKML transconjugant yielded 2.6 mg BMPs per liter of culture, and 95% conversion of iron into magnetite was obtained, at a nitrate concentration of 1.4 mM. Continuous feeding of iron as ferric quinate significantly enhanced growth and total magnetic production. Final cell concentration of 1.8 x 10(9) cells/mL and 6 mg per liter of culture was obtained. Magnetite production by fed-batch culture of AMB-1 was about 3 times that obtained by batch culture. There were no significant differences in BMPs yield between recombinant AMB-1 cultivated by fed-batch culture and wild type of AMB-1.