Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum

Transketolase is a key enzyme of the nonoxidative pentose phosphate pathway. The effect of its overexpression on aromatic amino acid production was investigated in Corynebacterium glutamicum, a typical amino-acid-producing organism. For this purpose, the transketolase gene of the organism was cloned on the basis of its ability to complement a C. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. The gene was shown by deletion mapping and complementation analysis to be located in a 3.2-kb XhoI-SalI fragment of the genome. Amplification of␣the gene by use of low-, middle-, and high-copy-number vectors in a C. glutamicum strain resulted in overexpression of transketolase activities as well as a␣protein of approximately 83kDa in proportion to the copy numbers. Introduction of the plasmids into a tryptophan and lysine co-producer resulted in copy-dependent increases in tryptophan production along with concomitant decreases in lysine production. Furthermore, the presence of the gene in high copy numbers enabled tyrosine, phenylalanine and tryptophan producers to accumulate 5%–20% more aromatic amino acids. These results indicate that overexpressed transketolase activity operates to redirect the glycolytic intermediates toward the nonoxidative pentose phosphate pathway in vivo, thereby increasing the intracellular level of erythrose 4-phosphate, a precursor of aromatic biosynthesis, in the aromatic-amino-acid-producing C. glutamicum strains. Received: 27 July 1998 / Received last revision: 12 October 1998 / Accepted: 24 October 1998     

Induction of only limited elongation instead of filamentation by inhibition of cell division in Corynebacterium glutamicum

In order to characterize the cell-division mechanism of coryneform bacteria, we tried to isolate cell-division mutants from Corynebacterium glutamicum after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, such as Escherichia coli fts mutants, which form long filaments at the restrictive temperatures. At the non-permissive temperature, most of the mutants formed club-shaped or dumbbell-shaped, elongated rod cells, but no filament formers were isolated. Then we examined the effects of cell division inhibitors on this organism. Cephalexin and sparfloxacin, which are the inhibitors of septation and DNA synthesis respectively, and are known to cause cell filamentation in E. coli, did not cause filamentation in C. glutamicum but induced morphological changes that were similar to those observed with the temperature-sensitive ts mutants of C.␣glutamicum. These results suggest that C. glutamicum has a unique regulation mechanism, that is, the inhibition of cell division leads to cessation of cell elongation. Received: 5 February 1998 / Received revision: 6 April 1998 / Accepted: 27 April 1998     

Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene

Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction (PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicumpc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified. Received: 16 December 1997 / Received revision: 31 March 1998 / Accepted: 19 April 1998     

Flux through the tetrahydrodipicolinate succinylase pathway is dispensable for l-lysine production in Corynebacterium glutamicum

The N-succinyl-ll-diaminopimelate desuccinylase gene (dapE) in the four-step succinylase branch of the l-lysine biosynthetic pathway of Corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. This mutant strain grew and utilized glucose from minimal medium at the same rate as the parental strain. In addition, the dapE  ⁻ strain produced lysine at the same rate as its parent strain. Transformation of the parental and dapE  ⁻ strains with the amplified meso-diaminopimelate dehydrogenase gene (ddh) on a plasmid did not affect lysine production in either strain, despite an eightfold amplification of the activity of the enzyme. These results indicate that the four-step succinylase pathway is dispensable for lysine overproduction in shake-flask culture. In addition, the one-step meso-diaminopimelate dehydrogenase pathway does not limit lysine flux in Corynebacterium under these conditions. Received: 20 May 1998 / Received revision: 12 August 1998 / Accepted: 3 September 1998     

Elucidation of anaplerotic pathways in Corynebacterium glutamicum via 13C-NMR spectroscopy and GC-MS

We have obtained direct evidence indicating the presence of pyruvate-carboxylating activity in Corynebacterium glutamicum, a lysine-overproducing bacterium. This evidence was obtained through the use of ¹³C nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectrometry (GC-MS) of secreted metabolites in a lysine fermentation. The distribution of ¹³C label after multiple turns in the tricarboxylic acid cycle was accounted for properly to obtain predictions for [¹³C] metabolite enrichments that were employed in the interpretation of ¹³C-NMR and GC-MS data. Of critical importance in arriving at the conclusions was the use of C. glutamicum mutants with deletions of the pyruvate kinase and/or phosphoenolpyruvate carboxylase enzymes. Our results demonstrate the presence of pyruvate-carboxylating pathway(s) in C.␣glutamicum operating simultaneously with phosphoenolpyruvate carboxylase, with the latter enzyme contributing approximately 10 % of the total oxaloacetate synthesis during the lysine-production phase with pyruvate and gluconate as carbon sources. These findings are important for developing strategies to increase the total carbon flux for synthesis of amino acids of the aspartate family through metabolic engineering. Received: 11 June 1996 / Received revision: 30 October 1996 / Accepted: 15 November 1996     

A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA

An improved method for the electrotransformation of wild-type Corynebacterium glutamicum (ATCC 13032) is described. The two crucial alterations to previously developed methods are: cultivation of cells used for electrotransformation at 18 °C instead of 30 °C, and application of a heat shock immediately following electrotransformation. Cells cultivated at sub optimal temperature have a 100-fold improved transformation efficiency (10⁸ cfu μg⁻¹) for syngeneic DNA (DNA isolated from the same species). A heat shock applied to these cells following electroporation improved the transformation efficiency for xenogeneic DNA (DNA isolated from a different species). In combination, low cultivation temperature and heat shock act synergistically and increased the transformation efficiency by four orders of magnitude to 2.5 × 10⁶ cfu μg⁻¹ xenogeneic DNA. The method was used to generate gene disruptions in C. glutamicum. Received: 26 March 1999 / Received revision: 9 June 1999 / Accepted: 11 June 1999     

Antimould activity of sourdough lactic acid bacteria: identification of a mixture of organic acids produced by Lactobacillus sanfrancisco CB1

Sourdough lactic acid bacteria, cultivated in wheat flour hydrolysate, produced antimould compounds. The antimould activity varied greatly among the strains and was mainly detected within obligately heterofermentative Lactobacillus spp. Among these, Lb. sanfrancisco CB1 had the largest spectrum. It inhibited moulds related to bread spoilage such as Fusarium, Penicillium, Aspergillus and Monilia. A mixture of acetic, caproic, formic, propionic, butyric and n-valeric acids, acting in a synergistic way, was responsible for the antimould activity. Caproic acid played a key role in inhibiting mould growth. Received: 20 January 1998 / Received revision: 17 April 1998 / Accepted: 27 April 1998     

l-Glutamate and l-lysine: traditional products with impetuous developments

 Amino acids have been produced with the aid of microorganisms for nearly 40 years now. The economic importance of these cellular building blocks is enormous. Demand for them is rising continuously and currently more than 10⁶ tonnes/year are required. Continual efforts to increase production performance are directed towards the microorganisms themselves, as well as towards technical improvements of the respective processes. A special position within the amino-acid-producing microorganisms is traditionally occupied by Corynebacterium glutamicum. Molecular research in conjunction with NMR studies of flux has revealed fascinating new properties of this particular organism, including the existence of a new type of exporter and reverse fluxes within the anaplerosis. The knowledge gained will enable the further improvement of production strains and furthermore extend fundamental insights into metabolite flux management within bacteria in general. Received: 8 December 1998 / Received revision: 1 March 1999 / Accepted: 5 March 1999     

Mechanism for phenol tolerance in phenol-degrading Comamonas testosteroni strain

Comamonas testosteroni P15 and its mutant strain E23 can tolerate and utilize phenol as the sole source of carbon and energy at up to 15 mM and 20 mM, respectively. Compared to the wild type P15, mutant E23 showed higher values of K _s and K _i but a lower μ_max value, and had lower phenol hydroxylase and catechol 2,3-dioxygenase activities. Without phenol exposure, mutant E23 demonstrated a two-fold greater amount of cardiolipin than the wild type P15. Upon exposure to phenol, an increase in cardiolipin at the expense of phosphatidylethanolamine was observed in the wild type P15. However, there was no significant difference in major phospholipid contents between mutant E23 cells grown in the presence or absence of phenol. It was noted that the ratio of trans/cis fatty acids of phosphatidylethanolamine and cardiolipin in mutant E23 was 65–70% higher than that in the wild type P15. In the absence of phenol, the degree of saturation of cardiolipin in mutant E23 was 33% higher than that in wild type P15. In contrast to earlier findings, an increase in C16:1 9trans with a simultaneous decrease in C18:1 11cis instead of C16:1 9cis was observed in specific classes of phospholipids. Received: 30 July 1998 / Received last revision: 16 November 1998 / Accepted: 12 December 1998     

Fungal biosolubilization of Rhenish brown coal monitored by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide

Residues and coal fractions that remained after the biosolubilization of Rhenish brown coal by strains of Lentinula edodes and Trametes versicolor have been studied by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide (NEt₄OH) at 610 °C. To differentiate methyl derivatives of esters and ethers from free or bound hydroxyl and carboxyl groups NEt₄OH was used in the thermochemolysis experiments instead the commonly used tetramethylammonium hydroxide. A comparison of humic acid fractions before and after fungal attack shows considerable alteration of the soluble macromolecules of coal. Depending on the coal fraction studied and the fungi used, the assortment of fatty acid esters released during the pyrolysis varies significantly. Furthermore, dicarbonic acid ethyl diesters as well as ethyl derivatives of aromatic ethers and acids yield information about humic acid structure and the biosolubilization of brown coal. Variations in the mixture produced are possibly caused by differences in the pattern of extracellular enzymes secreted that attack the macromolecular structural elements of brown coal. Therefore pyrolysis of native and microbiologically altered geomacromolecules using NEt₄OH allows one to differentiate between free hydroxyl groups as well as substances that are attached to humic substances via ester or ether bridges, and their methylated counterparts. Received: 13 July 1998 / Received revision: 12 October 1998 / Accepted: 16 October 1998     

The formation of 1-hydroxymethylnaphthalene and 6-hydroxymethylquinoline by both oxidative and reductive routes in Cunninghamella elegans

Extraction of medium after incubation of the fungus, Cunninghamella elegans, with 0.03% (w/v) 1-methylnaphthalene produced mainly 1-hydroxymethylnaphthalene together with some 1-naphthoic acid and hydroxynaphthoic acid. Higher concentrations of substrate were inhibitory to biotransformation. Similar incubations with 1-naphtoic acid as substrate resulted in reduction of the carboxyl group to give 1-hydroxymethylnaphthalene. When 6-methylquinoline was used, the main product was 6-hydroxymethylquinoline but also some quinoline-6-carboxylic acid and some 6-methylquinoline-N-oxide were identified. In a 2-l fermenter 2.5 g substrate was transformed in 324 h. The 6-hydroxymethylquinoline was also produced by reduction of quinoline-6-carboxylic acid by the organism. Received: 9 March 1998 / Received revision: 15 June 1998 / Accepted: 19 June 1998     

The Corynebacterium glutamicum insertion sequence ISCg2 prefers conserved target sequences located adjacent to genes involved in aspartate and glutamate metabolism

An IS element, termed ISCg2, was identified in the chromosome of Corynebacterium glutamicum ATCC 13032. After screening a cosmid library of the C. glutamicum ATCC 13032 genome, six copies of ISCg2 including their flanking regions were sequenced and analyzed. ISCg2 is 1636 bp in length and has 26-bp imperfect inverted repeats flanked by 3-bp direct repeats. By comparisons with other IS elements, ISCg2 was classified as a member of the IS30 family of insertion sequences. The six copies of ISCg2 were identical at the nucleotide level and were located in intergenic, AT-rich regions of the chromosome. The regions in which the six copies of ISCg2 were inserted displayed significant similarities. This similarity extends over a region of 65 bp, which was assumed to be the target region for ISCg2. Interestingly, five of the six copies of ISCg2 were located adjacent to genes that may be involved in aspartate and glutamate metabolism or its regulation. Investigation of the distribution of ISCg2 showed that the IS element is restricted to certain C. glutamicum strains. Analysis of various integration regions indicates active transposition of ISCg2 in C. glutamicum. Received: 7 April 1999 / Accepted: 17 June 1999     

Biosynthesis of polyhydroxyalkanoates from low-rank coal liquefaction products by Pseudomonas oleovorans and Rhodococcus ruber

A screening identified several bacteria that were able to use chemically heterogeneous low-rank coal liquefaction products as complex carbon sources for growth. Pseudomonas oleovorans and Rhodococcus ruber accumulated polyhydroxyalkanoic acids (PHA) amounting to 2%–8% of the cell dry weight when the cells were cultivated on these liquefaction products in the absence of any other carbon source. R. ruber accumulated, in addition to PHA, small amounts of triacylglycerols. The accumulated PHA consisted of 3-hydroxyhexanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (P. oleovorans) or 3-hydroxybutyric acid and 3-hydroxyvaleric acid (R. ruber). Low-rank coal liquefaction products obtained from Trichoderma atroviride were better substrates for P. oleovorans than chemically produced fulvic acids. Received: 13 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost fermentation medium based on corn steep water

To improve the economic competitiveness of the acetone/butanol/ethanol fermentation process, glucose/corn steep water (CSW) medium was used on a pilot scale for the production of solvents. The production of butanol by the Clostridium beijerinckii NCIMB 8052 parent strain and the solvent-hyperproducing BA101 mutant was compared. In a 20-l fermentation using 5% glucose/CSW medium,  C. beijerinckii 8052 produced 8.5 g butanol/l and 5 g acetone/l, while  C. beijerinckii BA101 produced 16 g butanol/l and 7.5 g acetone/l. Further studies were carried out on a larger scale using an optimized 6% glucose/CSW medium. In a 200-l pilot-scale fermentor,  C. beijerinckii 8052 produced 12.7 g butanol/l and 6 g acetone/l following 96 h of fermentation.  C. beijerinckii BA101 produced 17.8 g/l and 5.5 g/l butanol and acetone respectively, following 130 h of fermentation. These results represent a 40% increase in final butanol concentration by the C. beijerinckii BA101 mutant strain when compared to the 8052 parent strain. The total solvents (acetone, butanol, and ethanol) produced by C. beijerinckii NCIMB 8052 and BA101 in a 200-l fermentation were 19.2 g/l and 23.6 g/l respectively. This is the first report of pilot-scale butanol production by the solvent-hyperproducing C. beijerinckii BA101 mutant employing an inexpensive glucose/CSW medium. Received: 26 May 1998 / Received revision: 21 September 1998 / Accepted: 11 October 1998     

Effect of biofilm formation by Pseudomonas 8909N on the bioavailability of solid naphthalene

The effect of biofilm formation by Pseudomonas 8909N (DSM no. 11634) on the dissolution and biodegradation rates of solid naphthalene was quantified. Biofilms were cultivated on solid naphthalene as a model polycyclic aromatic hydrocarbon in continuous cultures. After different periods of incubation, the dissolution rate of naphthalene was determined by batch dissolution tests with active or inactivated biofilms and without biofilms. Results show that the naphthalene dissolution rate to the bulk liquid phase was reduced by over 90% after 7 days of biofilm formation. The degradation of naphthalene in the biofilm proved to be insignificant compared to the decrease in the bulk liquid conversion of naphthalene, and the overall biodegradation rate of the solid naphthalene decreased. Received: 26 January 1998 / Received revision: 16 April 1998 / Accepted: 19 April 1998     

Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101

Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C. beijerinckii NCIMB 8052. C. beijerinckii BA101 demonstrated a greater increase in solvent production than C. beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium. In 1-l batch fermentations, C. beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose. To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture. Received: 29 September 1998 / Received revision: 13 February 1999 / Accepted: 26 February 1999     

Improved ethanol production by glycerol-3-phosphate dehydrogenase mutants of Saccharomyces cerevisiae

The anaerobic performance of gpd1Δ and gpd2Δ mutants of Saccharomyces cerevisiae was characterized and compared to that of a wild-type strain under well-controlled conditions by using a high-performance bioreactor. There was a 40% reduction in glycerol level in the gpd2Δ mutant compared to the wild-type. Also the gpd1Δ mutant showed a slight decrease in glycerol formation but to a much lesser degree. As a consequence, ethanol formation in the gpd2Δ mutant was elevated by 13%. In terms of growth, the gpd1Δ mutant and the wild-type were indistinguishable. The gpd2Δ mutant, on the other hand, displayed an extended lag phase as well as a reduced growth rate under the exponential phase. Even though glycerol-3-phosphate dehydrogenase 2 (GPD2) is the important enzyme under anaerobic conditions it can, at least in part, be substituted by GPD1. This was indicated by the higher expression level of GPD1 in the gpd2Δ mutant compared to the wild type. These results also show that the cells are able to cope and maintain redox balance under anaerobic conditions even if glycerol formation is substantially reduced, as observed in the gpd2Δ mutant. One obvious way of solving the redox problem would be to make a biomass containing less protein, since most of the excess NADH originates from amino acid biosynthesis. However, the gpd2Δ mutant did not show any decrease in the protein content of the biomass. Received: 16 February 1998 / Received revision: 16 March 1998 / Accepted: 1 June 1998     

Production of (R )-3-pentyn-2-ol through stereospecific hydrolysis of racemic 3-pentyn-2-ol esters with microbial enzymes

Microorganisms and commercial enzymes were screened for their ability to produce (R)-3-pentyn-2-ol from racemic 3-pentyn-2-ol esters through stereospecific hydrolysis. Among the esters formed with acetic acid, propionic acid, hexanoic acid and benzoic acid, the acetate was most effectively hydrolyzed by microbial cells and commercial lipases with high stereospecificity. Rhodococcus rubropertinctus AKU NOC082 was a good catalyst for (R)-3-pentyn-2-ol production through the hydrolytic resolution of racemic 3-pentyn-2-yl acetate. With 15%, 25% and 50% (v/v) racemic 3-pentyn-2-yl acetate as the substrate, 42.6%, 40.8% and 40.0% was hydrolyzed in 5 h, 10 h and 98 h respectively, under the optimized conditions (pH 7.0, 30 °C, 7.5% wet cell concentration), the (R) enantiomer of 3-pentyn-2-ol being formed with an optical purity of 97.8%, 98.0% and 94.2% respectively. Received: 2 June 1998 / Received revision: 3 August 1998 / Accepted: 3 September 1998     

Characterization of Lactobacillus collinoides response to heat, acid and ethanol treatments

Tolerance to stress and cross-protection in Lactobacillus collinoides were examined after exposure to ethanol, acid or heat shock. Ethanol and heat-adapted cells demonstrate induced homologous␣tolerance and cross-resistance to acid stress. No cross-protection of acid-adapted cells against ethanol and heat stresses was observed. Heat was the only pretreatment leading to cross-protection against the two other stresses. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins; the synthesis of some of these polypeptides being induced by more than one condition. The greatest overlap was observed between ethanol and heat treatments. Ten proteins were found to be common to these stresses. Received: 7 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999     

Aerobic degradation of a hydrocarbon mixture in natural uncontaminated potting soil by indigenous microorganisms at 20 °C and 6 °C

A hydrocarbon mixture containing p-xylene, naphthalene, Br-naphthalene and straight aliphatic hydrocarbons (C₁₄ to C₁₇) was aerobically degraded without lag phase by a natural uncontaminated potting soil at 20 °C and 6 °C. Starting concentrations were approximately 46 ppm for the aromatic and 13 ppm for the aliphatic compounds. All aliphatic hydrocarbons were degraded within 5 days at 20 °C, to levels below detection (ppb levels) but only down to 10% of initial concentration at 6 °C. Naphthalene was degraded within 12 days at 20 °C and unaffected at 6 °C. At 20 °C p-xylene was degraded within 20 days, but no degradation occurred at 6 °C. Br-naphthalene was only removed down to 30% of initial concentration at 20 °C, with no significant effect at 6 °C. The biodegradation was monitored with head space solid-phase microextraction and gas chromatography–mass spectrometry. Received: 5 October 1998 / Received revision: 4 December 1998 / Accepted: 5 December 1998