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     

Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw

The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg²⁺ and Ca²⁺ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by CelA. Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998     

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     

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50 805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica. Received: 19 May 1997 / Received last revision: 22 September 1997 / Accepted: 14 October 1997     

A transketolase mutant of Corynebacterium glutamicum

A transketolase mutant was first isolated from Corynebacterium glutamicum, an organism of industrial importance. The mutant strain exhibited an absolute requirement for shikimic acid or the aromatic amino acids and vitamins for growth, and also failed to grow on ribose or gluconic acid as sole carbon source, even with the aromatic supplement. All of these defective properties were fully restored in spontaneous revertants, indicating the existence of a single transketolase in C. glutamicum that was indispensable both for aromatic biosynthesis and for utilization of these carbohydrates in vivo. The transketolase mutant accumulated ribulose extracellularly when cultivated in glucose medium with shikimic acid, but no ribose was detected. Received: 10 April 1998 / Received revision: 26 May 1998 / Accepted: 14 June 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     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 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     

Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11 xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome. Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998     

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     

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40° C. Received: 6 February 1998 / Accepted: 6 November 1998     

Properties of a cold-active protease from psychrotrophic Flavobacterium balustinum P104

Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 °C, which was isolated from salmon (Oncorhynchus keta) intestine. The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographyies. The molecular mass of the protease was 70 kDa, and its isoelectric point was close to 3.5. Maximal activity toward azocasein was observed at 40 °C and from pH 7.0 to 9.0. The activity was strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease. The n-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu-Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Glu-Asn. A search through the database for sequence homology yielded no significant match. The initial cleavage sites for oxidized insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate. Received: 17 April 1998 / Received last revision: 17 June 1998 / Accepted: 10 July 1998     

Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose

Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase. Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999     

Characterization of a novel Streptococcus thermophilus rolling-circle plasmid used for vector construction

The complete nucleotide sequence of pER371, a native plasmid in Streptococcus thermophilus ST137, was determined. A putative open reading frame coding for a replication protein, Rep371, was identified. A characteristic promoter sequence and ribosome-binding site were found upstream of rep371. Rep371 (247 amino acid residues) does not show homology with RepA and RepS of the small S. thermophilus cryptic plasmids pST1-No.29 and pST1 respectively. The plus-origin sequence and Rep371 are highly homologous to the corresponding elements of the Staphylococcus aureus plasmids pC194 and pSK89. A novel 140-nucleotide palindromic minus-origin sequence, which is structurally similar but does not show sequence homology to the palA region of pC194, was identified in pER371. A palindromic sequence capable of forming a putative hairpin structure was identified and subsequently recognized as being highly conserved among several lactococcal rolling-circle plasmids. Cloning vectors derived from pER371 should provide valuable gene-delivery vehicles for the genetic engineering of lactic acid bacteria. Received: 25 November 1997 / Received last revision: 13 April 1998 / Accepted: 19 April 1998     

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M _r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression. Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998     

Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61

The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal DNA of Pseudomonas stutzeri AK61 into Escherichia coli. The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334 amino acids with a calculated molecular mass of 37 518 Da. The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively. A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic activity, was conserved in cyanidase. The active form of cyanidase was successfully expressed by a DNA clone containing the cyanidase gene in E.coli. Its productivity was approximately 230 times larger than that of P. stutzeri AK61. The characteristics of the expressed cyanidase, including optimum pH, optimum temperature, Michaelis constant (K _m) for cyanide and specific activity, were similar to those of the native enzyme from P. stutzeri AK61. Received: 24 October 1997 / Received last revision: 17 March 1998 / Accepted: 20 March 1998     

trans activation of the Escherichia coliato structural genes by a regulatory protein from Bacillus megaterium : potential use in polyhydroxyalkanoate production

A Bacillus megaterium genomic fragment, which encoded an activator homologous to σ⁵⁴ regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B.␣megaterium screened for β-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains. Received: 20 October 1997 / Received revision: 18 February 1998 / Accepted: 23 February 1998     

Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining

The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca²⁺ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect. Received: 25 February 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase. Received: 16 June 1998 / Received last revision: 21 August 1998 / Accepted: 3 September 1998     

Evidence for and expression of a laccase gene in three basidiomycetes degrading humic acids

The majority of lignin-degrading basidiomycetes are able to depolymerize humic acids. In this presentation the relationship and possible similarities between enzymes involved in lignin degradation and humic acid depolymerization were examined on the genetic level. We have cloned fragments of the gene encoding the extracellular ligninolytic enzyme laccase from Clitocybula dusenii, Nematoloma frowardii and a fungal strain designated i63-2, and compared the three sequences with those of several other published laccase genes. The sequenced fragments displayed a high homology both on the DNA (97%–77%) and amino acid (100%–85%) level. Furthermore, the expression of this gene in the above-mentioned fungi was demonstrated by a nested polymerase chain reaction with cDNA as template. Received: 3 February 1998 / Received revision: 31 August 1998 / Accepted: 3 September 1998