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Using methanol as an alternative non-food feedstock for biotechnological production offers several advantages in line with a methanol-based bioeconomy. The Gram-positive, facultative methylotrophic and thermophilic bacterium Bacillus methanolicus is one of the few described microbial candidates with a potential for the conversion of methanol to value-added products. Its capabilities of producing and secreting the commercially important amino acids L-glutamate and L-lysine to high concentrations at 50 °C have been demonstrated and make B. methanolicus a promising target to develop cell factories for industrial-scale production processes. B. methanolicus uses the ribulose monophosphate cycle for methanol assimilation and represents the first example of plasmid-dependent methylotrophy. Recent genome sequencing of two physiologically different wild-type B. methanolicus strains, MGA3 and PB1, accompanied with transcriptome and proteome analyses has generated fundamental new insight into the metabolism of the species. In addition, multiple key enzymes representing methylotrophic and biosynthetic pathways have been biochemically characterized. All this, together with establishment of improved tools for gene expression, has opened opportunities for systems-level metabolic engineering of B. methanolicus. Here, we summarize the current status of its metabolism and biochemistry, available genetic tools, and its potential use in respect to overproduction of amino acids.

Poly(ε-L-lysine) (ε-PL) producer strain Streptomyces albulus PD-1 secreted a novel polymeric substance into its culture broth along with ε-PL. The polymeric substance was purified to homogeneity and identified. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance spectroscopy as well as other analytical techniques revealed that the substance was poly(L-diaminopropionic acid) (PDAP). PDAP is an L-α,β-diaminopropionic acid oligomer linking between amino and carboxylic acid functional groups. The molecular weight of PDAP ranged from 500 to 1500 Da, and no co-polymers composed of L-diaminopropionic acid and L-lysine were present in the culture broth. Compared with ε-PL, PDAP exhibited stronger inhibitory activities against yeasts but weaker activities against bacteria. ε-PL and PDAP co-production was also investigated. Both ε-PL and PDAP were synthesized during the stationary phase of growth, and the final ε-PL and PDAP concentration reached 21.7 and 4.8 g L(-1), respectively, in fed-batch fermentation. Citric acid feeding resulted in a maximum ε-PL concentration of 26.1 g L(-1) and a decrease in the final concentration of PDAP to 3.8 g L(-1). No studies on ε-PL and PDAP co-production in Streptomyces albulus have been reported previously, and inhibition of by-products such as PDAP is potentially useful in ε-PL production.

We constructed beta-glucosidase (BGL)-displaying Corynebacterium glutamicum, and direct L-lysine fermentation from cellobiose was demonstrated. After screening active BGLs, Sde1394, which is a BGL from Saccharophagus degradans, was successfully displayed on the C. glutamicum cell surface using porin as an anchor protein, and cellobiose was directly assimilated as a carbon source. The optical density at 600 nm of BGL-displaying C. glutamicum grown on cellobiose as a carbon source reached 23.5 after 48 h of cultivation, which was almost the same as that of glucose after 24 h of cultivation. Finally, Sde1394-displaying C. glutamicum produced 1.08 g/l of L-lysine from 20 g/l of cellobiose after 4 days of cultivation, which was about threefold higher than the amount of produced L-lysine using BGL-secretory C. glutamicum strains (0.38 g/l after 5 days of cultivation). This is the first report on amino acid production using cellobiose as a carbon source by BGL-expressing C. glutamicum.

Plants as well as pro- and eukaryotic microorganisms are able to synthesise lysine via de novo synthesis. While plants and bacteria, with some exceptions, rely on variations of the meso-diaminopimelate pathway for lysine biosynthesis, fungi exclusively use the α-aminoadipate pathway. Although bacteria and fungi are, in principle, both suitable as lysine producers, current industrial fermentations rely on the use of bacteria. In contrast, fungi are important producers of β-lactam antibiotics such as penicillins or cephalosporins. The synthesis of these antibiotics strictly depends on α-aminoadipate deriving from lysine biosynthesis. Interestingly, despite the resulting industrial importance of the fungal α-aminoadipate pathway, biochemical reactions leading to α-aminoadipate formation have only been studied on a limited number of fungal species. In this respect, just recently an essential isomerisation reaction required for the formation of α-aminoadipate has been elucidated in detail. This review summarises biochemical pathways leading to lysine production, discusses the suitability of interrupting lysine biosynthesis as target for new antibacterial and antifungal compounds and emphasises on biochemical reactions involved in the formation of α-aminoadipate in fungi as an essential intermediate for both, lysine and β-lactam antibiotics production.

Corynebacterium glutamicum is a biotin auxotrophic Gram-positive bacterium that is used for large-scale production of amino acids, especially of L-glutamate and L-lysine. It is known that biotin limitation triggers L-glutamate production and that L-lysine production can be increased by enhancing the activity of pyruvate carboxylase, one of two biotin-dependent proteins of C. glutamicum. The gene cg0814 (accession number YP_225000) has been annotated to code for putative biotin protein ligase BirA, but the protein has not yet been characterized. A discontinuous enzyme assay of biotin protein ligase activity was established using a 105aa peptide corresponding to the carboxyterminus of the biotin carboxylase/biotin carboxyl carrier protein subunit AccBC of the acetyl CoA carboxylase from C. glutamicum as acceptor substrate. Biotinylation of this biotin acceptor peptide was revealed with crude extracts of a strain overexpressing the birA gene and was shown to be ATP dependent. Thus, birA from C. glutamicum codes for a functional biotin protein ligase (EC 6.3.4.15). The gene birA from C. glutamicum was overexpressed and the transcriptome was compared with the control strain revealing no significant gene expression changes of the bio-genes. However, biotin protein ligase overproduction increased the level of the biotin-containing protein pyruvate carboxylase and entailed a significant growth advantage in glucose minimal medium. Moreover, birA overexpression resulted in a twofold higher L-lysine yield on glucose as compared with the control strain.

Nε-acetyl-β-lysine is an archaeal compatible solute whose synthesis is mediated by the sequential reactions of the lysine-2,3-aminomutase AblA and the acetyltransferase AblB. α-Lysine serves as the precursor and is converted by AblA to β-lysine, and AblB then acetylates this intermediate to N(ε)-acetyl-β-lysine. The biochemical and biophysical properties of N(ε)-acetyl-β-lysine have so far not been studied intensively due to restrictions in the supply of this compound. A search for ablAB-like genes in the genomes of members of the family Bacillaceae revealed the yodP-kamA genes that encode a AblA-related lysine-2,3-aminomutase and AblB-related putative acetyltransferase. In Bacillus subtilis, the yodP-kamA genes are part of a transcriptional unit (yodT-yodS-yodR-yodQ-yodP-kamA) whose expression is upregulated during sporulation and controlled by the mother-cell-specific transcription factor SigE. N(ε)-acetyl-β-lysine was not detectable in vegetatively growing or osmotically stressed B. subtilis cells, and the deletion of the yodT-yodS-yodR-yodQ-yodP-kamA region had no noticeable effects on growth in rich or minimal media or osmotic stress resistance. However, when we expressed the yodP-kamA genes outside their natural genetic context from an isopropyl β-D-1-thiogalactopyranoside-inducible promoter on a plasmid in B. subtilis, the recombinant strain synthesized considerable amounts (0.28 μmol/mg protein) of N(ε)-acetyl-β-lysine. The data reported here thus open the bottleneck for the large-scale production of N(ε)-acetyl-β-lysine to investigate its properties as a compatible solute.

We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial L-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the L-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by L-threonine and L-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l L-lysine and 59 g/l L-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l L-lysine and 69 g/l of L-glutamate, while a sixfold higher L-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced L-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric L-lysine yield and the L-lysine yield on methanol, and caused significantly reduced volumetric L-glutamate yield and L: -glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving L-lysine overproduction, PC activity plays an important role in controlling L-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for L-lysine overproduction by B. methanolicus.

Nicotinamide adenine dinucleotide phosphate (NADP) is synthesized by phosphorylation of either oxidized or reduced nicotinamide adenine dinucleotide (NAD/NADH). Here, the cg1601/ppnK gene product from Corynebacterium glutamicum genome was purified from recombinant Escherichia coli and enzymatic characterization revealed its activity as a polyphosphate (PolyP)/ATP-dependent NAD kinase (PPNK). PPNK from C. glutamicum was shown to be active as homotetramer accepting PolyP, ATP, and even ADP for phosphorylation of NAD. The catalytic efficiency with ATP as phosphate donor for phosphorylation of NAD was higher than with PolyP. With respect to the chain length of PolyP, PPNK was active with short-chain PolyPs. PPNK activity was independent of bivalent cations when using ATP, but was enhanced by manganese and in particular by magnesium ions. When using PolyP, PPNK required bivalent cations, preferably manganese ions, for activity. PPNK was inhibited by NADP and NADH at concentrations below millimolar. Overexpression of ppnK in C. glutamicum wild type slightly reduced growth and ppnK overexpression in the lysine producing strain DM1729 resulted in a lysine product yield on glucose of 0.136 +/- 0.006 mol lysine (mol glucose)(-1), which was 12% higher than that of the empty vector control strain.

Carbohydrates exclusively serve as feedstock for industrial amino acid production with Corynebacterium glutamicum. Due to the industrial interest, knowledge about the pathways for carbohydrate metabolization in C. glutamicum steadily increases, enabling the rational design of optimized strains and production processes. In this review, we provide an overview of the metabolic pathways for utilization of hexoses (glucose, fructose), disaccharides (sucrose, maltose), pentoses (D-ribose, L-arabinose, D-xylose), gluconate, and beta-glucosides present in C. glutamicum. Recent approaches of metabolic engineering of L: -lysine production strains based on the known pathways are described and evaluated with respect to L: -lysine yields.

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes alpha-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40 degrees C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37 degrees C, respectively. The alpha-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.

For the biotechnological production of L: -lysine, mainly strains of Corynebacterium glutamicum are used, which have been obtained by classical mutagenesis and screening or selection or by metabolic engineering. Gene targets for the amplification and deregulation of the lysine biosynthesis pathway, for the improvement of carbon precursor supply and of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) regeneration, are known. To identify novel target genes to improve lysine production, the transcriptomes of the classically obtained lysine producing strain MH20-22B and several other C. glutamicum strains were compared. As lysine production by the classically obtained strain, which possesses feedback-resistant aspartokinase and is leucine auxotrophic, exceeds that of a genetically defined leucine auxotrophic wild-type derivative possessing feedback-resistant aspartokinase, additional traits beneficial for lysine production are present. NCgl0855, putatively encoding a methyltransferase, and the amtA-ocd-soxA operon, encoding an ammonium uptake system, a putative ornithine cyclodeaminase and an uncharacterized enzyme, were among the genes showing increased expression in the classically obtained strain irrespective of the presence of feedback-resistant aspartokinase. Lysine production could be improved by about 40% through overexpression of NCgl0855 or the amtA-ocd-soxA operon. Thus, novel target genes for the improvement of lysine production could be identified in a discovery-driven approach based on global gene expression analysis.

Intracellular precursor supply is a critical factor for amino acid productivity of Corynebacterium glutamicum. To test for the effect of improved pyruvate availability on L-lysine production, we deleted the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex (PDHC) in the L-lysine-producer C. glutamicum DM1729 and characterised the resulting strain DM1729-BB1 for growth and L-lysine production. Compared to the host strain, C. glutamicum DM1729-BB1 showed no PDHC activity, was acetate auxotrophic and, after complete consumption of the available carbon sources glucose and acetate, showed a more than 50% lower substrate-specific biomass yield (0.14 vs 0.33 mol C/mol C), an about fourfold higher biomass-specific L-lysine yield (5.27 vs 1.23 mmol/g cell dry weight) and a more than 40% higher substrate-specific L-lysine yield (0.13 vs 0.09 mol C/mol C). Overexpression of the pyruvate carboxylase or diaminopimelate dehydrogenase genes in C. glutamicum DM1729-BB1 resulted in a further increase in the biomass-specific L-lysine yield by 6 and 56%, respectively. In addition to L-lysine, significant amounts of pyruvate, L-alanine and L-valine were produced by C. glutamicum DM1729-BB1 and its derivatives, suggesting a surplus of precursor availability and a further potential to improve L-lysine production by engineering the L-lysine biosynthetic pathway.

Corynebacterium glutamicum owns a citrate synthase and two methylcitrate synthases. Characterization of the isolated enzymes showed that the two methylcitrate synthases have comparable catalytic efficiency, k (cat)/K (m), as the citrate synthase with acetyl-CoA as substrate, although these enzymes are only synthesized during growth on propionate-containing media. Thus, the methylcitrate synthases have a relaxed substrate specifity, as also demonstrated by their activity with butyryl-CoA, whereas the citrate synthase does not accept acyl donors other than acetyl-CoA. A double mutant deleted of the citrate synthase gene gltA and one of the methylcitrate synthase genes, prpC1, was made unable to grow on glucose. From this mutant, a collection of suppressor mutants could be isolated which were demonstrated to have regained citrate synthase activity due to the relaxed specificity of the methylcitrate synthase PrpC2. Molecular characterization of these mutants showed that the regulator PrpR (Cg0800) located downstream of prpC1 is mutated with mutations likely to effect the secondary structure of the regulator, thus, resulting in expression of prpC2. This expression results in a citrate synthase activity, which is lower than that due to gltA in the original strain and results in increased L-lysine accumulation.

Streptomyces albulus NBRC14147 produces epsilon-poly-L: -lysine (epsilon-PL), which is an amino acid homopolymer antibiotic. Despite the commercial importance of epsilon-PL, limited information is available regarding its biosynthesis; the L: -lysine molecule is directly utilized for epsilon-PL biosynthesis. In most bacteria, L: -lysine is biosynthesized by an aspartate pathway. Aspartokinase (Ask), which is the first enzyme in this pathway, is subject to complex regulation such as through feedback inhibition by the end-product amino acids such as L: -lysine and/or L: -threonine. S. albulus NBRC14147 can produce a large amount of epsilon-PL (1-3 g/l). We therefore suspected that Ask(s) of S. albulus could be resistant to feedback inhibition to provide sufficient L: -lysine for epsilon-PL biosynthesis. To address this hypothesis, in this study, we cloned the ask gene from S. albulus and investigated the feedback inhibition of its gene product. As predicted, we revealed the feedback resistance of the Ask; more than 20% relative activity of Ask was detected in the assay mixture even with extremely high concentrations of L: -lysine and L: -threonine (100 mM each). We further constructed a mutated ask gene for which the gene product Ask (M68V) is almost fully resistant to feedback inhibition. The homologous expression of Ask (M68V) further demonstrated the increase in epsilon-PL productivity.

A critical factor in the biotechnological production of L: -lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP(+) via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by approximately 10% on glucose, approximately 70% on fructose, approximately 50% on glucose plus fructose, and even by approximately 300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type.

Amino acids are among the major products in biotechnology in both volume and value, and the global market is growing. Microbial fermentation is the dominant method used for industrial production, and today the most important microorganisms used are Corynebacteria utilizing sugars. For low-prize bulk amino acids, the possibility of using alternative substrates such as methanol has gained considerable interest. In this mini review, we highlight the unique genetics and favorable physiological traits of thermotolerant methylotroph Bacillus methanolicus, which makes it an interesting candidate for overproduction of amino acids from methanol. B. methanolicus genes involved in methanol consumption are plasmid-encoded and this bacterium has a high methanol conversion rate. Wild-type strains can secrete 58 g/l of L: -glutamate in fed-batch cultures at 50 degrees C and classical mutants secreting 37 g/l of L: -lysine have been selected. The relative high growth temperature is an advantage with respect to both reactor cooling requirements and low contamination risks. Key genes in L: -lysine and L: -glutamate production have been cloned, high-cell density methanol fermentation technology established, and recently a gene delivery method was developed for this organism. We discuss how this new knowledge and technology may lead to the construction of improved L: -lysine and L: -glutamate producing strains by metabolic engineering.

Oxygen supply is a key parameter in aerobic fermentation processes like the industrial production of amino acids. Although the oxygen transfer rate (OTR; or the volumetric oxygen transfer coefficient k(L)a) is routinely analyzed by engineers during stirred tank fermentations, it is often not taken into account by biologists conducting screening experiments in shake flasks. To show the importance of knowing how to avoid oxygen transfer limitations during primary screenings, Corynebacterium glutamicum ATCC 13032 (wild-type strain) and DSM 12866 (lysine-producing strain) were cultivated in shake flasks with different culture liquid volumes and under different shaking conditions. With the Respiration Activity Monitoring System, the OTR was determined quasi-continuously. Optical density as well as concentrations of lysine and byproducts (lactate, acetate, succinate) were determined off-line and correlated with the OTR signal. From the results, design criteria for improved screening in shaken bioreactors that help to avoid selection of suboptimal strains during early process development steps can be derived. Finally, the suitability of DSM 12866 as a strain for industrial processes with a high space-time yield is discussed.

We previously found by transcriptome analysis that global induction of amino acid biosynthetic genes occurs in a classically derived industrial L-lysine producer, Corynebacterium glutamicum B-6. Based on this stringent-like transcriptional profile in strain B-6, we analyzed the relevant mutations from among those identified in the genome of the strain, with special attention to the genes that are involved in amino acid biosynthesis and metabolism. Among these mutations, a Gly-456-->Asp mutation in the 3-isopropylmalate dehydratase large subunit gene (leuC) was defined as a useful mutation. Introduction of the leuC mutation into a defined L-lysine producer, AHD-2 (hom59 and lysC311), by allelic replacement led to the phenotype of a partial requirement for L-leucine and approximately 14% increased L-lysine production. Transcriptome analysis revealed that many amino acid biosynthetic genes, including lysC-asd operon, were significantly upregulated in the leuC mutant in a rel-independent manner.

We report here a sensitive and specific polymerase chain reaction (PCR) detection assay for the pathogenic Candida yeast based on the novel LYS1 [encoding saccharopine dehydrogenase (SDH)] and LYS5 [encoding phosphopantetheinyl transferase (PPTase)] gene sequences of the fungal unique lysine biosynthetic pathway. Both LYS1 and LYS5 DNA-specific PCR primers SG1, SG2 and SG3, SG4, respectively, amplified predicted 483 and 648-bp fragments from Candida albicans genomic DNA but not from other selected fungal, bacterial, or human DNA. The 18S rDNA control primers exhibited positive amplifications in all PCR assays. The LYS1-and LYS5-specific primers strongly amplified C. albicans and Candida tropicalis target sequences; however, the LYS1 primers also weakly amplified fragments from Candida kefyr and Candida lusitaniae DNA. Both sets of primers amplified target sequences from less than 10 pg of serially diluted C. albicans DNA, and the LYS1 specific primers also detected DNA isolated from serially diluted 50 C. albicans cells. The PCR primers reported here are sufficiently sensitive and specific for the potential early detection of Candida infections with no possibility of false positive results from cross-contamination with bacterial or human DNA.

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2(+) gene, which is homologous to Saccharomyces cerevisiae LYS4(+). We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2(+)-based cloning vector pRH3. In addition, we deleted the S. pombe his7(+) gene with a lys2(+) -marked polymerase chain reaction (PCR) product and the S. pombe lys2(+) gene with a his7(+)-marked PCR product. Strains carrying these deletions of lys2(+) or his7(+) serve as relatively efficient hosts for the deletion of the ade6(+) gene by lys2(+)-- or his7(+)--marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2(+) as a selectable marker, along with improved strains for the use of his7(+) to mark gene deletions.

The target of rapamycin (TOR) signaling pathway is an essential regulator of cell growth in eukaryotic cells. In Saccharomyces cerevisiae, TOR controls the expression of many genes involved in a wide array of distinct nutrient-responsive metabolic pathways. By exploring the TOR pathway under different growth conditions, we have identified novel TOR-regulated genes, including genes required for branched-chain amino acid biosynthesis as well as lysine biosynthesis (LYS genes). We show that TOR-dependent control of LYS gene expression occurs independently from previously identified LYS gene regulators and is instead coupled to cAMP-regulated protein kinase A (PKA). Additional genome-wide expression analyses reveal that TOR and PKA coregulate LYS gene expression in a pattern that is remarkably similar to genes within the ribosomal protein and "Ribi" regulon genes required for ribosome biogenesis. Moreover, this pattern of coregulation is distinct from other clusters of TOR/PKA coregulated genes, which includes genes involved in fermentation as well as aerobic respiration, suggesting that control of gene expression by TOR and PKA involves multiple modes of crosstalk. Our results underscore how multiple signaling pathways, general growth conditions, as well as the availability of specific nutrients contribute to the maintenance of appropriate patterns of gene activity in yeast.

Introduction: Enzymatic and glycation-induced nonenzymatic cross-links play important roles in the expression of bone strength. The cross-link pattern is affected by tissue maturation and senescence. The aim of our study was to understand the distinctive posttranslational modifications of collagen in areas with different degrees of mineralization with and without hip fracture., Methods: Sixteen female cases of intracapsular hip fracture (78+/-6 years) and 16 age- and gender-matched postmortem controls (76+/-6 years) were included in this study. A sample of each femoral neck cortex was fractionated into low (1.7 to 2.0 g/ml) and high (>2.0 g/ml) density portions. The contents of enzymatic cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, lysinonorleucine, pyridinoline, and deoxypyridinoline) and nonenzymatic cross-links (pentosidine) and the extent of lysine (Lys) hydroxylation were determined in each fraction., Results: In the controls, there was no significant difference in the contents of enzymatic cross-links between low- and high-mineralized bone fractions whereas pentosidine content was significantly higher in high-mineralized bone compared with low-mineralized bone (p=0.0014). When comparing enzymatic cross-link contents between controls and fracture cases, a trend toward lower (p=0.0961) cross-link content in low-mineralized bone and a significant reduction (p<0.0001) in high-mineralized bone were observed. Pentosidine content of low-mineralized bone was significantly higher in fracture cases than in controls (p<0.0001). The extent of Lys hydroxylation was significantly higher in fracture cases than in controls (p<0.001). The higher hydroxylation of Lys in collagen from fracture cases relative to controls was associated with significantly higher values of hydroxylysine-derived cross-link such that the enzymatic cross-link patterns correlated with the extent of Lys hydroxylation in the collagen molecules., Conclusions: These results suggest that reductions in the degree of mineralization and enzymatic cross-links and excessive formation of pentosidine may play an important role in explaining poor bone quality in osteoporosis.

Carbonic anhydrase catalyzes the interconversion of CO(2) and bicarbonate. We focused on this enzyme in the amino acid-producing organism Corynebacterium glutamicum in order to assess the availability of bicarbonate for carboxylation reactions essential to growth and for those required for L-lysine overproduction. A whole-genome sequence revealed two genes encoding putative beta-type and gamma-type carbonic anhydrases in C. glutamicum. These genes encode polypeptides containing zinc ligands strictly conserved in each type of carbonic anhydrase and were designated bca and gca, respectively. Internal deletion of the chromosomal bca gene resulted in a phenotype showing severely reduced growth under atmospheric conditions (0.04% CO(2)) on both complete and minimal media. The growth defect of the Delta bca strain was restored under elevated CO(2) conditions (5% CO(2)). Introduction of the red alga Porphyridium purpureum carbonic anhydrase gene ( pca) could compensate for the bca deletion, allowing normal growth under an atmospheric level of CO(2). In contrast, the Delta gca strain behaved identically to the wild-type strain with respect to growth, irrespective of the CO(2) conditions. Attempts to increase the dosage of bca, gca, and pca in the defined L-lysine-producing strain C. glutamicum AHD-2 led to no discernable effects on growth and production. Northern blot analysis indicated that the bca transcript in strain AHD-2 and another L-lysine producer, C. glutamicum B-6, was present at a much higher level than in the wild-type strain, particularly during exponential growth phases. These results indicate that: (1) the bca product is essential to achieving normal growth under ordinary atmospheric conditions, and this effect is most likely due to the bca product's ability to maintain favorable intracellular bicarbonate/CO(2) levels, and (2) the expression of bca is induced during exponential growth phases and also in the case of L-lysine overproduction, both of which are conditions of higher bicarbonate demand.

We have recently developed a new L-lysine-producing mutant of Corynebacterium glutamicum by "genome breeding" consisting of characterization and reconstitution of a mutation set essential for high-level production. The strain AHP-3 was examined for L-lysine fermentation on glucose at temperatures above 35 degrees C, at which no examples of efficient L-lysine production have been reported for this organism. We found that the strain had inherited the thermotolerance that the original coryneform bacteria was endowed with, and thereby grew and produced L-lysine efficiently up to 41 degrees C. A final titer of 85 g/l after only 28 h was achieved at temperatures around 40 degrees C, indicating the superior performance of the strain developed by genome breeding. When compared with the traditional 30 degrees C fermentation, the 40 degrees C fermentation allowed an increase in yield of about 20% with a concomitant decrease in final growth level, suggesting a significant transition of carbon flux distribution in glucose metabolism. DNA array analysis of metabolic changes between the 30 degrees C and 40 degrees C fermentations identified several differentially expressed genes in central carbon metabolism although we could not find stringent control-like global induction of amino-acid-biosynthetic genes in the 40 degrees C fermentation. Among these changes, two candidates were picked out as the potential causes of the increased production at 40 degrees C; decreased expression of the citrate synthase gene gltA and increased expression of malE, the product of which involves regeneration of pyruvate and NADPH.

The non-proteinogenic amino acid, alpha-aminoadipate, defines the biosynthetic branch-point of lysine and penicillin biosynthesis in the filamentous fungus, Aspergillus nidulans. Regulation of both pathways was analysed in response to amino acid limitation. The lysF-encoded homoaconitase acts upstream of the alpha-aminoadipate branch point, whereas the lysA gene product, saccharopine dehydrogenase, catalyses the ultimate step of the lysine-specific branch. The lysA gene from A. nidulans was identified and isolated. Amino acid starvation resulted in significantly increased transcription of lysA but not lysF. Starvation-dependent changes in transcription levels of lysA were dependent on the presence of the central transcriptional activator of the cross-pathway control (CPCA). The effect of amino acid starvation under penicillin-producing conditions was analysed in A. nidulans strains with reporter genes for the penicillin-biosynthesis genes, acvA and ipnA, and genetically altered activity of the cross-pathway control. Overproduction of CPCA decreased expression of ipnAand acvA reporter genes and even more drastically reduced penicillin production. This work suggests that, upon amino acid starvation, the cross-pathway control overrules secondary metabolite biosynthesis and favours the metabolic flux towards amino acids instead of penicillin in A. nidulans.

Classical whole-cell mutagenesis has achieved great success in development of many industrial fermentation strains, but has the serious disadvantage of accumulation of uncharacterized secondary mutations that are detrimental to their performance. In the post-genomic era, a novel methodology which avoids this drawback presents itself. This "genome-based strain reconstruction" involves identifying mutations by comparative genomic analysis, defining mutations beneficial for production, and assembling them in a single wild-type background. Described herein is an initial challenge involving reconstruction of classically derived L-lysine-producing Corynebacterium glutamicum. Comparative genomic analysis for the relevant terminal pathways, the efflux step, and the anaplerotic reactions between the wild-type and production strains identified a Val-59-->Ala mutation in the homoserine dehydrogenase gene (hom), a Thr-311-->Ile mutation in the aspartokinase gene (lysC), and a Pro-458-->Ser mutation in the pyruvate carboxylase gene (pyc). Introduction of the hom and lysC mutations into the wild-type strain by allelic replacement resulted in accumulation of 8 g and 55 g of L-lysine/l, respectively, indicating that both these specific mutations are relevant to production. The two mutations were then reconstituted in the wild-type genome, which led to a synergistic effect on production (75 g/l). Further introduction of the pyc mutation resulted in an additional contribution and accumulation of 80 g/l after only 27 h. This high-speed fermentation achieved the highest productivity (3.0 g l(-1) h(-1)) so far reported for microbes producing L-lysine in fed-batch fermentation.

The Lys14 regulatory protein of Saccharomyces cerevisiae activates the expression of the LYS genes involved in the lysine biosynthetic pathway. Studies with a fused Lys14-green fluorescent protein reveal that Lys14p is localised to the nucleus, even under growth conditions leading to the absence of LYS gene expression. Lys14p nuclear localisation is mediated by a tripartite sequence made up of three short basic motifs located on the C-terminal side of the Zn cluster domain of Lys14p. Substitution of basic residues by alanines in any of the three motifs partially prevents the nuclear import of the protein. Simultaneous mutations in the three basic domains are required to completely abolish the entry into the nucleus and to impair the Lys14 function.

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.

The unique alpha-aminoadipate pathway for lysine biosynthesis is present only in fungi and involves eight enzyme steps. alpha-Aminoadipate semialdehyde dehydrogenase, commonly called alpha-aminoadipate reductase (AAR), catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic semialdehyde by a novel mechanism. Two genes, LYS2 and LYS5, encode the heterodimeric enzyme in Saccharomyces cerevisiae. The LYS2 gene of Candida albicans was shown to be contained in the 4.8-kb insert of the plasmid pCaLYS2. This plasmid complemented lys2 mutants of both S. cerevisiae and C. albicans. The S. cerevisiae and C. albicans Lys2(+) transformants exhibited 138% and 160% of wild-type AAR activity, respectively. The DNA-sequence analysis of the 4.8-kb region in plasmid pCaLYS2 and a PCR product from genomic DNA which overlapped with the 4.8-kb insert revealed a continuous ORF of 4173 nucleotides encoding 1391 amino-acid residues. The C. albicans LYS2 ORF exhibited 63.0% identity at the nucleotide level and 56.2% identity at the amino-acid level to the LYS2 gene of S. cerevisiae. The ORF is preceded by consensus sequences for the TATA-, CAAT- and GCN4-box elements. An S. cerevesiae-type transcription termination signal is seen in the 3' flanking region. The deduced amino-acid sequence revealed a motif for an AMP-binding site and also the highly conserved core sequences common to peptide antibiotic synthetases. The LYS2 mRNA and alpha-aminoadipate reductase activity were repressed to a higher level in YEPD-grown cells than in cells grown in the presence of lysine or minimal medium. Additionally, AAR was shown to be feedback-inhibited by lysine and the lysine analog, thialysine. The results of the present report reveal the molecular characteristics of the LYS2 gene of C. albicans, its homology to peptide antibiotic synthetases, its divergence from the LYS2 gene of S. cerevisiae, and the regulation of AAR in C. albicans.

The amino acid L-lysine is produced on a large scale using mutants of Corynebacterium glutamicum. However, as yet recombinant DNA techniques have not succeed in improving strains selected for decades by classic mutagenesis for high productivity. We here report that seven biosynthetic enzymes were assayed and oversynthesis of the dihydrodipicolinate synthase resulted in an increase of lysine accumulation from 220 mM to 270 mM. The synthase, encoded by dapA, is located at the branch point of metabolite distribution to either lysine or threonine and competes with homoserine dehydrogenase for the common substrate aspartate semialdehyde. When graded dapA expression was used, as well as quantification of enzyme activities, intracellular metabolite concentrations and flux rates, a global response of the carbon metabolism to the synthase activity became apparent: the increased flux towards lysine was accompanied by a decreased flux towards threonine. This resulted in a decreased growth rate, but increased intracellular levels of pyruvate-derived valine and alanine. Therefore, modulating the flux at the branch point results in an intrinsically introduced growth limitation with increased intracellular precursor supply for lysine synthesis. This does not only achieve an increase in lysine yield but this example of an intracellularly introduced growth limitation is proposed as a new general means of increasing flux for industrial metabolite over-production.

Auxotrophic yeast cells were starved on solid media for their respective essential amino acid in the course of "adaptive mutation" experiments. Thereby, high proportions of mitochondrially respiratory deficient (rho-) mutants accumulated among the cells stressed on selective plates. Using a strain with a plus-four frameshift mutation in a chromosomal gene involved in lysine biosynthesis, we observed that many of the revertant colonies which arose late under the selective pressure were composed of mixtures of rho+ and rho- cells, indicating that they originated from founder cells containing intact as well as defective mitochondrial genomes. We show that in spite of the slower growth of rho- cells the late-appearing colonies cannot be interpreted as descending from rho- revertants present before selective plating.

A 2.9-kb SacI fragment containing the ask-asd operon, encoding aspartokinase and aspartatesemialdehyde dehydrogenase, was cloned from an aminoethylcysteine-resistant, lysine-producing Corynebacterium lactofermentum strain. Enzymatic analysis showed that the aspartokinase (ASK) activity was completely resistant to inhibition by mixtures of lysine and threonine. Comparison of the deduced amino acid sequence of the beta submit of the ask gene showed three amino acid residue changes with ask gene encoding wild-type, feedback-sensitive enzymes. Three C. lactofermentum strains, one being aspartokinase-negative, one carrying two ask genes on the chromosome and one having a sixfold higher specific ASK activity than the parental strain, were constructed by transconjugation and electroporation, and used to analyse the role of ASK in the lysine production by C. lactofermentum. The results indicate that, in this study, feed-back-resistant ASK is necessary for high-level lysine production, but dispensable for lysine and diaminopimelate synthesis required for cell growth.

The LYS5 and LYS2 genes of Saccharomyces cerevisiae are required for the synthesis of alpha-aminoadipate reductase in the lysine pathway. The LYS5 gene, originally cloned as a DNA insert of the plasmid pSC5, has been subcloned on a 3.2 kb SphI-Sau3AI DNA fragment of the recombinant plasmid pSR7. An internal 2.1 kb HpaI-HpaI DNA fragment of the subclone, upon Southern hybridization, exhibits homology with HpaI-restricted wild-type S. cerevisiae genomic DNA. The lys5+ transformants exhibited alpha-aminoadipate reductase activity similar to that of wild-type cells. S1 nuclease analysis localizes the transcription initiation site relative to the detailed restriction map, and reveals the direction of transcription, as well as the transcript size of the LYS5 gene which can be no greater than 1.65 kb. From this it is estimated that the encoded polypeptide is appreciably smaller than the 4 kb LYS2 gene product. These results provide a physical and biochemical characterization of the cloned LYS5 gene. Based on these observations, it is concluded that the LYS5 gene encodes a relatively small polypeptide of the large heteropolymeric alpha-aminoadipate reductase.

Six of the eight enzymes of the alpha-aminoadipate pathway for the biosynthesis of lysine in Saccharomyces cerevisiae were examined for repressibility to lysine and for susceptibility to the general control of amino acid biosynthesis. All of the enzymes exhibited a 2 to 4 fold lower level of specific activity in the wildtype strain X2180 when grown in lysine supplemented medium as compared to minimal medium. However, levels of only three of the enzymes, alpha-aminoadipate reductase, saccharopine reductase, and saccharopine dehydrogenase, were derepressed in the leaky lysine mutant 7305d and leaky arginine mutant 7853-6c when grown in minimal medium. These observations are characteristic of enzymes under general control of amino acid biosynthesis. The remaining three enzymes, homocitrate synthetase, homoaconitase and homoisocitrate dehydrogenase were repressed in 7305d cells grown in minimal or lysine supplemented medium.

Derepression of lysine biosynthetic enzymes of Saccharomyces cerevisiae was investigated in lys9 auxotrophs which lack saccharopine reductase activity. Five enzymes (homocitrate synthase, homoisocitrate dehydrogenase, alpha-aminoadipate aminotransferase, alpha-aminoadipate reductase and saccharopine dehydrogenase) were constitutively derepressed in all lys9 mutants with up to eight-fold higher enzyme levels than in isogenic wild-type cells. Levels of these enzymes in lys2, lys14, and lys15 mutants were the same or lower than those in wild-type cells. The regulatory property of lys9 mutants exhibited recessiveness to the wild-type gene in heterozygous diploids. Unlike the mating type effect, homozygous diploids resulting from crosses between lys9 auxotrophs exhibited even higher levels of derepressed enzymes than the haploid mutants. Addition of a higher concentration of lysine to the growth medium resulted in reduction of enzyme levels although they were still derepressed. These results suggest that lys9 mutants represent a lesion for the saccharopine reductase and may represent a repressor mutation which in the wild-type cells simultaneously represses unlinked structural genes that encode for five of the lysine biosynthetic enzymes.

The plasmid pSC4 which carries a 7.8 kb yeast DNA insert at the BamHI site of the Vector YEp13, complemented simultaneously MO-59-13c lys4, LU75 lys15 and LU32 lys4lys15 (double) mutations of Saccharomyces cerevisiae. The 1.9 kb BamHI-XbaI DNA insert of the subclone pSO51 complemented the LU75 lys15 mutation. The 2.8 kb Xhol-XhoI DNA insert of the pSO52 subclone, like pSC4, complemented all three mutations. The 1.9 kb BamHI-XbaI DNA and the 2.8 kb Xhol-XhoI DNA were 100 bp apart in the pSC4 DNA insert and exhibited no homology with each other upon Southern hybridization. The 1.9 kb BamHI-XbaI DNA insert exhibited homology with the pSC4 and pSO51 DNA as well as the genomic DNA of MO-59-13c lys4, LU75 lys15, LU32 lys4lys15, and RC1 (LYS) when digested with appropriate restriction enzymes. The 2.8 kb XhoI-XhoI DNA insert exhibited homology with the pSC4 and pSO52 DNA as well as MO-59-13c lys4, LU75 lys15, LU32 lys4lys15, and RC1 (LYS) genomic DNA, when digested with XhoI enzyme. The 2.8 kb DNA probe also hybridized with ply(A)+ RNA from RC1 and lys4+ transformant but not that from MO-59-13c lys4 mutant.

We have isolated five mutants of Candida maltos, which lack the 2-aminoadipate reductase activity, an enzyme involved in the lysine biosynthesis. By means of complementation analysis using protoplast fusion, the isolated mutants were divided into two complementation groups. Thereof the C. maltosa strain G457 could be transformed by the plasmids pDP12 and pDP13, which contain the LYS2-coding gene of Saccharomyces cerevisiae. On the basis of our presented results obtained by studies on hybridization, stability, and recovery of plasmids from C. maltosa transformants, we suggest that transformation does proceed integratively.