Catabolism of Lysine by Mixed Rumen Bacteria

Metabolites arising from the catabolism of lysine by the mixed rumen bacteria were chromatographically examined by using radioactive lysine. After 6 hr incubation, 241 nmole/ ml of lysine was decomposed to give ether-soluble substances and CO₂ by the bacteria and 90 nmole/ml of lysine was incorporated unchanged into the bacteria. δ-Aminovalerate, cadaverine or pipecolate did not seem to be produced from lysine even after incubation of the bacteria with addition of those three amino compounds to trap besides lysine and radioactive lysine. Most of the ether-soluble substances produced from radioactive lysine was volatile fatty acids (VFAs). Fractionation of VFAs revealed that the peaks of butyric and acetic acids coincided with the strong radioactive peaks. Small amounts of radioactivities were detected in propionic acid peak and a peak assumed to be caproic acid. The rumen bacteria appeared to decompose much larger amounts of lysine than the rumen ciliate protozoa did.     

In Vitro Metabolism of the Stereoisomers of 2,6-Diaminopimelic Acid by Mixed Rumen Protozoa and Bacteria

Formation of lysine from stereoisomers (SI) of 2,6-diaminopimelic acid (DAP) and the epimerization between the three SI of DAP (DAP-SI) by rumen protozoa and bacteria were examined. Mixed rumen protozoa (P) and bacteria (B) were isolated from the rumen of goats given a concentrate and hay cubes and incubated separately with and without a mixture and a single one of the three DAP-SI. In P suspensions, mixed DAP-SI decreased by 10.59% as a whole and converted mainly to lysine by 8.41% during 12 h incubation. When meso-, L- and D-DAP were added singly to the media, the results showed that each DAP-SI interconverted and produced lysine. This means that mixed rumen protozoa have an ability to synthesize lysine from not only meso-DAP but also from D- and L-DAP, though probably via meso-DAP, and hence have DAP epimerase activities for the reversal conversion of each DAP-SI. This is the first discovery to show the interconversion of DAP-SI and synthesis of lysine from them by protozoa. In B suspensions, mixed DAP-SI decreased by 10.92% as a whole and converted to lysine by 4.20% during 12 h incubation. When a single DAP-SI was added to the media, meso-, L- and D-DAP were interconverted and then converted to lysine by the rumen bacteria as well as the protozoa. This also means that mixed rumen bacteria have DAP epimerase activities to interconvert DAP-SI and have an ability to synthesize lysine from not only meso-DAP but also from L- and D-DAP, and this is also the first finding in rumen bacteria. Received: 16 March 1996 / Accepted: 14 May 1996     

Engineering photosynthetic production of L-lysine

L-lysine and other amino acids are commonly produced through fermentation using strains of heterotrophic bacteria such as Corynebacterium glutamicum. Given the large amount of sugar this process consumes, direct photosynthetic production is intriguing alternative. In this study, we report the development of a cyanobacterium, Synechococcus sp. strain PCC 7002, capable of producing L-lysine with CO₂ as the sole carbon-source. We found that heterologous expression of a lysine transporter was required to excrete lysine and avoid intracellular accumulation that correlated with poor fitness. Simultaneous expression of a feedback inhibition resistant aspartate kinase and lysine transporter were sufficient for high productivities, but this was also met with a decreased chlorophyll content and reduced growth rates. Increasing the reductant supply by using NH₄⁺, a more reduced nitrogen source relative to NO₃^-, resulted in a two-fold increase in productivity directing 18% of fixed carbon to lysine. Given this advantage, we demonstrated lysine production from media formulated with a municipal wastewater treatment sidestream as a nutrient source for increased economic and environmental sustainability. Based on our results, we project that Synechococcus sp. strain PCC 7002 could produce lysine at areal productivities approaching that of sugar cane to lysine via fermentation using non-agricultural lands and low-cost feedstocks.     

Lysine biosynthesis in microbes: relevance as drug target and prospects for β-lactam antibiotics production

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.     

Important impacts of intestinal bacteria on utilization of dietary amino acids in pigs

Bacteria in pig intestine can actively metabolize amino acids (AA). However, little research has focused on the variation in AA metabolism by bacteria from different niches. This study compared the metabolism of AA by microorganisms derived from the lumen and epithelial wall of the pig small intestine, aiming to test the hypothesis that the metabolic profile of AA by gut microbes was niche specific. Samples from the digesta, gut wall washes and gut wall of the jejunum and ileum were used as inocula. Anaerobic media containing single AA were used and cultured for 24 h. The 24-h culture served as inocula for the subsequent 30 times of subcultures. Results showed that for the luminal bacteria, all AA concentrations except phenylalanine in the ileum decreased during the 24-h in vitro incubation with a increase of ammonia concentration, while 4 AA (glutamate, glutamine, arginine and lysine) in the jejunum decreased, with the disappearance rate at 60–95 %. For tightly attached bacteria, all AA concentrations were generally increased during the first 12 h and then decreased coupled with first a decrease and then an increase of ammonia concentration, suggesting a synthesis first and then a catabolism pattern. Among them, glutamate in both segments, histidine in the jejunum and lysine in the ileum increased significantly during the first 12 h and then decreased at 24 h. The concentrations of glutamine and arginine did not change during the first 12 h, but significantly decreased at 24 h. Jejunal lysine and ileal threonine were increased for the first 6 or 12 h. For the loosely attached bacteria, there was no clear pattern for the entire AA metabolism. However, glutamate, methionine and lysine in the jejunum decreased after 24 h of cultivation, while glutamine and threonine in the jejunum and glutamine and lysine in the ileum increased in the first 12 h. During subculture, AA metabolism, either utilization or synthesis, was generally decreased with disappearance rate around 20–40 % for most of AA and negligible for branch chained AA (BCAA). However, the disappearance rate of lysine in each group was around 90 % throughout the subculture, suggesting a high utilization of lysine by bacteria from all three compartments. Analysis of the microbial community during the 24-h in vitro cultivation revealed that bacteria composition in most AA cultures varied between different niches (lumen and wall-adherent fractions) in the jejunum, while being relatively similar in the ileum. However, for isoleucine and leucine cultures, bacteria diversity was similar between the luminal fraction and tightly attached fraction, but significantly higher than in the loosely attached fraction. For glutamine and valine cultures, bacteria diversity was similar between the luminal and loosely attached fractions, but lower than that of tightly attached bacteria. After 30 subcultures, bacteria diversity in arginine, valine, glutamine, and leucine cultures varied between niches in the jejunum while being relatively stable in the ileum, consistent with those in the 24-h in vitro cultures. The findings may suggest that luminal bacteria tended to utilize free AA, while tightly attached adherent bacteria seemed in favor of AA synthesis, and that small intestinal microbes contributed little to BCAA metabolism.     

Enrichment of fusobacteria from the rumen that can utilize lysine as an energy source for growth

Ruminal lysine degradation is a wasteful process that deprives the animal of an essential amino acid. Mixed ruminal bacteria did not deaminate lysine (50 mM) at a rapid rate, but lysine degrading bacteria could be enriched if Trypticase (5 mg/mL) was also added. Lysine degrading isolates produced acetate, butyrate and ammonia, were non-motile, stained Gram-negative and could also utilize lactate, glucose, maltose or galactose as an energy source for growth. Lactate was converted to acetate and propionate, and 16S rDNA indicated that their closest relatives were Fusobacterium necrophorum. Growing cultures produced ammonia at rates as high as 2400 nmol/mg protein/mL/min. Washed cell suspensions took up (14)C lysine (3 microM) at an initial rate of 6 nmol/mg protein/min, and glucose addition did not affect the transport. Cells washed aerobically had the same transport rate as those handled anaerobically, but only if the transport buffer contained sodium. The affinity constant for sodium was 8 mM, and sodium could not be replaced by lithium. Cells treated with the sodium/proton antiporter, monensin (5 microM), did not take up lysine, but a protonophore that inhibited growth (tetrachlorosalicylanilide, 10 microM) had no effect. An artificial membrane potential created by potassium diffusion did not increase the rate of lysine transport, and an Eadie-Hofstee plot indicated the transport rate was directly proportional to the lysine concentration. Decreasing the pH from 6.7 to 5.5 caused an 85% decrease in the rate of lysine transport. The addition of F. necrophorum JB2 (130 microg protein/mL) to mixed ruminal bacteria increased lysine degradation 10-fold, but only if the pH was 6.7 and monensin was not present. Further work will be needed to see if dietary lysine enriches fusobacteria in vivo.     

Formation of Lysine from, α, ε-Diaminopimelic Acid Contained in Rumen Bacterial Cell Walls by Rumen Ciliate Protozoa

Cell walls containing α,ε-diaminopimelate-l,7-¹⁴C (DAP) was prepared from Escherichia coli isolated from the rumen. After incubation of ciliates with the cell walls, 22.0% of DAP contained in cell walls of E. coli was converted to lysine and pipecolate. Heat-treated mixed rumen bacteria and heat-treated cell walls of mixed rumen bacteria added to the culture medium of rumen ciliates increased 0.572 and 0.934 μmole/ml of sum of lysine and pipecolate, respectively.

From these results, it is clear that rumen ciliate protozoa can form lysine from DAP contained in the mucopeptide of bacterial cell walls. One of the nutritional significance of inhabitation of ciliates in the rumen was revealed.     

Inhibition of L-lysine uptake by alpha-methyl lysine in Escherichia coli and Bacillus sphaericus

alpha-Methyl lysine was investigated as a potential inhibitor of lysine transport in Escherichia coli and Bacillus sphaericus. At equimolar concentrations, no inhibition was observed in either organism, but at 10X and 100X the lysine concentration, alpha-methyl lysine caused a 20-50% reduction in the initial rate of lysine uptake in both bacteria. A similar inhibitory effect was observed with epsilon-N-methyl lysine on lysine uptake in B. sphaericus, but not in E. coli. alpha-Methyl lysine had a reduced effect on ornithine uptake and no effect on arginine transport in either bacterium.     

Enzyme electrode for specific determination of L-lysine

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 is immobilized in a gelatin support and fixed on a pO(2) sensor. The enzyme electrode obtained is used in a continuous flow system in order to measure the concentration of L-lysine in a fermentor. The sample oxygen-content dependance of the signal is minimized because of the enzyme support properties. The enzyme electrode response is set for lysine concentration from 0.2mM to 4mM. The specificity of lysine is tested with other amino acids. The enzyme membrane for lysine electrode can be used 3000 times or stored six months with good stability.     

Methanogens with pseudomurein use diaminopimelate aminotransferase in lysine biosynthesis

Methanothermobacter thermautotrophicus uses lysine for both protein synthesis and cross-linking pseudomurein in its cell wall. A diaminopimelate aminotransferase enzyme from this methanogen (MTH0052) converts tetrahydrodipicolinate to l,l-diaminopimelate, a lysine precursor. This gene complemented an Escherichia coli diaminopimelate auxotrophy, and the purified protein catalyzed the transamination of diaminopimelate to tetrahydrodipicolinate. Phylogenetic analysis indicated this gene was recruited from anaerobic Gram-positive bacteria. These results expand the family of diaminopimelate aminotransferases to a diverse set of plant, bacterial and archaeal homologs. In contrast marine methanogens from the Methanococcales, which lack pseudomurein, appear to use a different diaminopimelate pathway for lysine biosynthesis.     

Factors affecting lysine degradation by ruminal fusobacteria

Fusobacterium necrophorum can readily be enriched from the rumen with lysine, and its deamination rate is very rapid. The addition of F. necrophorum JB2 to mixed ruminal bacteria significantly increased lysine degradation, but only if the ratio of ruminal fluid to basal medium was less than 25%. If more ruminal fluid (pH 6.1) was added, ammonia production decreased by as much as 80%. Clarified, autoclaved ruminal fluid was also inhibitory. When F. necrophorum JB2 was grown in a lysine-limited continuous culture (0.1 h(-1) dilution rate) and pH was decreased using HCl, optical density decreased linearly, and the culture washed out at pH 5.6. Batch cultures of F. necrophorum JB2 deaminated as much lysine at pH 6.1 as at pH 6.6, but only if fermentation acids were not present. Sodium acetate (100 mM) had little effect at pH 6.6, but the same concentration inhibited ammonia production by 80% at pH 6.1. The idea that fermentation acids could prevent the enrichment of fusobacteria in vivo was supported by the observation that dietary lysine supplementation did not enhance the lysine deamination rate of the mixed ruminal bacteria.     

Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis.

The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis.     

Metabolism of select amino acids in bacteria from the pig small intestine

This study investigated the metabolism of select amino acids (AA) in bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the jejunum and ileum of pigs. Cells were incubated at 37°C for 3 h in anaerobic media containing 0.5–5 mM select AA plus [U-¹⁴C]-labeled tracers to determine their decarboxylation and incorporation into bacterial protein. Results showed that all types of bacteria rapidly utilized glutamine, lysine, arginine and threonine. However, rates of the utilization of AA by pure cultures of E. coli and Klebsiella sp. were greater than those for mixed bacterial cultures or Streptococcus sp. The oxidation of lysine, threonine and arginine accounted for 10% of their utilization in these pure bacterial cultures, but values were either higher or lower in mixed bacterial cultures depending on AA, bacterial species and the gut segment (e.g., 15% for lysine in jejunal and ileal mixed bacteria; 5.5 and 0.3% for threonine in jejunal mixed bacteria and ileal mixed bacteria, respectively; and 20% for arginine in ileal mixed bacteria). Percentages of AA used for bacterial protein synthesis were 50–70% for leucine, 25% for threonine, proline and methionine, 15% for lysine and arginine and 10% for glutamine. These results indicate diverse metabolism of AA in small-intestinal bacteria in a species- and gut compartment-dependent manner. This diversity may contribute to AA homeostasis in the gut. The findings have important implications for both animal and human nutrition, as well as their health and well-beings.     

Expression from the Escherichia coli dapA promoter is regulated by intracellular levels of diaminopimelic acid

Dihydropicolinate synthase (DHDPS; E.C. 4.2.1.52) catalyses the first committed step of lysine biosynthesis in plants and bacteria. Plant DHDPS enzymes, which are responsible solely for lysine biosynthesis, are strongly inhibited by lysine (I0.5 =10 microM), whereas the bacterial enzymes which are less responsive or insensitive to lysine inhibition have the additional function of meso-diaminopimelate biosynthesis which is required for cell wall formation. Previous studies have suggested that expression of the Escherichia coli dapA gene, encoding DHDPS, is unregulated. We show here that this is not the case and that expression of LacZ from the dapA promoter (PdapA) increases in response to diaminopimelic acid limitation in E. coli K-12.     

Regulation of aspartokinase in Lactobacillus plantarum

As a rational approach to the genetic development of a stable lysine overproducing strain of Lactobacillus plantarum for the fermentation of 'ogi', a Nigerian fermented cereal porridge, regulation of lysine biosynthesis in this species was investigated. Spontaneous lysine overproducing mutants of Lact. plantarum were obtained and their aspartokinase activities compared with those of wild-type strains under different conditions. Results showed that aspartokinase activity of Lact. plantarum cell extracts was not inhibited by either lysine, threonine, methionine or combinations of lysine and threonine. Instead, methionine enhanced aspartokinase activity in vitro. Results indicated that lysine biosynthesis in Lact. plantarum could be regulated by lysine via the control of aspartokinase production in a way different to that described for other bacteria.     

The effect of replacing the axial methionine ligand with a lysine residue in cytochrome c-550 from Paracoccus versutus assessed by X-ray crystallography and unfolding

The structure of cytochrome c-550 from the nonphotosynthetic bacteria Paraccocus versutus has been solved by X-ray crystallography to 1.90 A resolution, and reveals a high structural homology to other bacterial cytochromes c(2). The effect of replacing the axial heme-iron methionine ligand with a lysine residue on protein structure and unfolding has been assessed using the M100K variant. From X-ray structures at 1.95 and 1.55 A resolution it became clear that the amino group of the lysine side chain coordinates to the heme-iron. Structural differences compared to the wild-type protein are confined to the lysine ligand loop connecting helices four and five. In the heme cavity an additional water molecule is found which participates in an H-bonding interaction with the lysine ligand. Under cryo-conditions extra electron density in the lysine ligand loop is revealed, leading to residues K97 to T101 being modeled with a double main-chain conformation. Upon unfolding, dissociation of the lysine ligand from the heme-iron is shown to be pH dependent, with NMR data consistent with the occurrence of a ligand exchange mechanism similar to that seen for the wild-type protein.     

Relationship between amino acid production and phosphate-dissolving capacity of bacteria

The ability of soil bacteria to produce amino acids (alanine, aspartic acid, leucine, arginine, glutamic acid, and lysine) was related to the ability to dissolve inorganic phosphate. With the exception of lysine, amino acid production increased with increasing ability to dissolve phosphate.     

Acyl-CoA: glycine N-acyltransferase: in vitro studies on the glycine conjugation of straight- and branched-chained acyl-CoA esters in human liver

Apparent kinetic constants (Km and Vmax values) were determined for human liver acyl-CoA: glycine acyltransferase (glycine-N-acylase) towards isobutyryl-CoA, 2-methyl butyryl-CoA, isovaleryl-CoA, butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, and decanoyl-CoA. These acyl-CoA esters were selected because of their relevance to the human diseases with cellular accumulation of these esters, i.e., especially to metabolic defects in the acyl-CoA dehydrogenation steps of the branched-chain amino acids, lysine, 5-hydroxy lysine, tryptophan, and fatty acid oxidation pathways. With the acyl-CoA ester as the fixed substrate, the Km value for glycine ranged from 0.5 to 2.9 mole/liter, and with glycine as fixed substrate, the Km values for the acyl-CoA esters varied from 0.3 to 5.6 mmole/liter. It is concluded that the substrate concentration is decisive for the glycine conjugate formation and that the occurrence in urine of acylglycines reflects an intramitochondrial accumulation of the corresponding acyl-CoA ester.