Phenotypic Diversity of Lactobacillus casei Group Isolates as a Selection Criterion for Use as Secondary Adjunct Starters

Autochthonous lactic acid bacteria (LAB) play a key role in the development of cheese flavor. As the pasteurization treatment on raw milk causes the elimination of LAB, secondary starter cultures are used in cheese manufacture to obtain cheeses with improved and standardized flavors. In this work, strains of the L. casei group isolated from traditional Italian cheeses were screened for their phenotypic features of technological interest for use as secondary starters. Their milk acidifying performance and the production of volatile compounds when grown in milk were evaluated. Simultaneously, the acetoin metabolic pathway presence was screened in the strains and assessed for its transcriptional activation. The results showed that the analyzed strains, despite belonging to taxonomically-related species, vary greatly according to the measured phenotypes. Four strains among the fourteen screened could be potentially used as adjunct cultures for cheese-making processes. The strain that showed the highest production of acetoin upregulated the aspartate pathway. An increased knowledge of volatile compounds’ production and acidifying properties of LAB strains isolated from traditional dairy products might guide the selection of strains for industrial applications.

Derailing the aspartate pathway of Mycobacterium tuberculosis to eradicate persistent infection

A major constraint for developing new anti-tuberculosis drugs is the limited number of validated targets that allow eradication of persistent infections. Here, we uncover a vulnerable component of Mycobacterium tuberculosis (Mtb) persistence metabolism, the aspartate pathway. Rapid death of threonine and homoserine auxotrophs points to a distinct susceptibility of Mtb to inhibition of this pathway. Combinatorial metabolomic and transcriptomic analysis reveals that inability to produce threonine leads to deregulation of aspartate kinase, causing flux imbalance and lysine and DAP accumulation. Mtb’s adaptive response to this metabolic stress involves a relief valve-like mechanism combining lysine export and catabolism via aminoadipate. We present evidence that inhibition of the aspartate pathway at different branch-point enzymes leads to clearance of chronic infections. Together these findings demonstrate that the aspartate pathway in Mtb relies on a combination of metabolic control mechanisms, is required for persistence, and represents a target space for anti-tuberculosis drug development.

A cautionary tale of structure-guided inhibitor development against an essential enzyme in the aspartate-biosynthetic pathway

The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate β-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Structural characterization of these enzyme–inhibitor complexes has provided detailed explanations for the barriers that interfere with optimal binding. Despite binding in the same active-site region, significant changes are observed in the orientation of these bound inhibitors that are caused by relatively modest structural alterations. Taken together, these studies present a cautionary tale for issues that can arise in the systematic approach to the modification of lead compounds that are being used to develop potent inhibitors.

The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis

The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

Analysis and Manipulation of Aspartate Pathway Genes for l-Lysine Overproduction from Methanol by Bacillus methanolicus

ABSTRACTWe investigated the regulation and roles of six aspartate pathway genes inl-lysine overproduction inBacillus methanolicus:dapG, encoding aspartokinase I (AKI);lysC, encoding AKII;yclM, encoding AKIII;asd, encoding aspartate semialdehyde dehydrogenase;dapA, encoding dihydrodipicolinate synthase; andlysA, encodingmeso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed thatin vivo lysCtranscription was repressed 5-fold byl-lysine and induced 2-fold bydl-methionine added to the growth medium. Surprisingly,yclMtranscription was repressed 5-fold bydl-methionine, while thedapG,asd,dapA, andlysAgenes were not significantly repressed by any of the aspartate pathway amino acids. We show that thel-lysine-overproducing classicalB. methanolicusmutant NOA2#13A52-8A66 has—in addition to ahom-1mutation—chromosomal mutations in thedapGcoding region and in thelysApromoter region. No mutations were found in itsdapA,lysC,asd, andyclMgenes. The mutantdapGgene product had abolished feedback inhibition bymeso-diaminopimelatein vitro, and thelysAmutation was accompanied by an elevated (6-fold)lysAtranscription levelin vivo. Moreover,yclMtranscription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important forl-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increasedl-lysine production levels.

Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus

ABSTRACT Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V max values (between 47 and 58 μmol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

A Genome-Wide Approach Identifies Variations in the Aspartate Metabolism Pathway That Are Associated with Asparaginase Sensitivity

Asparaginase is an important drug for acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in asparaginase sensitivity, we employed a genome-wide association approach using the HapMap lymphoblastoid cell lines from 87 individuals of European ancestry (CEU) and diagnostic ALL blasts from 42 newly diagnosed, genomically-determined white patients. In vitro sensitivity was based on IC50 values measured following 48 hour exposures to native E. coli asparaginase (0.003–100 IU/ml) in CEU cell lines and 96 hour exposures (0.003–10 IU/ml) in patient samples using the methylthiazol tetrazolium assay. For CEU cell lines, single nucleotide polymorphism (SNP) genotypes were downloaded from the International HapMap database (www.hapmap.org) and gene expression data (Affymetrix GeneChip Human Exon 1.0 ST Array) were downloaded from http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE7761. For patients with ALL, we used the 500K SNP arrays to interrogate germline DNA and Affymetrix U133A GeneChip Array to assess gene expression in ALL blasts. We tested whether 2,390,203 SNP genotypes were associated with asparaginase IC50 using a linear mixed effect model in CEU cell lines, setting a p value threshold of p < 0.001 for individual SNPs and p < 0.05 at the gene level. This approach yielded 329 SNPs representing 94 genes. Combining these germline SNPs with those representing genes whose expression was also associated with IC50 at the p < 0.05 level (1,706 genes), there were 6 SNPs representing 5 genes, two of which (rs8135371 and rs17001863, both in the ADSL gene) contributed to asparaginase sensitivity (p = 6.9 × 10−4 and 9.1 × 10− 4, respectively) through their effects on ADSL gene expression. The top ranked KEGG pathway overrepresented by the 94 top-ranked genes (329 SNPs) was that of aspartate metabolism, which may be directly linked to the mechanism of action of asparaginase. The two most highly ranked genes (ADSL and DARS) in this pathway encompassed 7 SNPs (rs8135371, rs17001863, rs3768998, rs2278683, rs11893318, rs2322725, and rs7587285), all with p < .001. Using multiple linear regression analysis, 32% of the variability in asparaginase IC50 among the CEU cell lines could be accounted for by these 7 SNPs (p = 5.9 × 10−7). To examine the overall contribution of the aspartate metabolism pathway to asparaginase IC50, we compared all SNPs (935 in cell lines, 717 in patients) representing the aspartate pathway with those SNPs representing other pathways, using a random forest model. We found that the SNP genotypes in the aspartate pathway explained significantly more variation in asparaginase IC50 in cell lines (11.4%, p = 6.9 × 10−4) and in ALL patient samples (11.2%, p = 0.02) than other pathways. The expression of ADSL differed among ALL subtypes, with more sensitive subtypes (hyperdiploid and TEL-AML1 ALL) having lower ADSL expression than more resistant subtypes (T-ALL) (p = 1.1 × 10−5 and 2.9 × 10−9, respectively). Genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited and acquired genomic interindividual variation in a plausible candidate pathway contribute to asparaginase sensitivity.

Determination of aspartate kinase activity in maize tissues

Lysine, threonine, methionine and isoleucine are synthesized from aspartate in a branched pathway in higher plants. Aspartate kinase plays a key role in the control of the aspartate pathway. The enzyme is very sensitive to manipulation and storage and the hydroxamate assay normally used to determine aspartate kinase activity has to be altered according to the plant species and tissue to be analyzed. We have optimized the assay for the determination of aspartate kinase in maize plants callus cell cultures. Among all the assay parameters tested, the concentration of ATP/Mg and temperature were critical for enzyme activity. In the case of temperature, 35°C was shown to be the optimum temperature for aspartate kinase activity.