Role of Aminotransferase IlvE in Production of Branched-Chain Fatty Acids by Lactococcus lactis subsp. lactis

The objective of this study was to determine the role of a lactococcalbranched-chain amino acid aminotransferase gene, ilvE, in theproduction of branched-chain fatty acids. Lactococcus lactissubsp. lactis LM0230 and an ilvE deletion mutant, JLS450, producedbranched-chain fatty acids from amino and ${alpha}$ -keto acids at levelsabove ${alpha}$ -keto acid spontaneous degradation and the fatty acids'flavor thresholds. The deletion mutant produced the same amountsof branched-chain fatty acids from precursor amino acids asdid the parent. This was not the case, however, for the productionof branched-chain fatty acids from the corresponding precursor ${alpha}$ -keto acids. The deletion mutant produced a set of fatty acidsdifferent from that produced by the parent. We concluded fromthese observations that ilvE plays a role in the specific typeof fatty acids produced but has little influence on the totalamount of fatty acids produced by lactococci.

INTRODUCTION

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Introduction

Lactic acid bacteria (LAB) are important for flavor productionin fermented milk products. Fatty acids (FAs) are an importantclass of compounds produced by these organisms, contributingto distinctive flavor profiles in cheese (7, 25). Typical flavorsof FAs at high concentrations are considered detrimental, butmixtures of FAs at low concentrations are beneficial to cheeseflavor (21), especially Italian varieties (17).

FA production occurs via three known mechanisms: (i) glycolysis(14), (ii) lipolysis (8), and (iii) microbial metabolism ofamino acids and ${alpha}$ -keto acids (16). Branched-chain fatty acids(BCFAs) cannot be produced via glycolysis or lipolysis becausemilk fat lacks BCFAs; yet their concentration increases duringaging. The mechanism for their production is unexplained.

The environment of cheese as a medium for bacteria is acidicand of suboptimal water activity and temperature, and it lacksfermentable carbohydrates. Stuart et al. (22) demonstrated thatlactococci metabolize amino acids during carbohydrate starvationunder laboratory conditions that partially mimic cheese. Presumably,this metabolism may produce FAs via amino acid catabolism. Thismetabolism is relevant because amino acid metabolism generatesATP via substrate level phosphorylation and regenerates protonsto maintain the cellular oxidation-reduction potential (15).Amino acid catabolism is one plausible explanation for the occurrenceof BCFAs in cheese due to the conditions and the metabolismof the associated bacteria.

Aminotransferases (ATases) initiate catabolism of aromatic,branched-chain, and sulfur-containing amino acids to cheeseflavor compounds (11, 28). ${alpha}$ -Keto acids are the intermediatesand amino group acceptors in an ATase reaction (28); consequently,they are of interest in determining the role of these reactionsin FA production. A direct role for the ATases in FA production,except for methionine catabolism (4, 10), is unknown in LAB.Analysis of the genome sequences of Lactococcus lactis IL-1403(2) (GenBank accession no. AE005176) and Lactococcus lactissubsp. cremoris SK11 (http://www.jgi.doe.gov/JGI_microbial/html/index.html)determined that each contains 12 ATases. However, detailed expressionstudies of each ATase in these organisms are lacking. OverlappingATase substrate specificity (6, 20, 27), the large number ofATases in the lactococcal genomes, and the lack of defined conditionsleading to expression increase the complexity involved in assigninga specific role for individual ATases.

Previous studies focused on relating lactococcal ATases to flavorcompound generation. Deletion of the lactococcal aromatic ATase(araT) reduces catabolism of leucine (20), while addition of ${alpha}$ -ketoglutarate leads to an increase in aroma compounds (26).These studies independently verified the activities of lactococcalATases or FA production but did not associate these two events,making it impossible to define the causal relationship of ATasesand FA production. To determine the influence of the IlvE ATasein L. lactis subsp. lactis LM0230, a deletion mutant was previouslytested for growth (1).

The role of individual ATases in the metabolism of amino andketo acids is unclear. Use of an ATase deletion mutant allowsa direct assessment of the impact of the loss of the ATase onthis metabolism. While other studies found IlvE to be involvedin anabolism and growth, ATases are bidirectional and they mayalso be involved in the catabolism of amino and ${alpha}$ -keto acids.Based on previous studies, we hypothesized that ilvE modulatesthe production of BCFAs in lactococci. This study found thatdeletion of ilvE did not significantly (P > 0.05) changethe total concentration of BCFAs produced from amino acids butdid significantly (P < 0.05) change the type of FAs producedat pH 5.2 with 4% NaCl and no carbohydrate in the assay buffer.

Strains and media.

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Strains and Media.

L. lactis subsp. lactis LM0230 was obtained from the Utah StateUniversity culture collection. The ilvE deletion mutant, JLS450(1), was kindly provided by James L. Steele, University of Wisconsinat Madison. Stock cultures were prepared by growing the organismstwice in 10 ml of Elliker broth (Difco laboratories, Detroit,Mich.) at 30°C for 24 h. Cultures were frozen at -70°Cin 10% nonfat dry milk containing 30% glycerol. Before eachexperiment, frozen stock cultures were thawed and subculturedtwice at 30°C for 24 h in 10 ml of Elliker broth.

Cell preparation for FA assays.

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Cell preparation for fa...

Amino acids, ${alpha}$ -keto acids, FA standards, and buffers were purchasedfrom Sigma (Sigma-Aldrich, St. Louis, Mo.). Cells were harvestedfrom 10 ml of broth by centrifugation (3,500 x g for 15 minat 4°C), washed twice with 10 ml of sterile 0.05 M potassiumphosphate buffer (pH 7.2), and resuspended in 5 ml of 0.15 M2-(N-morpholino)ethane-sulfonic acid-sodium salt buffer (pH5.2) containing 4% NaCl to a final cell density of 0.2 at A₆₀₀.The suspended cells were incubated in sterile buffer (negativecontrol), individual amino acids, individual ${alpha}$ -keto acids, andmixtures of the amino acids or ${alpha}$ -keto acids as described below.The amino acids used were glutamic acid, isoleucine, leucine,and valine. The ${alpha}$ -keto acids used were ${alpha}$ -ketoisocaproate (KIC), ${alpha}$ -ketoglutarate (KGL), ${alpha}$ -ketoisovalerate (KIV), and ${alpha}$ -keto-ß-methylvalerate(KMV). These components were used individually as substratesat a final concentration of 1 mM in the assay buffer containingthe cells.

Based on previous studies with LAB (18, 19) and previous work(9), the cell-substrate mixtures were incubated at 30°Cfor 3 h and filter sterilized with 0.2-µm-pore-size syringefilters (Nalge Company, Rochester, N.Y.). The collected filtrateswere used for FA analysis.

Gas chromatography.

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Gas chromatography.

FAs were extracted and determined in the sterile buffer filtratesas described by de Jong and Badings (3), and instrument operatingconditions were as described previously (9). Gas chromatographywas done on the Shimadzu (Columbia, Md.) gas chromatograph modelGC17-A, equipped with a flame ionization detector. Samples wereseparated with a fused silica DB-FFAP column (J & W Scientific,Folsom, Calif.). Peak areas were determined from chromatogramsby using the Shimadzu VP 4.2 software. FAs were identified byusing coinjection of known standards and quantified based oninternal standards as described previously (9). Analyses werereplicated twice, and the results were averaged before beingpresented as millimolar concentrations of FAs.

Statistical analysis.

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Statistical analysis.

Assays were replicated twice, and the data were averaged beforepresentation. Student's t test was used to determine significantdifferences in FA production between the parent and mutant andthe substrates. Results with P values of $<=$ 0.05 were consideredsignificantly different. The standard deviation for FA productionwas less than 10% between replicates for both strains and allproducts.

Results and discussion.

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Results and Discussion.

Previous experiments (9) demonstrated that lactococci producedmore FAs than lactobacilli from ${alpha}$ -keto acids, while the two generaproduced similar amounts of FAs from amino acids. These observationsand suggestions from other reports (11, 12) led us to supposethat the initiation of the catabolic route by an ATase may bethe limiting step in FA production. Despite the large numberof ATases in lactococcal genomes (2) (http://www.jgi.doe.gov/JGI_microbial/html/index.html)and the overlapping substrate specificity of ATases (6, 20,27), we hypothesized that deletion of a single enzyme woulddecrease the total concentration of FAs. Therefore, a directcomparison with a branched-chain ATase (ilvE) deletion mutant(JLS450) of L. lactis subsp. lactis LM0230 in an assay systemwas used to investigate FA production from branched-chain aminoacids (BCAAs) and ${alpha}$ -keto acids.

As noted previously (9), the cells incubated in a buffer withouta substrate produced FAs (Fig. 1). This is probably due to theutilization of intracellular proteins and amino acids in theabsence of external substrates. We also observed that the absoluteconcentrations of some products were higher than the substrateconcentrations both in this study and in previous studies (9).This is plausible, given the degradation of amino acids and ${alpha}$ -keto acids to shorter-chain FAs and BCFAs. If these data areanalyzed as mole fractions, the total amount of carbon is smallerafter the assay, despite the FA products having a higher concentrationvalue than amino or ${alpha}$ -keto acid substrates (9) (data not shown).Other intracellular intermediates, such as pyruvate, phosphoglycerate,or acyl coenzyme A derivatives, may be incorporated from otherpathways into FA production, leading to a higher concentrationfor products of smaller chain length (24).

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FIG. 1. Mean FA production by L. lactis subsp. lactis LM0230 from amino acids (A) and from ${alpha}$ -keto acids (B), respectively, and by L. lactis subsp. lactis JLS450 from amino acids (C) and from ${alpha}$ -keto acids (D), respectively, beyond ${alpha}$ -keto acid autodegradation. Values represent the averages of twice-replicated assays of FA production. NC, negative control (cells in plain buffer); Ile, isoleucine; Leu, leucine; Val, valine; Glu, glutamic acid; KMV, ${alpha}$ -keto-ß-methylvalerate; KIC, ${alpha}$ -ketoisocaproate; KIV, ${alpha}$ -ketoisovalerate; KGL, ${alpha}$ -ketoglutarate. The standard deviation of the total FA production and individual FAs was $<=$ 10% within both strains.

The impact of ilvE was assessed by using two approaches thatallowed analysis of the impact of ATases. First, the initiatingATase reaction was bypassed by adding ${alpha}$ -keto acid substratesto a culture of the parent (LM0230) in the assay buffer. Second,the ilvE deletion mutant (JLS450) was incubated with BCAAs underthe same conditions as those of the parent. Additions of anamino acid substrate to LM0230 and of the ${alpha}$ -keto acid substrateto JLS450 were treated as the controls for these experiments(Fig. 1B and C).

The treatment condition using ${alpha}$ -keto acids as the substrate forFA production by LM0230 produced approximately the same totalFA concentrations as the control of amino acids, which rangedfrom 5 to 8 mM. LM0230 produced isobutyric acid from all the ${alpha}$ -keto acid substrates and isovaleric acid from KIC. Bypassingthe initial ATase reaction with the addition of ${alpha}$ -keto acidsto the parent significantly (P < 0.05) changed the type ofBCFAs produced compared with those found in the control aminoacid substrates (Fig. 1A and B). A significantly smaller (P< 0.05) amount of acetic acid was produced from all ${alpha}$ -ketoacids than from the corresponding BCAAs. Isobutyric acid wasproduced in significantly (P < 0.05) higher amounts fromKIC and KIV than leucine and valine, respectively. Interestingly,the only condition that produced n-butyric acid was LM0230 withKGL. In the amino acid control, the total FA production rangedfrom 5 to 10 mM FAs (Fig. 1A) and isobutyric acid was the onlyBCFA produced (Fig. 1A). These observations allowed us to supposethat the ATase reactions were not the limiting step in the productionof FAs from BCAAs. However, this approach provided evidencethat ATases play a role in directing intermediates into specificpathways that lead to specific FA products. Subsequent to theseobservations, we also hypothesized that ATases play a role inthe types of FAs produced.

To verify the role of ilvE, the deletion mutant (JLS450) wasassayed for FA production with amino acid substrates. Deletionof ilvE did not significantly (P > 0.05) change the totalFA quantities (Fig. 1C). However, deletion of ilvE changed thespecific FAs produced from amino acids compared with the parentwith the same substrates (Fig. 1A). With either approach, thetotal FA production did not decrease significantly (P > 0.05).These data agree with the previous observations that ATaseswere not the limiting step for producing FAs and lend supportto this study's hypothesis.

The addition of ${alpha}$ -keto acids to JLS450 allowed comparison ofcatabolic products independent of ATase reactions. JLS450 producedsimilar FA products and quantities from all ${alpha}$ -keto acids, exceptfor KIV, in which case acetic acid was not produced. When theATase reactions were bypassed, the mutant produced no BCFAsfrom ${alpha}$ -keto acids. The mutant also produced significantly less(P < 0.05) acetic acid than it did from the precursor BCAAs(Fig. 1 C and D). These differences suggest that ${alpha}$ -keto acidsare catabolized via multiple routes and that the specific routesare ATase dependent. This is plausible, considering the numerouspossible pathways subsequent to the ATase reaction (5, 13, 15,23, 29).

To further assess the role of IlvE, a comparison of the catabolicproducts from ${alpha}$ -keto acids by the parent and the mutant was done(Fig. 1B and D). We expected that the deletion of ilvE and thebypassing of the ATase reactions would yield similar FAs from ${alpha}$ -keto acids. Interestingly, JLS450 produced significantly (P< 0.05) smaller amounts of BCFAs from ${alpha}$ -keto acids than didLM0230. Deletion of ilvE also abolished production of n-butyricacid from KGL. While it is unclear why n-butyric acid was producedfrom KGL by LM0230, it is possible that subsequent metabolismof these components followed different pathways via interconversionof ${alpha}$ -keto acids and acyl coenzyme A, leading to different products.These data indicate that ilvE is involved in the productionof n-butyric and isobutyric acids. These unexpected differencespoint toward the interactions of metabolic pathways that occursubsequent to the ATase reaction.

Atiles et al. (1) deleted ilvE and found that addition of ${alpha}$ -ketoacids did not support growth of JLS450; they hypothesized thatthe deletion blocked BCAA biosynthesis. While this study didnot test the role of ilvE in growth, we observed that aminoacids and ${alpha}$ -keto acids were catabolized to FAs in JLS450, suggestingthat ilvE plays a role in production of specific FAs via a complexweb of intermediates in addition to a role in growth on ${alpha}$ -ketoacids.

Yvon et al. (27) deleted bcaT of L. lactis subsp. cremoris,thought to be involved in BCAA catabolism, and expectedly observeda reduction in BCFA production. Conversely, this study foundthe ilvE deletion of L. lactis subsp. lactis led to an increasein isobutyric and isovaleric acids from isoleucine and leucinecompared with that produced by the parent. Observations fromthis study may be explained by the activities of at least 11other ATases (2) that may catabolize leucine and isoleucineto BCFAs (6).

Considering the observations from this study, the complementaryactivity of multiple ATases with BCAAs, and the bidirectionalactivity of ATases, it is plausible that IlvE and other ATasesdirect intermediates into different pathways that lead to specificFAs. Deletion or inactivation of any single component of themetabolic web changes the specific products without completelyabolishing the catabolic capabilities or reducing the totalamount of products.

The observations from this study indicate that the activityof ATases is not the limiting step in producing FAs from aminoacids or ${alpha}$ -keto acids. However, ATases appear to play a rolein directing intermediate substrates toward specific FAs. IlvEwas directly involved in the production of isobutyric, n-butyric,and isovaleric acids from amino and ${alpha}$ -keto acids in L. lactissubsp. lactis. Additional studies are needed to delineate theroles of other ATases in FA production.

ACKNOWLEDGMENTS

Mention of companies and products does not constitute endorsementby Utah State University or Utah Agricultural Experiment Stationover similar products not mentioned.

FOOTNOTES

* Corresponding author. Mailing address: Center for Integrated BioSystems, Utah State University, Logan, UT 84322-4700. Phone: (435) 797-2753. Fax: (435) 797-2357. E-mail: bcweimer{at}cc.usu.edu.

Contribution number 7537 of the Utah Agricultural ExperimentStation.

REFERENCES

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