Influence of carbon and nitrogen sources on GABA production by Levilactobacillus brevis CRL 2013 in chemically defined media.

CATALDO, P.G.; RÍOS COLOMBO, N.S.; SAVOY DE GIORI, G.; SAAVEDRA, L.; HEBERT, E.M.

Virtual

Congreso; Third Meeting & First Workshop of the Argentine Network of Enzymatic Technology (TEz Network); 2021

Gamma-aminobutyric acid (GABA) is an ubiquitous non-protein amino acidwidely distributed in nature, having diverse physiological functions and greatpotential health benefits. Due to its relevance, GABA is becoming recognized asan essential nutrient for a healthy and balanced diet. In this sense, LABstrains, mainly belonging to Levilactobacillus brevis species,constitute the most competitive and technologically relevant group ofmicroorganisms used to synthesize GABA since they are able to produce highlevels of this compound within a variety of food matrices. Glutamic aciddecarboxylase (GAD) system is responsible for glutamate decarboxylation andGABA secretion in bacteria and consists of two important elements: aglutamate/GABA antiporter GadC and a GAD enzyme, either GadA or GadB. Besides,most L. brevis strains encode a transcriptional regulator, gadR,immediately upstream of the gadCA operon which could positively regulateits expression. Our previous observations strikingly support that CRL 2013 isable to grow and produce high amounts of GABA in a fructose-supplemented MRSrich medium with conversion ratio about 99%. We have also proved that GABAsynthesis is impaired when this strain grows at the expense of pentoses as theonly carbon source, and this impairment was at least partially overcome withthe initial supplementation of ethanol to the MRS. Interestingly, CRL 2013 was unable to produce GABAafter incubation in an monosodium glutamate (MSG)- supplemented chemicallydefined medium (CDM). Thus, the aim of this study was to analyze the effect of carbohydrates(glucose-fructose and xylose) and nitrogen sources [casitone (C), vegetalpeptone (VP) and yeast extract (YE)] on the ability of L. brevis CRL2013 to grow and produce GABA. The addition of  nitrogen sources significantly stimulate thegrowth of CRL 2013 in both hexose and pentose- based CDM. Nevertheless, GABAsynthesis could only be triggered in the presence of YE, C or PV within a CDMwith hexoses as carbon sources. GABA production, detectable from 24 h offermentation, decreased in the order of YE> C> VP> CDM. In all cases,GABA synthesis was translated into a pH increase in the culture media.Conversely, with xylose as the only carbon source, the supplementation of the basalCDM with nitrogen sources did not allow GABA production and only low GABAlevels were detected after 72 h of incubation in the presence of YE. In anattempt to restore GABA production, ethanol (0,25 g/L) was added to thexylose-CDM . Interestingly, the addition of ethanol could restore GABAproduction in the presence of YE. In order to gain insight to thetranscriptional changes that could be associated to GABA production, theexpression of genes encoding key enzymes and regulatory elements within the GADsystem and of the ccpA gene, involved in catabolite repression, wasassessed through RT-qPCR using recA as the housekeeping gene. In thehexose- CDMs, the expression of gadR and gadA was significantlyincreased in the presence of C and YE in both exponential and stationary growthphases in comparison with the non-supplemented CDM. At 24 h, gadR was overexpressed112 and 90 times in the presence of YE and C respectively, when compared to thecontrol CDM. Moreover, the expression levels of gadA were 2800 and 1000times higher in the presence of YE and C respectively, when compared to thesame control. However, when transcriptionlevels were compared against the YE-supplemented xylose CDM (YE-xCDM) at 24 h, gadAand gadR were upregulated 1000 and 200 times respectively, in thepresence of hexoses. The addition of ethanol to the YE-xCDM was translated intosignificantly higher gadA and gadR transcript levels comparedwith the same medium without ethanol (190 and 275- fold change respectively).Furthermore, gadB did not experience significant changes in expression ccpAwas 50 times upregulated in the presence of glucose and fructose when comparedto the YE-xCDM, which was compatible with an active catabolite repression.Additionally, the expression profiles revealed that gadA and gadRwere upregulated and gadB downregulated in the stationary growth phase inthe presence of hexoses, whereas the opposite behavior was observed in thepresence of xylose. This pattern was reversed after the addition of ethanol toYE-xCDM. To our knowledge, this is the first report regarding the impairment ofthe GAD system in the presence of pentoses as sole carbon sources and therestoration of GABA production upon ethanol supplementation in a CDM contextwithin LAB. Taken together, these results contribute to the understanding ofthe regulation of the GAD system in LAB and highlight the potential use ofalternative carbon sources to produce high GABA levels.