Phenotypic and Genotypic Analysis of Amino Acid Auxotrophy in Lactobacillus helveticus CNRZ 32

Abstract

The conversion of amino acids into volatile and nonvolatile compounds by lactic acid bacteria in cheese is thought to represent the rate-limiting step in the development of mature flavor and aroma. Because amino acid breakdown by microbes often entails the reversible action of enzymes involved in biosynthetic pathways, our group investigated the genetics of amino acid biosynthesis in Lactobacillus helveticus CNRZ 32, a commercial cheese flavor adjunct that reduces bitterness and intensifies flavor notes. Most lactic acid bacteria are auxotrophic for several amino acids, and L. helveticus CNRZ 32 requires 14 amino acids. The reconstruction of amino acid biosynthetic pathways from a draft-quality genome sequence for L. helveticus CNRZ 32 revealed that amino acid auxotrophy in this species was due primarily to gene absence rather than point mutations, insertions, or small deletions, with good agreement between gene content and phenotypic amino acid requirements. One exception involved the phenotypic requirement for Asp (or Asn), which genome predictions suggested could be alleviated by citrate catabolism. This prediction was confirmed by the growth of L. helveticus CNRZ 32 after the addition of citrate to a chemically defined medium that lacked Asp and Asn. Genome analysis also predicted that L. helveticus CNRZ 32 possessed ornithine decarboxylase activity and would therefore catalyze the conversion of ornithine to putrescine, a volatile biogenic amine. However, experiments to confirm ornithine decarboxylase activity in L. helveticus CNRZ 32 by the use of several methods were unsuccessful, which indicated that this bacterium likely does not contribute to putrescine production in cheese.Flavor development in Cheddar and other bacterium-ripened cheeses is a dynamic and complex biochemical process that requires lactic acid bacteria (LAB) and enzymes. The LAB that contribute to this process include deliberately added starter cultures and adjunct cultures as well as nonstarter LAB that enter the cheese through the milk or the processing environment. Collectively, these microbes influence flavor development through several basic mechanisms that include lactose fermentation, conversion of milk proteins (primarily caseins) into peptides and free amino acids, catabolism of amino acids into volatile aroma compounds, lipase/esterase activity, and citrate catabolism (6). In particular, the conversion of amino acids into volatile and nonvolatile compounds by LAB in cheese is thought to represent the rate-limiting step in the development of mature flavor and aroma (28). The conversion of amino acids into volatile cheese flavor compounds in cheese may be catalyzed by starter, adjunct, and nonstarter cultures of LAB and may also occur via biochemical interactions between different bacteria (13). The basis for culture interactions is not fully understood, but LAB are typically auxotrophic for several amino acids, and amino acid breakdown by LAB often involves the reversible action of enzymes involved in anabolic pathways (28). Thus...

Evidence of Two Functionally Distinct Ornithine Decarboxylation Systems in Lactic Acid Bacteria

“…In the industrial cheese starter Lactobacillus helveticus CNRZ, a putative system of the same type was detected (accession numbers YP001577954 and YP001577955 on Fig. 2A and B, respectively), but the strain is not a putrescine producer (8). The data provided here suggest that these decarboxylases, due to their low catalytic efficiencies and the absence of a system for putrescine release, cannot account for the production of milligram amounts of putrescine.…”

Fermentation dynamics of Lactobacillus helveticus H9 revealed by ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry

“…Free amino acids are usually available at low concentrations in meju – a fermented soya bean paste in Korea (Namgung et al ., ) – or in ultrafiltration (UF) milk retentate (Cretenet et al ., ). Lactobacillus helveticus is nutritionally fastidious, and its growth requires extensive supplementation with exogenous nutrients including amino acids such as aspartic acid, tyrosine, glutamic acid, isoleucine, leucine, histidine, methionine, threonine and valine (Christiansen et al ., ). During the first few hours of fermentation, L .…”

“…Free amino acids are usually available at low concentrations in mejua fermented soya bean paste in Korea (Namgung et al, 2010)or in ultrafiltration (UF) milk retentate (Cretenet et al, 2011). Lactobacillus helveticus is nutritionally fastidious, and its growth requires extensive supplementation with exogenous nutrients including amino acids such as aspartic acid, tyrosine, glutamic acid, isoleucine, leucine, histidine, methionine, threonine and valine (Christiansen et al, 2008). During the first few hours of fermentation, L. helveticus H9 mostly likely used the free amino acids present in milk to sustain its growth (Christensen & Steele, 2003), which explains the decrease in relative quantities of leucine and valine observed from 0 to 8 h ( Table 2).…”

Translation-dependent bioassay for amino acid quantification using auxotrophic microbes as biocatalysts of protein synthesis

“…Among these analytical methods, amino acid bioassays have been popular over the past 70 years because of their low cost, easy manipulation, coverage of wide range of amino acids, and applicability to high-throughput analysis (Cardinal and Hedrick 1948;Steele et al 1949;Tamura et al 1952). These assays utilize lactic acid bacteria, many of which are auxotrophic to specific amino acids (Christiansen et al 2008), mixing them with an analyte and synthetic medium that contains all nutrients except the target amino acid. During a 24-72-h incubation period, growth of the auxotrophic bacterium depends on the amount of the target amino acid in the analyte.…”