Evaluation of ITS PCR and RFLP for Differentiation and Identification of Brewing Yeast and Brewery 'Wild' Yeast Contaminants.

A reference library of ITS PCR/RFLP profiles was collated and augmented to evaluate its potential for routine identification of domestic brewing yeast and known 'wild' yeast contaminants associated with wort, beer and brewing processes. This library contains information on band sizes generated by restriction digestion of the ribosomal RNA-encoding DNA (rDNA) internal transcribed spacer (ITS) region consisting of the 5.8 rRNA gene and two flanking regions (ITS1 and ITS2) with the endonucleases CfoI, HaeIII, HinfI and includes strains from 39 non-Saccharomyces yeast species as well as for brewing and non-brewing strains of Saccharomyces. The efficacy of the technique was assessed by isolation of 59 wild yeasts from industrial fermentation vessels and conditioning tanks and by matching their ITS amplicon sizes and RFLP profiles with those of the constructed library. Five separate, non-introduced yeast taxa were putatively identified. These included Pichia species, which were associated with conditioning tanks and Saccharomyces species isolated from fermentation vessels. Strains of the lager yeast S. pastorianus could be reliably identified as belonging to either the Saaz or Frohberg hybrid group by restriction digestion of the ITS amplicon with the enzyme HaeIII. Frohberg group strains could be further sub-grouped depending on restriction profiles generated with HinfI.

Candida tropicalis Etr1p and Saccharomyces cerevisiae Ybr026p (Mrf1'p), 2-enoyl thioester reductases essential for mitochondrial respiratory competence.

We report here on the identification and characterization of novel 2-enoyl thioester reductases of fatty acid metabolism, Etr1p from Candida tropicalis and its homolog Ybr026p (Mrf1'p) from Saccharomyces cerevisiae. Overexpression of these proteins in S. cerevisiae led to the development of significantly enlarged mitochondria, whereas deletion of the S. cerevisiae YBR026c gene resulted in rudimentary mitochondria with decreased contents of cytochromes and a respiration-deficient phenotype. Immunolocalization and in vivo targeting experiments showed these proteins to be predominantly mitochondrial. Mitochondrial targeting was essential for complementation of the mutant phenotype, since targeting of the reductases to other subcellular locations failed to reestablish respiratory growth. The mutant phenotype was also complemented by a mitochondrially targeted FabI protein from Escherichia coli. FabI represents a nonhomologous 2-enoyl-acyl carrier protein reductase that participates in the last step of the type II fatty acid synthesis. This indicated that 2-enoyl thioester reductase activity was critical for the mitochondrial function. We conclude that Etr1p and Ybr026p are novel 2-enoyl thioester reductases required for respiration and the maintenance of the mitochondrial compartment, putatively acting in mitochondrial synthesis of fatty acids.

Molecular cloning and characterization of the human mitochondrial 2,4-dienoyl-CoA reductase gene (DECR).

2,4-Dienoyl-CoA reductase (EC 1.3.1.34) is an auxiliary enzyme of beta-oxidation, and it participates in the metabolism of unsaturated fatty enoyl-CoA esters having double bonds in both even- and odd-numbered positions. In this article we describe the molecular cloning of the human gene for the 120-kDa isoform of mitochondrial 2,4-dienoyl-CoA reductase (DECR). The gene is approximately 30 kb and comprises 10 exons varying in size from 79 to 203 bp and 9 introns whose sizes vary from 95 bp to about 10 kb. The 5' UTR and 3' UTR are included in exons 1 and 10, respectively. The promoter region contains putative binding sites for several transcription factors, e.g., Sp1, AP-2, AP-4, and C/EBP, but no TATA box was found. Primer extension analysis and 5' RACE-PCR revealed variability in the length of the 5'-UTR, the longest being 72 bp. Through the use of FISH analysis on metaphase chromosomes with a genomic fragment of 2,4-dienoyl-CoA reductase, the gene was assigned to the chromosomal band 8q21.3.

Peroxisomal beta-oxidation and polyunsaturated fatty acids.

Peroxisomes are capable of oxidizing a variety of substrates including (poly)unsaturated enoyl-CoA esters. The beta-oxidation of unsaturated enoyl-CoA esters in peroxisomes, and also in mitochondria, is not just chain-shortening but also involves the metabolizing of pre-existing carbon-to-carbon double bonds. In addition to the enzymes of the beta-oxidation spiral itself, this metabolism requires the participation of auxiliary enzymes: delta 3, delta 2-enoyl-CoA isomerase; 2,4-dienoyl-CoA reductase; 2-enoyl-CoA hydratase 2 or 3-hydroxyacyl-CoA epimerase; and delta 3,5 delta 2,4-dienoyl-CoA isomerase. Many of these enzymes are present as isoforms, and can be found located in multiple subcellular compartments, for example, peroxisomes, mitochondria or the endoplasmic reticulum, while some of the activities are integral parts of multifunctional enzymes of beta-oxidation systems.

Isolation and characterization of cDNA for human 120 kDa mitochondrial 2,4-dienoyl-coenzyme A reductase.

2,4-Dienoyl-CoA reductase (EC 1.3.1.34) participates in beta-oxidation of (poly)unsaturated enoyl-CoAs and it appears in mammalian mitochondria as two isoforms with molecular masses of 120 and 60 kDa [Hakkola and Hiltunen (1993) Eur. J. Biochem. 215, 199-204]. The 120 kDa isomer is a homotetrameric enzyme, and here we report cDNA cloning of its subunit from human. cDNA clones were isolated by reverse transcriptase-PCR from a fibrosarcoma cell line and by screening from a human liver lambda gt11 cDNA library. The 1128 bp clone contained an open reading frame of 1008 bp encoding a polypeptide of 335 amino acid residues with a predicted molecular mass of 36066 Da. This polypeptide represents the immature monomer of the 120 kDa enzyme, and it contains a predicted N-terminal mitochondrial targeting signal. The amino acid (nucleotide) sequence of human 2,4-dienoyl-CoA reductase shows 82.7% (81.7%) similarity (identity) to the corresponding sequence from the rat. Northern-blot analysis gave a single mRNA species of 1.2 kb in several human tissues, the amounts present in the tissues tested ranking as follows: heart approximately liver approximately pancreas > kidney >> skeletal muscle approximately lung. Immunoblotting of human and rat liver samples with an antibody to the subunit of the rat 120 kDa isoform indicates that the mature human enzyme is larger than its counterpart in the rat. The comparison of amino acid sequences for rat and human enzymes proposes that the difference in the size is 10 amino acid residues. The results show that the rat and human reductases are similar in many characteristics and that the reductase is expressed in human tissues capable of beta-oxidation of fatty acids.

Peroxisomal beta-oxidation of polyunsaturated fatty acids.

Peroxisomes have been shown to play an important role in the oxidative degradation of (poly)unsaturated fatty acids, and contain the enzyme activities needed for the metabolism of double bonds of unsaturated fatty acids in connection with this physiological function. Our understanding of the metabolic pathways and enzyme activities involved in the degradation of unsaturated acyl-CoAs has undergone a re-evaluation recently, and though many open questions still remain significant progress has been made, especially concerning the reactions metabolizing double bonds. The enzyme activities to be discussed here are 2,4-dienoyl-CoA reductase; 3/2-enoyl-CoA isomerase; 2-enoyl-CoA hydratase 2; 5-enoyl-CoA reductase and 3,5/2,4-dienoyl-CoA isomerase. Some of these activities are integral parts of the multifunctional proteins of beta-oxidation systems, which must also be taken into account in this context.