PubMed · 6755183
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R M Sarmiento, J Burgos. 1982. Diacetyl reductase.. https://doi.org/10.1016/s0076-6879(82)89089-7
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An alpha,beta-dicarbonyl reductase activity was purified from Saccharomyces cerevisiae and identified as the cytosolic enzyme D-Arabinose dehydrogenase (ARA1) by MALDI-TOF/TOF. Size exclusion chromatography analysis of recombinant Ara1p revealed that this protein formed a homodimer. Ara1p catalyzed the reduction of the reactive alpha,beta-dicarbonyl compounds methylglyoxal, diacetyl, and pentanedione in a NADPH dependant manner. Ara1p had apparent Km values of approximately 14 mM, 7 mM and 4 mM for methylglyoxal, diacetyl and pentanedione respectively, with corresponding turnover rates of 4.4, 6.9 and 5.9 s(-1) at pH 7.0. pH profiling showed that Ara1p had a pH optimum of 4.5 for the diacetyl reduction reaction. Ara1p also catalyzed the NADP+ dependant oxidation of acetoin; however this back reaction only occurred at alkaline pH values. That Ara1p was important for degradation of alpha,beta-dicarbonyl substrates was further supported by the observation that ara1-Delta knockout yeast mutants exhibited a decreased growth rate phenotype in media containing diacetyl.
AIMS: A metabolic pathway for L-2,3-butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce L-BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and L-2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb. METHODS AND RESULTS: The meso-BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to L-acetoin (AC) and the L-BDH of Brevibacterium saccharolyticum was used to reduce L-AC to L-BD. The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb). The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production. CONCLUSIONS: L-BD (2200 mg l(-1)) was generated from 3000 mg l(-1) added of DA, which corresponded to a 73% conversion rate. Meso-BD as a by-product was mixed by 2% at most. SIGNIFICANCE AND IMPACT OF THE STUDY: An enzyme system for converting DA to L-BD was constructed with a view to using DA-producing bacteria in the future.
A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously. Downstream of the butA gene of L. pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L. pseudomesenteroides. Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L. pseudomesenteroides.