Catabolism Of Acetoin Research Articles

Production of acetoin and its derivative tetramethylpyrazine from okara hydrolysate with Bacillus subtilis

Okara, a renewable biomass resource, is a promising fermentative raw material for the bio-production of value-added chemicals due to its abundance and low-costs. we developed a process for the enzymatic hydrolysis of okara, and then engineered Bacillus subtilis to utilize mixed sugars to produce acetoin in okara hydrolysis without the addition of a supplemental nitrogen source. Okara was initially hydrolyzed with cellulase, β-glucosidase, and pectinase to obtain okara hydrolysate containing mixed sugars (32.78 ± 0.23 g/L glucose, 1.43 ± 0.064 g/L arabinose, 7.74 ± 0.11 g/L galactose) and amino acids. In this study, Bacillus subtilis 168 was used as the acetoin-producing strain, and the key genes bdhA and acoA of the acetoin catabolism pathway were knocked out to improve the fermentation yield of acetoin. In order to utilize the galactose in the hydrolysate, the recombinant strain BS03 (Bacillus subtilis168∆bdhA∆acoA) was used to overexpress the arabinose transporter-encoding gene (araE) drive heterologous expression of the Leloir pathway gene (galKTE). The corn dry powder concentration was optimized to 29 g/L in the reducing sugar okara hydrolysate. The results show that the recombinant bacterium BS03 could still synthesize 11.79 g/L acetoin without using corn dry powder as a nitrogen source. Finally, using okara enzymatic hydrolysate as the carbon and nitrogen source, 11.11 g/L and 29.7 g/L acetoin were obtained by batch fermentation and fed-batch fermentation, respectively, which was further converted to 5.33 g/L and 13.37 g/L tetramethylpyrazine (TTMP) by reaction with an ammonium salt.

Mechanism Underlying the Role of LuxR Family Transcriptional Regulator abaR in Biofilm Formation by Acinetobacter baumannii.

Our study attempted to explore the mechanism underlying the role of LuxR family transcriptional regulator abaR in biofilm formation by Acinetobacter baumannii. The abaR gene was knocked out in ATCC 17978 strain using homologous recombination method. The growth curve and biofilm formation were measured in the wild type and abaR gene knockdown strains. Transcriptome sequencing was performed in the wild type and abaR gene knockdown strains following 8 h of culture. The growth curve in the abaR gene knockdown strain was similar to that of the wild-type strain. Biofilm formation significantly declined in the abaR gene knockdown strain at 8 and 48 h after culture. A total of 137 differentially expressed genes (DEGs) were obtained including 20 downregulated DEGs and 117 upregulated DEGs. Genes with differential expression were closely related to viral procapsid maturation (GO:0046797), acetoin catabolism (GO:0045150), carbon metabolism (ko01200), and the glycolysis/gluconeogenesis (ko00010)-related pathways. The results of the eight verified expression DEGs were consistent with the results predicted by bioinformatics. AbaR gene knockdown significantly affected biofilm formation by A. baumannii ATCC 17978 strain. The glycolysis/gluconeogenesis pathways were significantly dysregulated and induced by abaR gene knockdown in A. baumannii.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00284-021-02654-y.

Trophic cooperation promotes bacterial survival of Staphylococcus aureus and Pseudomonas aeruginosa.

In the context of infection, Pseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated, particularly in cystic fibrosis (CF) patients. Within lungs, the two pathogens exhibit a range of competitive and coexisting interactions. In the present study, we explored the impact of S. aureus on the physiology of P. aeruginosa in the context of coexistence. Transcriptomic analyses showed that S. aureus significantly and specifically affects the expression of numerous genes involved in P. aeruginosa carbon and amino acid metabolism. In particular, 65% of the strains presented considerable overexpression of the genes involved in the acetoin catabolic (aco) pathway. We demonstrated that acetoin is (i) produced by clinical S. aureus strains, (ii) detected in sputa from CF patients and (iii) involved in P. aeruginosa's aco system induction. Furthermore, acetoin is catabolized by P. aeruginosa, a metabolic process that improves the survival of both pathogens by providing a new carbon source for P. aeruginosa and avoiding the toxic accumulation of acetoin on S. aureus. Due to its beneficial effects on both bacteria, acetoin catabolism could testify to the establishment of trophic cooperation between S. aureus and P. aeruginosa in the CF lung environment, thus promoting their persistence.

Quorum and Light Signals Modulate Acetoin/Butanediol Catabolism in Acinetobacter spp.

Acinetobacter spp. are found in all environments on Earth due to their extraordinary capacity to survive in the presence of physical and chemical stressors. In this study, we analyzed global gene expression in airborne Acinetobacter sp. strain 5-2Ac02 isolated from hospital environment in response to quorum network modulators and found that they induced the expression of genes of the acetoin/butanediol catabolism, volatile compounds shown to mediate interkingdom interactions. Interestingly, the acoN gene, annotated as a putative transcriptional regulator, was truncated in the downstream regulatory region of the induced acetoin/butanediol cluster in Acinetobacter sp. strain 5-2Ac02, and its functioning as a negative regulator of this cluster integrating quorum signals was confirmed in Acinetobacter baumannii ATCC 17978. Moreover, we show that the acetoin catabolism is also induced by light and provide insights into the light transduction mechanism by showing that the photoreceptor BlsA interacts with and antagonizes the functioning of AcoN in A. baumannii, integrating also a temperature signal. The data support a model in which BlsA interacts with and likely sequesters AcoN at this condition, relieving acetoin catabolic genes from repression, and leading to better growth under blue light. This photoregulation depends on temperature, occurring at 23°C but not at 30°C. BlsA is thus a dual regulator, modulating different transcriptional regulators in the dark but also under blue light, representing thus a novel concept. The overall data show that quorum modulators as well as light regulate the acetoin catabolic cluster, providing a better understanding of environmental as well as clinical bacteria.

Functional Study of AcoX, an Unknown Protein Involved in Acetoin Catabolism

The function of AcoX, encoded by acoX existing in aco clusters which are responsible for acetoin catabolism in some microbes from the Kingdoms of Bacteria and Archaea, remained unknown till today. Although some AcoXs were annotated as ATP-NAD kinases in bioinformatics databases, AcoXs were found to form an independent family during evolution in this study. By bioinformatic analysis and molecular comparison, AcoX was postulated to have the catalytic function of acetoin phosphorylation. The AcoX from Pseudomonas putida KT2440 was expressed in Escherichia coli using the pET28a vector. The His-tagged AcoX was purified and digested by thrombin. The deconvolution of circular dichroism spectra using K2d algorithm indicates a >28% and >14% content for alpha and beta structures in AcoX, respectively. But unfortunately, not only the activities of NAD kinase, pyruvate kinase, and methylglyoxal synthase, but also the postulated enzymatic activity of acetoin kinase was not detected under all the conditions studied.

The ATP-binding motif in AcoK is required for regulation of acetoin catabolism in Klebsiella pneumoniae CG43

Many bacterial species utilize acetoin as a carbon source. In Klebsiella pneumoniae, the utilization of acetoin is catalyzed by an acetoin dehydrogenase complex encoded by the acoABCD operon, which is positively regulated in the presence of acetoin by the transcriptional factor AcoK. AcoK contains a LuxR type DNA-binding domain at the C-terminal region and putative Walker A and B nucleotide-binding motifs in the N-terminal region. The comprehensive deletion and mutation study performed here shows that mutations in the putative Walker A motif result in a significant reduction of ATP hydrolysis and trans-activation by AcoK of acoABCD expression, presumably due to a loss of ATP-binding ability. AcoK was shown to bind specifically to nucleotides -66 to -36 of the acoABCD promoter, though the DNA-binding ability was not affected by the Walker A motif mutation. Thus, this study provides an additional example of how a member of the signal transduction ATPases with numerous domains family activates its target gene expression.

An acetoin-regulated expression system of Bacillussubtilis

An expression system, which is based on the promoter of the acoABCL operon of Bacillus subtilis was developed and characterized. The acoABCL operon codes for the acetoin dehydrogenase complex, which is the major enzyme system responsible for the catabolism of acetoin in B. subtilis. Besides weak organic acids, the neutral overflow metabolite acetoin is metabolized by the cells in the early stationary phase. Transcription of reporter gene fusions with the acoA promoter of this operon is strongly repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The co-expression of an additional copy of the regulator gene acoR led to more than twofold higher activity of the acoA promoter. It is demonstrated that the induction of this promoter in growing cells with acetoin is possible with non-phosphotransferase system sugars as carbon and energy source and in a ccpA mutant background. Moreover, it could be shown that the activity of the acoA-directed expression system correlates with the level of acetoin in the medium. During glucose limitation, the utilization of the alternative energy source acetoin keeps the protein synthesis machinery of B. subtilis cells active and thus allows for a long lasting acoA-controlled expression of recombinant genes.

Three distinct phases of isoprene formation during growth and sporulation of Bacillus subtilis.

During growth on a glucose-tryptone medium, Bacillus subtilis 6051 (Marburg strain) exhibited three phases of isoprene (2-methyl-1, 3-butadiene) formation, corresponding to (i) glucose catabolism and secretion of acetoin, (ii) catabolism of acetoin, and (iii) the early stages of sporulation. These results establish an experimental system for studying the biological role of isoprene formation.

Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus.

The gene locus acoE, which is involved in the utilization of acetoin in Alcaligenes eutrophus, was identified as the structural gene of an acetyl coenzyme A synthetase (acetate:coenzyme A ligase [AMP forming]; EC 6.2.1.1). This gene was localized on a 3.8-kbp SmaI-EcoRI subfragment of an 8.1-kbp EcoRI restriction fragment (fragment E) that was cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The 1,983 bp acoE gene encoded a protein with a relative molecular weight of 72,519, and it was preceded by a putative Shine-Dalgarno sequence. A comparison analysis of the amino acid sequence deduced from acoE revealed a high degree of homology to primary structures of acetyl coenzyme A synthetases from other sources (amounting to up to 50.5% identical amino acids). Tn5 insertions in two transposon-induced mutants of A. eutrophus, that were impaired in the catabolism of acetoin were mapped 481 and 1,159 bp downstream from the translational start codon of acoE. The expression of acoE in Escherichia coli led to the formation of an acyl coenzyme A synthetase that accepted acetate as the preferred substrate (100% relative activity) but also reacted with propionate (46%) and hydroxypropionate (87%); fatty acids consisting of four or more carbon atoms were not accepted. In addition, evidence for the presence of a second acyl coenzyme A synthetase was obtained; this enzyme exhibited a different substrate specificity. The latter enzyme is obviously required for the activation of propionate, e.g., during the formation of the storage compound poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) when propionate is provided as the sole carbon source. An analysis of mutants provided evidence that the expression of the uptake protein for propionate depends on the presence of alternate sigma factor sigma 54.

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

The N-terminal amino acid sequence of purified acetaldehyde dehydrogenase II (AcDH-II) from ethanol-grown cells of Alcaligenes eutrophus was determined. By using oligonucleotides deduced from this sequence the structural gene for AcDH-II, which was referred to as acoD, was localized on a 7.2-kbp EcoRI restriction fragment (fragment D), which has been cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). A 2.8-kbp PstI subfragment of D, which harbored acoD, was sequenced. It revealed an open reading frame of 1,518 bp, encoding a protein with a relative molecular weight of 54,819. The insertions of Tn5::mob of two transposon-induced mutants of A. eutrophus, which were impaired in the catabolism of acetoin, were mapped 483 or 1,359 bp downstream from the translational start codon of acoD. The structural gene was preceded by a putative Shine-Dalgarno sequence. The transcriptional start site 57 bp upstream of acoD was identified and was preceded by a sequence which exhibited a striking homology to the enterobacterial sigma 54-dependent promoter consensus sequence. This was in accordance with the observation that the expression of acoD and of other acetoin-catabolic genes depended on the presence of an intact rpoN-like gene. Alignments of the amino acid sequence deduced from acoD with the primary structures of aldehyde dehydrogenases from other sources revealed high degrees of homology, amounting to 46.5% identical amino acids.

Biochemical and genetic analyses of acetoin catabolism in Alcaligenes eutrophus

In genetic studies on the catabolism of acetoin in Alcaligenes eutrophus, we used Tn5::mob-induced mutants which were impaired in the utilization of acetoin as the sole carbon source for growth. The transposon-harboring EcoRI restriction fragments from 17 acetoin-negative and slow-growing mutants (class 2a) and from six pleiotropic mutants of A. eutorphus, which were acetoin-negative and did not grow chemolithoautotrophically (class 2b), were cloned from pHC79 gene banks. The insertions of Tn5 were mapped on four different chromosomal EcoRI restriction fragments (A, C, D, and E) in class 2a mutants. The native DNA fragments were cloned from a lambda L47 or from a cosmid gene bank. Evidence is provided that fragments A (21 kilobase pairs [kb]) and C (7.7 kb) are closely linked in the genome; the insertions of Tn5 covered a region of approximately 5 kb. Physiological experiments revealed that this region encodes for acetoin:dichlorophenol-indophenol oxidoreductase, a fast-migrating protein, and probably for one additional protein that is as yet unknown. In mutants which were not completely impaired in growth on acetoin but which grew much slower and after a prolonged lag phase, fragments D (7.2 kb) and E (8.1 kb) were inactivated by insertion of Tn5::mob. No structural gene could be assigned to the D or E fragments. In class 2b mutants, insertions of Tn5 were mapped on fragment B (11.3 kb). This fragment complemented pleiotropic hno mutants in trans; these mutants were impaired in the formation of a rpoN-like protein. The expression of the gene cluster on fragments A and C seemed to be rpoN dependent.

Utilization of methylacetoin by the strict anaerobe Pelobacter carbinolicus and consequences for the catabolism of acetoin

Utilization of methylacetoin by the strict anaerobe Pelobacter carbinolicus and consequences for the catabolism of acetoin

Utilization of methylacetoin by the strict anaerobePelobacter carbinolicusand consequences for the catabolism of acetoin

Abstract Pelobacter carbinolicus strain GraBd1 fermented methylacetoin, which is a good carbon source for growth ( μ = 0.16 h −1 ) of this strict anaerobic bacterium, to acetone, acetate and ethanol (main products), acetoin, 2,3-butanediol and methylbutanediol (minor products). During growth on 2,3-butanediol, acetoin and methyl-acetoin the formation of a protein exhibiting acetoin: DCPIP oxidoreductase activity is induced. This enzyme amounts to a substantial portion of the soluble proteins. In vitro, it cleaves acetoin into acetate and acetaldehyde but reacts also with diacetyl or methylacetoin. We discussed four different models for the degradation of acetoin in the cells and came to the conclusion that in vivo the oxidative-thiolytic acetoin cleavage model is most probably realized in P. carbinolicus .