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Noda Institute for Scientific Research GRANT
2012 Research Grant

Analysis of energy metabolism in acetic acid bacteria duringoxidative fermentation
Hiroyuki Arai
Department of Biotechnology, The University of Tokyo

Research aims

profiles with its parental wild-type strain.

Acetic acid bacteria are obligate aerobic α-proteobacteriawith the unique ability to incompletely oxidize various alcohols and sugars to form organic acids. The ability ofthese bacteria to incompletely oxidize ethanol to acetate has
been historically utilized for vinegar production. These organisms also cause spoilage of alcoholic beverages. Thus,acetic acid bacteria are industrially important. The incomplete oxidation reactions are catalyzed by membrane-boundpyrroloquinoline quinone (PQQ)-dependent dehydrogenases. Because the substrate specificities of these enzymes
are relatively low, acetic acid bacteria expressing these enzymes are used as living biocatalysts for the production ofmany useful materials such as l-sorbose and 6-amino-lsorbose, which are precursors of vitamin C and the anti-diabetic drug miglitol, respectively. Complete genome sequences of some acetic acid bacteria are now available andconsiderable biochemical research on these bacteria has
been published. However, there are relatively few reports
on molecular biological research such as gene regulation,or omics research such as transcriptome and metabolome
analyses, on these bacteria compared to other industrial microorganisms. In this work, we investigated the transcriptome profiles of acetic acid bacteria under various growth
conditions for a better understanding the specific regulatory
features of energy and central carbon metabolism, and todevelop novel applications for acetic acid bacteria.

Results

Methods
Transcriptome analysis
Acetobacter aceti NBRC14818, which is a wild-typestrain, and Acetobacter pasteurianus NBRC3283, which isa strain utilized for industrial vinegar production, were cultivated under various growth conditions. RNA samples
were isolated from cultures and analyzed in microarraystudies by using custom arrays designed and manufactured
by Roche-NimbleGen.
1.

Analysis of the role of the glyoxylate pathway
The aceA-glcB gene cluster encoding the glyoxylate
pathway enzymes isocitrate lyase and malate synthase of A.
aceti was knocked out by homologous recombination withthe tetracycline-resistance gene. The constructed mutant
strain was used to compare the growth and transcriptome
2.

Transcriptome profile of acetic acid bacteria
In both A. aceti and A. pasteurianus, the genes for the tricarboxylic acid (TCA) cycle enzymes were found to be significantly repressed when ethanol was present in the medium, even in the presence of glucose or acetate.1,2) Acetobacter species are able to produce proton motive force thatis used for ATP synthesis during incomplete oxidation ofethanol, since ethanol oxidation by PQQ-dependent alcohol
dehydrogenases is coupled with oxygen reduction by respiratory quinol oxidases via ubiquinone. Our results suggestthat acetic acid bacteria have a mechanism to repress the regeneration of NADH by the TCA cycle when ATP is sufficient to maintain viability.
In contrast to the TCA cycle genes, the expression levelsof genes for glycolysis were not significantly changed according to the growth conditions. In Escherichia coli, it is
known that metabolic flow through glycolysis is faster thanthat through the TCA cycle under aerobic conditions.
Therefore, excessive amounts of pyruvate and acetyl-CoA
build up, causing production of acetate from glucose. Thisphenomenon is called overflow metabolism. When the Acetobacter strains were grown in medium containing ethanol
and glucose, pyruvate and acetyl-CoA were expected to accumulate intracellularly due to the overflow metabolism of
glucose seen in E. coli (Fig. 1). Accumulation of acetylCoA in the cytoplasm might inhibit the utilization of acetate produced from ethanol in the periplasm. That wasprobably one of the major reasons for the temporal accumulation of acetate in the presence of ethanol. Some stressresponsive genes were upregulated during incomplete oxidation, probably because of production of toxic acetaldehyde as a result of overflow metabolism (Fig. 1). The
glyoxylate pathway genes (aceA-glcB) were significantly
upregulated in A. aceti cells when ethanol or acetate waspresent in the medium, suggesting that this pathway isimportant for acetate metabolism by A. aceti. Because
A. pasteurianus does not have these genes, presence or absence of the glyoxylate pathway might affect the phenotypic difference between the strains.
1.

2.

Role of the glyoxylate pathway
Published genome sequences and our preliminary analy-		31Fig. 1. Central carbon metabolic pathway of A. aceti. Dashed arrows indicate the glyoxylate pathway. Thick arrows indicate genes mediating reactions
that were significantly upregulated in the glyoxylate pathway-deficient mutant.

ses have shown that the acetic acid bacteria used for vinegar production lack the genes for the glyoxylate pathway.
This pathway consists of isocitrate lyase and malate synthase and functions as an anaplerotic shunt of the TCA
cycle to supply oxaloacetate. The glyoxylate pathway is required when carbon compounds that are metabolizedthrough acetyl-CoA as an obligate intermediate are utilized
as the only carbon sources. Because TCA cycle intermediates are used as precursors for synthesis of molecules suchas amino acids, nucleotides and other cofactors, the supplyof oxaloacetate is necessary for function of this pathway.
The aceA-glcB knockout mutant of A. aceti showed significantly slower growth in medium containing ethanol andglucose, although the glyoxylate pathway was not necessary under these conditions. Acetate productivity was notaffected by the mutation, but the acetate produced was retained in the medium for a longer period of time in the mutant culture, indicating that the lack of the glyoxylate pathway is advantageous for vinegar production. Transcriptome
analyses of the mutant strain suggested that the slower
growth and the delay of acetate overoxidation were due tothe significant repression of the TCA cycle enzymes andaccumulation of acetyl-CoA in the cytoplasm (Fig. 1).3)
Conclusion
Transcriptome analyses of Acetobacter strains showedthat the presence of ethanol in the medium significantly
downregulates the expression of the TCA cycle enzymes,

resulting in the repression of acetyl-CoA-consumption. Accumulation of acetyl-CoA in the cytoplasm might be themajor reason that acetate produced by incomplete oxidationof ethanol in the periplasm is not metabolized. Acetic acidbacteria are able to produce ATP by the incomplete oxidation of alcohols and sugars, because ubiquinols reduced by
PQQ-dependent dehydrogenases are reoxidized by terminal
oxidases using molecular oxygen, generating proton motiveforce. Our results indicate that incomplete oxidation byacetic acid bacteria is the result of preferential utilization
PQQ-dependent dehydrogenases substrates over the endogenous respiratory substrate NADH for energy generation.
Acetic acid bacteria might have a specific mechanism to repress the regeneration of NADH by the TCA cycle for thispurpose. Inactivation of the glyoxylate pathway, which also
reduced metabolic flow of the TCA cycle, was advantageous for acetate production, which is consistent with thefact that strains utilized for vinegar production tend to lack
this pathway.
References
1)

Sakurai, K., Arai, H., Ishii, M., and Igarashi, Y. (2011) Transcriptome response to different carbon sources in Acetobacter aceti. Microbiology 157: 899–910.
2) Sakurai, K., Arai, H., Ishii, M., and Igarashi, Y. (2012) Changes in
the gene expression profile of Acetobacter aceti during growth onethanol. J. Biosci. Bioeng. 113: 343–348.
3) Sakurai, K., Yamazaki, S., Ishii, M., Igarashi, Y., and Arai, H. (2013)
Role of the glyoxylate pathway in acetic acid production by Acetobacter aceti. J. Biosci. Bioeng. 115: 32–36.