Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Sep 6:8:1713.
doi: 10.3389/fmicb.2017.01713. eCollection 2017.

Improving Acetic Acid Production by Over-Expressing PQQ-ADH in Acetobacter pasteurianus

Xuefeng Wu  1   2 , Hongli Yao  1 , Lili Cao  1   2 , Zhi Zheng  1   2 , Xiaoju Chen  3 , Min Zhang  1 , Zhaojun Wei  1 , Jieshun Cheng  1   2 , Shaotong Jiang  1   2 , Lijun Pan  1   2 , Xingjiang Li  1   2
Affiliations

Improving Acetic Acid Production by Over-Expressing PQQ-ADH in Acetobacter pasteurianus

Xuefeng Wu et al. Front Microbiol. .

Abstract

Pyrroquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) is a key enzyme in the ethanol oxidase respiratory chain of acetic acid bacteria (AAB). To investigate the effect of PQQ-ADH on acetic acid production by Acetobacter pasteurianus JST-S, subunits I (adhA) and II (adhB) of PQQ-ADH were over-expressed, the fermentation parameters and the metabolic flux analysis were compared in the engineered strain and the original one. The acetic acid production was improved by the engineered strain (61.42 g L-1) while the residual ethanol content (4.18 g L-1) was decreased. Analysis of 2D maps indicated that 19 proteins were differently expressed between the two strains; of these, 17 were identified and analyzed by mass spectrometry and two-dimensional gel electrophoresis. With further investigation of metabolic flux analysis (MFA) of the pathway from ethanol and glucose, the results reveal that over-expression of PQQ-ADH is an effective way to improve the ethanol oxidation respiratory chain pathway and these can offer theoretical references for potential mechanism of metabolic regulation in AAB and researches with its acetic acid resistance.

Keywords: Acetobacter pasteurianus JST-S; Pyrroquinoline quinone-dependent alcohol dehydrogenase; metabolic flux analysis; two-dimensional gel electrophoresis.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Construction of recombinant plasmid PBBR-adhA-adhB.
Figure 2
Figure 2
PCR amplification products shown by agarose gel electrophoresis. Lanes M, marker (bp);1, Padh; 2, adhA; 3, adhB.
Figure 3
Figure 3
Verification of recombinant plasmids by restriction enzyme digeston. Lanes: M, 8-kbp marker, 1, PBBR-adhA digested with Bam HI and Spe I; 2, PBBR-adhB digested with Spe I and Xba I; and 3, PBBR-adhA-adhB digested with Bam H I.
Figure 4
Figure 4
SDS-PAGE of recombinant protiens over-expressed in the genetically engineered strain Lanes: M, molecular weight marker (KDa); 1 and 2, original strain; 3, engineered strain.
Figure 5
Figure 5
Comparison of fermentation parameters in the original and engineered strains. ADH activity of the original strain(□) and engineered strain(■) were compared under the different content of ethanol in (A). In (B), white triangle and black triangle represent total acid content of the original strain and engineered strain, respectively. Similarly, white circle and black circle represent ethanol content of the original strain and engineered strain, respectively. Error bars show standard deviations of three replicated measurements.
Figure 6
Figure 6
Comparison of 2D-PAGE patterns between original engineered strains. (A) Primary 2D-PAGE maps. (B) 2D-PAGE maps analyzed using PD Quest 8.0 software. Protein spots whose expression level changed more than 50% were identified as differentially expressed proteins.
Figure 7
Figure 7
The metabolic network of A. pasteurianus JST-S Glc, glucose; G6P, glucose-6-phhospahte; F6P, fructose-6-phosphate; Ru5P, ribulose 5-posphate; G3P, glyceraldehydes-3-phosphate; PEP, poshonolpyruvate; Pyr, pyruvate; Pyr, pyruvate; AC.H., Acetaldehyde; OAA, oxaloacetate; Acetyl-CoA, acetyl coenzyme A; CoA, Coenzyme A; Lac, Lactate; Ace, Acetate; EtOH, ethanol; Cit, citrate; Isoc, Isocitrate; α-Ket, α-ketoglutaric acid; Suc, succinic acid; Fum, fumarate; Mal, Malate; BM, biomass.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Andrés-Barrao C., Saad M. M., Chappuis M. L., Boffa M., Perret X., Pérez R. O., et al. . (2012). Proteome analysis of Acetobacter pasteurianus, during acetic acid fermentation. J. Proteomics 75, 1701–1717. 10.1016/j.jprot.2011.11.027 - DOI - PubMed
    1. Antoniewicz M. R. (2015). Methods and advances in metabolic flux analysis: a mini-review. J. Ind. Microbiol. Biotechnol. 42, 317–325. 10.1007/s10295-015-1585-x - DOI - PubMed
    1. Chen Y., Bai Y., Li D., Wang C., Xu N., Wu S., et al. . (2016). Correlation between ethanol resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria. Eur. Food. Res. Technol. 242, 837–847. 10.1007/s00217-015-2589-5 - DOI
    1. Chinnawirotpisan P., Theeragool G., Limtong S., Toyama H., Adachi O. O., Matsushita K. (2003). Quinoprotein alcohol dehydrogenase is involved in catabolic acetate production, while NAD-dependent alcohol dehydrogenase in ethanol assimilation in Acetobacter pasteurianusSKU1108. J. Biosci. Bioeng. 96, 564–571. 10.1016/S1389-1723(04)70150-4 - DOI - PubMed
    1. Dandekar T., Fieselmann A., Majeed S., Ahmed Z. (2014). Software applications toward quantitative metabolic flux analysis and modeling. Brief. Bionform. 15, 91–107. 10.1093/bib/bbs065 - DOI - PubMed