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Cited by 32Microbial Metabolic Engineering and BioproductionMicrobial metabolism and enzyme functionBiochemical and biochemical processes

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

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Wu, Xuefeng, Yao, Hongli, Cao, Lili, Zheng, Zhi, Chen, Xiaoju, Zhang, Min, Wei, Zhaojun, Cheng, Jieshun, Jiang, Shaotong, Pan, Lijun, Li, Xingjiang·2017·Frontiers in Microbiology
DOI 10.3389/fmicb.2017.01713PMC PMC5592214
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Abstract

Pyrroquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) is a key enzyme in the ethanol oxidase respiratory chain of acetic acid bacteria. 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 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 acetic acid bacteria and researches with its acetic acid resistance.

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Crossref · 10.3389/fmicb.2017.01713

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Figure 1 Construction of recombinant plasmid PBBR-adhA-adhB.
Figure 1 Construction of recombinant plasmid PBBR-adhA-adhB.CC BY · PubMed Central
Figure 2 PCR amplification products shown by agarose gel electrophoresis. Lanes M, marker (bp);1, Padh; 2, adhA; 3, adhB.
Figure 2 PCR amplification products shown by agarose gel electrophoresis. Lanes M, marker (bp);1, Padh; 2, adhA; 3, adhB.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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 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.CC BY · PubMed Central
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.
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.CC BY · PubMed Central

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Cited by

Metadata

Authors
Wu, X., Yao, H., Cao, L., Zheng, Z., Chen, X., Zhang, M., Wei, Z., Cheng, J., Jiang, S., Pan, L., & Li, X.
Affiliations
  • Hefei University of Technology (CN)
  • Chaohu University (CN)
Corresponding author
Li, Xingjiang
ORCID
Venue
Frontiers in Microbiology
Publisher
Frontiers Media SA
Published
2017-09-06
Volume
8
Pages
1713-1713
ISSN
1664-302X (ISSN-L)
DOI
10.3389/fmicb.2017.01713
PMC
PMC5592214
OpenAlex
W2748870868
Cited by
32
References
0
Citations by year
2026: 1 · 2025: 2 · 2024: 6 · 2023: 5 · 2022: 2 · 2021: 4 · 2020: 9 · 2019: 2 · 2018: 1
FWCI
1.34
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Frontiers in Microbiology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (31601465)
  • Hefei University
  • Hefei University of Technology
  • Natural Science Foundation of Anhui Province (1408085MKL17)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial metabolism and enzyme function
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Biochemical and biochemical processes
Keywords
acetic acid production, Acetobacter pasteurianus, ethanol oxidation, electron transport chain, metabolic flux analysis, protein expression profiling, acetic acid resistance

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