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

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

doi:10.3389/fmicb.2017.01713

Xuefeng Wu, Zhi Zheng + Show 9 more

Open Access

https://doi.org/10.3389/fmicb.2017.01713

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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 (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.

Highlights

Acetic acid bacteria (AAB) are employed in the industrial production of acetic acid
Acetobacter pasteurianus JST-S was previously identified in a screen performed in our laboratory from A. pasteurianus CICC 20001. 16S rDNA gene sequencing had been deposited in the GenBank database
AdhA, and adhB were amplified from the genomic DNA of A. pasteurianus JST-S by polymerase chain reaction (PCR) using previously published primers (Zheng et al, 2014)

Summary

Introduction

Acetic acid bacteria (AAB) are employed in the industrial production of acetic acid. Their characteristics directly affect the yield and flavor of vinegar (Quintero et al, 2009). The fermentative oxidation of ethanol to acetic acid has been shown to depend on two sequential reactions of membrane-bound pyrrpquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) and aldehyde dehydrogenase (ALDH), both of which are localized on the periplasmic side of the inner membrane (Toyama et al, 2004; Yakushi and Matsushita, 2010; Trcek and Matsushita, 2013). PQQ-ADH is a unique member of the alcohol dehydrogenase family and is essential for AAB oxidation of ethanol into acetic acid (Chen et al, 2016)

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