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

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.

Classification of acetic acid bacteria and their acid resistant mechanism

“…Overexpression corresponding genes of intracellular grpE and dnaKJ increased resistance towards the fermentation environment in AAB (Ishikawa et al 2010 ; Okamoto-Kainuma et al 2004 ). Employing two-dimensional electrophoresis to conduct a comprehensive study of intracellular protein levels in A. pasteurianus LMG 1262 T during acetic acid fermentation, it was found that fermentation increased the protein expression levels of GrpE, DnaK, DnaJ, GroES, GroEL, and ClpB to varying extents, with the expression level of GrpE being increased by 9.42 times compared with the early fermentation stage (Andrés-Barrao et al 2012 ; Wu et al 2017 ). Overall, the aforementioned studies showed that the universal stress mechanism mediated by HSPs is one of the ways by which AAB ensure smooth acetic acid fermentation (Fig.…”

Engineering Bacterial Cellulose by Synthetic Biology

“…Indeed, it has not been until recently that FBA and genetic engineering have finally been combined to metabolically engineer an acetobacter. This was first achieved in 2017, when an industrial acetic acid producer strain of Acetobacter pasteurianus was genetically modified to improve acetic acid yields [35]. FBA combined with plasmid-based overexpression of subunits adhA and adhB of pyrroquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH), led the researchers to understand how to improve the ethanol oxidase respiratory chain in their cells, leading to more ethanol being converted into the acetic acid product [35].…”

Streamlining the Fermentation Process Using Mixed Cultures

“…The fermentation processes use different types of microorganisms. The identification and use of new types of microorganisms is performed with the aim of obtaining new strains with superior properties and abilities to produce high-quality and highyield products [11][12][13][14][15]. Furthermore, various types of fermentation operations and modifications to the stages within these operations have been tested to determine the most simple and efficient methods capable of producing high yields because the operational procedures of acetic acid fermentation can be complicated and require a long time when multiple processes are required.…”