[4S02a] 03. Fermentation Physiology/ Technology - Part 2

2:30 PM - 3:00 PM JST(5:30 AM - 6:00 AM UTC)

[4Ferm-06-KL] Harnessing the Acidic Physiology of a Traditional Vinegar-Producing Bacterium for Recombinant Expression of Acid-Functional Enzymes

○Shinsuke Fujiwara¹ (1. School of Biological and Environmental Sciences, Kwansei-Gakuin University (Japan))

Keywords:

Acetic acid bacteria,Komagataeibacter europaeus,Recombinant protein expression,Intracellular pH,Acid-functional enzymes

Recombinant protein production has long relied on Escherichia coli as a standard host. Its widespread use reflects historical advantages: well-established cultivation conditions and the early development of versatile vector systems. However, this system presumes that most proteins function optimally in a neutral intracellular environment. Enzymes that evolved in acidic niches may require physicochemical conditions that are not readily reproduced in conventional hosts. This consideration prompted us to re-examine a fundamental premise in biotechnology: rather than adapting proteins to fit an established host, can we select a host whose intracellular physiology more closely matches the evolutionary background of the protein? Acetic acid bacteria provide a compelling model. Unlike most microorganisms, which tightly maintain intracellular pH despite extracellular acidification, acetic acid bacteria oxidize acetic acid through respiratory metabolism, and a decrease in environmental pH is accompanied by a partial reduction in intracellular pH (1). Their cytoplasmic proteins therefore operate under conditions of dynamic acid stress. Among them, Komagataeibacter europaeus has been safely used for more than a century in industrial vinegar fermentation, underscoring both its robustness and its long-standing association with food production. The strain harbors three endogenous plasmids that facilitate vector construction and stable gene maintenance (2), and it can be cultivated heterotrophically to biomass levels comparable to E. coli, making it an attractive yet underexplored recombinant host. Using the native groES/EL promoter of K. europaeus, we expressed UPV230, a restriction endonuclease derived from the acid-tolerant vaginal commensal bacterium Ureaplasma parvum, a microorganism implicated in infertility and adverse pregnancy outcomes. During functional characterization of recombinant UPV230 produced in the acetic acid bacterium, we discovered that the enzyme alters its DNA recognition sequence in a pH-dependent manner (3). This property had not been previously described and became evident through expression and activity analyses enabled by the mildly acidic intracellular context of the host system. The acetic acid bacterial platform therefore did not merely support expression; it enabled the identification of an intrinsic acid-responsive regulatory behavior of the enzyme. These findings indicate that the intracellular environment can influence not only protein stability but also the manifestation of latent functional properties. Our study demonstrates that an acetic acid bacterium long associated with traditional fermentation can serve as a viable recombinant host for acid-functional enzymes.References: (1) Ishii Y et al. J. Bacteriol. 203: e00162-21 (2021); (2) Akasaka N et al. J. Biosci. Bioeng. 119: 661–668 (2015); (3) Tanakura Y et al. J. Biotechnol. 380: 38–50 (2024).