Enhanced bacterial cellulose production in Komagataeibacter sucrofermentans: impact of different PQQ-dependent dehydrogenase knockouts and ethanol supplementation

Abstract

Background: Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation. Specifically, glucose oxidation to gluconic acid substantially lowers environmental pH and hinders BC production. Conversely, ethanol supplementation is known to enhance BC yields in Komagataeibacter spp. by promoting efficient glucose utilization.

Results: K. sucrofermentans ATCC 700178 was engineered, knocking out the four PQQ-mDHs, to assess their impact on BC production. The strain KS003, lacking PQQ-dependent glucose dehydrogenase (PQQ-GDH), did not produce gluconic acid and exhibited a 5.77-fold increase in BC production with glucose as the sole carbon source, and a 2.26-fold increase under optimal ethanol supplementation conditions. In contrast, the strain KS004, deficient in the PQQ-dependent alcohol dehydrogenase (PQQ-ADH), showed no significant change in BC yield in the single carbon source experiment but showed a restrained benefit from ethanol supplementation.

Conclusions: The results underscore the critical influence of PQQ-GDH and PQQ-ADH and clarify the effect of ethanol supplementation on BC production in K. sucrofermentans ATCC 700178. This study provides a foundation for further metabolic pathway optimization, emphasizing the importance of diauxic ethanol metabolism for high BC production.

Keywords: Komagataeibacter; Acetic acid; Acetic acid bacteria; Bacterial cellulose; Gluconic acid; Metabolic engineering; PQQ-dependent dehydrogenases.

Fig. 1

Overview of the key metabolic steps and pathways involved in BC production and energy generation in the presence of glucose and ethanol. The periplasmic oxidation of the sugars, alcohols, and sugar alcohols is linked to the electron transport chain (ETC)—the dehydrogenases transfer the reducing equivalents to ubiquinone (UQ—dark green) converting it to its reduced form, ubiquinol (UQ—light green). UQ can be further reoxidized by terminal oxidases in a process coupled with oxygen (O₂) reduction to water (H₂0) and the generation of a proton (H⁺) gradient. Then, F₁F₀ ATPase facilitates H⁺ translocation, owing to the H⁺ gradient, in a process coupled with ATP generation. In the periplasm, glucose can be partially oxidized to D-gluconate, 2-keto-D-gluconate, and 5-D-ketogluconate by PQQ-dependent glucose dehydrogenase (GDH), flavin-dependent gluconate-2-dehydrogenase (G2D), and PQQ-dependent gluconate-5-dehydrogenase (G5D). Glucose can also be imported and used for cellulose biosynthesis or metabolized through the Entner–Doudoroff (ED), Embden–Meyerhof (EMP), or Pentose Phosphate Pathway (PPP). Ethanol can be oxidized to acetaldehyde and further to acetic acid in the periplasm by PQQ-dependent alcohol dehydrogenase (ADH) and further to acetaldehyde by flavin-dependent aldehyde dehydrogenase (ALDH). When ethanol, acetaldehyde, or acetate are assimilated by the cell, the metabolites are converted to acetyl-CoA for biomass generation, or complete oxidation through the tricarboxylic acid cycle (TCA). This is also coupled with ATP generation at different points. Additionally, NADH can also be oxidized to NAD⁺ in the cytosolic membrane by NADH dehydrogenase (NADH), which is also linked to the ETC.

Fig. 2

Time-course growth and metabolic profiles of agitated cultures in HS-glucose medium for five K. sucrofermentans strains—A: KS001; B: KS002; C: KS003; D: KS004; E: KS005. For each strain, the left panel shows OD600 nm monitoring; the middle panel depicts glucose concentration (g/L); the right panel represents gluconic acid concentration (g/L). In all panels, Replicate 1 (orange lines with dot markers) and Replicate 2 (blue lines with cross markers) are shown

Fig. 3

pH variation during fermentations of K. sucrofermentans strains in different media–A: pH measurements for strains KS001 to KS005 over time in HS-glucose medium; B: pH values for the same strains in HS-ethanol medium

Fig. 4

Time-course growth and metabolic profiles of agitated cultures in HS-ethanol medium for five K. sucrofermentans strains—A: KS001; B: KS002; C: KS003; D: KS004; E: KS005. For each strain, the left panel shows OD600 nm monitoring; the middle panel depicts ethanol concentration (g/L); the Right panel represents acetic acid concentration (g/L). In all panels, Replicate 1 (orange lines with dot markers) and Replicate 2 (blue lines with cross markers) are shown

Fig. 5

BC production (g/L) of K. sucrofermentans strains KS001 to KS005 grown in HS-glucose (black bars) and HS-ethanol (white bars) media, in static culture. Error bars denote standard deviations. * Statistically significant difference in BC concentration compared to the KS001 strain for the corresponding carbon source (N = 2; p < 0.05)

Fig. 6

Comparative analysis of bacterial cellulose production efficiency in K. sucrofermentans strains in HS-glucose-ethanol media in a range of ethanol concentrations, in static culture—A: KS001; B: KS003; C: KS004. The top panel (VPE) represents volumetric production efficiency (g/L), the second panel (BPCG) shows bacterial cellulose production per consumed glucose (g/mol), the third panel (BPCE) denotes bacterial cellulose production per consumed ethanol (g/mol), and the bottom panel (BPCC) illustrates bacterial cellulose production per consumed carbon (g/mol). Red bars represent data at 7 days, while green bars denote data at 14 days. Error bars indicate the standard deviation. * Significance against strain KS001 for the same condition and time point (N = 2; p < 0.1). ** Significance when comparing 14 days to 7 days for a given condition (N = 2; p < 0.1)

Fig. 7

Metabolic profiles of K. sucrofermentans strains in HS-glucose-ethanol media. Each panel shows the residual concentration of (from top to bottom) glucose, gluconic acid, ethanol, and acetic acid (all in g/L) for strains KS001 A, KS003 B, and KS004 C. Red and green bars indicate measurements at 7 and 14 days, respectively; error bars represent standard deviation; the horizontal blue dashed line represents the initial glucose concentration (g/L). * Significant differences from strain KS001 under identical conditions (N = 2; p < 0.1). ** Significant differences between the time points for each strain (N = 2; p < 0.1)