Presentation Information
[3PL-01]Improvement of Cellulose Production by Deletion of the Glucose Dehydrogenase Gene in Acetic Acid Bacteria
○Miho Suginaka1, Shunsuke Nagai1, Ryo Takahama1, Yuma Ishido1, Chiaki Ogino2, Hirofumi Tani1, Kenji Tajima1 (1. Hokkaido University (Japan), 2. Kobe University (Japan))
Keywords:
bacterial cellulose,acetic acid bacteria,glucose dehydrogenase
Bacterial cellulose (BC), produced by acetic acid bacteria, has excellent mechanical properties, biocompatibility, and biodegradability. These characteristics make BC a promising environmentally friendly and recyclable material. In our laboratory, we have successfully achieved large-scale production of nanofibrillated bacterial cellulose (NFBC) by aerobically cultivating Komagataeibacter intermedius (K. intermedius) NEDO-01, a cellulose-producing acetic acid bacterium, in a medium containing a dispersant. However, further improvements in productivity are necessary for large-scale industrial applications. NEDO-01 synthesizes BC from sugars such as glucose. However, glucose can also be oxidized into organic acids by enzymes such as glucose dehydrogenase (Gcd), and this reaction is not essential for BC synthesis. Therefore, in this study, we aimed to enhance BC productivity by constructing a gcd1 gene-deficient strain (Δgcd1).
Since it was difficult to visually select deletion mutants was difficult using conventional methods, a blue pigment–based screening approach was applied to acetic acid bacteria for the first time. A markerless gene deletion plasmid was used to perform gene deletion in K. intermedius. NEDO-01. Mutants that underwent the first homologous recombination were selected based on ampicillin resistance and blue pigmentation caused by indigoidine synthesis. Double-recombination mutants were then isolated through counter-selection with 5-fluorocytosine (5-FC). Genomic DNA was extracted from the mutant strains obtained, and deletion of the gcd1 gene was confirmed by PCR and sequencing of the flanking regions.
To evaluate the effects of the gcd1 gene deletion, both static flask cultures and aerobic stirred fermentations using 10 L jar fermenters were conducted. Gluconic acid production was significantly suppressed in static culture, while the sugar conversion rate was improved in the Δgcd1 strain compared to the wild type (WT). In aerobic stirred culture, the Δgcd1 strain exhibited a higher final NFBC production and a higher sugar conversion rate than the WT. These results suggest that deleting the gcd1 gene successfully enhances NFBC production. However, the Δgcd1 strain consumed less sugar. Therefore, improving the strain's sugar uptake capacity is essential for enhancing NFBC production. Metabolic analysis can identify rate-limiting pathways in sugar utilization, and overexpressing these pathways can further improve production. In addition, solid-state NMR and AFM revealed that the chemical composition and morphology of NFBC remained largely unchanged in the Δgcd1 compared to the WT strain.
Overall, deleting the gcd1 gene suppressed gluconate production, resulting in a higher sugar conversion rate and more efficient BC production.
Since it was difficult to visually select deletion mutants was difficult using conventional methods, a blue pigment–based screening approach was applied to acetic acid bacteria for the first time. A markerless gene deletion plasmid was used to perform gene deletion in K. intermedius. NEDO-01. Mutants that underwent the first homologous recombination were selected based on ampicillin resistance and blue pigmentation caused by indigoidine synthesis. Double-recombination mutants were then isolated through counter-selection with 5-fluorocytosine (5-FC). Genomic DNA was extracted from the mutant strains obtained, and deletion of the gcd1 gene was confirmed by PCR and sequencing of the flanking regions.
To evaluate the effects of the gcd1 gene deletion, both static flask cultures and aerobic stirred fermentations using 10 L jar fermenters were conducted. Gluconic acid production was significantly suppressed in static culture, while the sugar conversion rate was improved in the Δgcd1 strain compared to the wild type (WT). In aerobic stirred culture, the Δgcd1 strain exhibited a higher final NFBC production and a higher sugar conversion rate than the WT. These results suggest that deleting the gcd1 gene successfully enhances NFBC production. However, the Δgcd1 strain consumed less sugar. Therefore, improving the strain's sugar uptake capacity is essential for enhancing NFBC production. Metabolic analysis can identify rate-limiting pathways in sugar utilization, and overexpressing these pathways can further improve production. In addition, solid-state NMR and AFM revealed that the chemical composition and morphology of NFBC remained largely unchanged in the Δgcd1 compared to the WT strain.
Overall, deleting the gcd1 gene suppressed gluconate production, resulting in a higher sugar conversion rate and more efficient BC production.
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