Table 3.
Effect of mutation on BNC biosynthesis and/or BNC properties
| Strain | Disrupted gene or a fragment | Mutation | Mutation conditions or type of mutagenesis | Phenotypic effects | References | |||
|---|---|---|---|---|---|---|---|---|
| Culture conditions or environment effect on the formation of cellulose-non-producing (Cel−) forms | ||||||||
| K. xylinus E25 | Cel− | Fragment downstream of ORF encoding phosphoglucomutase | A single nucleotide (T) deletion. Inhibited expression of phosphoglucomutase and glucose-1-phosphate uridylyltransferase | Repeated 3 times passages under agitated culture conditions (SH medium, 48 h, 30 °C, 90 rpm) of the parental strain | No BNC production | (Krystynowicz et al. 2005) | ||
| K. europaeus | LMG 18494 | bcsCI (cellulose synthase subunit CI) | Large deletion (1900-bp deletion) | Isolation from vinegar | No BNC production | (Andrés-Barrao et al. 2011) | ||
| LMG 18890 | bcsCI | 5-bp deletion | ||||||
| Komagataeibacter medellinensis | NBRC 3288 (parental strain) | bcsBI (cellulose synthase subunit BI) | 17-bp deletion | Frameshift. Gene disruption by stop codon introduction | Isolation from vinegar | No BNC production | (Ogino et al. 2011) | |
| R1 | Revertants | a1-bp (C) deletion at the 277486th position of the gene. Frameshift and BcsBI restoration | Repeated static culture of the parental strain | Restoration of BNC production | (Matsutani et al. 2015) | |||
| R2 | a1-bp (C) deletion at the 277491th position of the gene, frameshift and BcsBI restoration | |||||||
| K. oboediens |
MSKU 3 (wild type) |
bcsCI (cellulose synthase subunit CI) | – | Isolated from rotten fruit samples in Thailand; thermotolerant strain (growth up to 39 °C) | BNC yield of 0.33 g/L. BNC fibril diameter size: 70.52 nm; density: 0.43 ± 0.05 g cm−3. Mechanical properties of BNC; tensile strength: 73.94 ± 16.94 MPa, Young’s modulus: 5.83 ± 0.69 | (Taweecheep et al. 2019a, b) | ||
|
E3 (parental strain for revertants) |
bA single nucleotide (T) insertion at the 2135th position of bcsCI | Frameshift. Gene disruption by stop codon | Repeated static cultivation of MSKU 3 strain for 63 passages (YPGD1A medium containing increasing concentration of ethanol) | No BNC production. High acetic acid production ability | ||||
| R30-3 | Revertants | 2-bp (GC) insertion at the 2145th position. Frameshift and two amino acid alterations (N713E and Q714P) and one amino acid addition (A715) | Repeated static cultivation of E3 strain (YPGD1A3E medium) | Increased BNC yield (2.15 g/L). BNC fibril diameter size: 65.9 nm; density 0.42 ± 0.02 g cm−3. Reduced mechanical properties of BNC (ca. 2-fold ↓); tensile strength: 43.56 ± 10.98 MPa, Young’s modulus: 3.60 ± 0.40 | ||||
| R30-12 | 1-bp (C) deletion at the 2149th position. Frameshift and four amino acid substitution (N713E, Q714P, R715A and G716W) | Repeated static cultivation of E3 strain (YPGD medium) | Increased BNC yield (0.53 g/L). BNC fibril diameter size: 70.14 nm; density: 0.54 ± 0.01 g cm−3. Mechanical properties of BNC; tensile strength: 73.94 ± MPa; Young’s modulus: 5.14 ± 0.58 | |||||
| R37-4 | 1-bp (T) deletion at the 2132nd position. Frameshift and a single amino acid substitution (L711R) | Repeated static cultivation of E3 strain (YPGD medium) | Increased BNC yield (1.12 g/L). BNC fibrils diameter size: 59.14 nm; density: 0.72 ± 0.05 g cm−3. Increased mechanical properties of BNC (ca. 2-fold ↑); tensile strength: 158.72 ± 28.29 MPa; Young’s modulus: 8.75 ± 1.54 | |||||
| R37-9 | 1-bp (A) deletion at the 2139th position. Frameshift and a single amino acid substitution (N713D) | Increased BNC yield (0.7 g/L). BNC fibril diameter size: 34.58 nm; increased density (2-fold ↑): 0.85 ± 0.07 g cm−3. Increased mechanical properties of BNC (2-fold ↑); tensile strength: 159.47 ± 29.76 MPa; Young’s modulus: 9.83 ± 0.69 | ||||||
| Transposon mutagenesis | ||||||||
| K. hansenii ATCC 23769 | 5 | acsA (cellulose synthase subunit A) | Insertion site 1893 bp, gene disruption | Tn 5 transposon insertion mutagenesis | No or reduced BNC production | (Deng et al. 2013) | ||
| 10 | acsC (cellulose synthase subunit C) | Insertion site 1984 bp, gene disruption | ||||||
| I-13 | ccpAx (cellulose complementing protein Acetobacter xylinum) | Insertion site 40 bp, gene disruption | ||||||
| I-7 | dgc1 (diguanylate cyclase) | Insertion site 656 bp, gene disruption | ||||||
| V-31 | dgc1 (diguanylate cyclase) | Insertion site 242 bp, gene disruption | ||||||
| II-23 | crp− fnr (transcriptional regulator, Crp/Fnr family protein) | Insertion site 187 bp, gene disruption | ||||||
| K. hansenii ATCC 23769 | I-23 | AlaR (alanine racemase) | Insertion site 628 bp, gene disruption | Tn 5 transposon insertion mutagenesis | Reduced BNC production and crystallinity | (Deng et al. 2015) | ||
| #52 | LDC (lysine decarboxylase) | Insertion site 347 bp, gene disruption | ||||||
| Mutagenisation by chemical and/or physical agents | ||||||||
| K. xylinus ATCC 53582 | plr 15 | bcsA (cellulose synthase subunit AI) | Substitution of nucleotide G to A at position 1345 bp of the gene; A449T substitution in the BcsA (missense mutation) | EMS (ethyl methane sulfonate) mutagenesis and screening on a medium containing10 μM pellicin | Increased BNC synthesis rate; reduced crystallinity; resistance to pellicin | (Salgado et al. 2019) | ||
| K. hansenii | HDM 1–3 (wild type) | – | – | Isolated from rotten Actinida chinesis Planch | BNC yield of 1.43 g/L | (Li et al. 2016) | ||
| Br-12 | Three specific fragments were identified: TonB-dependent transport (TBDT, 779 bp, LCL 80534); exopolysaccharides output protein (PePr, 162 bp, LCL77813); unknown protein (380 bp, LCL57743) | AFLP-based analysis between mutant lines and the wild type | DES (diethyl sulphate) mutagenesis | Decreased BNC yield (0.56 g/L). Increased gluconic acid production (67.53%) | ||||
| Br-3 | nd | DES (diethyl sulphate) mutagenesis and screened and selection of mutant in a medium containing NaBr–NaBrO3 | Increased BNC yield (2.45 g/L). Decreased gluconic acid production (10.23%) | |||||
| Co-5 | Two specific fragments were identified: acsD and galE (UDP-galactose-4-epimerase) | Mutagenesis of the Br-3 mutant by 60Co-γ radiation and screened and selection of mutant in medium containing NaBr–NaBrO3 | Increased BNC yield (3.38 g/L). Decreased gluconic acid production (54.79%) | |||||
YPGD medium (0.5% of glucose, 1% of yeast extract, 1% of polypeptone and 2% of glycerol) containing 1% acetic acid and ethanol (YPGD1A3E)
aAll the mutations were found to occur in a small C-rich region (CCCGGCCC) in bcsBI
bAll the mutations were found to occur in a small region (TGCTGAACCAGCGTGGC) in bcsCI, which seems to be a sensitive region for spontaneous mutation