Table 4.
The results of gene editing
| Strains and genes | Mutation length (bp)a | Results (C/I/T)b | Efficiency (%) |
|---|---|---|---|
|
Gene deletion C. glutamicum ATCC 13032 | |||
| ldh | 568 | 15/5/20 | 75.0 |
| eutD | 877 | 19/3/22 | 86.4 |
| gabP | 930 | 19/4/23 | 82.6 |
| glnA1 | 730 | 13/10/23 | 56.5 |
| glnA2 | 728 | 16/7/23 | 69.6 |
| alaT | 614 | 17/6/23 | 73.9 |
| argR | 317 | 6/4/10 | 60.0 |
| gabTD | 1949 | 6/17/23 | 26.1 |
| aceAB | 3450 | 10/13/23 | 43.5 |
| poxB | 980 | 15/8/23 | 65.2 |
| C. glutamicum ATCC 14067 | |||
| gabP | 930 | 8/15/23 | 34.8 |
| C. glutamicum ssp. lactofermentum | |||
| gabP | 930 | 2/6/8 | 25.0 |
| Gene insertion (deletion/insertion) | |||
| Φ(PtacM-gdhA1) | 166/150 | 22/1/23 | 95.7 |
| eutD::speC | 877/2261 | 8/15/23 | 34.8 |
| gabP::gadB | 930/1557 | 7/16/23 | 30.4 |
| gabP::gadB2B1m | 930/3167 | 5/18/23 | 21.7 |
| eutD::gdhA | 877/1459 | 16/7/23 | 69.6 |
| alaT::gdhA (pCCG1) | 614/1459 | 6/17/23 | 26.1 |
| alaT::gdhA (pCCG2) | 614/1459 | 5/18/23 | 21.7 |
a, the mutation length is the length of deleted or/and inserted fragments. 166-bp region has been deleted from genome and 150-bp fragment was inserted when replace native gdhA1 promoter in C. glutamicum ATCC 13032; 877-bp region has been deleted from genome and 1459-bp fragment was inserted when insert gdhA into eutD; 877-bp region has been deleted from genome and 2261-bp fragment was inserted when insert speC into eutD; 930-bp region has been deleted from genome and 1557-bp fragment was inserted when insert gadB into gabP; 930-bp region has been deleted from genome and 3167-bp fragment was inserted when insert gene cluster gadB2B1m into gabP; 614-bp region has been deleted from genome and 1459-bp fragment was inserted when insert gdhA into alaT. The gene speC, gdhA and gadB are all under the control of PtacM promoter; b, T: the total number of colonies for PCR verification, C: the number of correct colonies, I: the number of incorrect colonies. The efficiency was calculated by (C/T) * 100%