Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (5): 290-299.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0979
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WANG Zhou1(), YU Jie1, WANG Jin-hua1,2,3, WANG Yong-ze1,2,3, ZHAO Xiao1,2,3()
Received:
2023-10-19
Online:
2024-05-26
Published:
2024-05-23
Contact:
ZHAO Xiao
E-mail:2797807952@qq.com;zhaoxiao1@hbut.edu.cn
WANG Zhou, YU Jie, WANG Jin-hua, WANG Yong-ze, ZHAO Xiao. Anaerobic Expression of Lactate Dehydrogenase to Improve the D-lactic Acid Optical Purity Procluced by Escherichia coli[J]. Biotechnology Bulletin, 2024, 40(5): 290-299.
菌株及质粒 Strain and plasmid | 特征 Characteristic | 来源 Source |
---|---|---|
HBUT-D | W ∆frdBC ∆pta ∆adhE ∆pflB ∆aldA | 本实验室保藏 |
HBUT-DP | HBUT-D carrying pUC19 | 本研究 |
HBUT-D3 | HBUT-D carrying pUC19-PLD | 本研究 |
HBUT-D5 | HBUT-D carrying pUC19-PNLD | 本研究 |
HBUT-D7 | HBUT-D carrying pUC19-NLD | 本研究 |
pUC19-PLD | pUC19::PpflBp6-lldD, Apr | 本研究 |
pUC19-NLD | pUC19::PnirB-lldD, Apr | 本研究 |
pUC19-PNLD | pUC19::PpflBp6-PnirB-lldD, Apr | 本研究 |
Table 1 Strains and plasmids in this study
菌株及质粒 Strain and plasmid | 特征 Characteristic | 来源 Source |
---|---|---|
HBUT-D | W ∆frdBC ∆pta ∆adhE ∆pflB ∆aldA | 本实验室保藏 |
HBUT-DP | HBUT-D carrying pUC19 | 本研究 |
HBUT-D3 | HBUT-D carrying pUC19-PLD | 本研究 |
HBUT-D5 | HBUT-D carrying pUC19-PNLD | 本研究 |
HBUT-D7 | HBUT-D carrying pUC19-NLD | 本研究 |
pUC19-PLD | pUC19::PpflBp6-lldD, Apr | 本研究 |
pUC19-NLD | pUC19::PnirB-lldD, Apr | 本研究 |
pUC19-PNLD | pUC19::PpflBp6-PnirB-lldD, Apr | 本研究 |
引物名称 Primer name | 序列 Sequence(5'-3') |
---|---|
P1 | CCGTGACTTAAGAAAATTTATAC |
P2 | CTGGCTGCGGAAATAATCATTTTTGCCTCGATTTCTTTTC |
P3 | GAAAAGAAATCGAGGCAAAAATGTCTTCCATGACAACAACTG |
P4 | ACATACAGCGCCGAACGGTC |
P5 | CAGCAATATACCCATTAAGGAGTAT |
P6 | AACGACTATGCCGCATTC |
P7 | TTCGCCTGATGCTCCC |
P8 | CTGGCTGCGGAAATAATCATACTAACTCTCTCTTTATTAAGTCGGC |
P9 | GCCGACTTAATAAAGAGAGAGTTAGTATGATTATTTCCGCAGCCAG |
P10 | ATATGCGGTGTGAAATACC |
P11 | GTTTTACAACGTCGTGACTTTGGATAATCAAATATTTACTCCGT |
P12 | TAAGGAGAAAATACCGCATCTATGCCGCATTCCCTTT |
P13 | AGTCACGACGTTGTAAAAC |
P14 | ATGCGGTATTTTCTCCTTA |
P15 | ATAGTTTAGCGGCCGCATTCTTATACAGATGCGTAAGGAGAAAA |
P16 | ATAAGAATGCGGCCGCTAAACTATTTTTCTCCTTACGCATCTGT |
P17 | GCTCATACCTGAATGCGCA |
P18 | CGGAAGAGCGCCCAAT |
P19 | CTTATCGCCACTGGCAGCC |
P20 | TGTAGGTCGTTTCGCTCCAA |
Table 2 Primers in this study
引物名称 Primer name | 序列 Sequence(5'-3') |
---|---|
P1 | CCGTGACTTAAGAAAATTTATAC |
P2 | CTGGCTGCGGAAATAATCATTTTTGCCTCGATTTCTTTTC |
P3 | GAAAAGAAATCGAGGCAAAAATGTCTTCCATGACAACAACTG |
P4 | ACATACAGCGCCGAACGGTC |
P5 | CAGCAATATACCCATTAAGGAGTAT |
P6 | AACGACTATGCCGCATTC |
P7 | TTCGCCTGATGCTCCC |
P8 | CTGGCTGCGGAAATAATCATACTAACTCTCTCTTTATTAAGTCGGC |
P9 | GCCGACTTAATAAAGAGAGAGTTAGTATGATTATTTCCGCAGCCAG |
P10 | ATATGCGGTGTGAAATACC |
P11 | GTTTTACAACGTCGTGACTTTGGATAATCAAATATTTACTCCGT |
P12 | TAAGGAGAAAATACCGCATCTATGCCGCATTCCCTTT |
P13 | AGTCACGACGTTGTAAAAC |
P14 | ATGCGGTATTTTCTCCTTA |
P15 | ATAGTTTAGCGGCCGCATTCTTATACAGATGCGTAAGGAGAAAA |
P16 | ATAAGAATGCGGCCGCTAAACTATTTTTCTCCTTACGCATCTGT |
P17 | GCTCATACCTGAATGCGCA |
P18 | CGGAAGAGCGCCCAAT |
P19 | CTTATCGCCACTGGCAGCC |
P20 | TGTAGGTCGTTTCGCTCCAA |
Fig. 1 Validation of different plasmids A: Validation of plasmid pUC19-PLD and plasmid pUC19-NLD(M:5000 marker;1:pUC19-PLD;2-3:pUC19;4:pUC19-PNLD). B: Validation of plasmid pUC19-PNLD(M:5000 marker;1:pUC19-NLD;2:pUC19)
Fig. 2 Plasmids expressing lldD from different promoters A: PnirB promoter recombinant plasmid. B: PpflBP6 promoter recombinant plasmid. C: PpflBP6-PnirB tandem promoter plasmid
Fig. 3 LldD specific enzyme activities from different strains Strain HBUT-D is the blank control, strain HBUT-DP was transferred to the plasmid pUC19 by HBUT-D, strain HBUT-D3 to plasmid pUC19-PLD, strain HBUT-D5 to plasmid pUC19-NLD by HBUT-D, and strain HBUT-D7 to plasmid pUC19-PNLD. Different lowercase letters indicate significant differences in LldD specific enzyme activities of different strains(P<0.05)
菌株 Strain | 酶活Enzyme activity/U | 蛋白量Protein content/mg | 比酶活Specific enzyme activity/(U·g-1) |
---|---|---|---|
HBUT-DP | 20±6 | 2 489 | 8.5±1.1 |
HBUT-D | 20±4 | 2 596 | 8.1±1.4 |
HBUT-D3 | 632±12 | 7 893 | 80.7±1.5 |
HBUT-D5 | 275 ±10 | 7 274 | 37.8±2.8 |
HBUT-D7 | 483±11 | 7 863 | 61.4±1.4 |
Table 3 LldD enzyme activity results for different strains
菌株 Strain | 酶活Enzyme activity/U | 蛋白量Protein content/mg | 比酶活Specific enzyme activity/(U·g-1) |
---|---|---|---|
HBUT-DP | 20±6 | 2 489 | 8.5±1.1 |
HBUT-D | 20±4 | 2 596 | 8.1±1.4 |
HBUT-D3 | 632±12 | 7 893 | 80.7±1.5 |
HBUT-D5 | 275 ±10 | 7 274 | 37.8±2.8 |
HBUT-D7 | 483±11 | 7 863 | 61.4±1.4 |
Fig. 4 Fermentation of different strains in inorganic salt medium A: L-lactate consumption curves of different strains. B: Acid production curves of D-lactic acid of different strains. C: Glucose consumption curves of different strains. D: Growth curves of different strains
菌株 Strain | L-乳酸消耗速率Rate of L-lactate depletion/(mg·L-1·h-1) | L-乳酸消耗量L-lactate depletion concentration/(g·L-1) | D-乳酸生产强度D-lactate productivity/(g·L-1) | 葡萄糖消耗速率Rate of glucose consumption/(g·L-1·h-1) | OD600 nm | D-乳酸糖酸转化率D-lactate yield/% | 初始L-乳酸浓度Initial L -lactate concentration/(g·L-1) |
---|---|---|---|---|---|---|---|
HBUT-D | 8±0.13d | 0.21±0.01d | 4.09±0.11a | 4.16±0.04a | 3.22±0.01a | 98.31±0.21 | 1.01±0.11 |
HBUT-D3 | 40±0.32a | 0.98±0.02a | 2.45±0.04c | 2.52±0.06d | 2.75±0.02b | 97.21±0.11 | 1.02±0.02 |
HBUT-D5 | 14±0.33c | 0.34±0.01c | 4.09±0.04a | 4.15±0.08b | 3.12±0.01a | 98.55±0.33 | 1.04±0.02 |
HBUT-D7 | 34±0.23b | 0.82±0.01b | 3.92±0.03b | 3.98±0.01c | 2.97±0.01a | 98.49±0.31 | 1.02±0.05 |
Table 4 Data on the fermentation of different strains in inorganic salt medium
菌株 Strain | L-乳酸消耗速率Rate of L-lactate depletion/(mg·L-1·h-1) | L-乳酸消耗量L-lactate depletion concentration/(g·L-1) | D-乳酸生产强度D-lactate productivity/(g·L-1) | 葡萄糖消耗速率Rate of glucose consumption/(g·L-1·h-1) | OD600 nm | D-乳酸糖酸转化率D-lactate yield/% | 初始L-乳酸浓度Initial L -lactate concentration/(g·L-1) |
---|---|---|---|---|---|---|---|
HBUT-D | 8±0.13d | 0.21±0.01d | 4.09±0.11a | 4.16±0.04a | 3.22±0.01a | 98.31±0.21 | 1.01±0.11 |
HBUT-D3 | 40±0.32a | 0.98±0.02a | 2.45±0.04c | 2.52±0.06d | 2.75±0.02b | 97.21±0.11 | 1.02±0.02 |
HBUT-D5 | 14±0.33c | 0.34±0.01c | 4.09±0.04a | 4.15±0.08b | 3.12±0.01a | 98.55±0.33 | 1.04±0.02 |
HBUT-D7 | 34±0.23b | 0.82±0.01b | 3.92±0.03b | 3.98±0.01c | 2.97±0.01a | 98.49±0.31 | 1.02±0.05 |
菌株 Strain | D+ | D- | C- | 排序Sort |
---|---|---|---|---|
HBUT-D | 0.334 3 | 0.139 7 | 0.294 8 | 4 |
HBUT-D3 | 0.141 2 | 0.334 3 | 0.702 9 | 2 |
HBUT-D5 | 0.271 6 | 0.154 2 | 0.362 2 | 3 |
HBUT-D7 | 0.070 0 | 0.296 3 | 0.808 8 | 1 |
Table 5 Euclidean distance D+ and D- between each target value and the ideal point and the relative closeness C- of each target
菌株 Strain | D+ | D- | C- | 排序Sort |
---|---|---|---|---|
HBUT-D | 0.334 3 | 0.139 7 | 0.294 8 | 4 |
HBUT-D3 | 0.141 2 | 0.334 3 | 0.702 9 | 2 |
HBUT-D5 | 0.271 6 | 0.154 2 | 0.362 2 | 3 |
HBUT-D7 | 0.070 0 | 0.296 3 | 0.808 8 | 1 |
菌株 Strain | D-乳酸糖酸转化率D-lactate yield/% | D-乳酸光纯D-lactate optical purty/% | D-乳酸生产强度D-lactate productivity/(g·L-1·h-1) | L-乳酸消耗速率Rate of L-lac consumption/(mg·L-1·h-1) | 初始L-乳酸浓度Initial L-lac concentration/(g·L-1) |
---|---|---|---|---|---|
HBUT-D | 97.64±0.01 | 99.22±0.02 | 4.24±0.04 | 10.41±0.01*** | 1.0±0.02 |
HBUT-D7 | 96.89±0.01 | 99.93±0.01 | 3.87±0.02 | 34.75±0.01*** | 1.0±0.02 |
Table 6 Data on fermentation of strain HBUT-D7 and HBUT-D in corn syrup medium
菌株 Strain | D-乳酸糖酸转化率D-lactate yield/% | D-乳酸光纯D-lactate optical purty/% | D-乳酸生产强度D-lactate productivity/(g·L-1·h-1) | L-乳酸消耗速率Rate of L-lac consumption/(mg·L-1·h-1) | 初始L-乳酸浓度Initial L-lac concentration/(g·L-1) |
---|---|---|---|---|---|
HBUT-D | 97.64±0.01 | 99.22±0.02 | 4.24±0.04 | 10.41±0.01*** | 1.0±0.02 |
HBUT-D7 | 96.89±0.01 | 99.93±0.01 | 3.87±0.02 | 34.75±0.01*** | 1.0±0.02 |
菌株 Strain | D-乳酸糖酸转化率D-lactate yield/% | D-乳酸光纯 D-lactate optical purty/% | L-乳酸消耗速率Rate of L-lactate consumption/(mg·L-1·h-1) | D-乳酸生产强度D-lactate productivity/(g·L-1·h-1) |
---|---|---|---|---|
HBUT-D | 61.82±0.01 | 99.01±0.01 | 6.87±0.02*** | 1.93 |
HBUT-D7 | 60.43±0.01 | 99.99±0.01 | 17.18±0.01*** | 1.88 |
Table 7 Data on the fermentation of strain HBUT-D and HBUT-D7 in molasses medium
菌株 Strain | D-乳酸糖酸转化率D-lactate yield/% | D-乳酸光纯 D-lactate optical purty/% | L-乳酸消耗速率Rate of L-lactate consumption/(mg·L-1·h-1) | D-乳酸生产强度D-lactate productivity/(g·L-1·h-1) |
---|---|---|---|---|
HBUT-D | 61.82±0.01 | 99.01±0.01 | 6.87±0.02*** | 1.93 |
HBUT-D7 | 60.43±0.01 | 99.99±0.01 | 17.18±0.01*** | 1.88 |
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