Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (2): 173-183.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0345
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KOU Hang1,2(), WANG Yan-mei1, LI Tong2, BO Ming-jing2, ZHANG Wei-cai2, XIONG Xiang-hua2(), LI Ming1()
Received:
2021-03-20
Online:
2022-02-26
Published:
2022-03-09
Contact:
XIONG Xiang-hua,LI Ming
E-mail:727846160@qq.com;xiongxianghua@sina.com;liming09@tust.edu.cn
KOU Hang, WANG Yan-mei, LI Tong, BO Ming-jing, ZHANG Wei-cai, XIONG Xiang-hua, LI Ming. Fermentation Optimization for PQQ Synthesis Based on the Genome-scale Metabolic Model of Methylovorus sp. J1-1[J]. Biotechnology Bulletin, 2022, 38(2): 173-183.
引物名称Primer name | 引物序列Primer sequence(5'-3') | 引物功能Primer function |
---|---|---|
glyA-F | GCCTGCAGGTCGACTCTAGATATAGCGCTCAACAAGGACCTC | glyA基因扩增 Gene amplification of glyA |
glyA-R | GTGAATTCGAGCTCGGTACCTTAAGCGCCGTATACCGGG | |
hps1-F | GCCTGCAGGTCGACTCTAGATATTGCTGACTTGAATAGCGCTAAT | hps1基因扩增 Gene amplification of hps1 |
hps1-R | GTGAATTCGAGCTCGGTACCTTAGTGAGCCAGCGAAGTGA | |
hps2-up-F | AGAAAAGATCAAAGGATCTTCGGATCCTGCGGAATTCCTTTTGTGG | hps2基因敲除质粒构建 Construction of hps2 knockout plasmid |
hps2-up-R | CACTATAGGGCGAATTGCTCGAGTGAAGTGGCACAGAACCAG | |
hps2-kan-F | CCCACAAAAGGAATTCCGCAGGATCCGAAGATCCTTTGATCTTTTC | |
hps2-kan-R | TAAAGCTCAGCGGGGCTGATACTAGTCAGGTGGCACTTTTCGG | |
hps2-down-F | TTCCCCGAAAAGTGCCACCTGACTAGTATCAGCCCCGCTGAGC | |
hps2-down-R | GAGCTCCACCGCGGTGGCGGCCGCATTGCCTTGCTGGGGG | |
hps2-CF | GCACCATGGGCCAGTCTGA | hps2敲除菌株鉴定 Identifying the strain with hps2 knockout |
hps2-CR | GGAGCACTTTCACCAGAAGCT | |
glyA-Y-F | AGTGGATGCTGGTGCCAACAT | 实时荧光定量PCR qRT-PCR |
glyA-Y-R | GCCGCTGGAAGTCGAACCTT | |
hps1-Y-F | ATCGGTTCGCCAGCCATCAC | |
hps1-Y-R | GCCTTGGTCGCAGCAATCAC | |
fdh-Y-F | AGCTGCACGTACCAAGGAAGTT | |
fdh-Y-R | TGTTGGCACCAGCATCCACTAC | |
fae-Y-F | GCCGCTGGAAGTCGAACCTT | |
fae-Y-R | GCCACGCAGGTTGATCCACTT |
Table 1 Primers used in this study
引物名称Primer name | 引物序列Primer sequence(5'-3') | 引物功能Primer function |
---|---|---|
glyA-F | GCCTGCAGGTCGACTCTAGATATAGCGCTCAACAAGGACCTC | glyA基因扩增 Gene amplification of glyA |
glyA-R | GTGAATTCGAGCTCGGTACCTTAAGCGCCGTATACCGGG | |
hps1-F | GCCTGCAGGTCGACTCTAGATATTGCTGACTTGAATAGCGCTAAT | hps1基因扩增 Gene amplification of hps1 |
hps1-R | GTGAATTCGAGCTCGGTACCTTAGTGAGCCAGCGAAGTGA | |
hps2-up-F | AGAAAAGATCAAAGGATCTTCGGATCCTGCGGAATTCCTTTTGTGG | hps2基因敲除质粒构建 Construction of hps2 knockout plasmid |
hps2-up-R | CACTATAGGGCGAATTGCTCGAGTGAAGTGGCACAGAACCAG | |
hps2-kan-F | CCCACAAAAGGAATTCCGCAGGATCCGAAGATCCTTTGATCTTTTC | |
hps2-kan-R | TAAAGCTCAGCGGGGCTGATACTAGTCAGGTGGCACTTTTCGG | |
hps2-down-F | TTCCCCGAAAAGTGCCACCTGACTAGTATCAGCCCCGCTGAGC | |
hps2-down-R | GAGCTCCACCGCGGTGGCGGCCGCATTGCCTTGCTGGGGG | |
hps2-CF | GCACCATGGGCCAGTCTGA | hps2敲除菌株鉴定 Identifying the strain with hps2 knockout |
hps2-CR | GGAGCACTTTCACCAGAAGCT | |
glyA-Y-F | AGTGGATGCTGGTGCCAACAT | 实时荧光定量PCR qRT-PCR |
glyA-Y-R | GCCGCTGGAAGTCGAACCTT | |
hps1-Y-F | ATCGGTTCGCCAGCCATCAC | |
hps1-Y-R | GCCTTGGTCGCAGCAATCAC | |
fdh-Y-F | AGCTGCACGTACCAAGGAAGTT | |
fdh-Y-R | TGTTGGCACCAGCATCCACTAC | |
fae-Y-F | GCCGCTGGAAGTCGAACCTT | |
fae-Y-R | GCCACGCAGGTTGATCCACTT |
Fig. 1 Distributions of reactions in GSMM AM(Amino Acids Metabolism),BOSM(Biosynthesis of Other Secondary Metabolism),CM(Carbohydrates Metabolism),EM(Energy Metabolism),GBM(Glycan Synthesis and Metabolism),LM(Lipids Metabolism),CV(Cofactors and Vitamins Metabolism),OAM(Other Amino Acid Metabolism),NM(Nucl-eotides Metabolism),TP(Terpenoids and Polyketides),TS(Transport system),and UM(Unclassified Reactions)
底物Substrate | 实验值In vivo | 模拟值In silico |
---|---|---|
碳源 Carbon source | ||
葡萄糖 Glucose | + | + |
甲醇 Methanol | + | + |
乙醇 Ethanol | + | + |
果糖 Fructose | + | + |
氮源 Nitrogen source | ||
尿素 Urea | + | + |
硝酸钠 Sodium nitrate | + | + |
硝酸铵 Ammonium nitrate | + | + |
氯化铵 Ammonium chloride | + | + |
硫酸铵 Ammonium sulfate | + | + |
Table 2 Simulation of growing in carbon and nitrogen sources
底物Substrate | 实验值In vivo | 模拟值In silico |
---|---|---|
碳源 Carbon source | ||
葡萄糖 Glucose | + | + |
甲醇 Methanol | + | + |
乙醇 Ethanol | + | + |
果糖 Fructose | + | + |
氮源 Nitrogen source | ||
尿素 Urea | + | + |
硝酸钠 Sodium nitrate | + | + |
硝酸铵 Ammonium nitrate | + | + |
氯化铵 Ammonium chloride | + | + |
硫酸铵 Ammonium sulfate | + | + |
氨基酸Amino acids | OD600 | PQQ/(mg·L-1) |
---|---|---|
对照 Control | 2.57±0.21 | 106.18±2.35 |
谷氨酸 Glutamic acid | 2.94±0.08 | 117.15±0.56 |
谷氨酰胺 Glutamine | 2.69±0.18 | 106.26±1.25 |
脯氨酸 Proline | 2.91±0.06 | 130.46±0.56 |
Table 3 Effects of amino acid addition on cell growth and PQQ synthesis
氨基酸Amino acids | OD600 | PQQ/(mg·L-1) |
---|---|---|
对照 Control | 2.57±0.21 | 106.18±2.35 |
谷氨酸 Glutamic acid | 2.94±0.08 | 117.15±0.56 |
谷氨酰胺 Glutamine | 2.69±0.18 | 106.26±1.25 |
脯氨酸 Proline | 2.91±0.06 | 130.46±0.56 |
Fig. 4 Identification of pCM66-glyA and pCM66-hps1 strain culture liquid by PCR M:Trans 2k plus II DNA marker.1-2:pCM66- hps1 transformants. 5:pCM66- glyA transformants. 3 and 6:Positive control. 4 and 7:Negative control
菌株Strain | OD600 | PQQ/(mg·L-1) |
---|---|---|
J1-1/pCM66 | 2.63±0.15 | 82.51±1.56 |
J1-1/pCM66-glyA | 2.86±0.18 | 99.48±2.15 |
J1-1/pCM66- hps1 | 2.91±0.09 | 94.58±3.14 |
Table 4 Phenotype analysis of each overexpression strain
菌株Strain | OD600 | PQQ/(mg·L-1) |
---|---|---|
J1-1/pCM66 | 2.63±0.15 | 82.51±1.56 |
J1-1/pCM66-glyA | 2.86±0.18 | 99.48±2.15 |
J1-1/pCM66- hps1 | 2.91±0.09 | 94.58±3.14 |
Fig.6 PCR verification of J1-1△hps2 strain M:Trans 2K plus II DNA marker. 1:pCR verification of hps2 with Up-kan-fragment knockout. 2:Up-kan-fragment PCR verification of J1-1 strain. 3:PCR verification of hps2 strain with kan-Down-fragment knowckout. 4:kan-Down-fragment PCR validation of J1-1 strain. 5:Negative control
菌株Strain | OD600 | PQQ/(mg·L-1) |
---|---|---|
J1-1 | 3.15±0.21 | 130.45±2.35 |
J1-1∆hps2 | 3.46±0.08 | 140.84±3.41 |
Table 5 Phenotype analysis of hps2 gene knockout strain
菌株Strain | OD600 | PQQ/(mg·L-1) |
---|---|---|
J1-1 | 3.15±0.21 | 130.45±2.35 |
J1-1∆hps2 | 3.46±0.08 | 140.84±3.41 |
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