Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (11): 227-237.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0280
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GUO Yu-fei(), YAN Rong-mei, ZHANG Xiao-ru, CAO Wei, LIU Hao()
Received:
2022-03-06
Online:
2022-11-26
Published:
2022-12-01
Contact:
LIU Hao
E-mail:guoyufeis@163.com;liuhao@tust.edu.cn
GUO Yu-fei, YAN Rong-mei, ZHANG Xiao-ru, CAO Wei, LIU Hao. Metabolic Engineering Modification of Aspergillus niger for the Production of D-glucaric Acid[J]. Biotechnology Bulletin, 2022, 38(11): 227-237.
年份Year | 菌株Strain | 基因改造Genes madified | 碳源Carbon source | 产量Yield/(g·L-1) | 引用文献Reference |
---|---|---|---|---|---|
2009 | E. coli BL21 Star(DE3) | ino1,MIOX,udh | Glucose | 1.13 | [ |
2010 | E. coli BL21 Star(DE3) | ino1,MIOX,udh | Glucose | 2.50 | [ |
2014 | E. coli MG1655 | ino1,SUMO-MIOX,udh | Myo-inositol | 4.85 | [ |
2016 | S. cerevisiae CEN. PK2-1D | ino1,inm,MIOX,udh | Glucose | 1.60 | [ |
2017 | E. coli L19S | ino1,MIOX,udh | Glucose | 0.80 | [ |
2017 | S. cerevisiae CEN. PK2-1C | MIOX,udh,ino1,pfk1 | Glucose | 0.23 | [ |
2018 | E. coli BL21 Star(DE3) | cscB,cscA,cscK,ino1, MIOX,udh,suhB | Sucrose | 1.42 | [ |
2018 | Pichia pastoris GS115 | MIOX,udh | Glucose | 2.60 | [ |
2018 | S. cerevisiae BY4741 | ino1,MIOX,udh | Glucose,myo-inositol | 6.00 | [ |
2020 | E. coli BL21 Star(DE3) | ino1,MIOX,udh,suhB | Glucose | 5.35 | [ |
2020 | S. cerevisiae | ino1,inm,MIOX,udh,vgb | Glucose | 6.38 | [ |
Table 1 Research progress of producing gluconic acid by microbial fermentation
年份Year | 菌株Strain | 基因改造Genes madified | 碳源Carbon source | 产量Yield/(g·L-1) | 引用文献Reference |
---|---|---|---|---|---|
2009 | E. coli BL21 Star(DE3) | ino1,MIOX,udh | Glucose | 1.13 | [ |
2010 | E. coli BL21 Star(DE3) | ino1,MIOX,udh | Glucose | 2.50 | [ |
2014 | E. coli MG1655 | ino1,SUMO-MIOX,udh | Myo-inositol | 4.85 | [ |
2016 | S. cerevisiae CEN. PK2-1D | ino1,inm,MIOX,udh | Glucose | 1.60 | [ |
2017 | E. coli L19S | ino1,MIOX,udh | Glucose | 0.80 | [ |
2017 | S. cerevisiae CEN. PK2-1C | MIOX,udh,ino1,pfk1 | Glucose | 0.23 | [ |
2018 | E. coli BL21 Star(DE3) | cscB,cscA,cscK,ino1, MIOX,udh,suhB | Sucrose | 1.42 | [ |
2018 | Pichia pastoris GS115 | MIOX,udh | Glucose | 2.60 | [ |
2018 | S. cerevisiae BY4741 | ino1,MIOX,udh | Glucose,myo-inositol | 6.00 | [ |
2020 | E. coli BL21 Star(DE3) | ino1,MIOX,udh,suhB | Glucose | 5.35 | [ |
2020 | S. cerevisiae | ino1,inm,MIOX,udh,vgb | Glucose | 6.38 | [ |
菌株Strain | 特征描述Characteristics | 来源Source |
---|---|---|
Escherichia coli JM109 | - | 本实验室保存 |
Agrobacterium tumerfaciens AGL-1 | - | 本实验室保存 |
S834 | A.niger ATCC1015,Tet-on::cre,ΔoahA,ΔcexA | 本实验室保存 |
S985 | ΔoahA,ΔcexA,OEppudh | 本研究 |
S1223 | ΔoahA,ΔcexA,OEppudh,OEanmioxA | 本研究 |
S1877 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1 | 本研究 |
S2086 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1,OEaninoA | 本研究 |
S2390 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1,OEaninoA,OEllnox | 本研究 |
S2603 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1,OEaninoA,OEllnox,RNAipfkA | 本研究 |
S2635 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1,OEaninoA,OEllnox,RNAipfkA,RNAizwf | 本研究 |
Table 2 Strains used in this study
菌株Strain | 特征描述Characteristics | 来源Source |
---|---|---|
Escherichia coli JM109 | - | 本实验室保存 |
Agrobacterium tumerfaciens AGL-1 | - | 本实验室保存 |
S834 | A.niger ATCC1015,Tet-on::cre,ΔoahA,ΔcexA | 本实验室保存 |
S985 | ΔoahA,ΔcexA,OEppudh | 本研究 |
S1223 | ΔoahA,ΔcexA,OEppudh,OEanmioxA | 本研究 |
S1877 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1 | 本研究 |
S2086 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1,OEaninoA | 本研究 |
S2390 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1,OEaninoA,OEllnox | 本研究 |
S2603 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1,OEaninoA,OEllnox,RNAipfkA | 本研究 |
S2635 | ΔoahA,ΔcexA,OEppudh,OEanmioxA,OEscJEN1,OEaninoA,OEllnox,RNAipfkA,RNAizwf | 本研究 |
质粒Plasmid | 特征描述Characteristics | 来源Source |
---|---|---|
pLH454 | loxP-hph-loxP,gpdA promoter,trpC terminator,hygr,kanr | 本实验室保存 |
pLH509 | loxP-hph-loxP,pkiA promoter,trpC terminator,hygr,kanr | 本实验室保存 |
pLH756 | loxP-hph-loxP,mbfA promoter,trpC terminator,hygr,kanr | 本实验室保存 |
pLH1418 | loxP-hph-loxP,glaA promoter,gfp loop,trpC terminator,hygr,kanr | 本实验室保存 |
pLH1453 | loxP-hph-loxP,pkiA promoter,gfp loop,trpC terminator,hygr,kanr | 本实验室保存 |
pLH690 | loxP-hph-loxP,gpdA promoter,ppudh gene,trpC terminator,hygr,kanr | 本研究 |
pLH737 | loxP-hph-loxP,pkiA promoter,anmioxA gene,trpC terminator,hygr,kanr | 本研究 |
pLH782 | loxP-hph-loxP,gpdA promoter,scJEN1 gene,trpC terminator,hygr,kanr | 本研究 |
pLH1020 | loxP-hph-loxP,gpdA promoter,aninoA gene,trpC terminator,hygr,kanr | 本研究 |
pLH1317 | loxP-hph-loxP,mbfA promoter,llnox gene,trpC terminator,hygr,kanr | 本研究 |
pLH1421 | loxP-hph-loxP,glaA promoter,sense sequence of zwf gene,gfp loop,antisense sequence of zwf gene,trpC terminator,hygr,kanr | 本研究 |
pLH1454 | loxP-hph-loxP,pkiA promoter,sense sequence of pfkA gene,gfp loop,antisense sequence of pfkA gene,trpC terminator,hygr,kanr | 本研究 |
Table 3 Plasmids used in this study
质粒Plasmid | 特征描述Characteristics | 来源Source |
---|---|---|
pLH454 | loxP-hph-loxP,gpdA promoter,trpC terminator,hygr,kanr | 本实验室保存 |
pLH509 | loxP-hph-loxP,pkiA promoter,trpC terminator,hygr,kanr | 本实验室保存 |
pLH756 | loxP-hph-loxP,mbfA promoter,trpC terminator,hygr,kanr | 本实验室保存 |
pLH1418 | loxP-hph-loxP,glaA promoter,gfp loop,trpC terminator,hygr,kanr | 本实验室保存 |
pLH1453 | loxP-hph-loxP,pkiA promoter,gfp loop,trpC terminator,hygr,kanr | 本实验室保存 |
pLH690 | loxP-hph-loxP,gpdA promoter,ppudh gene,trpC terminator,hygr,kanr | 本研究 |
pLH737 | loxP-hph-loxP,pkiA promoter,anmioxA gene,trpC terminator,hygr,kanr | 本研究 |
pLH782 | loxP-hph-loxP,gpdA promoter,scJEN1 gene,trpC terminator,hygr,kanr | 本研究 |
pLH1020 | loxP-hph-loxP,gpdA promoter,aninoA gene,trpC terminator,hygr,kanr | 本研究 |
pLH1317 | loxP-hph-loxP,mbfA promoter,llnox gene,trpC terminator,hygr,kanr | 本研究 |
pLH1421 | loxP-hph-loxP,glaA promoter,sense sequence of zwf gene,gfp loop,antisense sequence of zwf gene,trpC terminator,hygr,kanr | 本研究 |
pLH1454 | loxP-hph-loxP,pkiA promoter,sense sequence of pfkA gene,gfp loop,antisense sequence of pfkA gene,trpC terminator,hygr,kanr | 本研究 |
引物Primer | 序列Sequence(5'-3') |
---|---|
Primers related to ppudh gene | |
YZ-ppudh-F | ACCACCACCCCCTTCAAT |
YZ-ppudh-R | TCAGTGATGGTGGTGATGGT |
qPCR-ppudh-F | GCAACCACACCATCGGCTTCTAC |
qPCR-ppudh-R | CCATAGCGGTCGAAGTAGAAGGAGG |
Primers related to anmioxA gene | |
anmioxA-F | ATCAATCATCCGTCAAGATGGAATTCGCACCAGTCGCTGTGTCTCCT |
anmioxA-R | CCAGATCTCTGCAGGGTACCGAGCTCCTACCACTTGATCACCTTGTTGG |
qPCR-anmioxA-F | GAAGCTCAACACCCTCATCGACG |
qPCR-anmioxA-R | ATGAGACCGGTCAGCTGCATC |
Primers related to scJEN1 gene | |
YZ-scJEN1-F | ATGTCCTCCAGCATTACCGAT |
YZ-scJEN1-R | GACCTTGGCGTAATCTTCCTT |
qPCR-scJEN1-F | GACCACGAGAAGCTGTACCATAACCC |
qPCR-scJEN1-R | GGGCTTATCTTCTTCGTCCTCCTCG |
Primers related to aninoA gene | |
aninoA-F | CACATCTAAACAATGGAATTCATGGCTCCCCACGCAAGC |
aninoA-R | AGTGGATCCCTGCAGGGTACCCTAGAACAGCTTGTGCTCCAG |
qPCR-aninoA-F | GGCCACCAACTACCACTTCAAGG |
qPCR-aninoA-R | CCACAGAGCCGTAGTAGTTGGAGG |
Primers related to llnox gene | |
YZ-llnox-F | TCATCGGCACCAACCACG |
YZ-llnox-R | ACGGTCATGTAGTTGTAGGGG |
qPCR-llnox-F | ATCTGGTCATCAACTGCATCGG |
qPCR-llnox-R | CCAGGGCGATGTAGGTGAAATC |
Primers related to zwf gene | |
zwf-S-F | TACACCTCAGCAATGGAGCTCAGAAGGAGCAGAACAGAATCT |
zwf-S-R | GCTCCTGGAAGATCTGAGCTCATCCAGAGACTTTCCGTACT |
zwf-A-F | CGCATCGAGCTGAAGGGTACCATCCAGAGACTTTCCGTACT |
zwf-A-R | AAGTGGATCGCATGCGGTACCAGAAGGAGCAGAACAGAATCT |
YZ-PglaA-F | ACCTGCGTTATAGCTTCCCG |
YZ-gfp-F | AGGAGCGCACCATCTTCTTC |
YZ-gfp-R | CTCGATGCGGTTCACCAGG |
YZ-TtrpC-R | CTCCGGAGCTGACATCGAC |
qPCR-zwf-F | GAACGAGAGGTGGGACGGTG |
qPCR-zwf-R | CAGGCAGCTTGGAGTTCATCTTG |
Primers related to pfkA gene | |
pfkA-S-F | ATCAATCATCCGTCAAGATGGAATTCACTATGATGACACCCCGATCT |
pfkA-S-R | GGTGCGCTCCTGGAGGTACCGAGCTCCAAAGTTGTCACGCAGGAAGT |
pfkA-A-F | TGAACCGCATCGAGCTGAAGCTGCAGCAAAGTTGTCACGCAGGAAGT |
pfkA-A-R | TGATTTCAGTAACGTTAAGTCTGCAGACTATGATGACACCCCGATCT |
YZ-PpkiA-F | GCGAGGAGAAAATTCAGCACC |
YZ-gfp-F | AGGAGCGCACCATCTTCTTC |
YZ-gfp-R | CTCGATGCGGTTCACCAGG |
YZ-TtrpC-R | CTCCGGAGCTGACATCGAC |
qPCR-pfkA-F | ACCATTTCCAACAACGTGCCG |
qPCR-pfkA-R | TGTCACGCAGGAAGTCAATGTCG |
Primers related to actA gene | |
qPCR-actA-F | TCCTCACCCTCAGATACCC |
qPCR-actA-R | CACCGTCACCAGAGTCCA |
Table 4 Primers used in this study
引物Primer | 序列Sequence(5'-3') |
---|---|
Primers related to ppudh gene | |
YZ-ppudh-F | ACCACCACCCCCTTCAAT |
YZ-ppudh-R | TCAGTGATGGTGGTGATGGT |
qPCR-ppudh-F | GCAACCACACCATCGGCTTCTAC |
qPCR-ppudh-R | CCATAGCGGTCGAAGTAGAAGGAGG |
Primers related to anmioxA gene | |
anmioxA-F | ATCAATCATCCGTCAAGATGGAATTCGCACCAGTCGCTGTGTCTCCT |
anmioxA-R | CCAGATCTCTGCAGGGTACCGAGCTCCTACCACTTGATCACCTTGTTGG |
qPCR-anmioxA-F | GAAGCTCAACACCCTCATCGACG |
qPCR-anmioxA-R | ATGAGACCGGTCAGCTGCATC |
Primers related to scJEN1 gene | |
YZ-scJEN1-F | ATGTCCTCCAGCATTACCGAT |
YZ-scJEN1-R | GACCTTGGCGTAATCTTCCTT |
qPCR-scJEN1-F | GACCACGAGAAGCTGTACCATAACCC |
qPCR-scJEN1-R | GGGCTTATCTTCTTCGTCCTCCTCG |
Primers related to aninoA gene | |
aninoA-F | CACATCTAAACAATGGAATTCATGGCTCCCCACGCAAGC |
aninoA-R | AGTGGATCCCTGCAGGGTACCCTAGAACAGCTTGTGCTCCAG |
qPCR-aninoA-F | GGCCACCAACTACCACTTCAAGG |
qPCR-aninoA-R | CCACAGAGCCGTAGTAGTTGGAGG |
Primers related to llnox gene | |
YZ-llnox-F | TCATCGGCACCAACCACG |
YZ-llnox-R | ACGGTCATGTAGTTGTAGGGG |
qPCR-llnox-F | ATCTGGTCATCAACTGCATCGG |
qPCR-llnox-R | CCAGGGCGATGTAGGTGAAATC |
Primers related to zwf gene | |
zwf-S-F | TACACCTCAGCAATGGAGCTCAGAAGGAGCAGAACAGAATCT |
zwf-S-R | GCTCCTGGAAGATCTGAGCTCATCCAGAGACTTTCCGTACT |
zwf-A-F | CGCATCGAGCTGAAGGGTACCATCCAGAGACTTTCCGTACT |
zwf-A-R | AAGTGGATCGCATGCGGTACCAGAAGGAGCAGAACAGAATCT |
YZ-PglaA-F | ACCTGCGTTATAGCTTCCCG |
YZ-gfp-F | AGGAGCGCACCATCTTCTTC |
YZ-gfp-R | CTCGATGCGGTTCACCAGG |
YZ-TtrpC-R | CTCCGGAGCTGACATCGAC |
qPCR-zwf-F | GAACGAGAGGTGGGACGGTG |
qPCR-zwf-R | CAGGCAGCTTGGAGTTCATCTTG |
Primers related to pfkA gene | |
pfkA-S-F | ATCAATCATCCGTCAAGATGGAATTCACTATGATGACACCCCGATCT |
pfkA-S-R | GGTGCGCTCCTGGAGGTACCGAGCTCCAAAGTTGTCACGCAGGAAGT |
pfkA-A-F | TGAACCGCATCGAGCTGAAGCTGCAGCAAAGTTGTCACGCAGGAAGT |
pfkA-A-R | TGATTTCAGTAACGTTAAGTCTGCAGACTATGATGACACCCCGATCT |
YZ-PpkiA-F | GCGAGGAGAAAATTCAGCACC |
YZ-gfp-F | AGGAGCGCACCATCTTCTTC |
YZ-gfp-R | CTCGATGCGGTTCACCAGG |
YZ-TtrpC-R | CTCCGGAGCTGACATCGAC |
qPCR-pfkA-F | ACCATTTCCAACAACGTGCCG |
qPCR-pfkA-R | TGTCACGCAGGAAGTCAATGTCG |
Primers related to actA gene | |
qPCR-actA-F | TCCTCACCCTCAGATACCC |
qPCR-actA-R | CACCGTCACCAGAGTCCA |
Fig. 2 Effect of ppudh gene expression on glucaric acid production A:HPLC chromatogram of 100 mg/L glucaric acid standard and different strains fermentation. B:Glucaric acid yield of the modified strain
Fig. 3 Effect of anmioxA gene expression on glucaric acid production A:The relative expression of anmioxA gene in the modified strain. B:Glucaric acid yield of the modified strain
Fig. 4 Effect of scJEN1 gene expression on glucaric acid production A:Expression of scJEN1 gene in the modified strain. B:Glucaric acid yield of the modified strain
Fig. 5 Effect of aninoA gene expression on glucaric acid production A:Relative expression of aninoA gene in the modified strain. B:Glucaric acid yield of the modified strain
Fig. 6 Effects of llnox gene expression on glucaric acid production A:Expression of llnox gene in the modified strain;B:Glucaric acid yield of the modified strain
Fig. 7 Effect of interfering pfkA and zwf gene on glucaric acid production A:Branch metabolism of glucose-6-phosphate(Red arrows indicate weakened expression and green bold arrows indicate enhanced expression). B:Glucaric acid yield of the modified strain
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