生物技术通报 ›› 2022, Vol. 38 ›› Issue (2): 173-183.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0345
寇航1,2(), 王艳梅1, 李彤2, 薄明井2, 张惟材2, 熊向华2(), 黎明1()
收稿日期:
2021-03-20
出版日期:
2022-02-26
发布日期:
2022-03-09
作者简介:
寇航,男,硕士研究生,研究方向:微生物发酵;E-mail: 基金资助:
KOU Hang1,2(), WANG Yan-mei1, LI Tong2, BO Ming-jing2, ZHANG Wei-cai2, XIONG Xiang-hua2(), LI Ming1()
Received:
2021-03-20
Published:
2022-02-26
Online:
2022-03-09
摘要:
通过构建Methylovorus sp. J1-1基因组尺度代谢网络模型(genome scale of metabolic network model,GSMM),发掘能够提升吡咯喹啉醌(pyrroloquinoline quinone,PQQ)产量的发酵策略和相关靶基因。基于其注释的基因组和生化信息,构建J1-1的GSMM。再通过COBRApy预测可能使PQQ产量提高的氨基酸和潜在的靶基因并进行验证。构建了Methylovorus sp. J1-1的GSMM模型iKH584,共有584个基因,779个生化反应,121个交换反应与765个代谢产物,且可以用于后续模拟。根据iKH584模拟,外源添加谷氨酸、谷氨酰胺和脯氨酸、过表达glyA与hps1以及敲除hps2基因,均具有促进PQQ合成效果。结果表明:添加谷氨酸与脯氨酸时,PQQ的产量分别提高了10.4%与22.9%;过表达基因glyA与hps1,PQQ产量分别提高20.6%与14.6%;敲除基因hps2,PQQ产量最高可达140.84 mg/L,提高了8.0%,这与模拟结果基本一致,表明构建的模型基本正确。最后,在5 L发酵罐进行J1-1△hps2的分批补料发酵,PQQ的产量为812.64 mg/L,比亲本菌株提高了11.1%。建立的模型可以用来指导J1-1的发酵和菌株改造,提高PQQ产量。
寇航, 王艳梅, 李彤, 薄明井, 张惟材, 熊向华, 黎明. 基于Methylovorus sp. J1-1基因组尺度代谢网络优化吡咯喹啉醌合成[J]. 生物技术通报, 2022, 38(2): 173-183.
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 |
表1 本实验所用引物
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 |
图1 基因组代谢网络的反应分布 AM(氨基酸代谢);BOSM(其他次级代谢);CM(碳水化合物代谢);EM(能量代谢);GBM(糖合成代谢);LM(脂质代谢);CV(辅因子和维生素代谢);OAM(其他氨基酸代谢);NM(核苷酸代谢);TP(萜类和多酮类代谢);TS(运输系统);UM(未分类反应)
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 | + | + |
表2 J1-1在不同碳源和氮源上的生长模拟
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 |
表3 氨基酸添加对菌体生长和PQQ合成的影响
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 |
图4 pCM66-glyA与pCM66-hps1菌液PCR鉴定 M:Trans 2k plus II DNA marker;1-2:pCM66- hps1转化子;5:pCM66-gl- yA转化子;3,6:阳性对照;4,7:阴性对照
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 |
表4 过表达菌株表型分析
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 |
图6 J1-1△hps2菌株的PCR验证 M:Trans 2K plus II DNA marker;1:hps2敲除菌-Up-kan-片段PCR验证;2:J1-1菌株Up-kan-片段PCR验证;3:hps2敲除菌-kan-Down-片段PCR验证;4:J1-1菌株kan-Down-片段PCR验证;5:阴性对照
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 |
表5 hps2基因敲除菌株表型分析
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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