启动子PpetE与Pcpc560对集胞藻PCC 6803生物合成乙醇的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0241

摘要/Abstract

摘要：

为了增加工程集胞藻PCC 6803的乙醇合成产量,通过选用强启动子Pcpc560 驱动并提高外源乙醇合成基因（pdc,yqhD）的表达,从而促进乙醇的生产。具体方法利用同源双交换引入来源于运动型发酵单胞菌的丙酮酸脱羧酶基因（pdc）与来源于大肠杆菌的NADPH依赖型醛还原酶基因（yqhD）并选用不同的启动子来驱动其表达。通过逆转录定量PCR分析,比较在不同启动子驱动的情况下,外源乙醇合成基因（pdc,yqhD）的表达情况并检测相应突变株的乙醇产量。结果显示相较于中等启动子,铜离子诱导启动子PpetE,来源于集胞藻PCC 6803的光强启动子Pcpc560显著促进了外源乙醇合成基因（pdc,yqhD）的表达,并增加了工程菌株乙醇合成的产量。超强启动子Pcpc560搭配pdc,yqhD的组合表达,显著提高了工程菌株的乙醇合成产量。

关键词: 乙醇, 启动子, 集胞藻PCC 6803, 合成, 逆转录

Abstract:

To increase the synthesized ethanol yield of engineered Synechocystis sp. PCC 6803,the strong promoter Pcpc560 was selected to drive and enhance the expression of exogenous ethanol-producing genes（pdc,and yqhD）,thus ethanol yield was enhanced. The specific methods were that by homologous double exchange,the pyruvate decarboxylase gene（pdc）derived from Zymomonas mobilis and NADPH-dependent aldehyde reductase（yqhD）derived from Escherichia coli were introduced and driven by different promoters. By reverse transcription quantitative PCR analysis,the expressions of exogenous ethanol-producing genes（pdc and yqhD）and the ethanol yields of corresponding engineered strains under the driven of different promoters were detected and compared. The results showed that the light intensity promoter,Pcpc560 derived from the Synechocystis sp. PCC 6803 significantly enhanced the expressions of the exogenous ethanol-producing genes（pdc and yqhD）and increased synthesized ethanol output compared with the medium copper ion-induced promoter PpetE. The combined expression of super-strong promoter Pcpc560 with pdc and yqhD has significantly improved the ethanol production of the engineered strain.

Key words: ethanol, promoter, Synechocystis sp. PCC 6803, synthesis, reverse transcription

叶鹏林, KwasiKyere-Yeboah, 高恶斌. 启动子PpetE与Pcpc560对集胞藻PCC 6803生物合成乙醇的影响[J]. 生物技术通报, 2022, 38(2): 141-149.

YE Peng-lin, Kwasi Kyere-Yeboah, GAO E-bin. Effects on the Biosynthesis of Ethanol by Promoters PpetE and Pcpc560 in Synechocystis sp. PCC 6803[J]. Biotechnology Bulletin, 2022, 38(2): 141-149.

图/表 12

图1 集胞藻PCC 6803的乙醇合成路线 PEP：Phosphoenolpyruvate（磷酸烯醇丙酮酸）,Pyruvate：丙酮酸,Acetal-dehyde：乙醛,Ethanol：乙醇,CBB cycle：卡尔文循环,RuBP：1,5-diphosphate ribulose（1,5-二磷酸核酮糖）,G3P：glyceraldehyde-3-phosphate（三磷酸甘油醛）,PGA：phosphoglyceric acid（磷酸甘油酸）,F6P：fructose 6 Phosphate（磷酸果糖）,NADP：烟酰胺腺嘌呤二核苷酸磷酸,NADPH：还原型烟酰胺腺嘌呤二核苷酸磷酸,pdc：丙酮酸脱羧酶基因,yqhD：NADPH依赖型醛还原酶基因

Fig.1 Ethanol synthesis route in Synechocystis sp. PCC 6803

表1 本文使用的质粒

Table 1 Plasmids used in this study

名称 Name	特点 Characteristic
pMD18-T	氨苄霉素/Amp^R
pMD0168	氨苄霉素/Amp^R将slr0168的上下游600 bp基因扩增并克隆到质粒pMD18-T的SphI/MluI和XbaI/KpnI位点
pBE406	壮观霉素/ Sp^R将壮观霉素基因扩增并克隆到所构建质粒pMD0168的XhoI/XbaI位点
pBE407	壮观霉素/ Sp^R将启动子PpetE扩增并克隆到所构建质粒pBE406的BamHI/SalI位点,将终止子TrbcL扩增并克隆到所构建质粒pBE406的SalI/HindIII位点
pBE407-pdc	壮观霉素/ Sp^Rpdc基因被扩增并和质粒pBE407双酶切,通过NdeI/BamHI位点相连接获得质粒pBE407-pdc
pBE01	壮观霉素/ Sp^RyqhD 基因被扩增并和质粒pBE407-pdc双酶切,通过XbaI/KpnI位点相连接获得质粒pBE01
pBE408	壮观霉素/ Sp^R将启动子Pcpc560扩增并克隆到所构建质粒pBE406的BamHI/SalI位点,将终止子TrbcL扩增并克隆到所构建质粒pBE406的SalI/HindIII位点
pBE408-pdc	壮观霉素/ Sp^Rpdc基因被扩增并和质粒pBE408双酶切,通过NdeI/BamHI位点相连接获得质粒pBE408-pdc
pBE02	壮观霉素/ Sp^RyqhD 基因被扩增并和质粒pBE408-pdc双酶切,通过XbaI/KpnI位点相连接获得质粒pBE02

表2 本文使用的引物

Table 2 Primers used in this study

引物序列Sequence of a primer	大小Size
SP-F：5'-CCACGCGTAAGCTTGGATCCGCTCACGCAACTGGTCCAGAA-3' SP-R：5'-CGGGAGCTCGAATTCTAGAGTGCTTAGTGCATCTAACGC-3'	1.1 kb
Pcpc-F：5'-CGTCTAGAGGATCCCCTGTAGAGAAGAGTCCCTG-3' Pcpc-R：5'-TTTCTCCTCTTTTGAATTAATCTCCTACTTGACTTTATGAG-3'	550 bp
PetE-F：5'-GCTCTAGACAAGGATTCATAGCGGTTGCCCAATC-3' PetE-R：5'-GCTGCCTAGGATTCTGGCGAAAGGGGGATGTG-3'	200 bp
TrbcL-F：5'-CGCGTCGACCGGTGTTTGGATTGTCGGAGT-3' TrbcL-R：5'-CCGACGCGTAAGCTTCCGGTAATTGGTAAATTGCTGTC-3'	250 bp
Pdc-F：5'-CCGAGATCTCATATGTCCTACACCGTGGGCACCT-3' Pdc-R：5'-CGCGGATCCTGCAGCTCGAGTCTAGATTACAACAATTTGTTCACGGGT-3'	1.7 kb
YqhD-F：5'-CAAACTCGAGTCTAGATGAACAACTTTAACTTGCACACCCCCAC-3' YqhD-R：5'-CGGGGTACCTGCAGTTAGCGGGCGGCTTCGTATATACGGC-3'	1.2 kb
slr0168Up-F：5'-GGCATGCCGAGCGGCACCACGGGGCACCACCGC-3' slr0168Up-R：5'-GACGCGTCGGCGCACAGCAGCGTGCGACGTGTG-3'	600 bp
slr0168Dw-F：5'-CTCTAGAGTGCCACTACCTGGCGTGCCGCTACC-3' slr0168Dw-R：5'-GGGGTACCCCGCATGACCAGCTGCCGCCCCAGC-3'	600 bp

图2 SYN01菌株的构建示意图与PCR分析 M：DNA Marker,A：提取的转化子基因组DNA,W：提取的野生型集胞藻DNA,1：slr0168上游基因上游引物slr0168Up-F,2：pdc基因上游引物Pdc-F,3：pdc基因下游引物Pdc-R,4：yqhD基因上游引物YqhD-F,5：yqhD基因下游引物YqhD-R,6：slr0168下游基因下游引物slr0168DW-R,16S：扩增编码16S rRNA的内源基因（600 bp）,1+3：使用引物1和引物3进行扩增（3.6 kb）,2+5：使用引物2和引物5进行扩增（2.9 kb）,4+6：使用引物4和引物6进行扩增（2 kb）

Fig.2 Construction diagram and PCR analysis of the SYN01 strain M：DNA marker. A：Extracted DNA from transformant genome. W：Extracted DNA from wild-type Synechocystis. 1：slr0168 upstream gene upstream primer slr0168Up-F. 2：pdc gene upstream primer Pdc-F. 3：pdc gene downstream primer Pdc-R. 4：yqhD gene upstream primer YqhD-F. 5：yqhD gene downstream primer YqhD-R. 6：slr0168 downstream gene downstream primer slr0168DW-R. 16S：Amplify the endogenous gene encoding 16S rRNA（600 bp）. 1+3：Using primer 1 and primer 3 for amplification（3.6 kb）. 2+5：Using primer 2 and primer 5 for amplification（2.9 kb）. 4+6：Using primer 4 and primer 6 for amplification（2 kb）

图3 SYN02菌株的构建示意图与PCR分析 M：DNA Marker,A：提取的转化子基因组DNA,W：提取的野生型集胞藻DNA,1：slr0168上游基因上游引物slr0168Up-F,2：pdc基因上游引物Pdc-F,3：pdc基因下游引物Pdc-R,4：yqhD基因上游引物YqhD-F,5：yqhD基因下游引物YqhD-R,6：slr0168下游基因下游引物slr0168DW-R,16S：扩增编码16S rRNA的内源基因（600 bp）,1+3：使用引物1和引物3进行扩增（3.9 kb）,2+5：使用引物2和引物5进行扩增（2.9 kb）,4+6：使用引物4和引物6进行扩增（2 kb）

Fig.3 Construction diagram and PCR analysis of the SYN02 strain M：DNA marker. A：Extracted DNA from transformant genome. W：Extracted DNA from wild-type Synechocystis. 1：slr0168 upstream gene upstream primer slr0168Up-F. 2：pdc gene upstream primer Pdc-F. 3：pdc gene downstream primer Pdc-R. 4：yqhD gene upstream primer YqhD-F. 5：yqhD gene downstream primer YqhD-R. 6：slr0168 downstream gene downstream primer slr0168DW-R. 16S：Amplify the endogenous gene encoding 16S rRNA（600 bp）. 1+3：Using primer 1 and primer 3 for amplification（3.9 kb）. 2+5：Using primer 2 and primer 5 for amplification（2.9 kb）. 4+6：Using primer 4 and primer 6 for amplification（2 kb）

表3 逆转录定量PCR的结果

Table 3 Reverse transcription of quantitative PCR results

基因Gene	菌株Strain	16S	CT₁	CT₂	CT₃	平均 CT Average CT	∆CT	2^-∆∆CT	标准偏差SD
Pdc	SYN01	22.85	22.97	22.90	22.98	22.95	0.10	2.55	0.0252
Pdc	SYN02	22.85	21.60	21.57	21.63	21.60	-1.25	2.55	0.0173
YqhD	SYN01	22.85	23.76	23.72	24.04	23.84	0.99	2.31	0.1007
YqhD	SYN02	22.85	22.60	22.66	22.63	22.63	-0.22	2.31	0.0173

图4 外源乙醇合成基因的表达情况

Fig.4 Expressions of exogenous ethanol-producing genes

图5 乙醇的液相色谱检测

Fig.5 HPLC detection of ethanol

图6 乙醇的生产情况

Fig.6 Production of ethanol under the autotrophic conditions

图7 自养条件下菌株的生长情况

Fig.7 Growth condition of the strain under the autotrophic conditions

图8 优化培养下菌株的生长情况实线为通入5% CO2+95%空气,虚线为通入100%空气,下同

Fig.8 Growth condition of the strains under the optimized culture The solid line refers to 5% CO2+95% air,and the dashed line refers to 100% air. The same below

图9 优化培养下菌株的乙醇生产情况

Fig.9 Ethanol production of the strains under the optim-ized culture

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