Effects on the Biosynthesis of Ethanol by Promoters PpetE and Pcpc560 in Synechocystis sp. PCC 6803

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

Abstract

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

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.

Figures/Tables 12

References 25

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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

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

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

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

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