敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因对生物合成乙醇的影响

doi:10.19586/j.2095-2341.2021.0058

生物技术进展 ›› 2022, Vol. 12 ›› Issue (1): 105-111.DOI: 10.19586/j.2095-2341.2021.0058

敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因对生物合成乙醇的影响

许慧露¹^,²(), 徐岷¹, 张炜¹()

^1.江苏大学附属医院，江苏镇江 212000
^2.江苏大学医学院，江苏镇江 212000

收稿日期:2021-04-18 接受日期:2021-06-01 出版日期:2022-01-25 发布日期:2022-01-26
通讯作者: 张炜
作者简介:许慧露E-mail：xhl933600@163.com；
基金资助:
江苏省重点研发计划项目(BE2018689)

The Effect of Deleting the slr1556 Gene Encoding Lactate Dehydrogenase on Ethanol Biosynthesize in Synechocystis sp. PCC 6803

Huilu XU¹^,²(), Min XU¹, Wei ZHANG¹()

^1.The Affiliated Hospital of Jiangsu University，Jiangsu Zhenjiang 212000，China
^2.School of Medicine，Jiangsu University，Jiangsu Zhenjiang 212000，China

Received:2021-04-18 Accepted:2021-06-01 Online:2022-01-25 Published:2022-01-26
Contact: Wei ZHANG

摘要/Abstract

摘要：

探究在集胞藻PCC 6803中引入外源乙醇合成基因并敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因对生物合成乙醇的影响。在集胞藻PCC 6803中引入来源于运动型发酵单胞菌的丙酮酸脱羧酶基因（pdc）与大肠杆菌的NADPH依赖型醛还原酶基因（yqhD）光强启动子PrbcL的驱动下组合表达，生物合成乙醇。在此基础上进一步敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因，以提高乙醇合成前体丙酮酸含量，促进乙醇的生产。结果显示敲除slr1556基因可以提高丙酮酸含量并显著增加乙醇的产量。竞争性丙酮酸转化乳酸代谢途径的阻断可以有效促进丙酮酸的累积，进而促进乙醇的生产。

关键词: 乙醇, 乳酸, 集胞藻PCC 6803, 前体, 丙酮酸

Abstract:

To explore the effect of ethanol synthesize in Synechocystis sp. PCC 6803， the exogenous ethanol-producing genes were introduced and the competitive metabolic pathway of pyruvate to lactate was deleted. The pyruvate decarboxylase gene （pdc） from Synechocystis sp. PCC 6803 and the NADPH-dependent aldehyde reductase gene （yqhD） from Escherichia coli in this study were introduced and combined for expression under the drove of the light intensity promoter PrbcL to synthesize ethanol in Synechocystis sp. PCC 6803. Based of this， the slr1556 gene encoding lactate dehydrogenase in Synechocystis sp. PCC6803 was further knocked out to increase the content of pyruvate， the precursor for ethanol synthesis and promote the ethanol production. The results showed that by knocking out the slr1556 gene， the content of pyruvate was improved and the synthesized ethanol output was significantly increased. The block of the competitive metabolic pathway of the conversion of pyruvate to lactic acid can effectively promote the accumulation of pyruvate and then promote the production of ethanol.

Key words: ethanol, lactate, Synechocystis sp. PCC 6803, precursor, pyruvate

中图分类号:

Q943.2

许慧露, 徐岷, 张炜. 敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因对生物合成乙醇的影响[J]. 生物技术进展, 2022, 12(1): 105-111.

Huilu XU, Min XU, Wei ZHANG. The Effect of Deleting the slr1556 Gene Encoding Lactate Dehydrogenase on Ethanol Biosynthesize in Synechocystis sp. PCC 6803[J]. Current Biotechnology, 2022, 12(1): 105-111.

图/表 8

参考文献 23

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引物名称	引物序列（5'→3'）	大小/bp
SP⁃F	5'⁃CCACGCGTAAGCTTGGATCCGCTCACGCAACTGGTCCAGAA⁃3'	1 100
SP⁃R	5'⁃CGGGAGCTCGAATTCTAGAGTGCTTAGTGCATCTAACGC⁃3'	1 100
Prbc⁃F	5'⁃GCCTCGAGTTGGCTCTAGATCACCATTTGGACAAAACATCAGG⁃3'	200
Prbc⁃R	5'⁃CCGAGATCTGGTACCGCGGATCCATATGCATGCACGCGT⁃3'	200
TrbcL⁃F	5'⁃CGCGTCGACCGGTGTTTGGATTGTCGGAGT⁃3'	250
TrbcL⁃R	5'⁃CCGACGCGTAAGCTTCCGGTAATTGGTAAATTGCTGTC⁃3'	250
Pdc⁃F	5'⁃CCGAGATCTCATATGTCCTACACCGTGGGCACCT⁃3'	1 700
Pdc⁃R	5'⁃CGCGGATCCTGCAGCTCGAGTCTAGATTACAACAATTTGTTCACGGGT⁃3'	1 700
YqhD⁃F	5'⁃CAAACTCGAGTCTAGATGAACAACTTTAACTTGCACACCCCCAC⁃3'	1 200
YqhD⁃R	5'⁃CGGGGTACCTGCAGTTAGCGGGCGGCTTCGTATATACGGC⁃3'	1 200
slr1556Up⁃F	5'⁃ACATGCATGCCAGTTAGCCTAGGCAGTGGTTC⁃3'	600
slr1556Up⁃R	5'⁃CGACGCGTCGACCGCCCCCTAAATCCCCCAATA⁃3'	600
slr1556Dw⁃F	5'⁃CTAGTCTAGAGTCGACCATTGCCGCAACGACACTG⁃3'	600
slr1556Dw⁃R	5'⁃CGAGCTCGGTACCACCAAAGAGTTATTGGCGGCTA⁃3'	600
slr0168Up⁃F	5'⁃GGCATGCCGAGCGGCACCACGGGGCACCACCGC⁃3'	600
slr0168Up⁃R	5'⁃GACGCGTCGGCGCACAGCAGCGTGCGACGTGTG⁃3'	600
slr0168Dw⁃F	5'⁃CTCTAGAGTGCCACTACCTGGCGTGCCGCTACC⁃3'	600
slr0168Dw⁃R	5'⁃GGGGTACCCCGCATGACCAGCTGCCGCCCCAGC⁃3'	600

引物名称	引物序列（5'→3'）	大小/bp
SP⁃F	5'⁃CCACGCGTAAGCTTGGATCCGCTCACGCAACTGGTCCAGAA⁃3'	1 100
SP⁃R	5'⁃CGGGAGCTCGAATTCTAGAGTGCTTAGTGCATCTAACGC⁃3'	1 100
Prbc⁃F	5'⁃GCCTCGAGTTGGCTCTAGATCACCATTTGGACAAAACATCAGG⁃3'	200
Prbc⁃R	5'⁃CCGAGATCTGGTACCGCGGATCCATATGCATGCACGCGT⁃3'	200
TrbcL⁃F	5'⁃CGCGTCGACCGGTGTTTGGATTGTCGGAGT⁃3'	250
TrbcL⁃R	5'⁃CCGACGCGTAAGCTTCCGGTAATTGGTAAATTGCTGTC⁃3'	250
Pdc⁃F	5'⁃CCGAGATCTCATATGTCCTACACCGTGGGCACCT⁃3'	1 700
Pdc⁃R	5'⁃CGCGGATCCTGCAGCTCGAGTCTAGATTACAACAATTTGTTCACGGGT⁃3'	1 700
YqhD⁃F	5'⁃CAAACTCGAGTCTAGATGAACAACTTTAACTTGCACACCCCCAC⁃3'	1 200
YqhD⁃R	5'⁃CGGGGTACCTGCAGTTAGCGGGCGGCTTCGTATATACGGC⁃3'	1 200
slr1556Up⁃F	5'⁃ACATGCATGCCAGTTAGCCTAGGCAGTGGTTC⁃3'	600
slr1556Up⁃R	5'⁃CGACGCGTCGACCGCCCCCTAAATCCCCCAATA⁃3'	600
slr1556Dw⁃F	5'⁃CTAGTCTAGAGTCGACCATTGCCGCAACGACACTG⁃3'	600
slr1556Dw⁃R	5'⁃CGAGCTCGGTACCACCAAAGAGTTATTGGCGGCTA⁃3'	600
slr0168Up⁃F	5'⁃GGCATGCCGAGCGGCACCACGGGGCACCACCGC⁃3'	600
slr0168Up⁃R	5'⁃GACGCGTCGGCGCACAGCAGCGTGCGACGTGTG⁃3'	600
slr0168Dw⁃F	5'⁃CTCTAGAGTGCCACTACCTGGCGTGCCGCTACC⁃3'	600
slr0168Dw⁃R	5'⁃GGGGTACCCCGCATGACCAGCTGCCGCCCCAGC⁃3'	600

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敲除集胞藻PCC 6803中编码乳酸脱氢酶的slr1556基因对生物合成乙醇的影响

The Effect of Deleting the slr1556 Gene Encoding Lactate Dehydrogenase on Ethanol Biosynthesize in Synechocystis sp. PCC 6803

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