基于紫色杆菌素生物合成途径的L-色氨酸生物传感器的构建

doi:10.13560/j.cnki.biotech.bull.1985.2023-0271

摘要/Abstract

摘要：

结合生物传感器进行高通量筛选是鉴定L-色氨酸高产菌株的有力工具，但现有的L-色氨酸生物传感器普遍存在工作范围和动态范围都较低的缺点。在大肠杆菌中表达紫色杆菌素生物合成途径，测试不同来源的VioA酶，结合RBS工程，开发了一种新型酶偶联L-色氨酸生物传感器。测试发现来自Chromobacterium violaceum的VioA酶可使该传感器的检测上限扩大至10 g/L外源添加的L-色氨酸；将其RBS的初始翻译速率降低到约2 000时，传感器的动态范围扩大到55倍；通过肉眼可直观地区分L-色氨酸产量不同的大肠杆菌菌株。这种新型的L-色氨酸生物传感器可结合高通量筛选等手段，在鉴定L-色氨酸及其高附加值衍生物高产菌株等方面发挥巨大作用。

关键词: 大肠杆菌, 紫色杆菌素, L-色氨酸, 生物传感器

Abstract:

High-throughput screening combining with biosensors is a powerful tool for identifying high-yield L-tryptophan producing strains, but current L-tryptophan biosensors generally have the disadvantage of low operational and dynamic ranges. By expressing the violacein biosynthetic pathway in Escherichia coli and testing different sources of VioA enzyme and RBS engineering, a novel enzyme-coupled L-tryptophan biosensor was developed. It was found that the VioA enzyme from Chromobacterium violaceum expanded detection limit of the biosensor up to 0-10 g/L of exogenously added L-tryptophan. Reducing its translation initiation rate to approximately 2 000 expanded the dynamic range of the biosensor by 55-fold. Different strains of E. coli with varying L-tryptophan yields could be visually distinguished with the naked eye. This novel L-tryptophan biosensor can play a significant role in identifying high-yield L-tryptophan and its high-value derivatives producing strains through combining methods such as high-throughput screening.

Key words: Escherichia coli, violacein, L-tryptophan, biosensor

李仁瀚, 张乐乐, 刘春立, 刘秀霞, 白仲虎, 杨艳坤, 李业. 基于紫色杆菌素生物合成途径的L-色氨酸生物传感器的构建[J]. 生物技术通报, 2023, 39(10): 80-92.

LI Ren-han, ZHANG Le-le, LIU Chun-li, LIU Xiu-xia, BAI Zhong-hu, YANG Yan-kun, LI Ye. Development of an L-tryptophan Biosensor Based on the Violacein Biosynthesis Pathway[J]. Biotechnology Bulletin, 2023, 39(10): 80-92.

图/表 13

参考文献 39

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质粒Plasmid	描述Description	来源Source
pVio	在pRSFDuet载体的多克隆位点2内插入vioABCDE基因簇，卡那霉素抗性 pRSFDuet with vioABCDE gene cluster in MCS-2, Kan^R	[26]
pBL_401	删除了pVio中的vioD基因 pVio deleted vioD	本研究构建This study
pBL_403-407, 421-423	更换pBL_401中的RBSb区域 pBL_401 changed RBSb region	本研究构建This study
pBL_415	将pBL_401的Duganella sp. B2 vioA基因从多克隆位点2移到多克隆位点1，在多克隆位点前添加一个T7终止子 Duganella sp. B2 vioA of pBL_401 was moved to MCS-1 from MCS-2, and a T7 terminator was added before MCS-2	本研究构建This study
pBL_416	将pBL_415的Duganella sp. B2 vioA替换为Janthinobacteriun violaceinigrum vioA Duganella sp. B2 vioA of pBL_415 was replaced by Janthinobacteriun violaceinigrum vioA	本研究构建This study
pBL_417	将pBL_415的Duganella sp. B2 vioA替换为Pseudoduganella violaceinigra vioA Duganella sp. B2 vioA of pBL_415 was replaced by Pseudoduganella violaceinigra vioA	本研究构建This study
pBL_418	将pBL_415的Duganella sp. B2 vioA替换为Myxococcus xanthus vioA Duganella sp. B2 vioA of pBL_415 was replaced by Myxococcus xanthus vioA	本研究构建This study
pBL_419	将pBL_415的Duganella sp. B2 vioA替换为Chromobacterium violaceinum vioA Duganella sp. B2 vioA of pBL_415 was replaced by Chromobacterium violaceinum vioA	本研究构建This study
pBL_420	将pBL_415的Duganella sp. B2 vioA替换为Duganella sp. HH105 vioA Duganella sp. B2 vioA of pBL_415 was replaced by Duganella sp. HH105 vioA	本研究构建This study
pBL_425	更换pBL_415中的RBSa区域 pBL_415 changed RBSa region	本研究构建This study
pBL_429	更换pBL_416中的RBSa区域 pBL_416 changed RBSa region	本研究构建This study
pBL_437-439	更换pBL_419中的RBSa区域 pBL_419 changed RBSa region	本研究构建This study
pED_RBS1-RBS4	携带trpE^fbrD和不同RBS的pACYCDuet，氯霉素抗性 pACYCDuet with trpE^fbrD and different RBSs, Cm^R	[17]

质粒Plasmid	描述Description	来源Source
pVio	在pRSFDuet载体的多克隆位点2内插入vioABCDE基因簇，卡那霉素抗性 pRSFDuet with vioABCDE gene cluster in MCS-2, Kan^R	[26]
pBL_401	删除了pVio中的vioD基因 pVio deleted vioD	本研究构建This study
pBL_403-407, 421-423	更换pBL_401中的RBSb区域 pBL_401 changed RBSb region	本研究构建This study
pBL_415	将pBL_401的Duganella sp. B2 vioA基因从多克隆位点2移到多克隆位点1，在多克隆位点前添加一个T7终止子 Duganella sp. B2 vioA of pBL_401 was moved to MCS-1 from MCS-2, and a T7 terminator was added before MCS-2	本研究构建This study
pBL_416	将pBL_415的Duganella sp. B2 vioA替换为Janthinobacteriun violaceinigrum vioA Duganella sp. B2 vioA of pBL_415 was replaced by Janthinobacteriun violaceinigrum vioA	本研究构建This study
pBL_417	将pBL_415的Duganella sp. B2 vioA替换为Pseudoduganella violaceinigra vioA Duganella sp. B2 vioA of pBL_415 was replaced by Pseudoduganella violaceinigra vioA	本研究构建This study
pBL_418	将pBL_415的Duganella sp. B2 vioA替换为Myxococcus xanthus vioA Duganella sp. B2 vioA of pBL_415 was replaced by Myxococcus xanthus vioA	本研究构建This study
pBL_419	将pBL_415的Duganella sp. B2 vioA替换为Chromobacterium violaceinum vioA Duganella sp. B2 vioA of pBL_415 was replaced by Chromobacterium violaceinum vioA	本研究构建This study
pBL_420	将pBL_415的Duganella sp. B2 vioA替换为Duganella sp. HH105 vioA Duganella sp. B2 vioA of pBL_415 was replaced by Duganella sp. HH105 vioA	本研究构建This study
pBL_425	更换pBL_415中的RBSa区域 pBL_415 changed RBSa region	本研究构建This study
pBL_429	更换pBL_416中的RBSa区域 pBL_416 changed RBSa region	本研究构建This study
pBL_437-439	更换pBL_419中的RBSa区域 pBL_419 changed RBSa region	本研究构建This study
pED_RBS1-RBS4	携带trpE^fbrD和不同RBS的pACYCDuet，氯霉素抗性 pACYCDuet with trpE^fbrD and different RBSs, Cm^R	[17]

菌株Strain	描述Description	来源 Source
E. coli JM109	用于构建质粒Construct plasmids	实验室保存Lab stored
E. coli BL21（DE3）	用于生物传感器宿主菌The host of the biosensor	实验室保存Lab stored
E. coli B8	敲除trpR tnaA pheA的E. coli BL21（DE3）E. coli BL21（DE3）ΔtrpR ΔtnaA ΔpheA	[26]
EBL_400	携带pVio质粒的大肠杆菌BL21（DE3）E. coli BL21（DE3）with plasmid pVio	本研究构建This study
EBL_401-439	携带相应质粒的大肠杆菌BL21（DE3）E. coli BL21（DE3）with corresponding plasmids	本研究构建This study

菌株Strain	描述Description	来源 Source
E. coli JM109	用于构建质粒Construct plasmids	实验室保存Lab stored
E. coli BL21（DE3）	用于生物传感器宿主菌The host of the biosensor	实验室保存Lab stored
E. coli B8	敲除trpR tnaA pheA的E. coli BL21（DE3）E. coli BL21（DE3）ΔtrpR ΔtnaA ΔpheA	[26]
EBL_400	携带pVio质粒的大肠杆菌BL21（DE3）E. coli BL21（DE3）with plasmid pVio	本研究构建This study
EBL_401-439	携带相应质粒的大肠杆菌BL21（DE3）E. coli BL21（DE3）with corresponding plasmids	本研究构建This study

引物Primer	序列 Sequence（5'-3'）	用途Purpose
401_F	ACTTGGAAGGGTAAACTAAGCTGACCGCCAGCTTCG	扩增pVio以构建pBL_401 Amplifying pVio for pBL_401
401_R	CTTAGTTTACCCTTCCAAGTTTGTACCAAACGT	扩增pVio以构建pBL_401 Amplifying pVio for pBL_401
403_F	GTATAAGAGAGAATTTTAATGACAAATTATTCTGACATTTG	扩增pBL_401以构建pBL_403 Amplifying pBL_401 for pBL_403
403_R	TTGTCATTAAAATTCTCTCTTATACTTAACTAATATACTAAGATGGG	扩增pBL_401以构建pBL_403 Amplifying pBL_401 for pBL_403
404_F	GCAGGAAGTAAGTATGACAAATTATTCTGACATTTG	扩增pBL_401以构建pBL_404 Amplifying pBL_401 for pBL_404
404_R	TTGTCATACTTACTTCCTGCTTATACTTAACTAATATACTAAGATGGG	扩增pBL_401以构建pBL_404 Amplifying pBL_401 for pBL_404
405_F	TAGTTAAGTATAAGCGGGGGCTCGAGATGACAAATTATTCTGACATTTG	扩增pBL_401以构建pBL_405 Amplifying pBL_401 for pBL_405
405_R	CCCCCGCTTATACTTAACTAATATACTAAGATGGG	扩增pBL_401以构建pBL_405 Amplifying pBL_401 for pBL_405
406_F	TCGACACACATGACAAATTATTCTGACATTTG	扩增pBL_401以构建pBL_406 Amplifying pBL_401 for pBL_406
406_R	TAATTTGTCATGTGTGTCGAAACCCCTTATACTTAACTAATATACTAAGATGGG	扩增pBL_401以构建pBL_406 Amplifying pBL_401 for pBL_406
407_F	AGAACCGAGAATTATCTCATGACAAATTATTCTGACATTTG	扩增pBL_401以构建pBL_407 Amplifying pBL_401 for pBL_407
407_R	ATGAGATAATTCTCGGTTCTTATACTTAACTAATATACTAAGATGGG	扩增pBL_401以构建pBL_407 Amplifying pBL_401 for pBL_407
415V_F	ATAAGAAGGAGATATACATAATGAGCATTCTCGACTTTCC	扩增pBL_401以构建pBL_415 Amplifying pBL_401 for pBL_415
415V_R	GTCATGGTATATCTCCTTATTAAAGTTAAACAAAATTATT	扩增pBL_401以构建pBL_415 Amplifying pBL_401 for pBL_415
T7terA_F	GAGCGCGCATGAGGCAGCAGCCATCAC	扩增pBL_401的T7终止子序列以构建pBL_415 Amplifying the T7 terminator sequence of pBL_401 for pBL_415
T7terA_R	CCGTTTAGAGGCCCCAAGGGGTTATGCTAGATACGATTACTTTCTGTTCGACT
T7terB_F	CCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGTGTACACGGCCGCAT
T7terB_R	TATGTATATCTCCTTCTTATACTTAACTAATATACTAAGA
415_VioA_F	TAAGGAGATATACCATGACAAATTATTCTGACATTTGCAT	扩增pBL_401的Dug.B2_vioA序列以构建pBL_415 Amplifying Dug.B2_vioA sequence of pBL_401 for pBL_415
415_VioA_R	CTGCTGCCTCATGCGCGCTCAGTAAGA
416-420V_F	GGCAGCAGCCATCACCAT	扩增pBL_415以作为pBL_416-420的载体 Amplifying pBL_415 as their vectors for pBL_416-420
416-420V_R	GGTATATCTCCTTATTAAAGTTAAACAAAATTATTTCTACAGGGG
416_VioA_F	TTAACTTTAATAAGGAGATATACCATGAGCACCTATAGCGATATTTGCATTG	扩增Jan_vioA以构建pBL_416 Amplifying Jan_vioA for pBL_416
416_VioA_R	ATGGTGATGGCTGCTGCCTTACGCGCGTTCGGTGC	扩增Jan_vioA以构建pBL_416 Amplifying Jan_vioA for pBL_416
417_VioA_F	TTAACTTTAATAAGGAGATATACCATGACCAACTATAGCGATATTTGCATTATTGGC	扩增Pse_vioA以构建pBL_417 Amplifying Pse_vioA for pBL_417
417_VioA_R	ATGGTGATGGCTGCTGCCTTACGCGCTTTCCGCCG	扩增Pse_vioA以构建pBL_417 Amplifying Pse_vioA for pBL_417
418_VioA_F	TTAACTTTAATAAGGAGATATACCATGACCCATCATAGCGATATTTGTATTGTTGG	扩增Myx_vioA以构建pBL_418 Amplifying Myx_vioA for pBL_418
418_VioA_R	ATGGTGATGGCTGCTGCCTTACGGGGTGCGATACGCAAT	扩增Myx_vioA以构建pBL_418 Amplifying Myx_vioA for pBL_418
419_VioA_F	TTAACTTTAATAAGGAGATATACCATGAAACATAGCAGCGATATTTGCATTGT	扩增Chr_vioA以构建pBL_419 Amplifying Chr_vioA for pBL_419
419_VioA_R	ATGGTGATGGCTGCTGCCTTACGCCGCAATGCGCTG	扩增Chr_vioA以构建pBL_419 Amplifying Chr_vioA for pBL_419
420_VioA_F	TTAACTTTAATAAGGAGATATACCATGGGCCGCCGCTTTC	扩增Dug.HH105_vioA以构建pBL_420 Amplifying Dug.HH105_vioA for pBL_420
420_VioA_R	ATGGTGATGGCTGCTGCCTTACACGCGTTCCGCGGT
421_F	AGTATAAGGCATCGTAGAAGGGATGACAAATTATTCTGACATTTG	扩增pBL_401以构建pBL_421 Amplifying pBL_401 for pBL_421
421_R	CTTCTACGATGCCTTATACTTAACTAATATACTAAGATGGG	扩增pBL_401以构建pBL_421 Amplifying pBL_401 for pBL_421
422_F	TATAAGACCTGGGACACAAATGACAAATTATTCTGACATTTG	扩增pBL_401以构建pBL_422 Amplifying pBL_401 for pBL_422
422_R	TTTGTGTCCCAGGTCTTATACTTAACTAATATACTAAGATGGG	扩增pBL_401以构建pBL_422 Amplifying pBL_401 for pBL_422
423_F	CTAAACCCTTTTGCAAAATGACAAATTATTCTGACATTTG	扩增pBL_401以构建pBL_423 Amplifying pBL_401 for pBL_423
423_R	CATTTTGCAAAAGGGTTTAGCTTATACTTAACTAATATACTAAGATGG	扩增pBL_401以构建pBL_423 Amplifying pBL_401 for pBL_423
425_F	CTGTAGAAATAATTTTGTTTAACTTTAATAAGGGGTCCAGAACATGACAAATTATTCTGACATTTGCATAGTT	扩增pBL_415以构建pBL_425 Amplifying pBL_415 for pBL_425
429_F	CTGTAGAAATAATTTTGTTTAACTTTAATAGGTAGGGGGACATGAGCACCTATAGCGATATTTGC	扩增pBL_416以构建pBL_429 Amplifying pBL_416 for pBL_429
437_F	CTGTAGAAATAATTTTGTTTAACTTTAATTCGGAGAGAAAACACATGAAACATAGCAGCGATATTTGCA	扩增pBL_419以构建pBL_437 Amplifying pBL_419 for pBL_437
438_F	CTGTAGAAATAATTTTGTTTAACTTTAATAGGGTAAACACATGAAACATAGCAGCGATATTTGCA	扩增pBL_419以构建pBL_438 Amplifying pBL_419 for pBL_438
439_F	CTGTAGAAATAATTTTGTTTAACTTTAATTACAGTATTGACATGAAACATAGCAGCGATATTTGCA	扩增pBL_419以构建pBL_439 Amplifying pBL_419 for pBL_439
adj_RBSAll_R	ATTAAAGTTAAACAAAATTATTTCTACAGGGGAATT	425_F, 429_F, 437_F, 438_F和439_F的反向引物 The reverse primer for 425_F, 429_F, 437_F, 438_F and 439_F