Engineering a Lactobacillus Lysine Riboswitch to Dynamically Control Metabolic Pathways for Lysine Production in Corynebacterium glutamicum

被引:4
作者
Jiang, Qingwei [1 ]
Geng, Feng [2 ]
Shen, Juan [1 ]
Zhu, Ping [1 ]
Lu, Zhaoxin [1 ]
Zhou, Libang [1 ]
Lu, Fengxia [1 ]
机构
[1] Nanjing Agr Univ, Coll Food Sci & Technol, Nanjing 210095, Peoples R China
[2] Binzhou Med Univ, Coll Pharm, Yantai 264003, Peoples R China
基金
中国国家自然科学基金;
关键词
gene expression; lysine riboswitch; dynamic control; metabolic pathway; C; glutamicum; FMN-RIBOSWITCH; INSIGHTS; BIOSYNTHESIS; EXPRESSION; TRANSPORT; STRAIN;
D O I
10.3390/microorganisms12030606
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Knock-out of genes of metabolic pathways is conventionally used in the metabolic engineering of microorganisms, but it is not applicable for genes of essential pathways. In order to avoid undesirable effects caused by gene deletion, it is attractive to develop riboswitches to dynamically control the metabolic pathways of microbial cell factories. In this regard, the aim of this study is to utilize the lysine riboswitch to control gene expressions of the biosynthetic pathways and by-pathways and thus improve lysine production in Corynebacterium glutamicum. To achieve this, a natural lysine riboswitch from Lactobacillus plantarum (LPRS) was first detected and then fused with RFP to test its functionality. After that, engineered lysine-activated (Lys-A) and lysine-repressed (Lys-R) riboswitches were successfully screened by dual genetic selection. Furthermore, the optimized A263 and R152 were applied to control the expression of aspartate kinase III and homoserine dehydrogenase in the lysine-producing strain C. glutamicum QW45, respectively. In contrast with QW45, the growth of the resulting A263-lysC mutant QW48 was similar to that of QW45; however, the growth of the resulting R357-hom mutant QW54 was slightly inhibited, indicating an inhibition of threonine biosynthesis caused by the riboswitch upon binding of intracellular lysine. Importantly, the lysine production of QW48 and QW54 was, respectively, 35% and 43% higher than that of the parent strain QW45, implying more metabolic flux directed into the lysine synthesis pathway. Finally, the engineered A263 and R357 were simultaneously applied to the same mutant QW55, which greatly improved lysine production. Thus, the approach demonstrated in this work could be principally used as a powerful tool to dynamically control any other undesired metabolic pathways.
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页数:14
相关论文
共 43 条
[21]   Posttranscriptional Signal Integration of Engineered Riboswitches Yields Band-Pass Output [J].
Muranaka, Norihito ;
Yokobayashi, Yohei .
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2010, 49 (27) :4653-4655
[22]   Recent Advances in Metabolic Engineering, Protein Engineering, and Transcriptome-Guided Insights Toward Synthetic Production of Taxol [J].
Mutanda, Ishmael ;
Li, Jianhua ;
Xu, Fanglin ;
Wang, Yong .
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, 2021, 9
[23]   RNA-guided single/double gene repressions in Corynebacterium glutamicum using an efficient CRISPR interference and its application to industrial strain [J].
Park, Jaehyun ;
Shin, Hyojung ;
Lee, Sun-Mi ;
Um, Youngsoon ;
Woo, Han Min .
MICROBIAL CELL FACTORIES, 2018, 17
[24]   Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique [J].
Pfeifer-Sancar, Katharina ;
Mentz, Almut ;
Rueckert, Christian ;
Kalinowski, Joern .
BMC GENOMICS, 2013, 14
[25]   C-di-AMP Is a Second Messenger in Corynebacterium glutamicum That Regulates Expression of a Cell Wall-Related Peptidase via a Riboswitch [J].
Reich, Sebastian J. ;
Goldbeck, Oliver ;
Lkhaasuren, Tsenguunmaa ;
Weixler, Dominik ;
Weiss, Tamara ;
Eikmanns, Bernhard J. .
MICROORGANISMS, 2023, 11 (02)
[26]   Regulation of lysine biosynthesis and transport genes in bacteria: yet another RNA riboswitch? [J].
Rodionov, DA ;
Vitreschak, AG ;
Mironov, AA ;
Gelfand, MS .
NUCLEIC ACIDS RESEARCH, 2003, 31 (23) :6748-6757
[27]  
Sambrook J., 1989, Molecular cloning. Laboratory manual, p1.25
[28]   SMALL MOBILIZABLE MULTIPURPOSE CLONING VECTORS DERIVED FROM THE ESCHERICHIA-COLI PLASMIDS PK18 AND PK19 - SELECTION OF DEFINED DELETIONS IN THE CHROMOSOME OF CORYNEBACTERIUM-GLUTAMICUM [J].
SCHAFER, A ;
TAUCH, A ;
JAGER, W ;
KALINOWSKI, J ;
THIERBACH, G ;
PUHLER, A .
GENE, 1994, 145 (01) :69-73
[29]   Structural insights into amino acid binding and gene control by a lysine riboswitch [J].
Serganov, Alexander ;
Huang, Lili ;
Patel, Dinshaw J. .
NATURE, 2008, 455 (7217) :1263-U76
[30]   Amino acid recognition and gene regulation by riboswitches [J].
Serganov, Alexander ;
Patel, Dinshaw J. .
BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, 2009, 1789 (9-10) :592-611