A cross-species inducible system for enhanced protein expression and multiplexed metabolic pathway fine-tuning in bacteria DOI Creative Commons
Yang Li, Yaokang Wu, Xianhao Xu

et al.

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 53(2)

Published: Dec. 26, 2024

Abstract Inducible systems are crucial to metabolic engineering and synthetic biology, enabling organisms that function as biosensors produce valuable compounds. However, almost all inducible strain-specific, limiting comparative analyses applications across strains rapidly. This study designed presented a robust workflow for developing the cross-species system. By applying this approach, two reconstructed (a 2,4-diacetylphloroglucinol-inducible system PphlF3R1 an anhydrotetracycline-inducible Ptet2R2*) were successfully developed demonstrated in three model microorganisms, including Escherichia coli, Bacillus subtilis Corynebacterium glutamicum. To enhance their practicality, both subsequently placed on plasmid genome detailed characterization determine optimal expression conditions. Furthermore, more efficient Ptet2R2* was employed express various reporter proteins gene clusters these strains. Moreover, aTc-inducible Ptet2R2*, combined with T7 RNA polymerase dCas12a, utilized develop single-input genetic circuit enables simultaneous activation repression of expression. Overall, serves stringent, controllable effective tool protein pathway control different bacteria.

Language: Английский

CRISPR/Cas13X-assisted programmable and multiplexed translation regulation for controlled biosynthesis DOI Creative Commons
X. Xu, Xueqin Lv, Yanfeng Liu

et al.

Nucleic Acids Research, Journal Year: 2025, Volume and Issue: 53(1)

Published: Jan. 7, 2025

Abstract Developing efficient gene regulation tools is essential for optimizing microbial cell factories, but most existing only modulate expression at the transcriptional level. Regulation translational level provides a faster dynamic response, whereas developing programmable, and multiplexed tool remains challenge. Here, we have developed CRISPRi CRISPRa systems based on hfCas13X that can regulate translation in Bacillus subtilis. First, constructed system to catalytically deactivated (dhfCas13X). Second, designed unique mRNA–crRNA pairs construct DiCRISPRa (degradation-inhibited CRISPRa) TsCRISPRa (translation-started systems, which activate downstream by enhancing mRNA stability or initiating translation. In addition, found fusing dhfCas13X with RNA-binding chaperone BHfq significantly improved activation efficiency of (43.2-fold). Finally, demonstrated CRISPR could be used optimize metabolic networks two biotechnologically relevant compounds, riboflavin 2′-fucosyllactose, increasing their titers 3- 1.2-fold, respectively. The here provide new offer ideas construction systems.

Language: Английский

Citations

0
Genome-Wide A → G and C → T Mutations Induced by Functional TadA Variants in Escherichia coli DOI
Hao Wang,

Zhengxin Dong,

Jingyi Shi

et al.

ACS Synthetic Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 9, 2025

The fusion expression of deoxyribonucleic acid (DNA) replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity among the population. Most previous studies have focused on cytosine deaminase, which produces only C → T mutations, significantly limiting variety mutation types. In this study, we developed a system by combining DnaG (RNA primase) adenine TadA-8e (DnaG-TadA) Escherichia coli, is capable rapidly introducing A G into E. coli genome, resulting 664-fold increase terms rate. Additionally, tested dual-functional TadA variant, TadAD, and then fused it DnaG. This construct introduced both rate increased 370-fold upon coexpression uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA DnaG-TadAD-UGI systems to adaptive laboratory evolution for Cd2+ kanamycin resistance, achieving an 8.0 mM 200 μg/mL tolerance within just 17 days 132 h, respectively. Compared conventional methods, final levels were 320 266%, Our work offers novel strategy mutagenesis potentially other prokaryotic species.

Language: Английский

Citations

0
T7 RNA Polymerase-Guided Base Editor for Accelerated Continuous Evolution in Bacillus subtilis DOI Creative Commons

Bin Wang,

Yaokang Wu, Xueqin Lv

et al.

Synthetic and Systems Biotechnology, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Language: Английский

Citations

0
Improving Bacillus subtilis as Biological Chassis Performance by the CRISPR Genetic Toolkit DOI
Xingzhong Cao, Xiaojuan Wang, Ruirui Chen

et al.

ACS Synthetic Biology, Journal Year: 2025, Volume and Issue: unknown

Published: March 4, 2025

Bacillus subtilis is the model Gram-positive and industrial chassis bacterium; it has blossomed as a robust promising host for enzyme, biochemical, or bioflocculant production. However, synthetic biology metabolic engineering technologies of B. have lagged behind most widely used Saccharomyces cerevisiae Escherichia coli. CRISPR (an acronym clustered regularly interspaced short palindromic repeats) enables efficient, site-specific, programmable DNA cleavage, which revolutionized manner genome editing. In 2016, technology was first introduced into been intensely upgraded since then. this Review, we discuss recently developed key additions to toolkit design in with gene editing, transcriptional regulation, enzyme modulation. Second, advances efficient biochemicals proteins are discussed. Finally, conclude perspectives on challenges opportunities CRISPR-based biotechnology subtilis, wishing that can be comparable traditional microorganisms such E. coli S. someday soon.

Language: Английский

Citations

0
Genome-wide A to G and C to T Mutations Induced by Functional TadA Variants in Escherichia coli DOI Open Access
Hao Wang,

Zhengxin Dong,

Jingyi Shi

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 30, 2024

Abstract The fusion expression of DNA replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity among population. Most previous studies have focused on cytosine deaminase, which produces only C→T mutations, significantly limiting the variety mutation types. In this study, we developed a system by combining DnaG (RNA primase) adenine TadA-8e (DnaG-TadA) Escherichia coli , is capable rapidly introducing A→G into E. genome, resulting 664-fold increase terms rate. Additionally, engineered dual-functional TadA variant, TadAD, and then fused it DnaG. This construct introduced both rate further increased 370-fold upon co-expression an uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA DnaG-TadAD-UGI systems to adaptive laboratory evolution for Cd 2+ kanamycin resistance, achieving 8.0 mM 200 μg/mL tolerance within just 17 days 132 hours, respectively. Compared conventional methods, final levels were 320% 266%, Our work offers novel strategy mutagenesis potentially other prokaryotic species. Figure For TOC

Language: Английский

Citations

0
A cross-species inducible system for enhanced protein expression and multiplexed metabolic pathway fine-tuning in bacteria DOI Creative Commons
Yang Li, Yaokang Wu, Xianhao Xu

et al.

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 53(2)

Published: Dec. 26, 2024

Abstract Inducible systems are crucial to metabolic engineering and synthetic biology, enabling organisms that function as biosensors produce valuable compounds. However, almost all inducible strain-specific, limiting comparative analyses applications across strains rapidly. This study designed presented a robust workflow for developing the cross-species system. By applying this approach, two reconstructed (a 2,4-diacetylphloroglucinol-inducible system PphlF3R1 an anhydrotetracycline-inducible Ptet2R2*) were successfully developed demonstrated in three model microorganisms, including Escherichia coli, Bacillus subtilis Corynebacterium glutamicum. To enhance their practicality, both subsequently placed on plasmid genome detailed characterization determine optimal expression conditions. Furthermore, more efficient Ptet2R2* was employed express various reporter proteins gene clusters these strains. Moreover, aTc-inducible Ptet2R2*, combined with T7 RNA polymerase dCas12a, utilized develop single-input genetic circuit enables simultaneous activation repression of expression. Overall, serves stringent, controllable effective tool protein pathway control different bacteria.

Language: Английский

Citations

0