Optogenetic control of Corynebacterium glutamicum gene expression DOI Creative Commons
Deng Chen,

Ruijie Xin,

Xingjian Li

et al.

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 28, 2024

Abstract Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth product synthesis crucial enhancing production efficiency. Developing dynamic, broadly applicable, minimally toxic gene regulation tools C. remains challenging, as optogenetic ideal dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled expression system using RNA-binding proteins (RBP), first glutamicum. We established system, ‘LightOnC.glu’, utilizing the RBP to construct transcription factors in Simultaneously, we developed high-performance interference CRISPR/Cpf1 tools. The network was designed enable of chitin oligosaccharides (CHOSs) chondroitin sulphate A (CSA) time Additionally, bioreactor constructed, achieving CHOSs concentration 6.2 g/L, highest titer recorded biosynthesis date. Herein, programmable light-responsive genetic circuit glutamicum, advancing theory based on light signaling. breakthrough has potential applications optimizing modules other chassis cells synthesizing compounds.

Language: Английский

Reshaping Phosphatase Substrate Preference for Controlled Biosynthesis Using a “Design–Build–Test–Learn” Framework DOI Creative Commons

Jiangong Lu,

Xueqin Lv,

Wenwen Yu

et al.

Advanced Science, Journal Year: 2024, Volume and Issue: 11(22)

Published: March 19, 2024

Abstract Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural with limited catalytic performance often need be engineered meet specific needs through time‐consuming trial‐and‐error process. This study presents quantum mechanics (QM)‐incorporated design–build–test–learn (DBTL) framework rationally design phosphatase BT4131, an enzyme ambiguous substrate spectrum involved N ‐acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) designed using force field‐based methods, resulting 1.4‐fold increase preference ( k cat / K m ) toward GlcNAc‐6‐phosphate (GlcNAc6P). QM calculations indicate that shift caused by 13.59 kcal mol −1 reduction activation energy. Furthermore, iterative computer‐aided conducted stabilize transition state. As result, M4 (I49Q/L129Q/G172L) 9.5‐fold cat‐GlcNAc6P m‐GlcNAc6P 59% decrease cat‐Glc6P m‐Glc6P highly desirable compared wild type GlcNAc‐producing chassis. The GlcNAc titer increases 217.3 g L yield 0.597 (g glucose) 50‐L bioreactor, representing highest reported level. Collectively, this DBTL provides easy yet fascinating approach rational for industrially viable biocatalysts.

Language: Английский

Citations

7
Customizing biocatalysts by reducing ΔG‡: Integrating ground-state destabilization and transition-state stabilization DOI
Xin Xu, Wanqing Wei, Yiwen Zhou

et al.

Chem Catalysis, Journal Year: 2025, Volume and Issue: unknown, P. 101323 - 101323

Published: March 1, 2025

Language: Английский

Citations

0
Biosensor-Assisted Multitarget Gene Fine-Tuning for N-Acetylneuraminic Acid Production in Escherichia coli with Sole Carbon Source Glucose DOI
Bin Qi, Jianing Zhang, Wenlong Ma

et al.

Journal of Agricultural and Food Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: April 10, 2025

N-Acetylneuraminic acid (NeuAc) is widely used in the food and medical industries. Microbial fermentation has become one of most important approaches for NeuAc production. However, current research on confronted with challenges, including high production costs, interference from competitive pathways, low conversion efficiency, all which impede its efficient In this study, an engineered Escherichia coli capable utilizing glucose as sole carbon source was constructed by optimizing utilization pathway, redox balance NADH/NAD+. Subsequently, pathway genes were systematically upregulated to identify key target improving biosynthesis. The gene cluster glmSA*-glmM-SeglmU identified engineering target. To achieve multitarget coordinated optimization vivo, a highly responsive biosensor developed, exhibiting maximum response ratio 10.62-fold. By construction random mutation libraries integration NeuAc-responsive high-throughput screening using flow cytometry, expression levels three synergistically optimized. As result, NeuAc-producing strain A39 successfully obtained. 3-L bioreactor, achieved titer 58.26 g·L-1 productivity 0.83 g·L-1·h-1, representing highest reported source.

Language: Английский

Citations

0
Enhancing Substrate Preference of Iridoid Synthase via Focused Polarity-Steric Mutagenesis Scanning DOI Creative Commons

Huifen Yu,

Cuifang Ye, Yong Wang

et al.

Chem & Bio Engineering, Journal Year: 2024, Volume and Issue: 1(10), P. 826 - 835

Published: May 18, 2024

Nepetalactol serves as the scaffold for most iridoids, which exhibit a wide range of biological and pharmacological activities. Iridoid synthase (ISY) plays crucial role in vivo synthesis nepetalactol from 8-oxogeranial. However, substrate promiscuity ISY could result deviation flux toward off-target routes. In this work, preference (SP, ratio activity 8-oxogeranial to geranial) was improved by directed evolution. First, strategy focused polarity-steric mutagenesis scanning (FPSMS) performed construct small mutant library with NmISY2 Nepeta mussinii an object. Four amino acid residues varying polarity steric hindrance, including alanine, aspartic acid, serine, arginine, were incorporated scan hot spots. Consequently, four sites W109, M217, K343, W345 significant impact on found. Then, combined combinatorial active-site saturation test/iterative (CAST/ISM) strategy. As result, W345D/K343M/W109Y (3M+) obtained significantly increased SP value 6 8.5 293.1. Molecular dynamics simulations revealed that hindrance tunnel played pivotal roles NmISY2. Notably, upon integration 3M+ into Pichia pastoris, de novo titer 24.9 times, reaching 15.8 mg/L. This study offers strategic approach improving enzymes.

Language: Английский

Citations

2
Multi-method analysis revealed the mechanism of substrate selectivity in NHase: A gatekeeper residue at the activity center DOI

Yiwei Meng,

Łukasz Pepłowski, Tong Wu

et al.

International Journal of Biological Macromolecules, Journal Year: 2024, Volume and Issue: 279, P. 135426 - 135426

Published: Sept. 7, 2024

Language: Английский

Citations

2
Crystal structure and structure-guided tunnel engineering in a bacterial β-1,4-galactosyltransferase DOI

Guocong Luo,

Zhaolin Huang,

Yingying Zhu

et al.

International Journal of Biological Macromolecules, Journal Year: 2024, Volume and Issue: 279, P. 135374 - 135374

Published: Sept. 10, 2024

Language: Английский

Citations

1
Optogenetic control of Corynebacterium glutamicum gene expression DOI Creative Commons
Deng Chen,

Ruijie Xin,

Xingjian Li

et al.

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 28, 2024

Abstract Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth product synthesis crucial enhancing production efficiency. Developing dynamic, broadly applicable, minimally toxic gene regulation tools C. remains challenging, as optogenetic ideal dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled expression system using RNA-binding proteins (RBP), first glutamicum. We established system, ‘LightOnC.glu’, utilizing the RBP to construct transcription factors in Simultaneously, we developed high-performance interference CRISPR/Cpf1 tools. The network was designed enable of chitin oligosaccharides (CHOSs) chondroitin sulphate A (CSA) time Additionally, bioreactor constructed, achieving CHOSs concentration 6.2 g/L, highest titer recorded biosynthesis date. Herein, programmable light-responsive genetic circuit glutamicum, advancing theory based on light signaling. breakthrough has potential applications optimizing modules other chassis cells synthesizing compounds.

Language: Английский

Citations

0