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

Customized Design of Host-Independent T7 Expression System (HITES) for a Broad Host Range DOI

Mingxin Cui,

Okei Wong,

Kexin Shi

et al.

Journal of Biotechnology, Journal Year: 2025, Volume and Issue: 398, P. 202 - 214

Published: Jan. 6, 2025

Language: Английский

Citations

1
OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG DOI Creative Commons

Meizi Liu,

Zuhui Li,

Jianfeng Huang

et al.

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(13), P. 8003 - 8016

Published: June 11, 2024

Abstract Optogenetics’ advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon system, commonly used in Escherichia coli, relies on the binding or isopropyl β-d-1-thiogalactopyranoside (IPTG) repressor protein LacI, playing a pivotal role operon. Here, we harnessed light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as tool control engineered LacI into two variants, OptoLacIL OptoLacID. These variants exhibit direct responsiveness darkness, respectively, eliminating need IPTG. Building upon OptoLacI, constructed light-controlled E. coli gene expression systems, OptoE.coliLight system OptoE.coliDark system. systems enable bifunctional regulation through manipulation show superior controllability compared IPTG-induced systems. We applied production metabolic flux control. Protein levels are comparable those induced by Notably, titers dark-induced 1,3-propanediol (1,3-PDO) ergothioneine exceeded 110% 60% IPTG, respectively. development OptoLacI will contribute field optogenetic engineering, holding substantial potential applications across various fields.

Language: Английский

Citations

4
Efficient expression of recombinant proteins in Bacillus subtilis using a rewired gene circuit of quorum sensing DOI Open Access
Wenliang Hao, Shihao Yang,

Yan Sheng

et al.

Biotechnology Progress, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 19, 2025

Abstract Bacillus subtilis is a favored chassis for high productivity of several value‐added product in synthetic biology. Efficient production recombinant proteins critical but challenging using this because these expression systems use, such as constitutive and inducible systems, demand coordination cell growth with addition chemical inducers. These compete intracellular resources the host, eventually resulting dysfunction survival. To overcome problem, study, LuxRI quorum sensing (QS) system from Aliivibrio fischeri was functionally reconstituted B. achieving coordinated protein overproduction cell‐density‐dependent manner. Furthermore, output‐controlling promoter, P luxI , engineered through two rounds evolution, by which we identified four mutants, P22, P47, P56, P58 that exhibited elevated activity compared to original . By incorporating strong terminator (TB5) downstream target gene further enhanced level. The level surpasses commonly used promoter‐based like P43 ylbP QS proves be potent platform

Language: Английский

Citations

0
Developing Bacillus subtilis as cell factory for the production of the natural biocontrol compound pulcherrimin DOI Creative Commons
Taichi Chen,

Haris Uzunovic,

Stanley Brul

et al.

Bioresource Technology, Journal Year: 2025, Volume and Issue: unknown, P. 132433 - 132433

Published: March 1, 2025

Pulcherrimin, a natural metabolite produced by Bacillus subtilis, demonstrates range of biological activities, including its potential use as antimicrobial, antioxidant, or coloring agent. PS832 was selected the host cell from four B. subtilis strains. Transcriptome data revealed that leucine pathway has minimal impact on pulcherrimin titer, whereas enzymes encoded yvmC-cypX operon are essential for achieving high production. Alleviating transcriptional repression led to an increase in titer representing 9.5-fold enhancement 487 mg/l. The mutant BSP17 showed 65 % inhibition rate phytopathogen, revealing biocontrol Furthermore, optimizing iron concentration medium resulted titers 610 mg/l shake flasks and 811 1.5-l bioreactor. It is highest reported sets stage further metabolic engineering achieve industrial-scale production pulcherrimin.

Language: Английский

Citations

0
Innovations, Challenges and Future Directions of T7RNA Polymerase in Microbial Cell Factories DOI Creative Commons
Sefli Sri Wahyu Effendi, I‐Son Ng

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

Published: April 10, 2025

The study of "resource allocator" bacteriophage T7 RNA polymerase (T7RNAP) has garnered significant interest, particularly for optimizing transcriptional systems in microbial cell factories (MCFs). Most previous reviews have primarily focused on T7RNAP by dissecting specific aspects its molecular structure and functional dynamics; this critical review seeks to broaden the scope. We emphasize a comprehensive guide utilizing versatile variants, covering both fundamental principles fine-tuned circuit designs synthetic biology applications. Recent advancements engineered with enhanced specificity controllability are also highlighted. Furthermore, we discuss host compatibility considerations implementing sustainable bioproduction. Finally, key challenges regulatory complexities emerging opportunities next-generation technology discussed, reinforcing future directions improving MCF performance.

Language: Английский

Citations

0
Development and construction of a novel Bacillus subtilis autoinducible extracellular expression system based on a LuxI/R device DOI Creative Commons

Bin Wang,

Keyi Wang, Xin Zhao

et al.

Microbial Cell Factories, Journal Year: 2025, Volume and Issue: 24(1)

Published: April 19, 2025

Microbial chassis expression systems are valuable tools in biotechnology and synthetic biology, Bacillus subtilis is an important industrial microbial chassis. Quorum sensing (QS)-based dynamic regulation widely used to automatically activate gene response changes cell density. The main bottleneck currently limiting the use of exogenous QS B. for efficient autoinducible extracellular recombinant proteins their low level expression. A novel system based on LuxI/R-type (lux system) Vibrio fischeri was developed which lux enhanced by engineering module promoters. By promoter SPluxI core region (- 10 - 35 elements) critical (UP spacer elements), RPluxIR6 box copy number original LuxI/R device (S0-R0), high-expression Sc-R2 construct obtained. After shake flask 3-L fermenter fermentation, amylase activity obtained with 2.7- 3.1-fold greater, respectively, than that well-characterized Pveg. achieved 2.6-fold greater S0-R0 when either levansucrase or invertase as a reporter protein. Overall, this study showed good generalizability application potential industrial-scale fermentation. To our knowledge, first report sequence RPluxIR6. This further expands biology.

Language: Английский

Citations

0
Cloning Systems in Bacillus: Bioengineering of Metabolic Pathways for Valuable Recombinant Products DOI Creative Commons
Alexander Arsov, Nadya Armenova, Emanoel Gergov

et al.

Fermentation, Journal Year: 2024, Volume and Issue: 10(1), P. 50 - 50

Published: Jan. 9, 2024

Representatives of the genus Bacillus have been established as one most important industrial microorganisms in last few decades. Genetically modified B. subtilis and, to a lesser extent, licheniformis, amyloliquefaciens, and megaterium used for heterologous expression numerous proteins (enzymes, vaccine components, growth factors), platform chemicals, other organic compounds importance. Vectors designed work spp. dramatically increased number complexity. Today, they provide opportunities genetic manipulation on every level, from point mutations systems biology, that were impossible even ten years ago. The present review aims describe concisely latest developments shuttle, integrative, CRISPR-Cas9 vectors well their application large-scale bioengineering with prospect producing valuable an scale. Genetic manipulations promoters vectors, together impact secretory metabolic pathways, are discussed detail.

Language: Английский

Citations

2
Design and Model-Driven Analysis of Synthetic Circuits with the Staphylococcus aureus Dead-Cas9 (sadCas9) as a Programmable Transcriptional Regulator in Bacteria DOI
Davide De Marchi,

Roman Shaposhnikov,

Samy Gobaa

et al.

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(3), P. 763 - 780

Published: Feb. 20, 2024

Synthetic circuit design is crucial for engineering microbes that process environmental cues and provide biologically relevant outputs. To reliably scale-up complexity, the availability of parts toolkits central. Streptococcus pyogenes (sp)-derived CRISPR interference/dead-Cas9 (CRISPRi/spdCas9) widely adopted implementing programmable regulations in synthetic circuits, alternative CRISPRi systems will further expand our orthogonal components. Here, we showcase potential using engineered dCas9 from Staphylococcus aureus (sadCas9), not previously used bacterial attractive its low size high specificity. We designed a collection ∼20 increasingly complex circuits variants Escherichia coli, including with static function like one-/two-input logic gates (NOT, NAND), dynamic behavior incoherent feedforward loops (iFFLs), applied sadCas9 to fix T7 polymerase-based cascade. Data demonstrated specific efficient target repression (100-fold) qualitatively successful functioning all circuits. Other advantageous features included sadCas9-borne cell load orthogonality spdCas9. However, different showed quantitatively unexpected unreported steady-state responses: range, switch point, slope NOT/NAND changed output promoters, multiphasic was observed iFFLs, differing expected bell-shaped or sigmoidal curves. Model analysis explained curves by interplays among components, due reporter gene-borne regulator competition. Overall, CRISPRi/sadCas9 successfully expanded available toolkit engineering. Analysis depicted impact generally neglected effects modulating shape component dose–response curves, avoid drawing wrong conclusions on functioning.

Language: Английский

Citations

2
Establishment of the CRISPR-Cpf1 gene editing system in Bacillus licheniformis and multiplexed gene knockout DOI Creative Commons

Suxin Liu,

Fengxu Xiao,

Youran Li

et al.

Synthetic and Systems Biotechnology, Journal Year: 2024, Volume and Issue: 10(1), P. 39 - 48

Published: Aug. 8, 2024

is a significant industrial microorganism. Traditional gene editing techniques relying on homologous recombination often exhibit low efficiency due to their reliance resistance genes. Additionally, the established CRISPR technology, utilizing Cas9 endonuclease, faces challenges in achieving simultaneous knockout of multiple To address this limitation, CRISPR-Cpf1 system has been developed, enabling multiplexed across various microorganisms. Key efficient capability rigorous screening highly effective expression elements achieve conditional protein Cpf1. In study, we employed mCherry as reporter and harnessed P

Language: Английский

Citations

2
Synergistic regulation of chassis cell growth and screening of promoters, signal peptides and fusion protein linkers for enhanced recombinant protein expression in Bacillus subtilis DOI

Bin Wang,

Yaokang Wu,

Xueqin Lv

et al.

International Journal of Biological Macromolecules, Journal Year: 2024, Volume and Issue: 280, P. 136037 - 136037

Published: Sept. 25, 2024

Language: Английский

Citations

2