Discovery and remodeling of Vibrio natriegens as a microbial platform for efficient formic acid biorefinery

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Nov. 27, 2023

Language: Английский

---

Efficient natural plasmid transformation of Vibrio natriegens enables zero-capital molecular biology

PNAS Nexus, Journal Year: 2024, Volume and Issue: 3(2)

Published: Feb. 1, 2024

Abstract The fast-growing microbe Vibrio natriegens is capable of natural transformation where it draws DNA in from media via an active process under physiological conditions. Using engineered strain with a genomic copy the master competence regulator tfoX cholerae combination new minimal (MCM) that uses acetate as energy source, we demonstrate naturally competent cells which are created, transformed, and recovered entirely same media, without exchange or addition fresh media. Cells to plasmids, recombination linear DNA, cotransformation both select for scarless markerless edits. entire simple inexpensive, requiring no capital equipment room temperature (zero protocol, 104 cfu/μg), just incubator (high-efficiency 105−6 cfu/μg). These retain their state when frozen transformable immediately upon thawing like typical chemical electrochemical cell. Since optimized protocol requires only 50 min hands-on time, V. grows quickly even on plates, started at 9 AM yields abundant culturable single colonies by 5 PM. Further, because all stages occur can be arbitrarily scaled volume, this could ideal automated directed evolution applications. As result, compete Escherichia coli excellent chassis low-cost highly scalable synthetic biology.

Language: Английский

---

Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration

Biochemistry, Journal Year: 2024, Volume and Issue: 63(5), P. 587 - 598

Published: Feb. 15, 2024

Production of soluble proteins is essential for structure/function studies; however, this usually requires milligram amounts protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens emerged as a novel and alternative host platform production in high yields. Here, we used commercial strain derived from V. (Vmax X2) produce bacterial fungal scale, struggled achieve Escherichia coli. These include cholera toxin (CT) N-acetyl glucosamine-binding protein A (GbpA) cholerae, heat-labile enterotoxin (LT) E. coli nematotoxin CCTX2 Coprinopsis cinerea. CT, GbpA, LT are secreted by Type II secretion system their natural hosts. When these three were produced Vmax, they also could recovered growth media. This simplified downstream purification procedure resulted considerably higher yields compared (6- 26-fold increase). We tested Vmax perdeuteration using deuterated minimal media deuterium oxide solvent achieved 3-fold increase yield equivalent protocol good news, since isotopic labeling expensive often ineffective but represents necessary prerequisite some structural biology techniques. Thus, promising challenging targets suitable studies.

Language: Английский

---

Vibrio natriegens as a host for rapid biotechnology

Trends in biotechnology, Journal Year: 2021, Volume and Issue: 40(4), P. 381 - 384

Published: Nov. 16, 2021

Language: Английский

---

Rapid production of l‐DOPA by Vibrio natriegens, an emerging next‐generation whole‐cell catalysis chassis

Microbial Biotechnology, Journal Year: 2022, Volume and Issue: 15(5), P. 1610 - 1621

Published: Jan. 10, 2022

Summary 3, 4‐Dihydroxyphenyl‐ l ‐alanine ( ‐DOPA) is a compound of high medical value and considered effective as treatment for Parkinson’s disease. Currently, bioproduction ‐DOPA mainly carried out by whole‐cell catalysis mediated recombinant Escherichia coli carrying heterogeneous tyrosine phenol lyase. Vibrio natriegens increasingly attracting attention owing to its superiority, including extremely rapid growth soluble protein expression capacity. In this study, we attempt develop an efficient catalyst production using V. the chassis. The maximum was accomplished in 4 h at 37°C, which equivalent that achieved E. 16 16°C. Furthermore, productivity reached over 10.0 g −1 early stage biocatalysis, nearly two‐fold higher than previously reported. Approximately 54.0 obtained with catechol conversion rate greater 95%. conclusion, displays advantages, catalytic process production. These findings strongly suggest has remarkable potential chassis valuable chemicals.

Language: Английский

---

Recombinant Protein Expression Chassis Library of Vibrio natriegens by Fine-Tuning the Expression of T7 RNA Polymerase

ACS Synthetic Biology, Journal Year: 2023, Volume and Issue: 12(2), P. 555 - 564

Published: Jan. 31, 2023

Vibrio natriegens is the fastest-growing bacteria, and its doubling time less than 10 min. At present, T7 expression system has been introduced into V. for heterologous protein expression, including commercial strain Vmax1 variant VnDX,2 which a backup chassis of Escherichia coli BL21(DE3). However, strength existing not optimal every recombinant protein. The different strengths RNA polymerase (T7 RNAP) can be obtained by changing promoter ribosome binding site (RBS) sequences RNAP at transcription translation levels. In this work, we robust VnDX library with fine-tuning using industrially used enzyme glucose dehydrogenase (GDH) as reporter Among library, VnDX-tet, whose was changed from PlacUV5 to Ptet, showed that GDH activity increased 109% system. Similarly, variants levels were RBS upstream RNAP, results VnDX-RBS12/pGDH had highest activity, 12.6%. constructed in study provides choice expressing various proteins, greatly expanding application natriegens.

Language: Английский

---

Vibrio species as next-generation chassis for accelerated synthetic biology

Biotechnology and Bioprocess Engineering, Journal Year: 2024, Volume and Issue: 29(2), P. 241 - 253

Published: Feb. 19, 2024

Language: Английский

---

Vibrio natriegens as a superior host for the production of c-type cytochromes and difficult-to-express redox proteins

Scientific Reports, Journal Year: 2024, Volume and Issue: 14(1)

Published: March 13, 2024

Abstract C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be obtained through heterologous production. Heterologous production c-type Escherichia coli is hindered since the ccmABCDEFGH genes necessary for incorporation heme c are only expressed under conditions. Different strategies were devised to bypass this obstacle, such as co-expressing ccm from pEC86 vector. However, co-expression methods restrict choice expression host Here we describe first use Vibrio natriegens V max X2 recombinant difficult-to-express redox proteins extreme acidophile Acidithiobacillus ferrooxidans CCM4253, including three cytochromes. Co-expression was not required produce holo-c-type X2. E. T7 Express produced during able inner membrane cytochrome CycA. Additionally, cell extracts contained higher portions holo-proteins than extracts. All translocated intended compartment hosts. In conclusion, V. represents a promising alternative proteins.

Language: Английский

---

Unlocking the strength of inducible promoters in Gram‐negative bacteria

Microbial Biotechnology, Journal Year: 2023, Volume and Issue: 16(5), P. 961 - 976

Published: Feb. 3, 2023

Abstract Inducible bacterial promoters are ubiquitous biotechnology tools that have a consistent architecture including two key elements: the operator region recognized by transcriptional regulatory proteins, and −10 −35 consensus sequences required to recruit sigma (σ) 70 subunits of RNA polymerase initiate transcription. Despite their widespread use, leaky transcription in OFF state remains challenge. We updated lac tet improve strength, control portability adaptation sequence boxes strongly targeted σ , incorporation strong ribosome binding site broadly Gram‐negative bacteria, independent regulators constitutive promoters. To test promoters, we use far‐red fluorescent protein mCardinal, which significantly improves signal‐to‐background ratio promoter measurements over widely utilized green proteins. validate improvement inducibility demonstrating production toxic aggregate‐prone cocaine esterase enzyme CocE. further demonstrate additional species Pseudomonas putida Vibrio natriegens. Our results represent significant existing expression systems will enable advances for various applications.

Language: Английский

---

Innovations, Challenges and Future Directions of T7RNA Polymerase in Microbial Cell Factories

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

---