Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries

Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties.

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Heterologous Expression ofMycobacteriumAlkene Monooxygenases in Gram-Positive and Gram-Negative Bacterial Hosts

Applied and Environmental Microbiology ◽

10.1128/aem.00397-18 ◽

2018 ◽

Vol 84 (15) ◽

Cited By ~ 3

Author(s):

Victoria McCarl ◽

Mark V. Somerville ◽

Mai-Anh Ly ◽

Rebecca Henry ◽

Elissa F. Liew ◽

...

Keyword(s):

Heterologous Expression ◽

Stereoselective Synthesis ◽

Cell Activity ◽

Functional Expression ◽

Soluble Form ◽

Gram Negative ◽

Content Type ◽

Alkene Oxidation ◽

Peptide Mass ◽

Heterologous Expression Systems

ABSTRACTAlkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) fromMycobacterium chubuensestrain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved inMycobacterium smegmatismc2155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C2to C4), and their activity was much lower on liquid alkenes (C5to C8). Despite intensive efforts to express the complete EtnABCD enzyme inEscherichia coli, this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 μmol cytochromecreduced/min/mg protein). Cloning the EtnABCD gene cluster intoPseudomonas putidaKT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression ofcpn60chaperonins from eitherMycobacteriumspp. orE. coli. Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels.IMPORTANCEAlkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.

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Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria

mBio ◽

10.1128/mbio.01291-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 15

Author(s):

Jia Jia Zhang ◽

Xiaoyu Tang ◽

Michelle Zhang ◽

Darlene Nguyen ◽

Bradley S. Moore

Keyword(s):

Heterologous Expression ◽

Host Range ◽

Regulatory Elements ◽

Cloning And Expression ◽

Broad Host Range ◽

Implicit Assumption ◽

Gram Negative ◽

E Coli ◽

Expression Platform ◽

Violacein Production

ABSTRACTHeterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC fromPseudoalteromonas luteoviolacea2ta16 revealed robust production in two proteobacterial hosts,Pseudomonas putidaKT2440 andAgrobacterium tumefaciensLBA4404, but very little production of the antibiotic in various laboratory strains ofEscherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor fromP. luteoviolacea2ta16, PviR, that increases pathway transcription and violacein production inE. coliby ∼60-fold independently of acyl-homoserine lactone autoinducers. AlthoughE. coliharbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of variousE. colitranscription factors did not result in a statistically significant increase in violacein production, while overexpression of twoA. tumefaciensPviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.IMPORTANCEAlthough Gram-positive heterologous hosts such asStreptomyceshave been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs in Gram-negative proteobacterial expression hosts, and we leverage this platform to uncover regulatory elements that influence violacein expression fromPseudoalteromonas. Although it is often assumed that BGCs will be more successfully expressed in more closely related hosts, our work suggests that this may not be a general rule of thumb, as heterologous production of natural products can be influenced by specific host regulatory and/or biosynthetic elements, and the identity and effectiveness of those elements are difficult to predict. We argue for the use of a diverse set of heterologous hosts, which may also provide insights into the BGC biosynthetic mechanism and the biological function of BGCs.

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Improved broad-spectrum antibiotics against Gram-negative pathogens via darobactin biosynthetic pathway engineering

Chemical Science ◽

10.1039/d1sc02725e ◽

2021 ◽

Author(s):

Sebastian Groß ◽

Fabian Panter ◽

Domen Pogorevc ◽

Carsten E. Seyfert ◽

Selina Deckarm ◽

...

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Broad Spectrum ◽

Biosynthetic Pathway ◽

Pathway Engineering ◽

Gene Deletions ◽

Gram Negative ◽

E Coli ◽

Broad Spectrum Antibiotics

Heterologous expression of a synthetically engineered darobactin gene cluster in E. coli yields new darobactin derivatives with improved anti-Gram-negative activity. Targeted gene deletions provide first insights into biosynthetic steps.

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Eight genes are necessary and sufficient for biogenesis of Quinohemoprotein amine dehydrogenase

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab117 ◽

2021 ◽

Author(s):

Tadashi Nakai ◽

Katsuyuki Tanizawa ◽

Toshihide Okajima

Keyword(s):

Heterologous Expression ◽

Gram Negative Bacteria ◽

Post Translational Modification ◽

Expression Study ◽

Gram Negative ◽

Amine Dehydrogenase ◽

Necessary And Sufficient

Abstract Quinohemoprotein amine dehydrogenase (QHNDH) containing a peptidyl quinone cofactor, cysteine tryptophylquinone, is produced in the periplasm of Gram-negative bacteria through an intricate process of post-translational modification that requires at least eight genes including those encoding three nonidentical subunits and three modifying enzymes. Our heterologous expression study has revealed that the eight genes are necessary and sufficient for the QHNDH biogenesis.

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Heterologous Expression of the Marine-Derived Quorum Quenching Enzyme MomL Can Expand the Antibacterial Spectrum of Bacillus brevis

Marine Drugs ◽

10.3390/md17020128 ◽

2019 ◽

Vol 17 (2) ◽

pp. 128 ◽

Cited By ~ 5

Author(s):

Jingjing Zhang ◽

Jiayi Wang ◽

Tao Feng ◽

Rui Du ◽

Xiaorong Tian ◽

...

Keyword(s):

Heterologous Expression ◽

Bacterial Pathogens ◽

Quorum Quenching ◽

Plant Diseases ◽

Signal Molecules ◽

Bacterial Disease ◽

Gram Positive ◽

Bacillus Brevis ◽

Gram Negative ◽

Antibacterial Spectrum

Quorum sensing (QS) is closely associated with the production of multiple virulence factors in bacterial pathogens. N-acyl homoserine lactones (AHLs) are important QS signal molecules that modulate the virulence of gram-negative pathogenic bacteria. Enzymatic degradation of AHLs to interrupt QS, termed quorum quenching (QQ), has been considered a novel strategy for reduction of pathogenicity and prevention of bacterial disease. However, the low expression levels of QQ proteins in the original host bacteria has affected the applications of these proteins. Previously, we identified a novel marine QQ enzyme, named MomL, with high activity and promising biocontrol function. In this study, we linked the target fragment momL to pNCMO2, which provided a basis for the first heterologous expression of MomL in the antifungal and anti-gram-positive-bacteria biocontrol strain Bacillus brevis, and obtaining the recombinant strain named BbMomL. The QQ activity of BbMomL was confirmed using a series of bioassays. BbMomL could not only degrade the exogenous signal molecule C6-HSL, but also the AHL signal molecules produced by the gram-negative pathogens Pectobacterium carotovorum subsp. carotovorum (Pcc) and Pseudomonas aeruginosa PAO1. In addition, BbMomL significantly reduced the secretion of pathogenic factors and the pathogenicity of Pcc and P. aeruginosa PAO1. We tested the biocontrol function of BbMomL for prevention of plant diseases in vitro. The result indicates that BbMomL has a broad antibacterial spectrum. Compared with wild-type B. brevis, BbMomL not only inhibited fungi and gram-positive bacterial pathogens but also considerably inhibited gram-negative bacterial pathogens. Moreover, the Bacillus brevis expression system has good application prospects and is an ideal host for expression and secretion of foreign proteins.

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Rational construction of genome-reduced Burkholderiales chassis facilitates efficient heterologous production of natural products from proteobacteria

Nature Communications ◽

10.1038/s41467-021-24645-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jiaqi Liu ◽

Haibo Zhou ◽

Zhiyu Yang ◽

Xue Wang ◽

Hanna Chen ◽

...

Keyword(s):

Natural Products ◽

Heterologous Expression ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Wild Type ◽

Biosynthetic Gene Clusters ◽

Gram Negative ◽

Cell Autolysis ◽

Rational Construction ◽

Genomic Regions

AbstractHeterologous expression of biosynthetic gene clusters (BGCs) avails yield improvements and mining of natural products, but it is limited by lacking of more efficient Gram-negative chassis. The proteobacterium Schlegelella brevitalea DSM 7029 exhibits potential for heterologous BGC expression, but its cells undergo early autolysis, hindering further applications. Herein, we rationally construct DC and DT series genome-reduced S. brevitalea mutants by sequential deletions of endogenous BGCs and the nonessential genomic regions, respectively. The DC5 to DC7 mutants affect growth, while the DT series mutants show improved growth characteristics with alleviated cell autolysis. The yield improvements of six proteobacterial natural products and successful identification of chitinimides from Chitinimonas koreensis via heterologous expression in DT mutants demonstrate their superiority to wild-type DSM 7029 and two commonly used Gram-negative chassis Escherichia coli and Pseudomonas putida. Our study expands the panel of Gram-negative chassis and facilitates the discovery of natural products by heterologous expression.

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A Freeze-Etch Study of Acinetobacter SP. Grown on a Hydrocarbon Substrate

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050883 ◽

1974 ◽

Vol 32 ◽

pp. 174-175

Author(s):

C. L. Scott ◽

W. R. Finnerty

Keyword(s):

Membrane System ◽

Structural Distortion ◽

Intracytoplasmic Membrane ◽

Gram Negative ◽

Sectioned Material ◽

Acinetobacter Sp

Acinetobacter sp. HO-1-N, a gram-negative hydrocarbon oxidizing bacterium previously designated Micrococcus cerificans, has been shown to sequester the hydrocarbon into intracytoplasmic pools as a result of growth on this substrate. In hydrocarbon grown cells, an intracytoplasmic membrane system was also observed along with a doubling of cellular phospholipids (Z). However, using conventional dehydration and embedding procedures in preparing thin sectioned material, the hydrocarbon is extracted from the cells. This may lead to structural distortion, consequently, the freeze-etch technique was applied to preserve the integrity of the cell.

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Bacterial-Mucosal Cell Junctional Complexes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050731 ◽

1974 ◽

Vol 32 ◽

pp. 144-145

Author(s):

Roger C. Wagner

Keyword(s):

Intestinal Wall ◽

Rat Colon ◽

Mucosal Cell ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Mucosal Cells ◽

Mucosal Lining ◽

Degenerative Alterations ◽

Junctional Complexes ◽

Intestinal Mucosal Cells

Bacteria exhibit the ability to adhere to the apical surfaces of intestinal mucosal cells. These attachments either precede invasion of the intestinal wall by the bacteria with accompanying inflammation and degeneration of the mucosa or represent permanent anchoring sites where the bacteria never totally penetrate the mucosal cells.Endemic gram negative bacteria were found attached to the surface of mucosal cells lining the walls of crypts in the rat colon. The bacteria did not intrude deeper than 0.5 urn into the mucosal cells and no degenerative alterations were detectable in the mucosal lining.

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Microanatomy of the Periplasm of Bacillus Licheniformis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100065365 ◽

1971 ◽

Vol 29 ◽

pp. 262-263

Author(s):

B.K. Ghosh

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Bacillus Licheniformis ◽

External Environment ◽

Permeability Barrier ◽

Periplasmic Space ◽

Modified Technique ◽

Gram Positive ◽

Gram Negative ◽

Gram Positive Bacteria

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

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