Dataset Information

Engineering Tropism of Pseudomonas putida toward Target Surfaces through Ectopic Display of Recombinant Nanobodies.

ABSTRACT: Gram-negative bacteria are endowed with complex outer membrane (OM) structures that allow them to both interact with other organisms and attach to different physical structures. However, the design of reliable bacterial coatings of solid surfaces is still a considerable challenge. In this work, we report that ectopic expression of a fibrinogen-specific nanobody on the envelope of Pseudomonas putida cells enables controllable formation of a bacterial monolayer strongly bound to an antigen-coated support. To this end, either the wild type or a surface-naked derivative of P. putida was engineered to express a hybrid between the β-barrel of an intimin-type autotransporter inserted in the outer membrane and a nanobody (V_HH) moiety that targets fibrinogen as its cognate interaction partner. The functionality of the thereby presented V_HH and the strength of the resulting cell attachment to a solid surface covered with the cognate antigen were tested and parametrized with Quartz Crystal Microbalance technology. The results not only demonstrated the value of using bacteria with reduced OM complexity for efficient display of artificial adhesins, but also the potential of this approach to engineer specific bacterial coverings of predetermined target surfaces.

SUBMITTER: Fraile S

PROVIDER: S-EPMC8397431 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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Engineering Tropism of <i>Pseudomonas putida</i> toward Target Surfaces through Ectopic Display of Recombinant Nanobodies.

Fraile Sofía S Briones María M Revenga-Parra Mónica M de Lorenzo Víctor V Lorenzo Encarnación E Martínez-García Esteban E

ACS synthetic biology 20210802 8

Gram-negative bacteria are endowed with complex outer membrane (OM) structures that allow them to both interact with other organisms and attach to different physical structures. However, the design of reliable bacterial coatings of solid surfaces is still a considerable challenge. In this work, we report that ectopic expression of a fibrinogen-specific nanobody on the envelope of <i>Pseudomonas putida</i> cells enables controllable formation of a bacterial monolayer strongly bound to an antigen- ...[more]

PMID: 34337948

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Project description:Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.

Dataset Information

Engineering Tropism of Pseudomonas putida toward Target Surfaces through Ectopic Display of Recombinant Nanobodies.

Publications

Engineering Tropism of <i>Pseudomonas putida</i> toward Target Surfaces through Ectopic Display of Recombinant Nanobodies.

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