Dataset Information

Valorization of Lignin as an Immobilizing Agent for Bioinoculant Production using Azospirillum brasilense as a Model Bacteria.

ABSTRACT: Plant growth-promoting bacteria (PGPB) have been largely considered as beneficial in harsh and limiting environments given their effects on alleviating plant stress. For practical applications, most of the PGPB are prepared in immobilization matrices to improve the stability and benefits of bacteria. Despite the long list of immobilizing agents/carriers tested to date, a long list of desired requirements is yet to be achieved. Here, lignin stands as a scarcely tested immobilizer for bioinoculants with great potential for this purpose. The aim of this work was to demonstrate the feasibility of lignin as a carrier of the nitrogen-fixing Azospirillum brasilense. These bacteria were cultured in liquid media with recovered organosolv lignin added for bacterial immobilization. Then, lignin was recovered and the immobilized biomass was quantified gravimetrically by DNA extraction and serial dilution plating. Fluorescent microscopy as well as Congo red agar plating showed the immobilization of the bacterial cells in the lignin matrix and crystal violet dyeing showed the biofilms formation in lignin particles. A high number of cells were counted per gram of dried lignin. Lignin can be readily used as low-cost, health-safe bioinoculant carrier to be used in soil and agricultural applications.

SUBMITTER: Tapia-Olivares VR

PROVIDER: S-EPMC6943589 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Valorization of Lignin as an Immobilizing Agent for Bioinoculant Production using <i>Azospirillum brasilense</i> as a Model Bacteria.

Tapia-Olivares Victor Rogelio VR Vazquez-Bello Eimy Alejandra EA Aguilar-Garnica Efrén E Escalante Froylán M E FME

Molecules (Basel, Switzerland) 20191217 24

Plant growth-promoting bacteria (PGPB) have been largely considered as beneficial in harsh and limiting environments given their effects on alleviating plant stress. For practical applications, most of the PGPB are prepared in immobilization matrices to improve the stability and benefits of bacteria. Despite the long list of immobilizing agents/carriers tested to date, a long list of desired requirements is yet to be achieved. Here, lignin stands as a scarcely tested immobilizer for bioinoculant ...[more]

PMID: 31861075

Similar Datasets

Project description:Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae, do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.

Project description:Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 μM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

Dataset Information

Valorization of Lignin as an Immobilizing Agent for Bioinoculant Production using Azospirillum brasilense as a Model Bacteria.

Publications

Valorization of Lignin as an Immobilizing Agent for Bioinoculant Production using <i>Azospirillum brasilense</i> as a Model Bacteria.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets