Culturomics and Circular Agronomy: Two Sides of the Same Coin for the Design of a Tailored Biofertilizer for the Semi-Halophyte Mesembryanthemum crystallinum.

According to the EU, the global consumption of biomass, fossil fuels, metals, and minerals is expected to double by 2050, while waste will increase by 70%. In this context, the Circular Economy Action Plan (CEAP) intends to integrate development and sustainability. In this regard, tailored biofertilizers based on plant growth-promoting bacteria (PGPB) can improve plant yield with fewer inputs. In our project, an autochthonous halophyte of the Andalusian marshes, namely Mesembryanthemum crystallinum, was selected for its interest as a source of pharmaceuticals and nutraceuticals. The aim of this work was to use a culturomics approach for the isolation of specific PGPB and endophytes able to promote plant growth and, eventually, modulate the metabolome of the plant. For this purpose, a specific culture medium based on M. crystallinum biomass, called Mesem Agar (MA), was elaborated. Bacteria of three compartments (rhizosphere soil, root endophytes, and shoot endophytes) were isolated on standard tryptone soy agar (TSA) and MA in order to obtain two independent collections. A higher number of bacteria were isolated on TSA than in MA (47 vs. 37). All the bacteria were identified, and although some of them were isolated in both media (Pseudomonas, Bacillus, Priestia, Rosellomorea, etc.), either medium allowed the isolation of specific members of the M. crystallinum microbiome such as Leclercia, Curtobacterium, Pantoea, Lysinibacillus, Mesobacillus, Glutamicibacter, etc. Plant growth-promoting properties and extracellular degrading activities of all the strains were determined, and distinct patterns were found in both media. The three best bacteria of each collection were selected in order to produce two different consortia, whose effects on seed germination, root colonization, plant growth and physiology, and metabolomics were analyzed. Additionally, the results of the plant metabolome revealed a differential accumulation of several primary and secondary metabolites with pharmaceutical properties. Overall, the results demonstrated the feasibility of using "low cost media" based on plant biomass to carry out a culturomics approach in order to isolate the most suitable bacteria for biofertilizers. In this way, a circular model is established in which bacteria help plants to grow, and, in turn, a medium based on plant wastes supports bacterial growth at low prices, which is the reason why this approach can be considered within the model of "circular agronomy".

Microbiota-Liver-Bile Salts Axis, a Novel Mechanism Involved in the Contrasting Effects of Sodium Selenite and Selenium-Nanoparticle Supplementation on Adipose Tissue Development in Adolescent Rats.

Adolescence is a period during which body composition changes deeply. Selenium (Se) is an excellent antioxidant trace element related to cell growth and endocrine function. In adolescent rats, low Se supplementation affects adipocyte development differently depending on its form of administration (selenite or Se nanoparticles (SeNPs). Despite this effect being related to oxidative, insulin-signaling and autophagy processes, the whole mechanism is not elucidated. The microbiota-liver-bile salts secretion axis is related to lipid homeostasis and adipose tissue development. Therefore, the colonic microbiota and total bile salts homeostasis were explored in four experimental groups of male adolescent rats: control, low-sodium selenite supplementation, low SeNP supplementation and moderate SeNPs supplementation. SeNPs were obtained by reducing Se tetrachloride in the presence of ascorbic acid. Supplementation was received orally through water intake; low-Se rats received twice more Se than control animals and moderate-Se rats tenfold more. Supplementation with low doses of Se clearly affected anaerobic colonic microbiota profile and bile salts homeostasis. However, these effects were different depending on the Se administration form. Selenite supplementation primarily affected liver by decreasing farnesoid X receptor hepatic function, leading to the accumulation of hepatic bile salts together to increase in the ratio Firmicutes/Bacteroidetes and glucagon-like peptide-1 (GLP-1) secretion. In contrast, low SeNP levels mainly affected microbiota, moving them towards a more prominent Gram-negative profile in which the relative abundance of Akkermansia and Muribaculaceae was clearly enhanced and the Firmicutes/Bacteroidetes ratio decreased. This bacterial profile is directly related to lower adipose tissue mass. Moreover, low SeNP administration did not modify bile salts pool in serum circulation. In addition, specific gut microbiota was regulated upon administration of low levels of Se in the forms of selenite or SeNPs, which are properly discussed. On its side, moderate-SeNPs administration led to great dysbiosis and enhanced the abundance of pathogenic bacteria, being considered toxic. These results strongly correlate with the deep change in adipose mass previously found in these animals, indicating that the microbiota-liver-bile salts axis is also mechanistically involved in these changes.

A Culturomics-Based Bacterial Synthetic Community for Improving Resilience towards Arsenic and Heavy Metals in the Nutraceutical Plant Mesembryanthemum crystallinum.

Plant-growth-promoting bacteria (PGPB) help plants thrive in polluted environments and increase crops yield using fewer inputs. Therefore, the design of tailored biofertilizers is of the utmost importance. The purpose of this work was to test two different bacterial synthetic communities (SynComs) from the microbiome of Mesembryanthemum crystallinum, a moderate halophyte with cosmetic, pharmaceutical, and nutraceutical applications. The SynComs were composed of specific metal-resistant plant-growth-promoting rhizobacteria and endophytes. In addition, the possibility of modulating the accumulation of nutraceutical substances by the synergetic effect of metal stress and inoculation with selected bacteria was tested. One of the SynComs was isolated on standard tryptone soy agar (TSA), whereas the other was isolated following a culturomics approach. For that, a culture medium based on M. crystallinum biomass, called Mesem Agar (MA), was elaborated. Bacteria of three compartments (rhizosphere soil, root endophytes, and shoot endophytes) were isolated on standard TSA and MA media, stablishing two independent collections. All bacteria were tested for PGP properties, secreted enzymatic activities, and resistance towards As, Cd, Cu, and Zn. The three best bacteria from each collection were selected in order to produce two different consortiums (denominated TSA- and MA-SynComs, respectively), whose effect on plant growth and physiology, metal accumulation, and metabolomics was evaluated. Both SynComs, particularly MA, improved plant growth and physiological parameters under stress by a mixture of As, Cd, Cu, and Zn. Regarding metal accumulation, the concentrations of all metals/metalloids in plant tissues were below the threshold for plant metal toxicity, indicating that this plant is able to thrive in polluted soils when assisted by metal/metalloid-resistant SynComs and could be safely used for pharmaceutical purposes. Initial metabolomics analyses depict changes in plant metabolome upon exposure to metal stress and inoculation, suggesting the possibility of modulating the concentration of high-value metabolites. In addition, the usefulness of both SynComs was tested in a crop plant, namely Medicago sativa (alfalfa). The results demonstrate the effectiveness of these biofertilizers in alfalfa, improving plant growth, physiology, and metal accumulation.

Biofoundry-assisted expression and characterization of plant proteins.

Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterization has remained a technical bottleneck, often requiring significant effort to optimize expression and purification protocols. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimization and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using Escherichia coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification and improved expression/folding. We next optimized automated assembly of miniaturized cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11-amino acid tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.

A biofoundry workflow for the identification of genetic determinants of microbial growth inhibition.

Biofoundries integrate high-throughput software and hardware platforms with synthetic biology approaches to enable the design, execution and analyses of large-scale experiments. The unique and powerful combination of laboratory infrastructure and expertise in molecular biology and automation programming, provide flexible resources for a wide range of workflows and research areas. Here, we demonstrate the applicability of biofoundries to molecular microbiology, describing the development and application of automated workflows to identify the genetic basis of growth inhibition of the plant pathogen Streptomyces scabies by a Pseudomonas strain isolated from a potato field. Combining transposon mutagenesis with automated high-throughput antagonistic assays, the workflow accelerated the screening of 2880 mutants to correlate growth inhibition with a biosynthetic gene cluster within 2 weeks.

Phytobricks: Manual and Automated Assembly of Constructs for Engineering Plants.

Phytobricks are standardized DNA parts for plants that can be assembled hierarchically into transcriptional units and, subsequently, into multigene constructs. Phytobricks each contain the sequences of one or more functional elements that comprise eukaryotic transcription units, with sequence features that enable them to be used interchangeably in one-step cloning reactions to facilitate combinatorial assembly. The simplicity and efficiency of this one-step reaction has enabled Phytobrick assembly to be miniaturized and automated on liquid handing platforms. In this method, we describe how to design and construct new Phytobricks as well as how to assemble them in both manual and nanoscale automated one-step reactions. Finally, we describe a high-throughput method for sequence verification of assembled plasmids.