Time-resolved, deuterium-based fluxomics uncovers the hierarchy and dynamics of sugar processing by Pseudomonas putida.

Pseudomonas putida, a microbial host widely adopted for metabolic engineering, processes glucose through convergent peripheral pathways that ultimately yield 6-phosphogluconate. The periplasmic gluconate shunt (PGS), composed by glucose and gluconate dehydrogenases, sequentially transforms glucose into gluconate and 2-ketogluconate. Although the secretion of these organic acids by P. putida has been extensively recognized, the mechanism and spatiotemporal regulation of the PGS remained elusive thus far. To address this challenge, we adopted a dynamic 13C- and 2H-metabolic flux analysis strategy, termed D-fluxomics. D-fluxomics demonstrated that the PGS underscores a highly dynamic metabolic architecture in glucose-dependent batch cultures of P. putida, characterized by hierarchical carbon uptake by the PGS throughout the cultivation. Additionally, we show that gluconate and 2-ketogluconate accumulation and consumption can be solely explained as a result of the interplay between growth rate-coupled and decoupled metabolic fluxes. As a consequence, the formation of these acids in the PGS is inversely correlated to the bacterial growth rate-unlike the widely studied overflow metabolism of Escherichia coli and yeast. Our findings, which underline survival strategies of soil bacteria thriving in their natural environments, open new avenues for engineering P. putida towards efficient, sugar-based bioprocesses.

An automated workflow for multi-omics screening of microbial model organisms.

Multi-omics datasets are becoming of key importance to drive discovery in fundamental research as much as generating knowledge for applied biotechnology. However, the construction of such large datasets is usually time-consuming and expensive. Automation might enable to overcome these issues by streamlining workflows from sample generation to data analysis. Here, we describe the construction of a complex workflow for the generation of high-throughput microbial multi-omics datasets. The workflow comprises a custom-built platform for automated cultivation and sampling of microbes, sample preparation protocols, analytical methods for sample analysis and automated scripts for raw data processing. We demonstrate possibilities and limitations of such workflow in generating data for three biotechnologically relevant model organisms, namely Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida.

Automating the design-build-test-learn cycle towards next-generation bacterial cell factories.

Automation is playing an increasingly significant role in synthetic biology. Groundbreaking technologies, developed over the past 20 years, have enormously accelerated the construction of efficient microbial cell factories. Integrating state-of-the-art tools (e.g. for genome engineering and analytical techniques) into the design-build-test-learn cycle (DBTLc) will shift the metabolic engineering paradigm from an almost artisanal labor towards a fully automated workflow. Here, we provide a perspective on how a fully automated DBTLc could be harnessed to construct the next-generation bacterial cell factories in a fast, high-throughput fashion. Innovative toolsets and approaches that pushed the boundaries in each segment of the cycle are reviewed to this end. We also present the most recent efforts on automation of the DBTLc, which heralds a fully autonomous pipeline for synthetic biology in the near future.

Current landscape and future directions of synthetic biology in South America.

Synthetic biology (SynBio) is a rapidly advancing multidisciplinary field in which South American countries such as Chile, Argentina, and Brazil have made notable contributions and have established leadership positions in the region. In recent years, efforts have strengthened SynBio in the rest of the countries, and although progress is significant, growth has not matched that of the aforementioned countries. Initiatives such as iGEM and TECNOx have introduced students and researchers from various countries to the foundations of SynBio. Several factors have hindered progress in the field, including scarce funding from both public and private sources for synthetic biology projects, an underdeveloped biotech industry, and a lack of policies to promote bio-innovation. However, open science initiatives such as the DIY movement and OSHW have helped to alleviate some of these challenges. Similarly, the abundance of natural resources and biodiversity make South America an attractive location to invest in and develop SynBio projects.

Protocol for absolute quantification of proteins in Gram-negative bacteria based on QconCAT-based labeled peptides.

Mass-spectrometry-based absolute protein quantification uses labeled quantification concatamer (QconCAT) as internal standards (ISs). To calculate the amount of protein(s), the ion intensity ratio between the analyte and its cognate IS is compared in each biological sample. The present protocol describes a systematic workflow to design, produce, and purify QconCATs and to quantify soluble proteins in Pseudomonas putida KT2440. Our methodology enables the quantification of detectable peptide and serves as a versatile platform to produce ISs for different biological systems.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes.

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways for a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Building on this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-informed engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route-the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic P. putida auxotroph to rewire its metabolic network to restore C2 prototrophy via the PKT shunt. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff pathway and the synthetic PKT bypass for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

Role of the CrcB transporter of Pseudomonas putida in the multi-level stress response elicited by mineral fluoride.

The presence of mineral fluoride (F- ) in the environment has both a geogenic and anthropogenic origin, and the halide has been described to be toxic in virtually all living organisms. While the evidence gathered in different microbial species supports this notion, a systematic exploration of the effects of F- salts on the metabolism and physiology of environmental bacteria remained underexplored thus far. In this work, we studied and characterized tolerance mechanisms deployed by the model soil bacterium Pseudomonas putida KT2440 against NaF. By adopting systems-level omic approaches, including functional genomics and metabolomics, we gauged the impact of this anion at different regulatory levels under conditions that impair bacterial growth. Several genes involved in halide tolerance were isolated in a genome-wide Tn-Seq screening-among which crcB, encoding an F- -specific exporter, was shown to play the predominant role in detoxification. High-resolution metabolomics, combined with the assessment of intracellular and extracellular pH values and quantitative physiology experiments, underscored the key nodes in central carbon metabolism affected by the presence of F- . Taken together, our results indicate that P. putida undergoes a general, multi-level stress response when challenged with NaF that significantly differs from that caused by other saline stressors. While microbial stress responses to saline and oxidative challenges have been extensively studied and described in the literature, very little is known about the impact of fluoride (F- ) on bacterial physiology and metabolism. This state of affairs contrasts with the fact that F- is more abundant than other halides in the Earth crust (e.g. in some soils, the F- concentration can reach up to 1 mg gsoil -1 ). Understanding the global effects of NaF treatment on bacterial physiology is not only relevant to unveil distinct mechanisms of detoxification but it could also guide microbial engineering approaches for the target incorporation of fluorine into value-added organofluorine molecules. In this regard, the soil bacterium P. putida constitutes an ideal model to explore such scenarios, since this species is particularly known for its high level of stress resistance against a variety of physicochemical perturbations.

Dynamic flux regulation for high-titer anthranilate production by plasmid-free, conditionally-auxotrophic strains of Pseudomonas putida.

Anthranilate, an intermediate of the shikimate pathway, is a high-value aromatic compound widely used as a precursor in the production of dyes, fragrances, plastics and pharmaceuticals. Traditional strategies adopted for microbial anthranilate production rely on the implementation of auxotrophic strains-which requires aromatic amino acids or complex additives to be supplemented in the culture medium, negatively impacting production costs. In this work, we engineered the soil bacterium Pseudomonas putida for high-titer, glucose-dependent anthranilate production by repurposing elements of the Esa quorum sensing (QS) system of Pantoea stewartii. The PesaS promoter mediated a self-regulated transcriptional response that effectively knocked-down the expression of the trpDC genes. Next, we harnessed the synthetic QS elements to engineer a growth-to-anthranilate production switch. The resulting plasmid-free P. putida strain produced the target compound at 3.8 ± 0.3 mM in shaken-flask cultures after 72 h-a titer >2-fold higher than anthranilate levels reported thus far. Our results highlight the value of dynamic flux regulation for the production of intermediate metabolites within highly-regulated routes (such as the shikimate pathway), thereby circumventing the need of expensive additives.

Merging automation and fundamental discovery into the design-build-test-learn cycle of nontraditional microbes.

Major advances toward a bio-based industry enabled cost-efficient bioproduction of multiple chemicals, yet successful industrial processes are relatively scarce and limited to the use of few workhorse microbes as hosts. An in-depth understanding of the physiology and metabolism of nontraditional microorganisms is key to unleash their biotechnological potential. The inception of biofoundries multiplied the capacity of constructing and testing a large number of microbial strains tailored for bioproduction - and we argue that automation workflows therein can be adapted to gain fundamental knowledge of nontraditional hosts. Here, we propose a 'metabolism-centric' approach to the design-build-test-learn cycle of synthetic biology, supported by multi-omic analyses, to facilitate the deployment of microbial cell factories designed for bioproduction beyond the typical landscape of target products.

High-throughput dilution-based growth method enables time-resolved exo-metabolomics of Pseudomonas putida and Pseudomonas aeruginosa.

Understanding metabolism is fundamental to access and harness bacterial physiology. In most bacteria, nutrient utilization is hierarchically optimized according to their energetic potential and their availability in the environment to maximise growth rates. Low-throughput methods have been largely used to characterize bacterial metabolic profiles. However, in-depth analysis of large collections of strains across several conditions is challenging since high-throughput approaches are still limited - especially for non-traditional hosts. Here, we developed a high-throughput dilution-resolved cultivation method for metabolic footprinting of Pseudomonas putida and Pseudomonas aeruginosa. This method was benchmarked against a conventional low-throughput time-resolved cultivation approach using either a synthetic culture medium (where a single carbon source is present) for P. putida or a complex nutrient mixture for P. aeruginosa. Dynamic metabolic footprinting, either by sugar quantification or by targeted exo-metabolomic analyses, revealed overlaps between the bacterial metabolic profiles irrespective of the cultivation strategy, suggesting a certain level of robustness and flexibility of the high-throughput dilution-resolved method. Cultivation of P. putida in microtiter plates imposed a metabolic constraint, dependent on oxygen availability, which altered the pattern of secreted metabolites at the level of sugar oxidation. Deep-well plates, however, constituted an optimal cultivation set-up yielding consistent and comparable metabolic profiles across conditions and strains. Altogether, the results illustrate the usefulness of this technological advance for high-throughput analyses of bacterial metabolism for both biotechnological applications and automation purposes.