Biology subjects

Orsi, E.

Publications and source records attributed to Orsi, E..

7 recordsLinked to original sources

bioRxiv2026

Adaptive laboratory evolution rewires Pseudomonas putida for resource-efficient acetate assimilation

Acetate is an attractive renewable two-carbon substrate for microbial biotechnology, but its toxicity limits growth and carbon-use efficiency at process-relevant concentrations. Here, we used adaptive laboratory evolution to improve acetate tolerance in a genome-reduced strain of Pseudomonas putida and combined whole-genome sequencing, reverse engineering, transcriptomics, proteomics, and 13C-acetate fluxomics to resolve the underlying adaptation mechanisms. Evolution under increasing acetate concentrations selected recurrent mutations in gacA and fabB, which encode a global response regulator and a fatty acid biosynthesis enzyme, respectively. Reverse engineering of these mutations recovered most of the evolved phenotype, including shorter lag phase and substantially higher biomass yield from acetate. Multi-omic analyses showed repression of type VI secretion systems, carbohydrate storage functions, fatty acid metabolism, and oxidative stress-associated proteins, indicating resource reallocation away from costly stress and non-essential programs. Fluxomics further revealed reduced EDEMP cycling and increased glyoxylate shunt flux, consistent with improved acetate-carbon retention in biomass. These results establish acetate tolerance in P. putida as a resource-efficiency phenotype and identify gacA and fabB as actionable targets for acetate-based bioproduction.

synthetic biology↗

bioRxiv2026

Seven mutations unlock strict synthetic methylotrophy in engineered Pseudomonas putida

Methanol is a reduced, soluble one-carbon (C1) feedstock for sustainable bioproduction, but converting this potential into robust microbial growth remains difficult. Several synthetic C1 assimilation routes depend on autocatalytic cycles, whose operation requires coordinated control of redox balance, toxic intermediates, substrate regeneration, and host regulation. Here, we implemented the serine-threonine cycle (STC) in the soil bacterium Pseudomonas putida and used growth-coupled selection with adaptive laboratory evolution (ALE) to transition from mixotrophic C1 incorporation to strict methylotrophy. The evolved strain grew with methanol as the sole carbon and energy source under atmospheric CO2 with a doubling time of ca. 40 h. Whole-genome sequencing, reverse genetics, biosensors, isotope labelling, and comparative RNA sequencing showed that evolution repeatedly targeted native pyrroloquinoline quinone (PQQ)-dependent methanol oxidation, membrane-bound transhydrogenase activity, glycine regeneration, STC enzyme balance, and global regulatory nodes. Additional ALE under glycine-methanol co-feeding increased growth rates and exposed further targets for improving cycle flux. These results establish P. putida as a chassis for strict synthetic methylotrophy and define actionable engineering routes toward C1 biomanufacturing. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/739708v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1642d2dorg.highwire.dtl.DTLVardef@1d203d1org.highwire.dtl.DTLVardef@f8a5d7org.highwire.dtl.DTLVardef@4fe272_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

A microbial growth-coupled platform for in vivo interrogation of Rubisco oxygenase activity

Rubisco catalyzes the primary CO2-fixing reaction of the biosphere, yet its competing oxygenation reaction reduces net global carbon fixation and has resisted direct exploration in living cells. Here, we engineer an auxotrophic Escherichia coli strain in which 2-phosphoglycolate, the direct product of Rubisco oxygenation, becomes essential for growth, making bacterial fitness a quantitative proxy for oxygenation flux in vivo. This provides direct access to catalytic selectivity, something previously inaccessible to carboxylation-coupled assays. The platform enables screening of phylogenetically diverse Form II Rubisco and phosphoribulokinase (Prk) variants circumventing protein purification and extensive in vitro characterization. Adaptive laboratory evolution under oxygenation-selective pressure identified two mutations: Rubisco M115I genetically rebalances the in vivo carboxylation/oxygenation trade-off (resulting in 6-fold reduction in kcat,C), while Prk N216T improves overall flux without altering selectivity. This platform makes Rubiscos least-studied catalytic function selectable and evolvable in vivo, opening the carboxylation/oxygenation trade-off to systematic genetic dissection and engineering.

synthetic biology↗

bioRxiv2024

Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system

Cupriavidus necator H16 is a promising microbial platform strain for CO2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 hours after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with [~]70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator, facilitating its potential role in the transition to a bio-based economy.

bioengineering↗

bioRxiv2024

Expanding the biotechnological scope of metabolic sensors through computation-aided designs

Metabolic sensors are microbial strains modified so that biomass formation correlates with the availability of specific metabolites. These sensors are essential for bioengineering (e.g. in growth-coupled designs) but creating them is often a time-consuming and low-throughput process that can potentially be streamlined by in silico analysis. Here, we present the systematic workflow of designing, implementing, and testing versatile Escherichia coli metabolic sensor strains. Glyoxylate, a key metabolite in (synthetic) CO2 fixation and carbon-conserving pathways, served as the test molecule. Through iterative screening of a compact metabolic model, we identified non-trivial growth-coupled designs that resulted in six metabolic sensors with a wide sensitivity range for glyoxylate, spanning three orders of magnitude in detected concentrations. We further adapted these E. coli strains for sensing glycolate and demonstrated their utility in both pathway engineering (testing a key metabolic module via glyoxylate) and applications in environmental monitoring (quantifying glycolate produced by photosynthetic microalgae). The versatility and ease of implementation of this workflow make it suitable for designing and building multiple metabolic sensors for diverse biotechnological applications. TeaserA streamlined workflow enables the rapid design of versatile E. coli metabolic sensors for detecting key metabolites in bioengineering and monitoring.

synthetic biology↗

bioRxiv2023

Engineering the biological conversion of formate into crotonate in Cupriavidus necator

To advance the sustainability of the biobased economy, our society needs to develop novel bioprocesses based on truly renewable resources. The C1-molecule formate is increasingly proposed as carbon and energy source for microbial fermentations, as it can be efficiently generated electrochemically from CO2 and renewable energy. Yet, its biotechnological conversion into value-added compounds has been limited to a handful of examples. In this work, we engineered the natural formatotrophic bacterium C. necator as cell factory to enable biological conversion of formate into crotonate, a platform short-chain unsaturated carboxylic acid of biotechnological relevance. First, we developed a small-scale (150-mL working volume) cultivation setup for growing C. necator in minimal medium using formate as only carbon and energy source. By using a fed-batch strategy with automatic feeding of formic acid, we could increase final biomass concentrations 15-fold compared to batch cultivations in flasks. Then, we engineered a heterologous crotonate pathway in the bacterium via a modular approach, where each pathway section was assessed using multiple candidates. The best performing modules included a malonyl-CoA bypass for increasing the thermodynamic drive towards the intermediate acetoacetyl-CoA and subsequent conversion to crotonyl-CoA through partial reverse {beta}-oxidation. This pathway architecture was then tested for formate-based biosynthesis in our fed-batch setup, resulting in a two-fold higher titer, three-fold higher productivity, and five-fold higher yield compared to the strain not harboring the bypass. Eventually, we reached a maximum product titer of 148.0 {+/-} 6.8 mg/L. Altogether, this work consists in a proof-of-principle integrating bioprocess and metabolic engineering approaches for the biological upgrading of formate into a value-added platform chemical.

synthetic biology↗

bioRxiv2022

Engineered synthetic one-carbon fixation exceeds yield of the Calvin Cycle

One-carbon (C1) feedstocks derived from CO2 and renewable electricity, such as formate, are promising substrates for sustainable production of chemicals, food and fuels. Energetically more efficient, engineered C1-fixation pathways were proposed to increase biomass yields above their natural counterparts, but have so far not been shown to achieve this. Here, we replace the native energy-inefficient Calvin-Benson-Bassham (CBB) cycle in Cupriavidus necator by genomic integration of the synthetic reductive glycine pathway for growth on formate. Our final engineered strain reaches a higher biomass yield than the CBB-cycle-utilizing wild type, showing for the first time that efficiencies found in natural metabolism can be exceeded via a synthetic pathway. This yield increase demonstrates the potential of synthetic metabolism and is an important step towards realizing truly sustainable, economically feasible bio-based production.

synthetic biology↗