Biology subjects

Rainaldi, V.

Publications and source records attributed to Rainaldi, V..

6 recordsLinked to original sources

bioRxiv2026

Combining evolution and machine learning-guided pathway optimization to engineer a novel methylsuccinate module for synthetic C1 metabolism in vivo

De novo metabolic pathways open possibilities for sustainable biotransformations in microbes. However, the in vivo-implementation of such new-to-nature pathways is highly challenging and heavily relies on adaptive laboratory evolution (ALE) of the hosts native metabolic network. Here, we assess how much this need for host-centric ALE can be overcome and/or complemented through the informed design of the newly introduced pathway. Exemplifying for a synthetic CO2-fixation module via methyl-succinate, we established methylsuccinate-dependent growth of Escherichia coli over six months by ALE of E. colis native metabolism. In parallel, we developed a machine-learning guided workflow (MEVIS) for the automated engineering of the synthetic pathway, resulting in methylsuccinate-dependent growth within three weeks. Critically, performing MEVIS in the background of the ALE-evolved strain is necessary to further approach wild-type like growth, demonstrating how ALE in combination with machine-learning-guided lab automation holds great potential to accelerate and improve design-build-test-learn cycles in contemporary metabolic engineering.

bioengineering↗

bioRxiv2025

Design and analysis of synthetic carbon fixation pathways based on novel enzymatic reactions

Biological carbon fixation is currently limited to seven naturally occurring pathways, each with its own limitations and constraints. In recent years, computational analyses of known biochemical reaction networks have identified dozens of theoretical carbon fixation pathways, some of which may have the potential to outperform their natural counterparts. This mix-and-match approach, however, cannot account for those reactions that have not been reported to occur in nature, which heavily limits the possible solution space. Here, we use a bioretrosynthetic approach coupled with expert biochemical knowledge to identify several novel pathways that leverage enzyme promiscuity and the latent biochemical reaction space. We analyze the thermodynamic, stoichiometric, and kinetic parameters of these pathways and compare them to the ubiquitous Calvin-Benson-Bassham cycle and previously proposed synthetic CO2 fixation cycles, highlighting advantages and disadvantages. We identify several promising pathways that could potentially outcompete the Calvin cycle and other previously proposed synthetic CO2 fixation pathways in predicted biomass yield and/or overall pathway activity. In addition, unlike most of the previously proposed efficient mix-and-match pathways, the pathways proposed in this work do not require vitamin B12, which is an advantage for future implementation in plants or microalgae that typically lack B12 biosynthesis. This work highlights the need for enzyme engineering and design in the quest for efficient biological carbon fixation.

biochemistry↗

bioRxiv2025

Modular in vivo engineering of the reductive methylaspartate cycles for synthetic CO2 fixation

Biological carbon fixation is currently limited to seven naturally occurring pathways. Synthetic carbon fixation pathways have the potential to surpass aerobic natural pathways in efficiency, but none have been realized in living cells. Here, we present the reductive methylaspartate cycles (rMASP), a novel family of 4 energy-efficient aerobic CO2 fixation pathway variants. These cycles have the potential to outperform the yields and/or rates of the Calvin cycle and previously proposed synthetic CO2 fixation cycles. To realize these designs, we adopted a modular engineering approach in Escherichia coli. Several pathway modules up to a cascade of 11 enzymes were realized via engineering and evolution. We demonstrate crotonyl-CoA carboxylase dependent growth for both elevated and ambient CO2 conditions, and show in vitro activity of the other CO2-fixing 2-oxoglutarate carboxylase, which requires further activity optimization for mesophilic temperatures. This work demonstrates important steps toward realizing efficient synthetic carbon fixation pathways in living organisms.

synthetic biology↗

bioRxiv2025

A recursive pathway for isoleucine biosynthesis arises from enzyme promiscuity

Enzyme promiscuity can be the starting point for the evolution of new enzymatic activities and pathways. Previously Cotton et al. (2020) identified underground isoleucine biosynthesis routes that can replace the canonical route in Escherichia coli, after they deleted the enzymes that catalyze the formation of its precursor 2-ketobutyrate. Using this strain and short-term evolution we identify a new pathway for isoleucine biosynthesis based on the promiscuous activity of the native enzyme acetohydroxyacid synthase II. We demonstrate that this enzyme catalyzes the previously unreported condensation of glyoxylate with pyruvate to generate 2-ketobutyrate in vivo. The gene encoding this enzyme, ilvG, is inactivated by a frameshift mutation in the laboratory model strain E. coli K-12 MG1655. Its evolutionary reactivation we report here points to a potential natural role in isoleucine biosynthesis in E. coli. Isoleucine biosynthesis proceeds with a further condensation step of 2-ketobutyrate with pyruvate, again catalyzed by AHAS, giving the proposed pathway the unusual property of recursivity. The discovered enzyme activity uses glyoxylate and pyruvate as direct central metabolic precursors for isoleucine biosynthesis instead of its canonical indirect biosynthesis via the amino acid threonine. Unlike previously discovered underground isoleucine routes by Cotton et al., this route is more likely to play a role in natural isoleucine biosynthesis in E. coli due to the use of ubiquitous metabolites and its activity in aerobic conditions. The discovered route further expands the known metabolic space for isoleucine biosynthesis in E. coli and potentially other organisms, and could find applications in biotechnological isoleucine production.

microbiology↗

bioRxiv2022

Synthetic carbon fixation via the autocatalytic serine threonine cycle

Atmospheric CO2 poses a major threat to life on Earth by causing global warming and climate change. On the other hand, it is the only carbon source that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO2 to reduced one-carbon (C1) molecules (e.g. formate) using renewable energy are improving fast. Driven by the idea of creating sustainable bioproduction platforms, natural and synthetic C1-utilization pathways are engineered into industrially relevant microbes. The realization of synthetic C1-assimilation cycles in living organisms is a promising but challenging endeavour. Here, we engineer the autocatalytic serine threonine cycle, a synthetic C1-assimilation route in Escherichia coli. Our stepwise engineering approach in tailored selection strains combined with adaptive laboratory evolution experiments enabled the organism to grow on formate. The synthetic strain uses formate as the sole carbon and energy source and is capable of growing at ambient CO2 concentrations, demonstrating the feasibility of establishing synthetic C1-assimilation cycles over laboratory timescales.

synthetic biology↗

bioRxiv2022

Optimizing E. coli as a formatotrophic platform for bioproduction via the reductive glycine pathway

Microbial C1 fixation has a vast potential to support a sustainable circular economy. Hence, several biotechnologically important microorganisms have been recently engineered for fixing C1 substrates. However, reports about C1-based bioproduction with these organisms are scarce. Here, we describe the optimization of a previously engineered formatotrophic Escherichia coli strain. Short-term adaptive laboratory evolution enhanced biomass yield and accelerated growth of formatotrophic E. coli to 3.3 g-CDW/mol-formate and 6 hours doubling time, respectively. Genome sequence analysis revealed that manipulation of acetate metabolism is the reason for better growth performance, verified by subsequent reverse engineering of the parental E. coli strain. Moreover, the improved strain is capable of growing to an OD600 of 22 in bioreactor fed-batch experiments, highlighting its potential use for industrial bioprocesses. Finally, demonstrating the strains potential to support a sustainable, formate-based bioeconomy, lactate production from formate and CO2 was engineered. The optimized strain generated 1.2 mM lactate--10 % of the theoretical maximum--providing the first proof-of-concept application of the reductive glycine pathway for bioproduction.

synthetic biology↗