Biology subjects

Schulz-Mirbach, H.

Publications and source records attributed to Schulz-Mirbach, H..

7 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

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

Systematic engineering of synthetic serine cycles in Pseudomonas putida uncovers emergent topologies for methanol assimilation

The urgent need for a circular carbon economy has driven research into sustainable substrates, including one-carbon (C1) compounds. The non-pathogenic soil bacterium Pseudomonas putida is a promising host for exploring synthetic methylotrophy due to its versatile metabolism. In this work, we implemented synthetic serine cycle variants in P. putida for methanol assimilation combining modular engineering and growth-coupled selection, whereby methanol assimilation supported biosynthesis of the essential amino acid serine. The serine cycle forms acetyl-coenzyme A from C1 molecules without carbon loss but has bottlenecks that hinder engineering efforts. We adopted three synthetic variants (serine-threonine cycle, homoserine cycle, and modified serine cycle) that yield serine in a methanol-dependent fashion to overcome these challenges. By dividing these metabolic designs into functional modules, we systematically compared their performance for implementation in vivo. Additionally, we harnessed native pyrroloquinoline quinone-dependent dehydrogenases for engineering methylotrophy. Recursive rewiring of synthetic and native activities revealed novel metabolic topologies for methanol utilization, termed enhanced serine-threonine cycle, providing a blueprint for engineering C1 assimilation in non-model heterotrophic bacteria. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/638773v1_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@1bfccd0org.highwire.dtl.DTLVardef@11f77deorg.highwire.dtl.DTLVardef@f6a904org.highwire.dtl.DTLVardef@1d24a34_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

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↗

bioRxiv2022

Implementation of the β-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol

Ethylene glycol (EG) is a promising next generation feedstock for bioprocesses. It is a key component of the ubiquitous plastic polyethylene terephthalate (PET) and other polyester fibers and plastics, used in antifreeze formulations, and can also be generated by electrochemical conversion of syngas, which makes EG a key compound in a circular bioeconomy. The majority of biotechnologically relevant bacteria assimilate EG via the glycerate pathway, a wasteful metabolic route that releases CO2 and requires reducing equivalents as well as ATP. In contrast, the recently characterized {beta}-hydroxyaspartate cycle (BHAC) provides a more efficient, carbon-conserving route for C2 assimilation. Here we aimed at overcoming the natural limitations of EG metabolism in the industrially relevant strain Pseudomonas putida KT2440 by replacing the native glycerate pathway with the BHAC. We first prototyped the core reaction sequence of the BHAC in Escherichia coli before establishing the complete four-enzyme BHAC in Pseudomonas putida. Directed evolution on EG resulted in an improved strain that exhibits 35% faster growth and 20% increased biomass yield compared to a recently reported P. putida strain that was evolved to grow on EG via the glycerate pathway. Genome sequencing and proteomics highlight plastic adaptations of the genetic and metabolic networks in response to the introduction of the BHAC into P. putida and identify key mutations for its further integration during evolution. Taken together, our study shows that the BHAC can be utilized as plug-and-play module for the metabolic engineering of two important microbial platform organisms, paving the way for multiple applications for a more efficient and carbon-conserving upcycling of EG in the future.

synthetic biology↗

bioRxiv2022

On the flexibility of the cellular amination network in E. coli

Ammonium (NH4+) is essential to generate the nitrogenous building blocks of life. It gets assimilated via the canonical biosynthetic routes to glutamate and is further distributed throughout metabolism via a network of transaminases. To study the flexibility of this network, we constructed an Escherichia coli glutamate auxotrophic strain. This strain allowed us to systematically study which amino acids serve as amine source and found that several amino acids complement the auxotrophy, either by producing glutamate via transamination reactions or by their conversion to glutamate. In this network, we identified aspartate transaminase AspC as a major connector between many amino acids and glutamate. Additionally, we extended the transaminase network by the amino acids {beta}-alanine, alanine, glycine and serine as new amine sources and identified D-amino acid dehydrogenase (DadA) as an intracellular amino acid sink removing substrates from transaminase reactions. Finally, ammonium assimilation routes producing aspartate or leucine were introduced. Our study reveals the high flexibility of the cellular amination network, both in terms of transaminase promiscuity and adaptability to new connections and ammonium entry points.

systems biology↗

bioRxiv2021

Activating silent glycolysis bypasses in Escherichia coli

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal, the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal immediately operated in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary for implementing a functional methylglyoxal pathway. Furthermore, we engineered the serine shunt which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection we performed an adaptive laboratory evolution study using an enolase deletion strain. The evolved mutants were shown to use the serine shunt. Our study reveals the flexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism.

synthetic biology↗