Biology subjects

Erb, T. J.

Publications and source records attributed to Erb, T. J..

30 records · Page 2Linked to original sources

bioRxiv2023

An interdependent Metabolic and Genetic Network shows emergent properties in vitro

A hallmark of all living organisms is their ability for self-regeneration which requires a tight integration of metabolic and genetic networks. Here we constructed a metabolic and genetic linked in vitro network (MGLN) that shows life-like behavior outside of a cellular context and generates its own building blocks from non-living matter. To this end, we integrated the metabolism of the crotonyl-CoA/ethyl-malonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle with cell-free protein synthesis using recombinant elements (PURE). We demonstrate that the MGLN produces the essential amino acid glycine from inorganic carbon (CO2), and incorporates it into target proteins following DNA-encoded instructions. By programming genetically encoded response into metabolic networks our work opens new avenues for the development of advanced biomimetic systems with emergent properties, including decision-making, self-regeneration and evolution.

synthetic biology↗

bioRxiv2023

Structural Insights into the Iron Nitrogenase Complex

Nitrogenases are best known for catalysing the reduction of dinitrogen to ammonia at a complex metallic cofactor. Recently, nitrogenases were shown to reduce carbon dioxide (CO2) and carbon monoxide to hydrocarbons, offering a pathway to recycle carbon waste into hydrocarbon products. Among the nitrogenase family the iron nitrogenase is the isozyme with the highest wildtype activity for the reduction of CO2, but the molecular architecture facilitating these activities remained unknown. Here, we report a 2.35-[A] cryogenic electron microscopy structure of the Fe nitrogenase complex from Rhodobacter capsulatus, revealing an [Fe8S9C-(R)-homocitrate]-cluster in the active site. The enzyme complex suggests that the AnfG-subunit is involved in cluster stabilisation, substrate channelling and confers specificity between nitrogenase reductase and catalytic components. Moreover, the structure highlights a different interface between the two catalytic halves of the iron and the molybdenum nitrogenase, potentially influencing the intra-subunit communication and thus the nitrogenase mechanism.

biochemistry↗

bioRxiv2023

Ketosynthase engineering enhances activity and shifts specificity towards non-native extender units in type I linear polyketide synthase

Engineering modular type I polyketide synthases (PKS) for the targeted incorporation of non-natural substrates to create variations in the polyketide backbone is a long-standing goal of PKS research. Thus far, most approaches focused on engineering the acyltransferase domain (AT) of PKS, whereas the effects of other ubiquitous domains such as the ketosynthase domain (KS) have received much less attention. In this work, we investigated the effects of thirteen active site substitutions in the module 3 KS (KS3) of the 6-deoxyerythronolide B synthase (DEBS) on incorporation of non-natural extender units in vitro. Using a truncated and a complete DEBS assembly line, we show that substitutions of F263 in KS3 invert specificity up to 1,250-fold towards incorporation of non-natural extender units in the terminal position. In contrast, substitutions of I444 in KS3 show up to 8-fold increased production of 6-deoxyerythonolide B (6-dEB) analogues with non-natural extender units at internal positions. The latter notably without compromising overall productivity of the assembly line. Our study further elucidates the underlying mechanisms for these different behaviors, highlighting the potential of KS engineering for the production of designer polyketides in the future.

biochemistry↗

bioRxiv2022

Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides

Bioactive peptides are key molecules in health and medicine. Deep learning holds a big promise for the discovery and design of bioactive peptides. Yet, suitable experimental approaches are required to validate candidates in high throughput and at low cost. Here, we established a cell- free protein synthesis (CFPS) pipeline for the rapid and inexpensive production of antimicrobial peptides (AMPs) directly from DNA templates. To validate our platform, we used deep learning to design thousands of AMPs de novo. Using computational methods, we prioritized 500 candidates that we produced and screened with our CFPS pipeline. We identified 30 functional AMPs, which we characterized further through molecular dynamics simulations, antimicrobial activity and toxicity. Notably, six de novo-AMPs feature broad-spectrum activity against multidrug-resistant pathogens and do not develop bacterial resistance. Our work demonstrates the potential of CFPS for production and testing of bioactive peptides within less than 24 hours and <10$ per screen.

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↗

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↗

bioRxiv2021

A versatile active learning workflow for optimization of genetic and metabolic networks

The study, engineering and application of biological networks require practical and efficient approaches. Current optimization efforts of these systems are often limited by wet lab labor and cost, as well as the lack of convenient, easily adoptable computational tools. Aimed at democratization and standardization, we describe METIS, a modular and versatile active machine learning workflow with a simple online interface for the optimization of biological target functions with minimal experimental datasets. We demonstrate our workflow for various applications, from simple to complex gene circuits and metabolic networks, including several cell-free transcription and translation systems, a LacI-based multi-level controller and a 27-variable synthetic CO2-fixation cycle (CETCH cycle). Using METIS, we could improve above systems between one and two orders of magnitude compared to their original setup with minimal experimental efforts. For the CETCH cycle, we explored the combinatorial space of [~]1025 conditions with only 1,000 experiments to yield the most efficient CO2-fixation cascade described to date. Beyond optimization, our workflow also quantifies the relative importance of individual factors to the performance of a system. This allows to identify so far unknown interactions and bottlenecks in complex systems, which paves the way for their hypothesis-driven improvement, which we demonstrate for the LacI multi-level controller that we were able to improve by 34-fold after having identified resource competition as limiting factor. Overall, our workflow opens the way for convenient optimization and prototyping of genetic and metabolic networks with customizable adjustments according to user experience, experimental setup, and laboratory facilities.

synthetic biology↗

bioRxiv2021

Emergence of NADP+-reducing enzymes in Escherichia coli central metabolism via adaptive evolution

The nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation towards the emergence of novel cofactor regeneration routes was not previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotroph strain. We continuously cultivated this strain under selective conditions. After 500-1100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD-dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wildtype variant with NAD+. In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP+ thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve towards reducing NADP+, which we attribute to several biochemical constraints such as unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context towards changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries. ImportanceIn the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism. Correspondingly, the ratio of the reduced to the oxidized form differs for NAD (low) and NADP (high), reflecting their distinct roles. We challenged the flexibility of E. colis central metabolism in multiple adaptive evolution experiments using an NADPH-auxotroph strain. We found several mutations in two enzymes, changing the cofactor preference of malic enzyme and dihydrolipoamide dehydrogenase. Upon deletion of their corresponding genes we performed additional evolution experiments which did not lead to the emergence of any additional mutants. We attribute this restricted number of mutational targets to intrinsic thermodynamic barriers: The high ratio of NADPH to NADP+ limits metabolic redox reactions which can regenerate NADPH, mainly by mass action constraints.

microbiology↗

bioRxiv2020

Awakening a latent carbon fixation cycle in Escherichia coli

Carbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner-Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP+

Efficient regeneration of cofactors is vital for the establishment of continuous biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO2). However, formate dehydrogenases (FDHs) are usual specific to NAD+, such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP+, but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP+, specificity towards NADP+, and affinity towards formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits no initial activity towards NADP+, we generate a library of >106 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 13-fold higher specificity than the best enzyme previously engineered, while retaining high affinity towards formate. By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how non-additive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify superior enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.

biochemistry↗

bioRxiv2020

Structural adaptation of oxygen tolerance in 4-hydroxybutyrl-CoA dehydratase, a key enzyme of archaeal carbon fixation

Autotrophic microorganisms that convert inorganic carbon into organic matter were key players in the evolution of life on Earth. As the early atmosphere became oxygenated, microorganisms needed to develop mechanisms for oxygen protection, especially those relying on enzymes containing oxygen-sensitive metal clusters (e.g., Fe-S). Here we investigated how 4-hydroxybutyryl-CoA dehydratase (4HBD) - the key enzyme of the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle for CO2-fixation - adapted as conditions shifted from anoxic to oxic. 4HBD is found in both anaerobic bacteria and aerobic ammonia-oxidizing archaea (AOA). The oxygen-sensitive bacterial 4HBD and oxygen-tolerant archaeal 4HBD share 59 % amino acid identity. To examine the structural basis of oxygen tolerance in archaeal 4HBD, we determined the atomic resolution structure of the enzyme. Two tunnels providing access to the canonical [4Fe-4S] cluster in oxygen-sensitive bacterial 4HBD were closed with four conserved mutations found in all aerobic AOA and other archaea. Further biochemical experiments and molecular dynamics simulations support our findings that restricting access to the active site is the key to oxygen tolerance, explaining how active site evolution drove a major evolutionary transition. Significance statementAutotrophy (primary production) was the first life strategy on Earth. Before photosynthesis (using solar energy to fix carbon dioxide), life relied on chemical reactions for energy. These chemosynthetic reactions are present in all domains of life, including archaea possessing the most energy-efficient carbon fixation pathway - the 3-hydroxypropionate/4-hydroxybutyrate cycle. This efficiency results from enzyme modifications, including enhanced enzyme stability and catalysis of multiple reactions. We reveal the first structure of aerobic 4-hydroxybutyryl-CoA dehydratase (4HBD) from ammonia-oxidizing archaea. These archaea are among the most abundant organisms on the planet, and their 4HBD active site evolved oxygen tolerance to support aerobic metabolism. This modification can provide further insight into enzyme evolution on early earth, as photosynthesis developed and began oxygenating the atmosphere.

evolutionary biology↗

bioRxiv2019

Coupled inter-subunit dynamics enable the fastest CO2-fixation by reductive carboxylases

Enoyl-CoA carboxylases/reductases (ECRs) belong to the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying ECRs extraordinary catalytic activity on the level of the protein assembly remain elusive. Here we used a combination of ambient temperature X-ray Free Electron Laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. K. setae ECR is a homo-tetramer that differentiates into a dimer of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in K. setae ECR across the pair of two dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intra-subunit communications of this remarkably efficient CO2-fixing enzyme during catalysis. Significance StatementFixation of CO2 offers real potential for reaching negative CO2 emissions in bioenergy, and bioproduct utilization. The capture and conversion of atmospheric CO2 remains a challenging task. Existing biological systems can be exploited and optimized for this use. Bacterial enoyl-CoA carboxylases/reductases (ECRs) encompass the fastest CO2-fixing enzymes found in nature to date. However, the mechanisms underlying ECRs extraordinary catalytic activity remain elusive. Our structural, computational, and biochemical results elucidate the dynamic structural organization of the ECR complex and describe how coupled motions of catalytic domains in the ECR tetramer drive carboxylation. This mechanistic understanding is critical for engineering highly efficient CO2-fixing biocatalysts for bioenergy and bioproduct applications.

biophysics↗