Cell‐Free Protein Synthesis for the Screening of Novel Azoreductases and Their Preferred Electron Donor

Rolf, Jascha; Ngo, Anna; Tischler, Dirk; Lütz, Stephan; Rosenthal, Katrin

doi:10.1002/cbic.202200121

Cited by 5 publications

(7 citation statements)

References 38 publications

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“…Depending on the assay applied, results can be obtained only 5 h after the reaction was started, thus allowing increased flexibility and handling. The capability of cell-free systems for those screening approaches was recently demonstrated by the synthesis and characterization of several novel azoreductases ( Rolf et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases

Walter

Zemella

Schramm

et al. 2022

Front. Bioeng. Biotechnol.

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Unspecific peroxygenases (UPOs, EC 1.11.2.1) are fungal enzymes that catalyze the oxyfunctionalization of non-activated hydrocarbons, making them valuable biocatalysts. Despite the increasing interest in UPOs that has led to the identification of thousands of putative UPO genes, only a few of these have been successfully expressed and characterized. There is currently no universal expression system in place to explore their full potential. Cell-free protein synthesis has proven to be a sophisticated technique for the synthesis of difficult-to-express proteins. In this work, we aimed to establish an insect-based cell-free protein synthesis (CFPS) platform to produce UPOs. CFPS relies on translationally active cell lysates rather than living cells. The system parameters can thus be directly manipulated without having to account for cell viability, thereby making it highly adaptable. The insect-based lysate contains translocationally active, ER-derived vesicles, called microsomes. These microsomes have been shown to allow efficient translocation of proteins into their lumen, promoting post-translational modifications such as disulfide bridge formation and N-glycosylations. In this study the ability of a redox optimized, vesicle-based, eukaryotic CFPS system to synthesize functional UPOs was explored. The influence of different reaction parameters as well as the influence of translocation on enzyme activity was evaluated for a short UPO from Marasmius rotula and a long UPO from Agrocybe aegerita. The capability of the CFPS system described here was demonstrated by the successful synthesis of a novel UPO from Podospora anserina, thus qualifying CFPS as a promising tool for the identification and evaluation of novel UPOs and variants thereof.

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Section: Discussionmentioning

confidence: 99%

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases

Walter

Zemella

Schramm

et al. 2022

Front. Bioeng. Biotechnol.

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“…Subsequently, high-throughput experimental methods can be applied to validate the simulation results in a synergistic manner. Multiple studies have shown that cell-free protein expression methods can nowadays allow efficient enzyme discovery because they can be directly coupled to an activity assay (Silverman et al 2020 ; Garenne et al 2021 ; Kwon and Jewett 2015 ; Tamiev et al 2021 ; Rolf et al 2019 ; Haslinger et al 2021 ; Rolf et al 2022 ). Moreover, they overcome the time-consuming constraints of working with cells and allow for rapid protein production bypassing most of the cloning, expression, and purification steps (Silverman et al 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Discovery and biochemical characterization of thermostable glycerol oxidases

Santema,

Rotilio,

Xiang

et al. 2024

Appl Microbiol Biotechnol

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Alditol oxidases are promising tools for the biocatalytic oxidation of glycerol to more valuable chemicals. By integrating in silico bioprospecting with cell-free protein synthesis and activity screening, an effective pipeline was developed to rapidly identify enzymes that are active on glycerol. Three thermostable alditol oxidases from Actinobacteria Bacterium, Streptomyces thermoviolaceus, and Thermostaphylospora chromogena active on glycerol were discovered. The characterization of these three flavoenzymes demonstrated their glycerol oxidation activities, preference for alkaline conditions, and excellent thermostabilities with melting temperatures higher than 75 °C. Structural elucidation of the alditol oxidase from Actinobacteria Bacterium highlighted a constellation of side chains that engage the substrate through several hydrogen bonds, a histidine residue covalently bound to the FAD prosthetic group, and a tunnel leading to the active site. Upon computational simulations of substrate binding, a double mutant targeting a residue pair at the tunnel entrance was created and found to display an improved thermal stability and catalytic efficiency for glycerol oxidation. The hereby described alditol oxidases form a valuable panel of oxidative biocatalysts that can perform regioselective oxidation of glycerol and other polyols. Key points • Rapid pipeline designed to identify putative oxidases • Biochemical and structural characterization of alditol oxidases • Glycerol oxidation to more valuable derivatives Graphical Abstract

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“…When using unpurified extract-based systems, endogenous proteins or metabolic pathways may compete with the biocatalytic activity of the target enzyme for substrates and reduce or completely mask its detectable activity. 5,6 In such a case, so-called PURE (protein synthesis using recombinant elements) systems can be used, which consist exclusively of components required for protein biosynthesis. 6 Furthermore, even though comparatively high protein concentrations up to mg mL À1 scale have already been achieved in CFPS systems, 7 the average yields and thus concentrations are significantly lower than in many applications using proteins obtained in vivo.…”

Section: Introductionmentioning

confidence: 99%

“…5,6 In such a case, so-called PURE (protein synthesis using recombinant elements) systems can be used, which consist exclusively of components required for protein biosynthesis. 6 Furthermore, even though comparatively high protein concentrations up to mg mL À1 scale have already been achieved in CFPS systems, 7 the average yields and thus concentrations are significantly lower than in many applications using proteins obtained in vivo. Typically, CFPS on a smallscale (<100 μL) is followed by further dilution with reaction buffer and substrates for the biocatalytic reaction.…”

Section: Introductionmentioning

confidence: 99%

“…However, various limitations to the applicability of CFPS have also been demonstrated. When using unpurified extract‐based systems, endogenous proteins or metabolic pathways may compete with the biocatalytic activity of the target enzyme for substrates and reduce or completely mask its detectable activity 5,6 . In such a case, so‐called PURE (protein synthesis using recombinant elements) systems can be used, which consist exclusively of components required for protein biosynthesis 6 .…”

Section: Introductionmentioning

confidence: 99%

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Amino acid balancing for the prediction and evaluation of protein concentrations in cell‐free protein synthesis systems

Rolf

Handke

Frank

et al. 2023

Biotechnology Progress

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Cell‐free protein synthesis (CFPS) systems are an attractive method to complement the usual cell‐based synthesis of proteins, especially for screening approaches. The literature describes a wide variety of CFPS systems, but their performance is difficult to compare since the reaction components are often used at different concentrations. Therefore, we have developed a calculation tool based on amino acid balancing to evaluate the performance of CFPS by determining the fractional yield as the ratio between theoretically achievable and experimentally achieved protein molar concentration. This tool was applied to a series of experiments from our lab and to various systems described in the literature to identify systems that synthesize proteins very efficiently and those that still have potential for higher yields. The well‐established Escherichia coli system showed a high efficiency in the utilization of amino acids, but interestingly, less considered systems, such as those based on Vibrio natriegens or Leishmania tarentolae, also showed exceptional fractional yields of over 70% and 90%, respectively, implying very efficient conversions of amino acids. The methods and tools described here can quickly identify when a system has reached its maximum or has limitations. We believe that this approach will facilitate the evaluation and optimization of existing CFPS systems and provides the basis for the systematic development of new CFPS systems.

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Cell‐Free Protein Synthesis for the Screening of Novel Azoreductases and Their Preferred Electron Donor

Cited by 5 publications

References 38 publications

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases

Vesicle-based cell-free synthesis of short and long unspecific peroxygenases

Discovery and biochemical characterization of thermostable glycerol oxidases

Amino acid balancing for the prediction and evaluation of protein concentrations in cell‐free protein synthesis systems

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