From Cell-Free Protein Synthesis to Whole-Cell Biotransformation: Screening and Identification of Novel α-Ketoglutarate-Dependent Dioxygenases for Preparative-Scale Synthesis of Hydroxy-l-Lysine

Rolf, Jascha; Nerke, Philipp; Britner, Annette; Krick, Sebastian; Lütz, Stephan; Rosenthal, Katrin

doi:10.3390/catal11091038

Cited by 11 publications

(12 citation statements)

References 38 publications

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“…The synthesis temperature for CFPS systems based on E. coli is often 30 to 37 °C due to the temperature optimum of the parent organism. The synthesis times at this temperature range from 2 to 8 h. Based on these parameters and own experiences, [17] the synthesis conditions of 37 °C and 4 h were selected for the first experiments with an in‐house E. coli extract‐based CFPS system. Unfortunately, the high background activity of the E. coli extract rendered this synthesis system useless for subsequent methyl red degradation assays.…”

Section: Resultsmentioning

confidence: 99%

“…Due to its open nature, CFPS offers the possibility of easy manipulation of the synthesis conditions and is, therefore, a valuable platform for the synthesis of difficult-to-express proteins, such as proteins with a toxic effect on host cell metabolism [16] or proteins that tend to be synthesized in insoluble form. [17] Thus, the applicability of CFPS for protein screenings and characterizations has already been shown in several studies. [18][19][20] In this study, we aimed to develop a screening setup using CFPS in combination with a subsequent in vitro activity assay in a multi-well microplate, which allowed the parallel screening of several putative genes.…”

Section: Introductionmentioning

confidence: 95%

“…These methods can also be applied to CFPS systems to increase the expression yields of soluble proteins. For example, chaperone-enriched cell extracts can be used [17,26] or due to the open environment, the exogenous addition of molecular chaperones can be easily performed. [27][28][29] Two commercially available chaperone mixes, the DnaK and GroE mix, were tested for the synthesis of RopaAzoR in this study.…”

Section: Chembiochemmentioning

confidence: 99%

“…Thus, protein concentrations at a milligram per milliliter scale can be achieved within a few hours of synthesis time. Due to its open nature, CFPS offers the possibility of easy manipulation of the synthesis conditions and is, therefore, a valuable platform for the synthesis of difficult‐to‐express proteins, such as proteins with a toxic effect on host cell metabolism [16] or proteins that tend to be synthesized in insoluble form [17] . Thus, the applicability of CFPS for protein screenings and characterizations has already been shown in several studies [18–20] …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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Azoreductases are potent biocatalysts for the cleavage of azo bonds. Various gene sequences coding for potential azoreductases are available in databases, but many of their gene products are still uncharacterized. To avoid the laborious heterologous expression in a host organism, we developed a screening approach involving cell-free protein synthesis (CFPS) combined with a colorimetric activity assay, which allows the parallel screening of putative azoreductases in a short time. First, we evaluated different CFPS systems and optimized the synthesis conditions of a model azoreductase. With the findings obtained, 10 azoreductases, half of them undescribed so far, were screened for their ability to degrade the azo dye methyl red. All novel enzymes catalyzed the degradation of methyl red and can therefore be referred to as azoreductases. In addition, all enzymes degraded the more complex and bulkier azo dye Brilliant Black and four of them also showed the ability to reduce p-benzoquinone. NADH was the preferred electron donor for the most enzymes, although the synthetic nicotinamide co-substrate analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) was also accepted by all active azoreductases. This screening approach allows accelerated identification of potential biocatalysts for various applications.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

Section: Chembiochemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2022

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“…Fe(II)- and 2-ketoglutarate-dependent dioxygenases (Fe/2-KG DOs) are excellent biocatalysts for the C–H hydroxylation of amino acids because of their remarkable oxygen transformation capabilities . Various l -proline hydroxylases have been explored to facilitate hydroxylation of substrates at different positions. − l -Lysine dioxygenase (KDO) has also been well studied for catalyzing hydroxylation. − As an efficient biocatalyst, GlbB can hydroxylate l -lysine with high regioselectivity in the preparation of key dipeptide fragments of griseofulvin and has received more attention regarding catalytic performance and thermostability in recent years. − In addition, PolL enables the sequential hydroxylation of α-amino-δ-carbamoylhydroxyvaleric acid .…”

Section: Introductionmentioning

confidence: 99%

Molecular Insights into the Regioselectivity of the Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Catalyzed C–H Hydroxylation of Amino Acids

Jing

et al. 2022

ACS Catal.

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l-Isoleucine dioxygenase (IDO) directly catalyzes the C–H bond hydroxylation of several hydrophobic aliphatic amino acids. However, the ambiguous selectivity of IDO prevents its application in chiral hydroxy amino acid production. The hydroxylation of l-norleucine by IDO, which produces 4-hydroxynorleucine and 5-hydroxynorleucine with obvious regioselectivity, was used to investigate the mechanism of IDO regioselectivity. Along with computational structural analysis and high-throughput screening, the IDO structure revealed single-site variants (T244A, T244G, and T244S) with enhanced regioselectivity; for example, the 4-hydroxynorleucine purity in regioisomeric products increased from 78.9% (by wild-type IDO) to 95.1%, 96.6%, and 95.3%, respectively. Molecular dynamics simulations showed that mutating T244 into smaller amino acids fine-tuned the substrate binding pose. For asymmetric catalysis requiring precise positioning, this change expanded the most frequent distances between the substrate C4 or C5 and Fe2+, giving a maximum 4-hydroxynorleucine purity of 96.6%. We improved the understanding of the regioselectivity of Fe(II)/2-ketoglutarate-dependent dioxygenases and provide a route for diversifying C–H hydroxylation-based active compounds.

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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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From Cell-Free Protein Synthesis to Whole-Cell Biotransformation: Screening and Identification of Novel α-Ketoglutarate-Dependent Dioxygenases for Preparative-Scale Synthesis of Hydroxy-l-Lysine

Cited by 11 publications

References 38 publications

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

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

Molecular Insights into the Regioselectivity of the Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Catalyzed C–H Hydroxylation of Amino Acids

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

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