scholarly journals A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases

2020 ◽  
Author(s):  
Pascal Püllmann ◽  
Anja Knorrscheidt ◽  
Judith Münch ◽  
Paul R. Palme ◽  
Wolfgang Hoehenwarter ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
High Throughput Screening ◽  
Secretion System ◽  
Signal Peptides ◽  
Golden Gate ◽  
Preparative Scale ◽  
One Step ◽  
Protein Tags ◽  
Fungal Kingdom ◽  
Enzyme Class

AbstractFungal unspecific peroxygenases (UPOs) are biocatalysts of outstanding interest. Providing access to novel UPOs using a modular secretion system was the central goal of this work. UPOs represent an enzyme class, catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and solely rely on hydrogen peroxide as the oxygen source. Fungal peroxygenases are widespread throughout the fungal kingdom and hence a huge variety of UPO gene sequences is available. However, the heterologous production of UPOs in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. Here, we developed and applied a modular Golden Gate-based secretion system, allowing the first yeast production of four active UPOs, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be broadly applicable and consists of the three module types: i) a signal peptide panel guiding secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. We show that optimal signal peptides could be selected for successful UPO secretion by combinatorial testing of 17 signal peptides for each UPO gene. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid workflow from putative UPO gene to preparative scale enantioselective biotransformations.

scholarly journals A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Pascal Püllmann ◽  
Anja Knorrscheidt ◽  
Judith Münch ◽  
Paul R. Palme ◽  
Wolfgang Hoehenwarter ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
High Throughput Screening ◽  
Yeast Species ◽  
Secretion System ◽  
Signal Peptides ◽  
Golden Gate ◽  
Preparative Scale ◽  
One Step ◽  
Protein Tags ◽  
Enzyme Class

AbstractFungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.

scholarly journals Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Javier Garrigós-Martínez ◽  
Astrid Weninger ◽  
José Luis Montesinos-Seguí ◽  
Christian Schmid ◽  
Francisco Valero ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Pichia Pastoris ◽  
Drug Development ◽  
High Throughput Screening ◽  
Cell Activity ◽  
Production Rates ◽  
Model Case ◽  
Preparative Scale ◽  
Whole Cell ◽  
Human Cytochrome

Abstract Background Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy. Results A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3′hydroxyibuprofen. Conclusions For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Microbiological transformations 35: Enantioselective one-step preparative scale synthesis of 1,3-dithiane-1-oxide via whole-cell bacterial oxidation

1996 ◽  
Vol 37 (34) ◽  
pp. 6117-6120 ◽  
Author(s):  
Véronique Alphand ◽  
Nicoletta Gaggero ◽  
Stefano Colonna ◽  
Roland Furstoss
Keyword(s):  
Bacterial Oxidation ◽  
Preparative Scale ◽  
Whole Cell ◽  
One Step

Enzymes in organic synthesis. 32. Stereospecific horse liver alcohol dehydrogenase-catalyzed oxidations of exo- and endo-oxabicyclic meso diols

1984 ◽  
Vol 62 (11) ◽  
pp. 2578-2582 ◽  
Author(s):  
J. Bryan Jones ◽  
Christopher J. Francis
Keyword(s):  
Alcohol Dehydrogenase ◽  
Organic Synthesis ◽  
Horse Liver Alcohol Dehydrogenase ◽  
Preparative Scale ◽  
Enantiomerically Pure ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver ◽  
One Step ◽  
Catalyzed Oxidation ◽  
Enzymes In Organic Synthesis

Preparative-scale horse liver alcohol dehydrogenase-catalyzed oxidation of mesoexo- and endo-7-oxabicyclo[2.2.1]heptane diols provides a direct one-step route to enantiomerically pure chiral γ-lactones of the oxabicyclic series.

scholarly journals One-Step Seeding of Neural Stem Cells with Vitronectin-Supplemented Medium for High-Throughput Screening Assays

2016 ◽  
Vol 21 (10) ◽  
pp. 1112-1124 ◽  
Author(s):  
Sheng Dai ◽  
Rong Li ◽  
Yan Long ◽  
Steve Titus ◽  
Jinghua Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Neuronal Cells ◽  
Drug Efficacy ◽  
Human Stem Cells ◽  
Induced Pluripotent Cells ◽  
One Step ◽  
Human Neuronal Cells

Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate precoating, hampering its applications in high-throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate precoating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high-throughput assays using neuronal cells differentiated from human stem cells for translational research.

scholarly journals Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals

2020 ◽  
Vol 104 (16) ◽  
pp. 7023-7035
Author(s):  
Samuel Sutiono ◽  
Bettina Siebers ◽  
Volker Sieber
Keyword(s):  
Racemic Mixture ◽  
Highly Active ◽  
Key Points ◽  
Deoxy Sugar ◽  
Weimberg Pathway ◽  
One Step ◽  
The One ◽  
Increased Activity ◽  
Enzyme Class

Abstract2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to α-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed in Escherichia coli. Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator (CnL-KdpD) and D-KdpD from Pseudomonas putida (PpD-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on CnL-KdpD, whereas increased activity and stability were observed for PpD-KdpD in the presence of Mg2+. Furthermore, CnL-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas PpD-KdpD was inhibited with D-xylonate (I50 of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP. CnL-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while PpD-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer. Key points• L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively.• Mg2+affected activity and stabilities of D-KdpDs, but not of L-KdpDs.• CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield.• Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield.

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