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 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

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1038
Author(s):  
Jascha Rolf ◽  
Philipp Nerke ◽  
Annette Britner ◽  
Sebastian Krick ◽  
Stephan Lütz ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Specific Activity ◽  
Value Added ◽  
Small Scale ◽  
Preparative Synthesis ◽  
Preparative Scale ◽  
Whole Cell ◽  
Free Protein ◽  
Starting Point ◽  
Cell Free Protein Synthesis

The selective hydroxylation of non-activated C-H bonds is still a challenging reaction in chemistry. Non-heme Fe2+/α-ketoglutarate-dependent dioxygenases are remarkable biocatalysts for the activation of C-H-bonds, catalyzing mainly hydroxylations. The discovery of new Fe2+/α-ketoglutarate-dependent dioxygenases with suitable reactivity for biotechnological applications is therefore highly relevant to expand the limited range of enzymes described so far. In this study, we performed a protein BLAST to identify homologous enzymes to already described lysine dioxygenases (KDOs). Six novel and yet uncharacterized proteins were selected and synthesized by cell-free protein synthesis (CFPS). The subsequent in vitro screening of the selected homologs revealed activity towards the hydroxylation of l-lysine (Lys) into hydroxy-l-lysine (Hyl), which is a versatile chiral building block. With respect to biotechnological application, Escherichia coli whole-cell biocatalysts were developed and characterized in small-scale biotransformations. As the whole-cell biocatalyst expressing the gene coding for the KDO from Photorhabdus luminescens showed the highest specific activity of 8.6 ± 0.6 U gCDW−1, it was selected for the preparative synthesis of Hyl. Multi-gram scale product concentrations were achieved providing a good starting point for further bioprocess development for Hyl production. A systematic approach was established to screen and identify novel Fe2+/α-ketoglutarate-dependent dioxygenases, covering the entire pathway from gene to product, which contributes to accelerating the development of bioprocesses for the production of value-added chemicals.

scholarly journals Biosynthesis of Chiral Amino Alcohols via an Engineered Amine Dehydrogenase in E. coli

2022 ◽  
Vol 9 ◽  
Author(s):  
Feifei Tong ◽  
Zongmin Qin ◽  
Hongyue Wang ◽  
Yingying Jiang ◽  
Junkuan Li ◽  
...  
Keyword(s):  
Reductive Amination ◽  
Asymmetric Reduction ◽  
Amino Alcohols ◽  
Saturation Mutagenesis ◽  
Preparative Scale ◽  
One Step ◽  
Amine Dehydrogenase ◽  
The One ◽  
Total Turnover ◽  
Chiral Amino Alcohols

Chiral amino alcohols are prevalent synthons in pharmaceuticals and synthetic bioactive compounds. The efficient synthesis of chiral amino alcohols using ammonia as the sole amino donor under mild conditions is highly desired and challenging in organic chemistry and biotechnology. Our previous work explored a panel of engineered amine dehydrogenases (AmDHs) derived from amino acid dehydrogenase (AADH), enabling the one-step synthesis of chiral amino alcohols via the asymmetric reductive amination of α-hydroxy ketones. Although the AmDH-directed asymmetric reduction is in a high stereoselective manner, the activity is yet fully excavated. Herein, an engineered AmDH derived from a leucine dehydrogenase from Sporosarcina psychrophila (SpAmDH) was recruited as the starting enzyme, and the combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was applied to improve the activity. After three rounds of mutagenesis in an iterative fashion, the best variant wh84 was obtained and proved to be effective in the asymmetric reductive amination of 1-hydroxy-2-butanone with 4-fold improvements in kcat/Km and total turnover number (TTN) values compared to those of the starting enzyme, while maintaining high enantioselectivity (ee >99%) and thermostability (T5015 >53°C). In preparative-scale reaction, the conversion of 100 and 200 mM 1-hydroxy-2-butanone catalyzed by wh84 was up to 91–99%. Insights into the source of an enhanced activity were gained by the computational analysis. Our work expands the catalytic repertoire and toolbox of AmDHs.

Optical fiber bioluminescent whole-cell microbial biosensors to genotoxicants

2000 ◽  
Vol 42 (1-2) ◽  
pp. 305-311 ◽  
Author(s):  
B. Polyak ◽  
E. Bassis ◽  
A. Novodvorets ◽  
S. Belkin ◽  
R.S. Marks
Keyword(s):  
Optical Fiber ◽  
Optical Fiber Sensor ◽  
Photon Counting ◽  
Genetically Engineered ◽  
Whole Cell ◽  
Light Production ◽  
Dna Damaging Agents ◽  
Sensor Module ◽  
One Step ◽  
Dose Dependent

Conservation of water resources calls for ever stricter regulatory measures and better monitoring systems. Whole-cell bacterial sensors have been genetically engineered to react to target toxicants by the induction of a selected promoter and the subsequent production of bioluminescent light through a recombinant lux reporter. In order to create a one-step assay, we have designed a new, self-contained, disposable optical fiber sensor module and a customized photodetector system that integrates these microorganisms. A photon-counting photomultiplier tube-based instrument was constructed. Optical fiber tip cores were covered with adlayer films consisting of calcium alginate containing bioluminescent bacterial sensors of genotoxicants. Multiplying these steps thickened the adlayer in increments, increasing the number of bacterial reporters attached to the optical fiber transducer. These whole cell optrodes are responsive to external traces of DNA damaging agents such as mitomycin C. Light production was shown to be dose-dependent and proportional to the number of bacterial layers.

scholarly journals Efficient Synthesis of Simvastatin by Use of Whole-Cell Biocatalysis

2007 ◽  
Vol 73 (7) ◽  
pp. 2054-2060 ◽  
Author(s):  
Xinkai Xie ◽  
Yi Tang
Keyword(s):  
High Performance ◽  
Fermentation Broth ◽  
Attractive Alternative ◽  
Acyl Donor ◽  
Whole Cell ◽  
Whole Cell Biocatalysis ◽  
Cholesterol Levels ◽  
Fed Batch Fermentation ◽  
One Step ◽  
Semisynthetic Derivative

ABSTRACT Simvastatin is a semisynthetic derivative of the fungal polyketide lovastatin and is an important drug for lowering cholesterol levels in adults. We have developed a one-step, whole-cell biocatalytic process for the synthesis of simvastatin from monacolin J. By using an Escherichia coli strain overexpressing the previously discovered acyltransferase LovD (X. Xie, K. Watanabe, W. A. Wojcicki, C. C. Wang, and Y. Tang, Chem. Biol. 13:1161-1169, 2006), we were able to achieve >99% conversion of monacolin J to simvastatin without the use of any chemical protection steps. The key finding was a membrane-permeable substrate, α-dimethylbutyryl-S-methyl-mercaptopropionate, that was efficiently utilized by LovD as the acyl donor. The process was scaled up for gram-scale synthesis of simvastatin. We also demonstrated that simvastatin synthesized via this method can be readily purified from the fermentation broth with >90% recovery and >98% purity as determined by high-performance liquid chromatography. Bioconversion using high-cell-density, fed-batch fermentation was also examined. The whole-cell biocatalysis can therefore be an attractive alternative to currently used multistep semisynthetic transformations.

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 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.

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