Design of recombinant whole-cell catalysts for double reduction of CC and CO bonds in enals and application in the synthesis of Guerbet alcohols as industrial bulk chemicals for lubricants

2017 ◽  
Vol 19 (2) ◽  
pp. 405-410 ◽  
Author(s):  
Marc Biermann ◽  
Daniel Bakonyi ◽  
Werner Hummel ◽  
Harald Gröger
Keyword(s):  
Double Reduction ◽  
Whole Cell ◽  
Unsaturated Aldehydes ◽  
Bulk Chemicals ◽  
Guerbet Alcohols

Whole-cell catalysts overexpressing two enzymes for a double reduction cascade in which aliphatic α-branched α,β-unsaturated aldehydes are converted into Guerbet alcohols as a highly demanded class of lubricants were constructed and applied in such biotransformations.

scholarly journals Efficient whole-cell oxidation of α,β-unsaturated alcohols to α,β-unsaturated aldehydes through the cascade biocatalysis of alcohol dehydrogenase, NADPH oxidase and hemoglobin

2021 ◽  
Author(s):  
Yan Qiao ◽  
Can Wang ◽  
Yin Zeng ◽  
Tairan Wang ◽  
Jingjing Qiao ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Nadph Oxidase ◽  
Selective Oxidation ◽  
Catalytic Performance ◽  
Whole Cell ◽  
E Coli ◽  
Unsaturated Alcohols ◽  
Unsaturated Aldehydes ◽  
Fusion Enzyme ◽  
Cascade Biocatalysis

Abstract Background: α,β-Unsaturated aldehydes are widely used in the organic synthesis of fine chemicals for application in products such as flavoring agents, fragrances and pharmaceuticals. In the selective oxidation of α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes, it remains challenging to overcome poor selectivity, overoxidation and a low atom efficiency in chemical routes. Results: An E. coli strain coexpressing the NADP+-specific alcohol dehydrogenase YsADH and the oxygen-dependent NADPH oxidase TkNOX was constructed; these components enabled the NADP+ regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with a yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin protein VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB were generated, which completely converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions for the cascade biocatalysis were optimized, in which supplementation with 0.2 mM FAD and 0.4 mM NADP+ was essential for maintaining high catalytic activity. Finally, the established whole-cell system could serve as a platform for the synthesis of valuable α,β-unsaturated aldehydes through the selective oxidation of various α,β-unsaturated alcohols. Conclusions: The construction of a strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB achieved efficient NADP+ regeneration and the selective oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes. Among the available redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the most recent successful example to improve catalytic performance in comparison with its separate components.

scholarly journals Efficient whole-cell oxidation of α,β-unsaturated alcohols to α,β-unsaturated aldehydes through cascade biocatalysis of alcohol dehydrogenase, NADPH oxidase and hemoglobin 

2020 ◽  
Author(s):  
Yan Qiao ◽  
Can Wang ◽  
Yin Zeng ◽  
Tairan Wang ◽  
Jingjing Qiao ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Nadph Oxidase ◽  
Selective Oxidation ◽  
Catalytic Performance ◽  
Whole Cell ◽  
E Coli ◽  
Unsaturated Alcohols ◽  
Unsaturated Aldehydes ◽  
Fusion Enzyme ◽  
Cascade Biocatalysis

Abstract Background: α,β-unsaturated aldehydes are widely used in as organic synthesis of fine chemicals such as flavor, fragrances and pharmaceuticals. The selective oxidation of α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes remains challenging to overcome poor selectivity, over-oxidation and low atom efficiency in chemical routes. Results: An E. coli strain co-expressing NADP+-specific alcohol dehydrogenase YsADH and oxygen-dependent NADPH oxidase TkNOX was constructed, which enabled the NADP+ regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with the yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield up to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB were achieved, which totally converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions of the cascade biocatalysis were optimized, in which the supplement of 0.2 mM FAD and 0.4 mM NADP+ was essential for maintaining high catalytic activity. Finally, the established whole-cell system could serve as a platform for the synthesis of valuable α,β-unsaturated aldehydes from selective oxidation of various α,β-unsaturated aldehydes. Conclusions: The construction of the strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB fulfilled efficient NADP+ regeneration and selective oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes. With the scope of redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the latest successful example to improve catalytic performance in comparison with separated counterparts.

scholarly journals Efficient Whole-Cell Oxidation of α,β-Unsaturated Alcohols to α,β-Unsaturated Aldehydes through Cascade Biocatalysis of Alcohol Dehydrogenase, NADPH Oxidase and Hemoglobin

2020 ◽  
Author(s):  
Yan Qiao ◽  
Can Wang ◽  
Yin Zeng ◽  
Tairan Wang ◽  
Jingjing Qiao ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Nadph Oxidase ◽  
Selective Oxidation ◽  
Catalytic Performance ◽  
Whole Cell ◽  
E Coli ◽  
Unsaturated Alcohols ◽  
Unsaturated Aldehydes ◽  
Fusion Enzyme ◽  
Cascade Biocatalysis

Abstract Background: α,β-unsaturated aldehydes are widely used in as organic synthesis of fine chemicals such as flavor, fragrances and pharmaceuticals. The selective oxidation of α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes remains challenging to overcome poor selectivity, over-oxidation and low atom efficiency in chemical routes.Results: An E. coli strain co-expressing NADP+-specific alcohol dehydrogenase YsADH and oxygen-dependent NADPH oxidase TkNOX was constructed, which enabled the NADP+ regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with the yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield up to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB was achieved, which totally converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions of the cascade biocatalysis were optimized, in which the supplement of 0.2 mM FAD and 0.4 NADP+ was essential for maintaining high catalytic activity. Finally, the established whole-cell system could serve as a platform for the synthesis of valuable α,β-unsaturated aldehydes from selective oxidation of various α,β-unsaturated aldehydes.Conclusions: The construction of the strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB fulfilled efficient NADP+ regeneration and selective oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes. With the scope of redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the latest successful example to improve catalytic performance in comparison with separated counterparts.

scholarly journals Efficient whole-cell oxidation of α,β-unsaturated alcohols to α,β-unsaturated aldehydes through the cascade biocatalysis of alcohol dehydrogenase, NADPH oxidase and hemoglobin

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Yan Qiao ◽  
Can Wang ◽  
Yin Zeng ◽  
Tairan Wang ◽  
Jingjing Qiao ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Nadph Oxidase ◽  
Selective Oxidation ◽  
Catalytic Performance ◽  
Whole Cell ◽  
E Coli ◽  
Unsaturated Alcohols ◽  
Unsaturated Aldehydes ◽  
Fusion Enzyme ◽  
Cascade Biocatalysis

Abstract Background α,β-Unsaturated aldehydes are widely used in the organic synthesis of fine chemicals for application in products such as flavoring agents, fragrances and pharmaceuticals. In the selective oxidation of α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes, it remains challenging to overcome poor selectivity, overoxidation and a low atom efficiency in chemical routes. Results An E. coli strain coexpressing the NADP+-specific alcohol dehydrogenase YsADH and the oxygen-dependent NADPH oxidase TkNOX was constructed; these components enabled the NADP+ regeneration and catalyzed the oxidation of 100 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal with a yield of 21.3%. The oxygen supply was strengthened by introducing the hemoglobin protein VsHGB into recombinant E. coli cells and replacing the atmosphere of the reactor with pure oxygen, which increased the yield to 51.3%. To further improve catalytic performance, the E. coli cells expressing the multifunctional fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB were generated, which completely converted 250 mM 3-methyl-2-buten-1-ol to 3-methyl-2-butenal after 8 h of whole-cell oxidation. The reaction conditions for the cascade biocatalysis were optimized, in which supplementation with 0.2 mM FAD and 0.4 mM NADP+ was essential for maintaining high catalytic activity. Finally, the established whole-cell system could serve as a platform for the synthesis of valuable α,β-unsaturated aldehydes through the selective oxidation of various α,β-unsaturated alcohols. Conclusions The construction of a strain expressing the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB achieved efficient NADP+ regeneration and the selective oxidation of various α,β-unsaturated alcohols to the corresponding α,β-unsaturated aldehydes. Among the available redox enzymes, the fusion enzyme YsADH-(GSG)-TkNOX-(GSG)-VsHGB has become the most recent successful example to improve catalytic performance in comparison with its separate components.

HVEM Analysis of Microfilament Patterns in The Margins of Tissue Cells

1980 ◽  
Vol 38 ◽  
pp. 808-811
Author(s):  
Carol Allen
Keyword(s):  
Cell Movement ◽  
Cell Spreading ◽  
Living Cells ◽  
Tissue Cell ◽  
Large Sample Size ◽  
Cell Periphery ◽  
Whole Cell ◽  
Critical Point Drying ◽  
Lamellar Region ◽  
Tissue Cells

When provided with a suitable solid substrate, tissue cells undergo a rapid conversion from the spherical form expressed in suspension culture to a characteristic flattened morphology. As a result of this conversion, called cell spreading, the cell nucleus and organelles come to occupy a central region of “deep cytoplasm” which slopes steeply into a peripheral “lamellar” region less than 1 pm thick at its outer edge and generally free of cell organelles. Cell spreading is accomplished by a continuous outward repositioning of the lamellar margins. Cell translocation on the substrate results when the activity of the lamellae on one side of the cell become dominant. When this occurs, the cell is “polarized” and moves in the direction of the “leading lamellae”. Careful analysis of tissue cell locomotion by time-lapse microphotography (1) has shown that the deformational movements of the leading lamellae occur in a repeating cycle of advance and retreat in the direction of cell movement and that the rate of such deformations are positively correlated with the speed of cell movement. In the present study, the physical basis for these movements of the cell margin has been examined by comparative light microscopy of living cells with whole-mount electron microscopy of fixed cells. Ultrastructural observations were made on tissue cells grown on Formvar-coated grids, fixed with glutaraldehyde, further processed by critical-point drying, and then photographed in the High Voltage Electron Microscope. This processing and imaging system maintains the 3-dimensional organization of the whole cell, the relationship of the cell to the substrate, and affords a large sample size which facilitates quantitative analysis. Comparative analysis of film records of living cells with the whole-cell micrographs revealed that specific patterns of microfilament organization consistently accompany recognizable stages of lamellar formation and movement. The margins of spreading cells and the leading lamellae of locomoting cells showed a similar pattern of MF repositionings (Figs. 1-4). These results will be discussed in terms of a working model for the mechanics of lamellar motility which includes the following major features: (a) lamellar protrusion results when an intracellular force is exerted at a locally weak area of the cell periphery; (b) the association of cortical MFs with one another determines the local resistance to this force; (c) where MF-to-MF association is weak, the cell periphery expands and some cortical MFs are dragged passively forward; (d) contact of the expanded area with the substrate then triggers the lateral association and reorientation of these cortical MFs into MF bundles parallel to the direction of the expansion; and (e) an active interaction between these MF bundles associated with the cortex of the expanded lamellae and the cortical MFs which remained in the sub-lamellar region then pulls the latter MFs forward toward the expanded area. Thus, the advance of the cell periphery on the substrate occurs in two stages: a passive phase in which some cortical MFs are dragged outward by the force acting to expand the cell periphery, and an active phase in which additional cortical MFs are pulled forward by interaction with the first set. Subsequent interactions between peripheral microfilament bundles and filaments in the deeper cytoplasm could then transmit the advance gained by lamellar expansion to the bulk of the cytoplasm.

Permeability Through Bacterial Porins Dictates Whole Cell Compound Accumulation

2020 ◽  
Author(s):  
Silvia Acosta Gutiérrez ◽  
Igor Bodrenko ◽  
Matteo Ceccarelli
Keyword(s):  
Cell Envelope ◽  
Scoring Function ◽  
Modern Medicine ◽  
New Drugs ◽  
Prediction Ability ◽  
Whole Cell ◽  
Virtual Libraries ◽  
Major Bottleneck ◽  
Global Threat ◽  
Bacterial Porins

The lack of new drugs for Gram-negative pathogens is a global threat to modern medicine. The complexity of their cell envelope, with an additional outer membrane, hinders internal accumulation and thus, the access of molecules to targets. Our limited understanding of the molecular basis for compound influx and efflux from these pathogens is a major bottleneck for the discovery of effective antibacterial compounds. Here we analyse the correlation between the whole-cell compound accumulation of ~200 molecules and their predicted porin permeability coefficient (influx), using a recently developed scoring function. We found a strong linear relationship (75%) between the two, confirming porins key role in compound penetration. Further, the remarkable prediction ability of the scoring function demonstrates its potentiality to guide the optimization of hits to leads as well as the possibility of screening ultra-large virtual libraries. Eventually, the analysis of false positives, molecules with high-predicted influx but low accumulation, provides new hints on the molecular properties behind efflux.<br>

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