Improvement of whole-cell transamination with Saccharomyces cerevisiae using metabolic engineering and cell pre-adaptation.

BACKGROUND: Whole-cell biocatalysis based on metabolically active baker's yeast with engineered transamination activity can be used to generate molecules carrying a chiral amine moiety. A prerequisite is though to express efficient ω-transaminases and to reach sufficient intracellular precursor levels. RESULTS: Herein, the efficiency of three different ω-transaminases originating from Capsicum chinense, Chromobacterium violaceum, and Ochrobactrum anthropi was compared for whole-cell catalyzed kinetic resolution of racemic 1-phenylethylamine to (R)-1-phenylethylamine. The gene from the most promising candidate, C. violaceum ω-transaminase (CV-TA), was expressed in a strain lacking pyruvate decarboxylase activity, which thereby accumulate the co-substrate pyruvate during glucose assimilation. However, the conversion increased only slightly under the applied reaction conditions. In parallel, the effect of increasing the intracellular pyridoxal-5'-phosphate (PLP) level by omission of thiamine during cultivation was investigated. It was found that without thiamine, PLP supplementation was redundant to keep high in vivo transamination activity. Furthermore, higher reaction rates were achieved using a strain containing several copies of CV-TA gene, highlighting the necessity to also increase the intracellular transaminase level. At last, this strain was also investigated for asymmetric whole-cell bioconversion of acetophenone to (S)-1-phenylethylamine using L-alanine as amine donor. Although functionality could be demonstrated, the activity was extremely low indicating that the native co-product removal system was unable to drive the reaction towards the amine under the applied reaction conditions. CONCLUSIONS: Altogether, our results demonstrate that (R)-1-phenylethylamine with >99% ee can be obtained via kinetic resolution at concentrations above 25 mM racemic substrate with glucose as sole co-substrate when combining appropriate genetic and process engineering approaches. Furthermore, the engineered yeast strain with highest transaminase activity was also shown to be operational as whole-cell catalyst for the production of (S)-1-phenylethylamine via asymmetric transamination of acetophenone, albeit with very low conversion.

Flavonoids as inhibitors of human carbonyl reductase 1.

Human carbonyl reductase 1 (CBR1), that is one of the enzymes responsible for the reduced efficiency of treatments by the antineoplastic agents anthracyclines, was functionally expressed in Saccharomyces cerevisiae. CBR1 was purified and kinetically characterised using daunorubicin as substrate. CBR1-catalysed reduction of daunorubicin followed an apparent Michaelis-Menten kinetics with K(M)=85.2+/-26.7microM and V(max)=3490+/-220micromol/(mingprotein). The type of inhibition for the flavonoid compound rutin was determined by studying initial reaction rates in the presence of rutin. The inhibition kinetics was found to follow an apparent mixed inhibition with K(ic)=1.8+/-1.2microM and K(iu)=2.8+/-1.6microM. IC50-values were also determined for a set of flavonoids in order to identify essential structure for inhibition activity. Computational docking experiments of the four best inhibitors to the catalytic site of CBR1 showed that the flavonoid skeleton structure was the binding part of the molecule. The presence of a sugar moiety in 1 and 2, or a sugar mimicking part in 9, directed the orientation of the flavonoid so that the sugars were pointing outwards, giving rise to a stabilising effect to the binding. Finally, additional binding epitopes that interacted with various parts of the flavonoid ligand were identified and could potentially be targeted for further improvement of inhibition activity. These included; hydrogen-binding sites surrounding Ser139 and Cys226, Met234 and Tyr193 or Trp229; aromatic-aromatic interaction with Tyr193, Trp229 or NADPH; van der Waals interactions with Ile140.

Mild detergent treatment of Candida tropicalis reveals a NADPH-dependent reductase in the crude membrane fraction, which enables the production of pure bicyclic exo-alcohol.

This study demonstrated the occurrence of a NADPH-dependent exo-alcohol reductase in the crude membrane fraction of Candida tropicalis. Cytosolic endo-alcohol reductase activity could be separated from the membrane-bound exo-alcohol activity by means of detergent treatment, enabling the preparation of pure exo-alcohol via the enzymatic conversion of the bicyclic diketone, bicyclo[2.2.2]octane-2,6-dione. The exo-alcohol reductase is, to our knowledge, the first membrane-bound NADPH-dependent reductase accepting a xenobiotic carbonyl substrate that was not a steroid. When C. tropicalis was grown on D-sorbitol, a two-fold increase in the exo-reductase activity was observed as compared to when grown on glucose. An in silico comparison at the protein level between putative xenobiotic carbonyl reductases in Candida albicans, C. tropicalis and Saccharomyces cerevisiae was performed to explain why Candida species are often encountered when screening yeasts for novel stereoselective reduction properties. C. albicans contained more reductases with the potential to reduce xenobiotic carbonyl compounds than did S. cerevisiae. C. tropicalis had many membrane-bound reductases (predicted with the bioinformatics program, TMHMM), some of which had no counterpart in the two other organisms. The exo-reductase is suspected to be either a beta-hydroxysteroid dehydrogenase or a polyol dehydrogenase from either the short chain dehydrogenase family or the dihydroflavonol reductase family.