Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes.
BACKGROUND
Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes.


RESULTS
Recombinant S. cerevisiae BY4741∆Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, α-methyl-trans-cinnamaldehyde, and trans-β-methyl-β-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity.


CONCLUSIONS
The OYE biocatalysts, based on recombinant S. cerevisiae expressing OYE genes from non-conventional yeasts, were able to differently reduce the activated double bond of enones, enals and nitro-olefins, exhibiting a wide range of substrate specificity. Moreover whole-cells biocatalysts bypassed the necessity of the cofactor recycling and, tuning reaction parameters, allowed the synthetic exploitation of endogenous carbonyl reductases. Molecular modeling studies highlighted key structural features for further improvement of catalytic properties of OYE enzymes.
Diego
D
Romano
Martina Letizia
ML
Contente
Francesco
F
Molinari
Ivano
I
Eberini
Erica
E
Ruvutuso
Cristina
C
Sensi
Alberto
A
Amaretti
Maddalena
M
Rossi
Dipartimento di Scienze della Vita, Università degli Studi di Modena e Reggio Emilia, Via G, Campi 183, 41125 Modena, Italy. stefano.raimondi@unimore.it.
Stefano
S
Raimondi
D000171:Acrolein / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000475:Alkenes / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D055672:Static Electricity; D013237:Stereoisomerism; D020058:Styrene / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
D000475:Alkenes; D005656:Fungal Proteins; D011994:Recombinant Proteins; D020058:Styrene; D000171:Acrolein; D010088:Oxidoreductases; C012843:cinnamaldehyde