Three-steps in one-pot: whole-cell biocatalytic synthesis of enantiopure (+)- and (−)-pinoresinol via kinetic resolution

Table 1 Concentrations of pinoresinol 3 and corresponding ee values achieved in the three-step one-pot system

Entry	Added concentration of 1 ^a	Addition of	Concentration of 3 (µM)	ee value (%)
1	1 x 1 mM^b	–	6 ± 5	74 [(+)-3a]
2	1 x 2.5 mM^b	–	32 ± 8	88 [(+)-3a]
3	1 x 10 mM^b	–	995 ± 119	4 [(+)-3a]
4	10 x 2.5 mM^b	1 every 1 h	2730 ± 10	25 [(+)-3a]
5	10 x 1 mM^b	1 every 1 h	1030 ± 70	37 [(+)-3a]
6	5 x 1 mM^b	1 every 2 h	190 ± 20	44 [(+)-3a]
7	3 x 1 mM^b	1 every 4 h	63 ± 9	≥99 [(+)-3a]
8	1 x 10 mM^c,d	–	1472 ± 16	1 [(+)-3a]
9	1 x 10 mM^c,e	C41_AtPrR2	822 ± 44	97 [(+)-3a]
10	1 x 10 mM^c,f	C41_AtPrR2	876 ± 21	98 [(+)-3a]
11	1 x 10 mM^c,g	C41_FiPLR	610 ± 19	97 [(−)-3b]
12	1 x 10 mM^c,e	C41_FiPLR	456 ± 19	95 [(−)-3b]
13	1 x 10 mM^c,h	C41_FiPLR	434 ± 40	92 [(−)-3b]

All reaction conditions tested yielded 100 % conversion of eugenol 1
^aReaction conditions: reaction buffer (50 mM KPi-buffer, pH 7.5, 100 µM IPTG), 2 % (v/v) dimethyl sulfoxide (DMSO), concentrations of eugenol 1 as indicated. Reactions were carried out for 24 h at 25 °C, 140 rpm
^bRecombinant E. coli C41(DE3) harbouring PsVAO, CgL1, and AtPrR2 resuspended in 10 ml reaction buffer with an adjusted cell wet weight (cww) of 70 g l⁻¹
^cRecombinant E. coli C41(DE3) harbouring PsVAO and CgL1 resuspended in 10 ml reaction buffer with 20 g l⁻¹ d-glucose (cww adjusted to 70 g l⁻¹)
^dWithout addition of recombinant E. coli cells harbouring AtPrR2 or FiPLR
^eAddition of recombinant E. coli cells harbouring AtPrR2 or FiPLR resuspended in 10 ml reaction buffer with 20 g l⁻¹ d-glucose (cww adjusted to 70 g l⁻¹) after 24 h; further incubation for 4 h
^fAs reaction e, but further incubation for 8 h
^gAs reaction e, but further incubation for 2 h
^hAs reaction e, but further incubation for 6 h

ISSN: 1475-2859