Tuning of the enzyme ratio in a neutral redox convergent cascade: A key approach for an efficient one‐pot/two‐step biocatalytic whole‐cell system

Abstract

The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer–Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε‐caprolactone and 3,4‐dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi‐enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter‐balanced by a higher concentration. Using a preconception‐free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1‐indanol (7.5 mM) at a 0.5‐L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε‐caprolactone, for the productivity of 244 mg·L −1·h −1) and that for 1‐indanol 60% (0.3 g 3,4‐dihydrocoumarin, for the productivity of 140 mg·L −1·h −1).

Rational Engineering of a Multi‐Step Biocatalytic Cascade for the Conversion of Cyclohexane to Polycaprolactone Monomers in Pseudomonas taiwanensis

“…Whereas the balancing of enzyme ratios in vitro is a rather straight‐forward approach, [ 2 ] in case of whole‐cell biocatalysis, this requires fine‐tuning of expression levels which, furthermore, should not drive the demand of resources beyond cellular capacities. [ 11 ] One possibility is the use of different plasmids to adjust the gene copy number, [ 37 ] which also can influence plasmid stability. [ 7 ] In our previous study, we varied copy number, RBS, and regulatory systems for Cyp gene expression.…”

“…The biocatalytic production of PCL or its precursors has been heavily investigated over the last years ( Table 3 ). Approaches based on isolated enzymes, [ 21,46–50 ] as well as whole cells, [ 23,37,51–53 ] have successfully been established. However, most of these approaches relied on cyclohexanol as a substrate, [ 37,47–53 ] which needs to be synthesized from cyclohexane employing an ecologically critical process.…”

“…Approaches based on isolated enzymes, [21,[46][47][48][49][50] as well as whole cells, [23,37,[51][52][53] have successfully been established. However, most of these approaches relied on cyclohexanol as a substrate, [37,[47][48][49][50][51][52][53] which needs to be synthesized from cyclohexane employing an ecologically critical process. [54] Additionally, inhibition of CHMO by cyclohexanol or substrate inhibition necessitated the development of suitable reaction concepts, for example, two-liquid phase [46] or fed-batch systems.…”

“…The highest -CL yield of 1.2 g -CL g biocatalyst with a high initial activity of 15 Ug CDW −1 was observed by Ménil et al in complex medium. [37] Compared to cyclohexanol, cyclohexane is an even more challenging substrate due to its high volatility and toxicity. In comparison to solvent-sensitive E. coli employed to convert cyclohexanol to 6HA, [53] we obtained a tenfold higher specific whole-cell activity and a similar yield on biocatalyst ( Table 3).…”

Divorce in the two-component BVMO family: the single oxygenase for enantioselective chemo-enzymatic Baeyer–Villiger oxidations

“…These enzymes are available in high numbers in protein databanks, easily detectable by consensus sequence research 23 and subsequently more frequently investigated and applied in many catalytic processes. 3,4,[24][25][26] In contrast, two-component systems referred to as type II BVMOs (from group C of FPMOs) are rare. Only two representatives are known so far, 2,5-diketocamphane 1,2-monooxygenase I (2,5-DKCMO; EC 1.14.14.108) and 3,6-diketocamphane 1,6-monooyxgenase (2,6-DKCMO; EC 1.14.14.155), [27][28][29][30][31][32] probably because a twocomponent system is not a trivial biocatalytic arrangement to identify.…”