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Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis

Abstract

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L−1 glycerol). Following induction of CHMO activity biomass concentrations of up to 6 gDCW L−1 were obtained. Cells from each fermentation were subsequently used for the Baeyer–Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L−1) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 μL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.

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Abbreviations

a Gas-liquid interfacial area:

(m−1)

a i Static gas-liquid interfacial area:

(m−1)

B Baffle width:

(m)

Bo Bond number:

(=ρ gd 2/W)

C Off-bottom clearance:

(m)

C LOxygen concentration in the liquid phase:

(kg m−3)

\(C^{*}_{\rm L}\) Saturation concentration of oxygen in the liquid phase:

(kg m−3)

ΔC Spacing between impellers:

(m)

D Impeller diameter:

(m)

D O2 Oxygen diffusion coefficient:

(m2 s−1)

d Well diameter:

(m)

d t Shaking diameter:

(m)

FL G Gas flow number:

(= Q G/ND 3)

Fr Froude number:

(= (d t 2π N )2/2g)

g Gravitational acceleration:

(m s−2)

H L Liquid height:

(m)

K L Overall mass transfer coefficient:

(m s−1)

k L Liquid-film mass transfer coefficient:

(m s−1)

N Impeller rotational speed:

(s−1)

OTR Oxygen transfer rate:

(kg m−3 s−1)

OUR Oxygen uptake rate:

(kg m−3 s−1)

P g Gassed power consumption:

(W)

Q G Gas flow rate:

(m3 s−1)

Re Reynolds number:

(= ρ Nd 2/μ)

Sc Schmidt number:

(= μ/ρ D O2)

T v Vessel internal diameter:

(m)

T Temperature:

(°C)

t b Baffle thickness:

(m)

V L Liquid volume:

(m3)

v s Superficial gas velocity:

(m s−1)

W Wetting tension:

(N m−1)

X final Final biomass concentration:

(kg m−3)

μ Dynamic viscosity:

(

kg m−1 s−1)

μmax Maximum growth rate:

(s−1)

ρ Liquid density:

(kg m−3)

96-DSW:

96 Deep square well plate

96-SRW:

96 Standard round well plate

BVMO:

Bayer–Villiger monooxygenase

CHMO:

Cyclohexanone monooxygenase

GC:

Gas chromatography

OD:

Optical density

U:

Unit of activity (1 μmol min−1)

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Acknowledgements

The authors would like to thank the UK Joint Infrastructure Fund (JIF), the Science Research Investment Fund (SRIF) and the Gatsbyharitable Foundation for funds to establish the UCL Centre for Micro Biochemical Engineering. We would also like to thank the UK Engineering and Physical Sciences Research Council (EPSRC) for support of the multidisciplinary Bioconversion–Chemistry–Engineering interface programme (BiCE, GR/S62505/01). Financial support from the Mexican Council of Science and Technology (CONACYT) in the form of a PhD studentship for Claudia Ferreira-Torres is also acknowledged.

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Correspondence to G. J. Lye.

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Ferreira-Torres, C., Micheletti, M. & Lye, G.J. Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis. Bioprocess Biosyst Eng 28, 83–93 (2005). https://doi.org/10.1007/s00449-005-0422-4

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Keywords

  • Microscale processing
  • E. coli bioconversion
  • Cyclohexanone monoxygenase
  • Baeyer–Villiger monoxygenase