Customized microscale approach for optimizing two-phase bio-oxidations of alkanes with high reproducibility

Abstract

BackgroundNumerous challenges remain to achieve industrially competitive space–time yields for bio-oxidations. The ability to rapidly screen bioconversion reactions for characterization and optimization is of major importance in bioprocess development and biocatalyst selection; studies at conventional lab scale are time consuming and labor intensive with low experimental throughput. The direct ω-oxyfunctionalization of aliphatic alkanes in a regio- and chemoselective manner is efficiently catalyzed by monooxygenases such as the AlkBGT enzyme complex from Pseudomonas putida under mild conditions. However, the adoption of microscale tools for these highly volatile substrates has been hindered by excessive evaporation and material incompatibility.ResultsThis study developed and validated a robust high-throughput microwell platform for whole-cell two-liquid phase bio-oxidations of highly volatile n-alkanes. Using microwell plates machined from polytetrafluoroethylene and a sealing clamp, highly reproducible results were achieved with no significant variability such as edge effects determined. A design of experiment approach using a response surface methodology was adopted to systematically characterize the system and identify non-limiting conditions for a whole cell bioconversion of dodecane. Using resting E. coli cells to control cell concentration and reducing the fill volume it is possible to operate in non-limiting conditions with respect to oxygen and glucose whilst achieving relevant total product yields (combining 1-dodecanol, dodecanal and dodecanoic acid) of up to 1.5 mmol gDCW−1.ConclusionsOverall, the developed microwell plate greatly improves experimental throughput, accelerating the screening procedures specifically for biocatalytic processes in non-conventional media. Its simplicity, robustness and standardization ensure high reliability of results.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0788-4) contains supplementary material, which is available to authorized users.

Improving Product Specificity of Whole‐Cell Alkane Oxidation in Nonconventional Media: A Multivariate Analysis Approach

“…In addition to the six cosolvents, control reactions were run without an additional cosolvent and with 0.1% Triton X‐100, as in previous research . The screening was carried out with 5% and 30% cosolvent in four different alkane substrates over 24 h giving a total of 56 reactions (Figure ).…”

“…Bioconversions were carried out in sealed, polytetrafluoroethylene (Radleys, UK) 24 deep square well MWPs using buffered resting cells according to Kolmar et al…”

“…For the control reaction without cosolvent, 70 μL of the alkane substrate was added to 280 µL of the aqueous buffer. Reactions were performed at conditions determined by Kolmar et al unless otherwise mentioned.…”

“…So far, the focus has been on developing microscale tools for aqueous single‐phase systems. The unique challenges associated with more complex nonconventional media result in only few examples of suitable specialized tools …”

“…Particular focus is on the overall product yield (refers to the sum of all products formed from the alkane oxidation in a specific reaction) and specificity (refers to the ratio of alcohol to acid product formed in a specific reaction) of the bio‐oxidation of four linear alkane substrates. In order to efficiently screen this wide range of experimental conditions, a high‐throughput microwell platform specifically customized for nonconventional media is used . In a second step, experimental data are combined with estimated physicochemical properties of the cosolvents in a multivariate PLS regression analysis.…”