Automated high-throughput approaches for the development and investigation of novel oxidative biocatalytic processes

Oxidative biocatalysts have a vast industrial and biotechnological potential in areas such as fine chemical and antibiotic synthesis. They offer an environmentally compatible and sustainable route to catalysis, often simpler and more specific than chemical alternatives. However, the routine use of biocatalysts in biopharmaceutical manufacture has been hindered by biocatalyst complexity and the experimental burden necessary for implementation. This thesis aims to investigate, using automated microscale technologies, how oxidative biocatalytic bioprocesses can be designed and developed at a reduced cost and timeframe compared to conventional laboratory scale experimentation. A robotic platform was used with 96-Deep square well microtiter plates to develop an effective bioprocess for investigating cyclohexanone monooxygenase (CHMO). E. coli cultivations for CHMO production, bioconversion, liquid-liquid metabolite extraction and analytic techniques were conducted using the developed microscale automated approach. Each step allowed rapid and reproducible collection of quantitative kinetic data over multiples runs achieving ‘walk away operation’. Whole bioprocess evaluation was achieved, whereby linking multiple unit operations enabled rapid assessment of process interactions. Factors influencing CHMO activity and bioconversion yields were investigated along with alternative bioconversion substrates. From identified limitations of the CHMO system an optimised process was developed where the processing time was almost halved and CHMO activity increased 5-fold. Two novel self-sufficient cytochrome P450 systems, P450SU1 and P450SU2 were investigated using an automated approach where factors limiting bioconversion were identified. Implementation of the required improvements resulted in a 5-fold improvement in enzymatic expression and 5-fold and 1.5-fold increase in product formation from cytochrome P450SU1 and P450SU2, respectively. A matched oxygen transfer coefficient approach was used for predictive scale-up. The optimised microscale CHMO and P450 processes were scaled to 75 L and 7.5 L bioreactor scale, respectively. Growth and bioconversion kinetics were found to be identical between scales for the CHMO system whereas differences were observed for the P450 systems. Results described in this thesis have demonstrated the benefits of microscale automated methodologies for the creation, investigation and predictive scale-up of oxidative biocatalytic bioprocesses. The established strategies evaluated in this work contribute to meeting the current demand to decrease developmental costs and timelines

The Progesterone Hydroxylase Cytochrome P450 Multicomponent System of Streptomyces roseochromogenes: Purification, Characterisation and Regulation

PhDStreptomyces roseochromogenes, NCIB 10984, hydroxylates exogenous progesterone to 16a hydroxyprogesterone and thereafter in a second phase bioconversion to 2ß, 16a-dihydroxyprogesterone. Characterisation of this reaction was carried out at the whole cell level. The cellular components responsible for this reaction were also purified to homogeneity. S. roseochromogenes contains a cytochrome P450 and two electron transfer proteins, roseoredoxin and roseoredoxin reductase. A reconstituted incubation containing these purified proteins and the natural electron donor, NADH. produced identical hydroxyprogesterone metabolites as intact cells. In sodium periodate (Na104) supported incubations, the initial rate of progesterone hydroxylation was marginally higher than in the natural reconstituted system but the product yield was significantly lower. The yield data showed that the reconstituted natural pathway, supported multiple rounds of hydroxylation in contrast to a likely single round by a minority of P450s in the periodate reaction. When S. roseochromogenes was incubated with exogenous progesterone for 25 h the major metabolite, 16a-hydroxyprogesterone was produced in 3.6 fold excess to the minor metabolite 2ß, 16a-dihydroxyprogesterone. In a reconstituted system containing highly purified progesterone 16a-hydroxylase cytochrome P450, roseoredoxin and roseoredoxin reductase, both metabolites were produced but in a 10: 1 ratio. When S. roseochromogenes was preincubated with progesterone and the purified components of the hydroxylase system assayed as before, the ratio of 16a-hydroxyprogesterone to 2ß, 16adihydroxyprogesterone produced, decreased to 2.8: 1, virtually identical to the ratio in whole cell biotransformations. Reconstitution assays containing all combinations of hydroxylase proteins purified from progesterone preincubated and control cells, identified roseoredoxin as solely responsible for the observed changes in in vitro metabolite ratios. The fact that the 2.8: 1 ratio was also obtained when S. roseochromogenes was exposed to cycloheximide prior to progesterone pre-incubation; pointed to post translation modification of roseoredoxin. Separation of two isoforms by 2-D isoelectric focusing supported this proposition. A partial 10 amino acid sequence was obtained for both the cytochrome P450 and roseoredoxin for the purpose of probe design for eventual cloning. An amino acid sequence search revealed this P450 to be unique and unlike any other known P450 sequence. These two proteins were also successfully crystallised by hanging drop vapour diffusion trials, giving isomorphous crystals. These crystals will be used for structure determinations pending further growth

Spectroscopic studies on cytochrome P450 11-beta-hydroxylase and model compounds

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1996.Includes bibliographical references.by Normand J. Cloutier.Ph.D

Evaluation of Single-Use Bioreactors for Rapid Development of Industrial Fermentation Processes

Microbial fermentation and whole cell biocatalysis have long been used in an industrial context for the generation of commercially valuable biomolecules. There is significant further potential if they can be made as economically attractive as competing chemical process routes. Improvements in genetic engineering techniques such as those pioneered in the area of synthetic biology, have provided access to novel products including therapeutic proteins and enzymes. In order to capitalise on these advances, there remains a requirement for rapid bioprocess development and scale up. Here, single use bioreactors are of interest due to the advantages over traditional stainless steel technologies. These include reduced need for validation and turnaround times, a reduction in capital expenditure and increased facility flexibility. To date, however, they have not been thoroughly investigated for use with microbial expression systems. The aim of this thesis is to; (i) evaluate the oxygen transfer capabilities of different single-use bioreactors with a view to determining suitability for microbial fermentation, and (ii) to define appropriate scale-up bases from high throughput microwell to laboratory and pilot scale bioreactors. Initial work focused on the characterisation, optimisation and scale-up of a whole cell P450 monooxygenase bioconversion in Escherichia coli. Three rounds of optimisation using a Design of Experiments (DoE) methodology resulted in a 3.3 fold increase in the bioconversion of 7-ethoxycoumarin to 7-hydroxycoumarin. Results from the 96 deep square well (DSW) plates were then scaled-up to a traditional, pilot scale stirred tank bioreactor, increasing titres 25 times over a 3000-fold scale increase. Peak oxygen demands of 25.8 mmolL-1min-1 were shown, with clear differences in oxygen consumption as a result of feeding and bioconversion during fermentation. The inability of microwell systems to support high biomass concentrations and oxidative bioconversion was also demonstrated. In addition, these studies helped provide fundamental insights into the mechanism for oxygen utilisation during microbial whole cell bioconversions. Prioritisation of oxygen utilisation for biomass accumulation over supplementary cellular activities such as bioconversion was seen in all cases. In some cases, oxygen demand as a result of growth was approximately 4 times greater than other contributions. This had previously been hypothesised in literature but not demonstrated. In order to better characterise oxygen mass transfer in microwell plate geometries, an improved method for quantification of the volumetric oxygen mass transfer coefficient (kLa) and oxygen uptake rate (OUR), based on the dynamic gassing out method, was subsequently developed. This method determines oxygen mass transfer parameters (kLa and OUR) during a fermentation to provide more representative values for OUR. Models for OUR and kLa were built in 24 DSW plates with maximum values of over 600 mgO2L-1h-1 and 103.5 h-1 respectively. The established models enable equivalent operating conditions for the different plate geometries to be determined. The applicability of two commercially available single use bioreactors (the Ambr®250 and the XDR-10) for microbial fermentation was evaluated, using a traditional pilot scale STR for comparison. This included building models which consider a number of factors likely to influence oxygen mass transfer simultaneously, as opposed to the empirical correlations which have been developed traditionally. The Ambr®250 was demonstrated as having similar oxygen mass transfer capability to the STR across the majority of the experimental ranges, reaching maximum kLa values of > 600 h-1. Analysis of a large number of industrial microbial fermentations (approximately 300) demonstrated that the Ambr®250 is capable of supporting microbial fermentation and bioconversion (or recombinant protein expression) in each, where the XDR-10 would not be suitable. After demonstrating the applicability of the Ambr®250 system for industrial microbial fermentation, a modelling tool was developed in the Python programming language capable of evaluating the cost and resource requirements of an Ambr®250 bioprocess development run. Preliminary sensitivity analysis highlights labour as the main influence on cost, followed by the replacement of single use bioreactors, each responsible for more than one third of the total run cost. Overall this work has established original, quantitative insights into oxygen utilisation in microbial expression systems and established engineering criteria for the selection and use of single-use bioreactor technologies for microbial cultivation. The methodologies developed here are considered generic and applicable to other expression systems with high specific oxygen demands such as yeast and heterotrophically cultured microalgae