Browsing by Author "Fenner, Caryn"

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    Beyond bottlenecks: expression of complementary enzymes and permeabilization of cell membranes to improve performance of CYP153A6 in Escherichia coli whole cell biocatalysis
    (2022) White, Bronwyn Elizabeth; Harrison, Susan; Smit, Martha S; Fenner, Caryn
    Cytochrome P450s are a diverse and versatile class of enzymes, able to carry out oxyfunctionalisation of hydrocarbons with exceptional regio- and stereospecificity. They show great promise within the medical and fine chemical industries, and could potentially be applied for the activation of linear alkanes into platform chemicals. However, bulk chemicals require higher titres, rates and yields than fine chemicals to be economically viable. Application of these enzymes in vitro is hampered by low biocatalyst stability, inhibitory upstream processing costs, and the need for costly cofactor supplementation. In vivo operation can overcome these issues, but brings its own set of limitations. This thesis presents research on the in vivo biotransformation of n-octane to 1-octanol by CYP153A6 and its natural redox partners, ferredoxin (Fdx) and ferredoxin reductase (FdR), from Mycobacterium sp. HXN-1500. These three proteins were heterologously expressed in Escherichia coli BL21DE3. Experiments were carried out at the millilitre scale, using resting cells. Potential limitations for the whole-cell biocatalyst include insufficient expression of the CYP153A6 or its redox partners, leading to a lack of biocatalytic “active sites”; lack of oxygen for biotransformation and cell metabolism; poor transport of n-octane from the organic phase into the cytoplasm, leading to limitations in substrate; toxicity resulting from the accumulation of substrate or product; and insufficient availability of NADH cofactor. With regards to heterologous enzyme expression, the decision was made to focus on CYP153A6 expression levels (with FdR and Fdx expression levels forming part of a related project). To investigate the effects of intracellular concentration of active CYP153A6, it was necessary to find a reliable method of varying P450 expression. The haem precursors δ-aminolevulinic acid (δ-ALA) and ferric chloride were applied during the growth and expression stage, and it was found that P450 expression could be optimised by varying the concentration of these chemicals, with co-addition of the two chemicals having a synergistic effect on expression levels. However, higher intracellular levels of active CYP153A6 did not lead to increased biotransformation rates or product titres in biotransformations carried out with whole cells. This demonstrated that the biotransformation of n-octane was not limited by the availability of active CYP153A6, even at concentrations as low as 0.3 µmolP450.gDCW-1 . Furthermore, these results suggest that optimising the expression of CYP153A6 in whole-cell biocatalysts does not equate to maximising its expression. Aminolevulinic acid constitutes a substantial portion of media formulation costs in the proposed whole-cell process, and these findings allow for more efficient application of this and other precursor chemicals. Based on an analysis of the literature, it was hypothesised that oxygen was unlikely to be a limiting substrate, due to the relatively low biotransformation rates being recorded, as well as the reduced oxygen demand of resting cells. Nevertheless, two sets of experiments were carried out to assess the effects of oxygen supply to the system. In the first set, the quantity of oxygen in the headspace was increased by enlarging the vials, while keeping cross-sectional area constant. In the second set, the headspace was replenished at regular intervals to increase the average concentration of oxygen in the headspace over the course of the biotransformation. In both sets of experiments, the differences between lower-oxygen vials and higher-oxygen vials were neither substantial nor consistent. This demonstrated that biotransformations by resting cell cultures at a density of 4 – 5 gDCW.L-1 were not limited by the quantity of oxygen available. The transport of hydrocarbons across the cell envelope affects the availability of n-octane substrate in the cytoplasm, and impacts on the in situ extraction of 1-octanol product. Thus, hydrocarbon transport has the potential not only to be a rate-limiting step, but also to control levels of cytotoxicity. To explore the significance of cross-membrane transport, cell membranes were permeabilised via the application of chemicals or via mechanical breakage, and biotransformations were also carried out using cell free extract. To investigate cofactor limitations, the NADH regeneration rate of E. coli was enhanced through over-expression of glycerol dehydrogenase (GLD) and supply of glycerol as sacrificial substrate. These interventions were implemented both separately and concurrently, and the results compared to biotransformations by low-GLD, non-permeabilised cells. The interactions between substrate transport and cofactor regeneration were found to be complex In cultures of intact or chemically permeabilised cells, strains with enhanced NADH regeneration capacity produced equal, and sometimes lower, titres than control cells carrying only the empty vector. The co-expression of GLD tended to reduce product titres in high-density cultures, by exacerbating instability of the whole-cell biocatalyst, which was already substantially reduced relative to low-density cultures. In low-density cultures, co-expression of GLD was not harmful, but it did not lead to any notable improvements in product formation rates. However, across numerous cultures, enhanced cofactor regeneration capacity correlated with enhanced CYP153A6 stability. Furthermore, in many low-density cultures, the co-expression of GLD provided marginal improvements in overall stability of the whole-cell biocatalyst when biotransformations were extended beyond 48 h. Thus, under the conditions tested, n-octane biotransformation rates were not limited by the availability of NADH. At the same time, the results suggest the possibility of harnessing enhanced cofactor regeneration to extend biotransformation times, presumably by improving the overall metabolic state of the whole cell – but also show that such a strategy has a ‘tipping point', related to cell density, beyond which the presence of additional dehydrogenase becomes harmful. The application of Triton X-100 or Polymyxin B substantially increased initial product formation rates relative to untreated cells. In low-density cultures, this led to chemically permeabilised cells achieving significantly higher final titres than untreated cells. However, the use of these chemicals in high-density cultures reduced the stability of the whole-cell biocatalyst; biotransformations levelled off rapidly, limiting the maximum titres that could be obtained. The co-expression of GLD was not beneficial in these cases. Conversely, the physical breakage of cell envelopes (via passage through a high-pressure homogeniser) improved product titres and space time yields relative to unpermeabilised cells, provided strains were co-expressing GLD. This clearly demonstrated the importance of balancing the supply of alkane and cofactor, with the expression of additional GLD compensating for reductions in metabolic capacity associated with the physically broken membranes. Mechanically permeabilised cells and cell free extract remained stable over extended periods of biotransformation, even though culture densities were high in these cases. Due to their capacity to maintain initial reaction rates at high cell density, mechanically permeabilised cells produced the highest final titres of any system tested in this study. If GLD was not co-expressed (or if CYP153A6 and GLD were expressed in separate cells), the biotransformation rates of mechanically permeabilised cells were substantially reduced. These results identified cross-membrane transport of hydrocarbons as the key limitation in the biotransformation of n-octane by whole-cell biocatalysts. Supply of substrate was a rate-limiting step in the reaction (hence the increase in product formation rates in low-density cultures of permeabilised cells). The degree of cell permeabilizationalso affected how the cell responded to substrate and product toxicity (hence the stability of high-density cultures of mechanically permeabilised cells). However, cofactor regeneration rates quickly became a limiting factor if the cofactor pool was disrupted (as was the case when the cell envelope was physically broken). To improve cross-membrane transport without destroying the cell membrane, E. coli strains were engineered to express AlkL, a passive membrane transporter protein that evolved to facilitate the growth of Pseudomonas putida on alkanes. The expression strategy included manipulating the supply of haem precursors δ-ALA and FeCl3, in a drive to achieve comparable expression of CYP153A6 in strains with and without AlkL. Even though the AlkL expression strategy was not fully optimised, cells co-expressing the transport protein demonstrated 50 – 100 % improvements on their yield on biomass after 48 h. These results confirmed the importance of overcoming the cross-membrane transport limitation. Overall, the findings highlight the importance of balancing reaction components. Cross-membrane transport of hydrocarbons emerged as the key limitation in biocatalysis by CYP153A6 in whole cells. This limitation could be alleviated by permeabilising the cell. Mechanical breakage of the cell envelope was particularly effective, but this in turn impacted negatively on the cell's ability to maintain its cofactor pool, necessitating the co-expression of glycerol dehydrogenase to boost NADH regeneration rates. In this regard, the membrane channel protein AlkL showed great promise, allowing cells to increase their rates of product formation even while relying on their native cofactor pool. These performance enhancements were achieved despite low intracellular concentrations of active CYP153A6 in cells co-expressing AlkL. Furthermore, in biotransformations with intact cells, intracellular concentration of CYP153A6 could be substantially reduced (via a reduction in the concentration of haem precursor), without any negative impact on biotransformation being observed. Increasing the cell density of biotransformation cultures allowed for rapid product accumulation, but substantially reduced the stability of the whole-cell biocatalyst. This instability correlated closely with the volumetric rate of product formation, suggesting that the effect was linked to reaction or product toxicity. Isolated improvements in enzyme expression, substrate supply, cofactor regeneration, or biomass loading did not translate into improved biocatalytic performance overall. This demonstrates that the creation of a useful P450-based biocatalyst will depend on the simultaneous optimisation of multiple components. In the field of biocatalysis, the focus tends to be on the characterisation and modification of the enzymes themselves. While this enzymatic understanding is crucial, the equally important task of understanding host physiology is often not explored in the same exacting detail. Unlocking the full potential of biocatalysis within the bulk chemical space requires an understanding of the cell architectures within which the enzymes of interest must function. This study has served to shed light on the links between cells, enzymes, and reactants – and on how much there still is to understand when it comes to whole cell performance.
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    Open Access
    The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6
    (BioMed Central Ltd, 2013) Olaofe, Oluwafemi; Fenner, Caryn; Gudiminchi, Rama Krishna; Smit, Martha; Harrison, Susan
    BACKGROUND: Biocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. This study focused on understanding the bioprocess limitations to identify factors that impact the expression of the terminal hydroxylase CYP153A6 and also influence the biocatalytic transformation of n-octane to 1-octanol using resting whole cells of recombinant E. coli expressing the CYP153A6 operon which includes the ferredoxin (Fdx) and the ferredoxin reductase (FdR). RESULTS: Specific hydroxylation activity decreased with increasing protein expression showing that the concentration of active biocatalyst is not the sole determinant of optimum process efficiency. Process physiological conditions including the medium composition, temperature, glucose metabolism and product toxicity were investigated. A fed-batch system with intermittent glucose feeding was necessary to ease overflow metabolism and improve process efficiency while the introduction of a product sink (BEHP) was required to alleviate octanol toxicity. Resting cells cultivated on complex LB and glucose-based defined medium with similar CYP level (0.20mumol gDCW-1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120h. This was influenced by differing glucose uptake rate which is directly coupled to cofactor regeneration and cell energy in whole cell biocatalysis. The maximum activity and biocatalyst efficiency achieved presents a significant improvement in the use of CYP153A6 for alkane activation. This biocatalyst system shows potential to improve productivity if substrate transfer limitation across the cell membrane and enzyme stability can be addressed especially at higher temperature. CONCLUSION: This study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.
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    Preliminary investigation of growth and antimicrobial production by streptomyces polyantibioticus : from shake flask to stirred tank bioreactor
    (2016) Matongo, Tarisayi Martin; Harrison, STL; Fenner, Caryn
    Resistance to antibiotics by microbial pathogens continues to be a major global health problem. Treatment of bacterial infections is becoming increasingly complex and expensive. Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, is affected by antibiotic resistance. In South Africa, the Western Province is the worst affected, with an increasing incidence of both multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis. Both resistant forms of TB increase the length of treatment to almost 24 months and cost by as much as 1400 times that of regular anti-tubercular chemotherapy. A potential solution to this problem is the discovery of new drugs, which can be obtained from natural sources. Actinomycetes are good sources of these drugs, with over 45% of current medicines derived from these bacteria. The actinobacterium Streptomyces polyantibioticus SPRT (SPRT) was locally isolated and first described by Le Roes (2006). It has been shown to produce bioactive molecules active against a range of bacteria, including compounds (drugs) that have anti-tubercular properties. One of the anti-tubercular molecules was identified as 2,5-diphenyloxazole (DPO). DPO is currently used as a component of scintillation fluid for its luminescent properties and is synthesised chemically in industry. SPRT is the only reported biological source of DPO, it is however not yet produced commercially via a biological route. The present study was performed to inform future process development of DPO production from SPRT. An investigation into the growth and production of antimicrobial compounds from submerged cultures of SPRT in shake flasks, and scale-up of the process into a laboratory stirred tank bioreactor (STR) was done in the present study. The work focused on obtaining growth kinetics and suitable operating conditions for cultivation. Characterisation of the growth profile of SPRT and determination of the kinetic growth parameters was carried out. Additionally, the antimicrobial production phases, and factors influencing their production was investigated. It was determined that the most reliable method of measuring biomass concentration was by dry cell gravimetric measurement of whole shake flasks following vacuum filtration, as it best suited the non-homogenous filamentous nature of SPRT.
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    Production and characterization of alkaliphilic amylases from Bacillus halodurans Alk36
    (2015) Mrisho, Latifa Mbwana; Harrison, STL; Fenner, Caryn
    Amylases are hydrolytic enzymes that cause the breakdown of starch and related polysaccharides to simple sugars. Amylases are applied in brewing, food, detergent and textile industries. Most commercial amylases are derived from fungi or bacteria. Bacterial amylases are desired for commercial use, due to their thermo-stability and faster production rates. Bacteria of the genus, Bacillus, are considered to be a good source of extracellular proteins because they have high growth rates and have a naturally high capacity for secretion of extracellular proteins. Bacillus halodurans Alk36 is an alkaliphilic, thermotolerant isolate that can grow over a wide pH and temperature range. Preliminary studies have shown that B. halodurans Alk36 can grown in EnBase® medium (at pH 8.5) containing starch as the carbon source, without the addition of a commercial amylase. The ability to grow on starch, in the absence of an external amylase, indicated that this strain produces endogenous alkaliphilic amylases, which may be exploited for a number of industrial applications. In the present study, the physiological and biochemical characterisation of B. halodurans Alk36 and its endogenous amylases were investigated.
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    Reactor design for the hydroxylation of n-octane using a cyp153a6-based biocatalytic system expressed in Escherichia coli
    (2013) Meissner, Murray Peter; Harrison, STL; Fenner, Caryn
    Large stockpiles of linear hydrocarbons have arisen as by-products from the global expansion of gas-to-liquid refining processes. Furthermore, these linear alkanes feature one of the strongest chemical bonds in nature and typically are of a low value due to their inertness. In an effort to valorise this resource, catalytic routes are being sought in order to improve their value by introducing functional groups into the inert carbon backbone of such linear alkanes. Biocatalytic approaches have thus far provided the most feasible route for industrial applications of this chemistry as they feature a uniquely high selectivity for a vast range of products and operate under mild processing conditions. This study focuses on the biological hydroxylation of n-octane to 1-octanol using a previously developed cytochrome P450 monooxygenase, CYP153A6, enzymatic system. The biocatalyst was expressed in Escherichia coli BL21(DE3) by using a pET28b-PFR1500 plasmid encoding the complete operon from Mycobacterium sp. HXN-1500 which included the ferrodoxin reductase (FdR) and ferrodoxin (Fdx) redox partner proteins. CYP153A6 offers several benefits over other biocatalysts such as a notable !95 regioselectivity for terminal carbon hydroxylation and lack of product degradation through overoxidation and by-product formation. Studies to date focussing on understanding interactions between physiological, molecular and bioprocess conditions have yielded maximum specific biocatalyst activities and biocatalyst concentrations in the range of 4.0-5.5 μmoloctanol•gDCW−1•min−1 and 0.18 μmolP450•gDCW−1 respectively. Thus far, this particular P450-based biocatalytic system for n-octane hydroxylation has only been applied in small-scale vials. The objective of this work was to scale-up the experimental apparatus of this system from 1 ml (working volume) vials to bioreactors with a working volume in excess of 1 l because reactors afford improved process stability, control and easier analyses than small-scale experimental setups. The scope of this study focussed on aspects of process configuration and the scale-up for this process. More specifically, optimal timings with respect to induction of gene expression and alkane addition were ascertained with a focus on process stability. Molecular changes to this system were not considered.