Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase.

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185-192 and 382-392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60°C and 65°C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55°C or 60°C for 1h. Interestingly, H192P was also 2-times more active than wild-type TK at 25°C. Optimal activity was achieved at 60°C for H192P compared to 55°C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.

Selective fractionation of Sugar Beet Pulp for release of fermentation and chemical feedstocks; optimisation of thermo-chemical pre-treatment.

The effect of time and pressure on the selective extraction of sugar beet pectin using steam pre-treatment on unprocessed Sugar Beet Pulp was evaluated using a design of experiments approach. This process gave the highest solubilisation of pectin oligomers at a relatively low pressure and longer time (5Bar, 24min), whilst leaving the majority of the cellulose fraction intact. This method of steam pre-treatment fits into the concept of a sugar beet biorefinery as it valorises an existing waste stream without requiring any further physical processing such as milling or dilution with water. The residual cellulose fraction was enriched in cellulose and could be effectively fermented into ethanol by yeast after enzymatic digestion, producing 0.48g ethanol per gram of glucose.

The protein fraction from wheat-based dried distiller's grain with solubles (DDGS): extraction and valorization.

Nowadays there is worldwide interest in developing a sustainable economy where biobased chemicals are the lead actors. Various potential feedstocks are available including glycerol, rapeseed meal and municipal solid waste (MSW). For biorefinery applications the byproduct streams from distilleries and bioethanol plants, such as wheat-based dried distiller's grain with solubles (DDGS), are particularly attractive, as they do not compete for land use. Wheat DDGS is rich in polymeric sugars, proteins and oils, making it ideal as a current animal feed, but also a future substrate for the synthesis of fine and commodity chemicals. This review focuses on the extraction and valorization of the protein fraction of wheat DDGS as this has received comparatively little attention to date. Since wheat DDGS production is expected to increase greatly in the near future, as a consequence of expansion of the bioethanol industry in the UK, strategies to valorize the component fractions of DDGS are urgently needed.

Engineering characterisation of a shaken, single-use photobioreactor for early stage microalgae cultivation using Chlorella sorokiniana.

This work describes the characterisation and culture performance of a novel, orbitally shaken, single-use photobioreactor (SUPBr) system for microalgae cultivation. The SUPBr mounted on an orbitally shaken platform was illuminated from below. Investigation of fluid hydrodynamics indicated a range of different flow regimes and the existence of 'in-phase' and 'out-of-phase' conditions. Quantification of the fluid mixing time (tm) indicated a decrease in tm values with increasing shaking frequency up to 90 rpm and then approximately constant tm values in the range 15-40 s. For batch cultivation of Chlorella sorokiniana, the highest biomass concentration achieved was 6.6 g L(-1) at light intensity of 180 µmol m2 s(-1). Doubling the total working volume resulted in 35-40% reduction in biomass yield while shaking frequency had little influence on culture kinetics and fatty methyl esters composition. Overall this work demonstrates the utility of the SUPBr for early stage development of algal cultivation processes.

On the fluid dynamics of a laboratory scale single-use stirred bioreactor.

The commercial success of mammalian cell-derived recombinant proteins has fostered an increase in demand for novel single-use bioreactor (SUB) systems that facilitate greater productivity, increased flexibility and reduced costs (Zhang et al., 2010). These systems exhibit fluid flow regimes unlike those encountered in traditional glass/stainless steel bioreactors because of the way in which they are designed. With such disparate hydrodynamic environments between SUBs currently on the market, traditional scale-up approaches applied to stirred tanks should be revised. One such SUB is the Mobius® 3 L CellReady, which consists of an upward-pumping marine scoping impeller. This work represents the first experimental study of the flow within the CellReady using a Particle Image Velocimetry (PIV) approach, combined with a biological study into the impact of these fluid dynamic characteristics on cell culture performance. The PIV study was conducted within the actual vessel, rather than using a purpose-built mimic. PIV measurements conveyed a degree of fluid compartmentalisation resulting from the up-pumping impeller. Both impeller tip speed and fluid working volume had an impact upon the fluid velocities and spatial distribution of turbulence within the vessel. Cell cultures were conducted using the GS-CHO cell-line (Lonza) producing an IgG4 antibody. Disparity in cellular growth and viability throughout the range of operating conditions used (80-350 rpm and 1-2.4 L working volume) was not substantial, although a significant reduction in recombinant protein productivity was found at 350 rpm and 1 L working volume (corresponding to the highest Reynolds number tested in this work). The study shows promise in the use of PIV to improve understanding of the hydrodynamic environment within individual SUBs and allows identification of the critical hydrodynamic parameters under the different flow regimes for compatibility and scalability across the range of bioreactor platforms.

Hybridisation and genetic diversity in introduced Mimulus (Phrymaceae).

Hybridisation among taxa with different ploidy levels is often associated with hybrid sterility. Clonal reproduction can stabilise these hybrids, but pervasive clonality may have a profound impact on the distribution of genetic diversity in natural populations. Here we investigate a widespread triploid taxon resulting from hybridisation between diploid Mimulus guttatus and tetraploid Mimulus luteus, two species that were introduced into the United Kingdom (UK) in the nineteenth century. This hybrid, Mimulus x robertsii, is largely sterile but capable of prolific vegetative propagation and has been recorded in the wild since 1872. We surveyed 40 Mimulus populations from localities across the UK to examine the current incidence of hybrids, and selected seventeen populations for genetic analysis using codominant markers. Cluster analyses revealed two main groups of genetically distinct individuals, corresponding to either diploid (M. guttatus) or polyploid (M. luteus and M. x robertsii) samples. Triploid hybrids were found in around 50% of sampled sites, sometimes coexisting with one of the parental species (M. guttatus). The other parent, M. luteus, was restricted to a single locality. Individual populations of M. x robertsii were genetically variable, containing multiple, highly heterozygous clones, with the majority of genetic variation distributed among- rather than within populations. Our findings demonstrate that this largely sterile, clonal taxon can preserve non-negligible amounts of genetic variation. The presence of genetically variable hybrid populations may provide the material for the continued success of asexual taxa in diverse environments.

Use of microwells to investigate the effect of quorum sensing on growth and antigen production in Bacillus anthracis Sterne 34F2.

AIM: The aim of this study was to investigate the role of quorum sensing in Bacillus anthracis growth and toxin production. METHODS AND RESULTS: A microwell plate culture method was developed to simulate the normal UK-licensed anthrax vaccine production run. Once established, sterile supernatant additions from a previous B. anthracis culture were made, and reductions in lag phase and early stimulation of the anthrax toxin component protective antigen (PA) were monitored using ELISA. The addition of the quorum-sensing inhibitor, fur-1, prolonged the lag phase and impeded PA production. Spin filters of various sizes were used to identify the molecular weight fraction of the sterile supernatant responsible for the autoinducer effect. A weight fraction between 5 and 10 kDa was responsible for the autoinducer effect; however, further identification using mass spectroscopy proved inconclusive. CONCLUSIONS: Quorum sensing mediated by the autoinducer two molecule plays a significant role in both B. anthracis growth and toxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: While genomic analysis has eluded to the importance of LuxS and quorum sensing in anthrax, this is the first analysis using a production strain of B. anthracis and a quorum-sensing inhibitor to monitor the effect on growth and toxin production. This gives insights into anthrax pathogenicity and vaccine manufacture.

Immobilised enzyme microreactor for screening of multi-step bioconversions: characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols.

Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His6-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to L-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of L-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation.

Reconstructing demographic events from population genetic data: the introduction of bumblebees to New Zealand.

Four British bumblebee species (Bombus terrestris, Bombus hortorum, Bombus ruderatus and Bombus subterraneus) became established in New Zealand following their introduction at the turn of the last century. Of these, two remain common in the United Kingdom (B. terrestris and B. hortorum), whilst two (B. ruderatus and B. subterraneus) have undergone marked declines, the latter being declared extinct in 2000. The presence of these bumblebees in New Zealand provides an unique system in which four related species have been isolated from their source population for over 100 years, providing a rare opportunity to examine the impacts of an initial bottleneck and introduction to a novel environment on their population genetics. We used microsatellite markers to compare modern populations of B. terrestris, B. hortorum and B. ruderatus in the United Kingdom and New Zealand and to compare museum specimens of British B. subterraneus with the current New Zealand population. We used approximate Bayesian computation to estimate demographic parameters of the introduction history, notably to estimate the number of founders involved in the initial introduction. Species-specific patterns derived from genetic analysis were consistent with the predictions based on the presumed history of these populations; demographic events have left a marked genetic signature on all four species. Approximate Bayesian analyses suggest that the New Zealand population of B. subterraneus may have been founded by as few as two individuals, giving rise to low genetic diversity and marked genetic divergence from the (now extinct) UK population.

Scale-down prediction of industrial scale pleated membrane cartridge performance.

Flat-sheet membrane discs represent the current standard format used for experimental prediction of the scale-up of normal flow filtration processes. Use of this format is problematic, however, since the scale-down results typically show a 40-55% difference in performance compared to large-scale cartridges depending upon the feedstock used. In this work, novel pleated scale-down devices (Am=1.51-15.1×10(-3) m2) have been designed and fabricated. It is shown that these can more accurately predict the performance of industrial scale single-use pleated membrane cartridges (Am=1.06 m2) commonly used within biopharmaceutical manufacture. The single-use scale-down cartridges retain the same pleat characteristics of the larger cartridges, but require a reduced feed volume by virtue of a substantially diminished number of active membrane pleats. In this study, a 1,000-fold reduction in feed volume requirement for the scale-down cartridge with the smallest membrane area was achieved. The scale-down cartridges were tested both with clean water and a pepsin protein solution, showing flux-time relationships within 10% of the large-scale cartridge in both cases. Protein transmission levels were also in close agreement between the different scale cartridges. The similarity in performance of the scale-down and the large-scale cartridges, coupled with the low feed requirement, make such devices an excellent method by which rapid scale-up can be achieved during early stage process development for biopharmaceutical products. This new approach is a significant improvement over using flat-sheet discs as the quantitative similarity in performance with the large-scale leads to reliable scale-up predictions while requiring especially small volumes of feed material.

Impacts of the use of nonnative commercial bumble bees for pollinator supplementation in raspberry.

Evidence for pollinator declines has led to concern that inadequate pollination services may limit crop yields. The global trade in commercial bumble bee (Bombus spp.) colonies provides pollination services for both glasshouse and open-field crops. For example, in the United Kingdom, commercial colonies of nonnative subspecies of the bumble bee Bombus terrestris L. imported from mainland Europe are widely used for the pollination of raspberries, Rubus idaeus L. The extent to which these commercial colonies supplement the services provided by wild pollinators has not been formally quantified and the impact of commercial bumble bees on native bees visiting the crop is unknown. Here, the impacts of allowing commercially available bumble bee colonies to forage on raspberry canes are assessed in terms of the yield of marketable fruit produced and the pollinator communities found foraging on raspberry flowers. No differences were found in the abundance, diversity, or composition of social bee species observed visiting raspberry flowers when commercial bumble bees were deployed compared with when they were absent. However, weight of marketable raspberries produced increased when commercial bees were present, indicating that wild pollinator services alone are inadequate for attaining maximum yields. The findings of the study suggests that proportional yield increases associated with deployment of commercial colonies may be small, but that nevertheless, investment in commercial colonies for raspberry pollination could produce very significant increases in net profit for the grower. Given potential environmental risks associated with the importation of nonnative bumble bees, the development of alternative solutions to the pollination deficit in raspberry crops in the United Kingdom may be beneficial.

Design and characterization of a prototype enzyme microreactor: quantification of immobilized transketolase kinetics.

In this work, we describe the design of an immobilized enzyme microreactor (IEMR) for use in transketolase (TK) bioconversion process characterization. The prototype microreactor is based on a 200-microm ID fused silica capillary for quantitative kinetic analysis. The concept is based on the reversible immobilization of His(6)-tagged enzymes via Ni-NTA linkage to surface derivatized silica. For the initial microreactor design, the mode of operation is a stop-flow analysis which promotes higher degrees of conversion. Kinetics for the immobilized TK-catalysed synthesis of L-erythrulose from substrates glycolaldehyde (GA) and hydroxypyruvate (HPA) were evaluated based on a Michaelis-Menten model. Results show that the TK kinetic parameters in the IEMR (V(max(app)) = 0.1 +/- 0.02 mmol min(-1), K(m(app)) = 26 +/- 4 mM) are comparable with those measured in free solution. Furthermore, the k(cat) for the microreactor of 4.1 x 10(5) s(-1) was close to the value for the bioconversion in free solution. This is attributed to the controlled orientation and monolayer surface coverage of the His(6)-immobilized TK. Furthermore, we show quantitative elution of the immobilized TK and the regeneration and reuse of the derivatized capillary over five cycles. The ability to quantify kinetic parameters of engineered enzymes at this scale has benefits for the rapid and parallel evaluation of evolved enzyme libraries for synthetic biology applications and for the generation of kinetic models to aid bioconversion process design and bioreactor selection as a more efficient alternative to previously established microwell-based systems for TK bioprocess characterization.

Cryptic differences in dispersal lead to differential sensitivity to habitat fragmentation in two bumblebee species.

Habitat loss has led to fragmentation of populations of many invertebrates, but social hymenopterans may be particularly sensitive to habitat fragmentation due to their low effective population sizes. The impacts of fragmentation depend strongly on dispersal abilities, but these are difficult to quantify. Here, we quantify and compare dispersal abilities of two bumblebee species, Bombus muscorum and Bombus jonellus, in a model island system. We use microsatellites to investigate population genetic structuring, dispersal and spatial patterns in genetic diversity. Populations of both species showed significant structuring, and isolation by distance, but this was markedly greater in B. muscorum (theta = 0.13) than in B. jonellus (theta = 0.034). This difference could reflect a higher effective population size in B. jonellus compared to B. muscorum, but this is not consistent with the observed abundance of the two species. We argue that it is more likely that B. jonellus has a higher propensity to disperse than B. muscorum. This will influence their relative susceptibility to habitat fragmentation and may in part explain differential declines of mainland populations of these and other bumblebee species.

Quantification of power consumption and oxygen transfer characteristics of a stirred miniature bioreactor for predictive fermentation scale-up.

Miniature parallel bioreactors are becoming increasingly important as tools to facilitate rapid bioprocess design. Once the most promising strain and culture conditions have been identified a suitable scale-up basis needs to be established in order that the cell growth rates and product yields achieved in small scale optimization studies are maintained at larger scales. Recently we have reported on the design of a miniature stirred bioreactor system capable of parallel operation [Gill et al. (2008); Biochem Eng J 39:164-176]. In order to enable the predictive scale-up of miniature bioreactor results the current study describes a more detailed investigation of the bioreactor mixing and oxygen mass transfer characteristics and the creation of predictive engineering correlations useful for scale-up studies. A Power number of 3.5 for the miniature turbine impeller was first established based on experimental ungassed power consumption measurements. The variation of the measured gassed to ungassed power ratio, P(g)/P(ug), was then shown to be adequately predicted by existing correlations proposed by Cui et al. [Cui et al. (1996); Chem Eng Sci 51:2631-2636] and Mockel et al. [Mockel et al. (1990); Acta Biotechnol 10:215-224]. A correlation relating the measured oxygen mass transfer coefficient, k(L)a, to the gassed power per unit volume and superficial gas velocity was also established for the miniature bioreactor. Based on these correlations a series of scale-up studies at matched k(L)a (0.06-0.11 s(-1)) and P(g)/V (657-2,960 W m(-3)) were performed for the batch growth of Escherichia coli TOP10 pQR239 using glycerol as a carbon source. Constant k(L)a was shown to be the most reliable basis for predictive scale-up of miniature bioreactor results to conventional laboratory scale. This gave good agreement in both cell growth and oxygen utilization kinetics over the range of k(L)a values investigated. The work described here thus gives further insight into the performance of the miniature bioreactor design and will aid its use as a tool for rapid fermentation process development.

Thermal profiling for parallel on-line monitoring of biomass growth in miniature stirred bioreactors.

Recently we have described the design and operation of a miniature bioreactor system in which 4-16 fermentations can be performed (Gill et al., Biochem Eng J 39:164-176, 2008). Here we report on the use of thermal profiling techniques for parallel on-line monitoring of cell growth in these bioreactors based on the natural heat generated by microbial culture. Results show that the integrated heat profile during E. coli TOP10 pQR239 fermentations followed the same pattern as off-line optical density (OD) measurements. The maximum specific growth rates calculated from off-line OD and on-line thermal profiling data were in good agreement, at 0.66+/-0.04 and 0.69+/-0.05 h(-1) respectively. The combination of a parallel miniature bioreactor system with a non-invasive on-line technique for estimation of culture kinetic parameters provides a valuable approach for the rapid optimisation of microbial fermentation processes.

Quantification of kinetics for enzyme-catalysed reactions: implications for diffusional limitations at the 10 ml scale.

The effects of different reaction scales [100 microl reactions in 96-standard round well (SRW) plates and 10 ml reactions in 24-square well (SW) plates] have been investigated using, as a model, transketolase (TK)-catalysed reaction producing L-erythrulose. Reactions were carried out under non-shaking, shaking and at 10 ml scale stirring conditions to assess the effect of diffusional limitations. Statistical analysis confirmed the significance of the observed difference in reaction rates under given conditions. Only when the laboratory scale system (10 ml) was well mixed did the reaction rate become comparable to that in the microwells, where there is negligible diffusional limitation. These findings have important implications for the scale-up (or scale-down) of enzyme-catalysed reactions.

Scale-up of Escherichia coli growth and recombinant protein expression conditions from microwell to laboratory and pilot scale based on matched k(L)a.

Fermentation optimization experiments are ideally performed at small scale to reduce time, cost and resource requirements. Currently microwell plates (MWPs) are under investigation for this purpose as the format is ideally suited to automated high-throughput experimentation. In order to translate an optimized small-scale fermentation process to laboratory and pilot scale stirred-tank reactors (STRs) it is necessary to characterize key engineering parameters at both scales given the differences in geometry and the mechanisms of aeration and agitation. In this study oxygen mass transfer coefficients are determined in three MWP formats and in 7.5 L and 75 L STRs. k(L)a values were determined in cell-free media using the dynamic gassing-out technique over a range of agitation conditions. Previously optimized culture conditions at the MWP scale were then scaled up to the larger STR scales on the basis of matched k(L)a values. The accurate reproduction of MWP (3 mL) E. coli BL21 (DE3) culture kinetics at the two larger scales was shown in terms of cell growth, protein expression, and substrate utilization for k(L)a values that provided effective mixing and gas-liquid distribution at each scale. This work suggests that k(L)a provides a useful initial scale-up criterion for MWP culture conditions which enabled a 15,000-fold scale translation in this particular case. This work complements our earlier studies on the application of DoE techniques to MWP fermentation optimization and in so doing provides a generic framework for the generation of large quantities of soluble protein in a rapid and cost-effective manner.

Decline and conservation of bumble bees.

Declines in bumble bee species in the past 60 years are well documented in Europe, where they are driven primarily by habitat loss and declines in floral abundance and diversity resulting from agricultural intensification. Impacts of habitat degradation and fragmentation are likely to be compounded by the social nature of bumble bees and their largely monogamous breeding system, which renders their effective population size low. Hence, populations are susceptible to stochastic extinction events and inbreeding. In North America, catastrophic declines of some bumble bee species since the 1990s are probably attributable to the accidental introduction of a nonnative parasite from Europe, a result of global trade in domesticated bumble bee colonies used for pollination of greenhouse crops. Given the importance of bumble bees as pollinators of crops and wildflowers, steps must be taken to prevent further declines. Suggested measures include tight regulation of commercial bumble bee use and targeted use of environmentally comparable schemes to enhance floristic diversity in agricultural landscapes.

Framework for the rapid optimization of soluble protein expression in Escherichia coli combining microscale experiments and statistical experimental design.

A major bottleneck in drug discovery is the production of soluble human recombinant protein in sufficient quantities for analysis. This problem is compounded by the complex relationship between protein yield and the large number of variables which affect it. Here, we describe a generic framework for the rapid identification and optimization of factors affecting soluble protein yield in microwell plate fermentations as a prelude to the predictive and reliable scaleup of optimized culture conditions. Recombinant expression of firefly luciferase in Escherichia coli was used as a model system. Two rounds of statistical design of experiments (DoE) were employed to first screen (D-optimal design) and then optimize (central composite face design) the yield of soluble protein. Biological variables from the initial screening experiments included medium type and growth and induction conditions. To provide insight into the impact of the engineering environment on cell growth and expression, plate geometry, shaking speed, and liquid fill volume were included as factors since these strongly influence oxygen transfer into the wells. Compared to standard reference conditions, both the screening and optimization designs gave up to 3-fold increases in the soluble protein yield, i.e., a 9-fold increase overall. In general the highest protein yields were obtained when cells were induced at a relatively low biomass concentration and then allowed to grow slowly up to a high final biomass concentration, >8 g.L-1. Consideration and analysis of the model results showed 6 of the original 10 variables to be important at the screening stage and 3 after optimization. The latter included the microwell plate shaking speeds pre- and postinduction, indicating the importance of oxygen transfer into the microwells and identifying this as a critical parameter for subsequent scale translation studies. The optimization process, also known as response surface methodology (RSM), predicted there to be a distinct optimum set of conditions for protein expression which could be verified experimentally. This work provides a generic approach to protein expression optimization in which both biological and engineering variables are investigated from the initial screening stage. The application of DoE reduces the total number of experiments needed to be performed, while experimentation at the microwell scale increases experimental throughput and reduces cost.

One-pot synthesis of amino-alcohols using a de-novo transketolase and beta-alanine: pyruvate transaminase pathway in Escherichia coli.

Biocatalysis continues to emerge as a powerful technique for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The power of biocatalysis can be enhanced if two or more reactions can be achieved by a single whole cell biocatalyst containing a pathway designed de-novo to facilitate a required synthetic sequence. The enzymes transketolase (TK) and transaminase (TAm) respectively catalyze asymmetric carbon--carbon bond formation and amine group addition to suitable substrate molecules. The ability of a transaminase to accept the product of the transketolase reaction can allow the two catalysts to be employed in series to create chiral amino-alcohols from achiral substrates. As proof of principle, the beta-alanine: pyruvate aminotransferase (beta-A:P TAm) from Pseudomonas aeruginosa has been cloned, to create plasmid pQR428, for overexpression in E.coli strain BL21gold(DE3). Production of the beta-A:P TAm alongside the native transketolase (overexpressed from plasmid pQR411), in a single E.coli host, has created a novel biocatalyst capable of the synthesis of chiral amino alcohols via a synthetic two-step pathway. The feasibility of using the biocatalyst has been demonstrated by the formation of a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT) product, in up to 21% mol/mol yield, by the beta-A:P TAm, via transamination of L-erythrulose synthesized by TK, from achiral substrates glycolaldehyde (GA) and beta-hydroxypyruvate (beta-HPA). ABT synthesis was achieved in a one-pot process, using either whole cells of the dual plasmid strain or cell lysate, while the dual alcohol-amine functionality of ABT makes it an excellent synthon for many pharmaceutical syntheses.