Whole-cell Systems Research Articles

Integration of spore-based genetically engineered whole-cell sensing systems into portable centrifugal microfluidic platforms

Bacterial whole-cell biosensing systems provide important information about the bioavailable amount of target analytes. They are characterized by high sensitivity and specificity/selectivity along with rapid response times and amenability to miniaturization as well as high-throughput analysis. Accordingly, they have been employed in various environmental and clinical applications. The use of spore-based sensing systems offers the unique advantage of long-term preservation of the sensing cells by taking advantage of the environmental resistance and ruggedness of bacterial spores. In this work, we have incorporated spore-based whole-cell sensing systems into centrifugal compact disk (CD) microfluidic platforms in order to develop a portable sensing system, which should enable the use of these hardy sensors for fast on-field analysis of compounds of interest. For that, we have employed two spore-based sensing systems for the detection of arsenite and zinc, respectively, and evaluated their analytical performance in the miniaturized microfluidic format. Furthermore, we have tested environmental and clinical samples on the CD microfluidic platforms using the spore-based sensors. Germination of spores and quantitative response to the analyte could be obtained in 2.5-3 h, depending on the sensing system, with detection limits of 1 x 10(-7) M for arsenite and 1 x 10(-6) M for zinc in both serum and fresh water samples. Incorporation of spore-based whole-cell biosensing systems on microfluidic platforms enabled the rapid and sensitive detection of the analytes and is expected to facilitate the on-site use of such sensing systems.

Export of Methyl Parathion Hydrolase to the Periplasm by the Twin-Arginine Translocation Pathway inEscherichia coli

The uptake of organophosphates (OPs) is a rate-limiting factor in whole-cell biocatalysis systems. Here, we report the periplasmic secretion of methyl parathion hydrolase (MPH) by employing the twin-arginine translocation (Tat) pathway in Escherichia coli. The twin-arginine signal peptide of trimethylamine N-oxide reductase (TorA) from E. coli was used for exporting MPH to the periplasm of E. coli, alleviating the substrate uptake limitation. A periplasmic expression vector, pUTM18, coding for TorA-MPH was constructed, and the periplasmic secretion and functionality of MPH were demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. The strain expressing periplasmic MPH showed 3-fold higher whole-cell activity than the control strain expressing cytoplasmic MPH. Suspended cultures also exhibited good stability, retaining almost 100% activity over a period of 2 weeks. Owing to their high activity and superior stability, these "live biocatalysts" are ideal for large-scale detoxification of OPs.

A cytochrome P450 class I electron transfer system from Novosphingobium aromaticivorans

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from Novosphingobium aromaticivorans are heme monooxygenases that catalyze the hydroxylation of a range of terpenoid compounds. CYP101D1 and CYP101D2 oxidized camphor to 5-exo-hydroxycamphor. CYP101B1 and CYP101C1 oxidized beta-ionone to predominantly 3-R-hydroxy-beta-ionone and 4-hydroxy-beta-ionone, respectively. CYP111A2 oxidized linalool to 8-hydroxylinalool. Physiologically, these CYP enzymes could receive electrons from Arx, a [2Fe-2S] ferredoxin equivalent to putidaredoxin from the CYP101A1 system from Pseudomonas putida. A putative ferredoxin reductase (ArR) in the N. aromaticivorans genome, with high amino acid sequence homology to putidaredoxin reductase, has been over-produced in Escherichia coli and found to support substrate oxidation by these CYP enzymes via Arx with both high activity and coupling of product formation to NADH consumption. The ArR/Arx electron-transport chain has been co-expressed with the CYP enzymes in an E. coli host to provide in vivo whole-cell substrate oxidation systems that could produce up to 6.0 g L(-1) of 5-exo-hydroxycamphor at rates of up to 64 microM (gram of cell dry weight)(-1) min(-1). These efficient biocatalytic systems have potential uses in preparative scale whole-cell biotransformations.

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Nootkatone—a biotechnological challenge

Due to its pleasant grapefruit-like aroma and various further interesting molecular characteristics, (+)-nootkatone represents a highly sought-after specialty chemical. (+)-Nootkatone is accumulated in its producer plants in trace amounts only, and the demand of the food, cosmetics and pharmaceutical industry is currently predominantly met by chemical syntheses. These typically require environmentally critical reagents, catalysts and solvents, and the final product must not be marketed as a "natural flavour" compound. Both the market pull and the technological push have thus inspired biotechnologists to open up more attractive routes towards natural (+)-nootkatone. The multifaceted approaches for the de novo biosynthesis or the biotransformation of the precursor (+)-valencene to (+)-nootkatone are reviewed. Whole-cell systems of bacteria, filamentous fungi and plants, cell extracts or purified enzymes have been employed. A prominent biocatalytic route is the allylic oxidation of (+)-valencene. It allows the production of natural (+)-nootkatone in high yields under mild reaction conditions. The first sequence data of (+)-valencene-converting activities have just become known.

Th-1 polarization is regulated by dendritic-cell comparison of MHC class I and class II antigens

AbstractIn the control of T-helper type I (Th-1) polarization, dendritic cells (DCs) must interpret a complex array of stimuli, many of which are poorly understood. Here we demonstrate that Th-1 polarization is heavily influenced by DC-autonomous phenomena triggered by the loading of DCs with antigenically matched major histocompatibility complex (MHC) class I and class II determinants, that is, class I and II peptide epitopes exhibiting significant amino acid sequence overlap (such as would be physiologically present during infectious processes requiring Th-1 immunity for clearance). Data were derived from 13 independent antigenic models including whole-cell systems, single-protein systems, and 3 different pairs of overlapping class I and II binding epitopes. Once loaded with matched class I and II antigens, these “Th-1 DCs” exhibited differential cytokine secretion and surface marker expression, a distinct transcriptional signature, and acquired the ability to enhance generation of CD8+ T lymphocytes. Mechanistically, tRNA-synthetases were implicated as components of a putative sensor complex involved in the comparison of class I and II epitopes. These data provide rigorous conceptual explanations for the process of Th-1 polarization and the antigenic specificity of cognate T-cell help, enhance the understanding of Th-1 responses, and should contribute to the formulation of more effective vaccination strategies.

BioXyce: an engineering platform for the study of cellular systems

Researchers use constructs from the field of electrical engineering for the modelling and analysis of biological systems, but few exploit parallels between electrical and biological circuits for simulation purposes. The authors discuss the development of BioXyce, a circuit-based biological simulation platform that uses Xyce, a large-scale electrical circuit simulator, as its simulation engine. BioXyce is capable of simulating whole-cell and multicellular systems. Simulation results for the central metabolism in Escherichia coli K12 and cellular differentiation in Drosophila sp. are presented.

A ternary complex of hydroxycinnamoyl-CoA hydratase–lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism

HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and is exploited in whole-cell systems for the bioconversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K(M) for the substrate ferulic acid, fulfilling its anticipated role as a factor in substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.

Advancing Biocatalysis through Enzyme, Cellular, and Platform Engineering

Biocatalysis offers opportunities for highly selective chemical reactions with high turnover rates under relatively mild conditions. Use of whole-cell or multi-enzyme systems enables transformations of complexity unmatched by nonbiological routes. However, advantages of biocatalysis are frequently compromised by poor enzymatic performance under non-native reaction conditions, the absence of enzymes with desired substrate or reaction specificities, and low metabolic fluxes or competing pathways. During the 234th National Meeting of the American Chemical Society, these issues were addressed in the "Advances in Biocatalysis" sessions. Protein engineering and metabolic pathway engineering were used to develop efficient enzymes and whole-cell catalysts. Novel strategies for the use of enzymes at solid interfaces and in nonaqueous environments were discussed, and efficient biotransformation platforms were demonstrated. These advances broaden the applications of biocatalysis in biofuels, pharmaceuticals, fine chemicals, and human health.

Construction of Spores for Portable Bacterial Whole-Cell Biosensing Systems

Whole-cell sensing systems based on living genetically engineered bacteria are known to have high sensitivity, selectivity, and rapid response times. Although these systems have found applications in biomedical and environmental analyses, their limited shelf life and transportability still restrict their use for on-site monitoring of analytes. To that end, we have developed a new method for the long-term preservation, storage, and transport of whole-cell biosensing systems that is based on bacterial spores, a dormant form of life. Specifically, we have employed spore-forming bacteria such as Bacillus subtilis and Bacillus megaterium for development of luminescent sensing systems for two model analytes, namely, arsenic and zinc. These sensing cells were converted to spores, which can then be "revived" (germinated) at a later time to generate viable and metabolically active cells. Herein, we demonstrate that these spore-based sensing systems retained their analytical performance, in terms of detection limit, dynamic range, and reproducibility, after storage at room temperature for at least 6 and 8 months, respectively, as well as after three cycles where the cells alternated between being dormant or active, i.e., sporulation-germination cycles. The ability to cycle the sensing cells between active and dormant states prolongs the cell's lifetimes and increases their robustness and ruggedness, thus making them more amenable for field applications. In addition, the small size of spores allows for their easy transport and incorporation in miniaturized portable devices. Finally, we envision that this novel strategy could expand the use of whole-cell biosensors for on-site sensing not only in mild environments but also in harsh environments and locations where there is no easy access to a laboratory, e.g., in developing countries.

Identification of the Cadmium-Inducible Hansenula polymorpha SEO1 Gene Promoter by Transcriptome Analysis and Its Application to Whole-Cell Heavy-Metal Detection Systems

The genomewide gene expression profiling of the methylotrophic yeast Hansenula polymorpha exposed to cadmium (Cd) allowed us to identify novel genes responsive to Cd treatment. To select genes whose promoters can be useful for construction of a cellular Cd biosensor, we further analyzed a set of H. polymorpha genes that exhibited >6-fold induction upon treatment with 300 muM Cd for 2 h. The putative promoters, about 1,000-bp upstream fragments, of these genes were fused with the yeast-enhanced green fluorescence protein (GFP) gene. The resultant reporter cassettes were introduced into H. polymorpha to evaluate promoter strength and specificity. The promoter derived from the H. polymorpha SEO1 gene (HpSEO1) was shown to drive most strongly the expression of GFP upon Cd treatment among the tested promoters. The Cd-inducible activity was retained in the 500-bp deletion fragment of the HpSEO1 promoter but was abolished in the further truncated 250-bp fragment. The 500-bp HpSEO1 promoter directed specific expression of GFP upon exposure to Cd in a dose-dependent manner, with Cd detection ranging from 1 to 900 muM. Comparative analysis of the Saccharomyces cerevisiae SEO1 (ScSEO1) promoter revealed that the ScSEO1 promoter has a broader specificity for heavy metals and is responsive to arsenic and mercury in addition to Cd. Our data demonstrate the potential use of the HpSEO1 promoter as a bioelement in whole-cell biosensors to monitor heavy metal contamination, particularly Cd.

Luciferase Detection during Stationary Phase in Lactococcus lactis

The luminescence signal of luxAB-encoded bacterial luciferase strongly depends on the metabolic state of the host cell, which restricts the use of this reporter system to metabolically active bacteria. Here we show that in stationary-phase cells of Lactococcus lactis, detection of luciferase is significantly improved by the addition of riboflavin or flavin mononucleotide to whole-cell assay systems.

A Window into Biocatalysis and Biotransformations

Eight papers were presented in this year's symposium "Advances in Biocatalysis" at the 232nd ACS National Meeting, accentuating the most recent development in biocatalysis. Researchers from both industry and academia are addressing several fundamental problems in biocatalysis, including the limited number of commercially available enzymes that can be provided in bulk quantities, the limited enzyme stability and activity in nonaqueous environments, and the permeability issue and cell localization problems in whole-cell systems. A trend that can be discerned from these eight talks is the infusion of new tools and technologies in addressing various challenges facing biocatalysis. Nanotechnology, bioinformatics, cellular membrane engineering and metabolic engineering (for engineering whole-cell catalysts), and protein engineering (to improve enzymes and create novel enzymes) are becoming more routinely used in research laboratories and are providing satisfactory solutions to the problems in biocatalysis. Significant progress in various aspects of biocatalysis from discovery to industrial applications was highlighted in this symposium.

Microbial Baeyer–Villiger oxidation of terpenones by recombinant whole-cell biocatalysts—formation of enantiocomplementary regioisomeric lactones

Recombinant whole-cell expression systems for Baeyer-Villiger monooxygenases of various bacterial origin were utilized in the regiodivergent biooxidation of cyclic terpenones enabling access to enantio- and regioisomeric lactones on preparative scale.

Towards engineered topogenesis of cytochrome b5 and P450 for in vivo transformation of xenobiotics

Nature is endowed with catalysts capable of an unprecedented diversity of biotransformations, beyond the capabilities of synthetic chemistries. In a biotechnological context, there is a growing and emerging need to tap this catalytic potential. CYP (cytochrome P450) represents a superfamily of enzymes capable of a diverse array of catalytic activities. Distinct members are engaged in biosynthetic reactions within many organisms, while others have a role in the detoxification of foreign compounds. The latter substrates include medicines, pollutants, pesticides, carcinogens, perfumes and herbicides, representing considerable applied importance for pharmacology and toxicology. CYPs show a high degree of stereo- and regio-specificity for their reactions, which have wide industrial applications. Recombinant CYPs are commonly expressed as active recombinant cytosolic forms in Escherichia coli. However, selective permeability of E. coli to many substrates and products can cause problems with product recovery when using whole-cell systems. To overcome these problems, we have been developing approaches to facilitate export of functional recombinant haemoproteins to the inner membrane, periplasm and the outer membrane of E. coli. Here, we describe the progress in relation to cytochrome b(5) and CYPs.

Outer membrane mutation effects on UDP-glucose permeability and whole-cell catalysis rate

In whole-cell biocatalysis, cell envelopes represent a formidable barrier for substrates to permeate. The present research addresses this critical issue by investigating the effects of outer membrane mutation on uridine diphosphate (UDP)-glucose-utilizing enzymes in whole-cell systems. Owing to the severe limitation in substrate permeability, the wild-type Escherichia coli cells only exhibited as low as 4% of available enzyme activities. The reduction of the barriers of the outer membrane permeability (by mutations in its structure) led to a striking acceleration (up to 14-fold) of the reaction rate in cells expressing UDP-glucose dehydrogenase. Mutations in the lipopolysaccharide synthesis pathway or Braun's lipoprotein are both effective. The acceleration was dependent upon the substrate concentrations as well as the enzyme expression level. In addition, the mutation has been demonstrated to be much more effective than the freeze-thaw permeabilizing method. An application of outer membrane mutants was illustrated with the synthesis of a disaccharide (N-acetyllactosamine) from UDP-glucose. Both reaction rate and product yield were enhanced significantly (more than twofold) in the lipoprotein mutant, demonstrating the importance of the outer membrane permeability barrier and the advantages of using outer membrane mutants in synthesis. This research and the results outlined in this paper point to a valid strategy in addressing permeability issues in whole-cell biocatalysis. It also highlights a need for an assessment of substrate permeability in biocatalysis research and development.

Metabolic engineering of microbes for oligosaccharide and polysaccharide synthesis

Metabolic engineering has recently been embraced as an effective tool for developing whole-cell biocatalysts for oligosaccharide and polysaccharide synthesis. Microbial catalysts now provide a practical means to derive many valuable oligosaccharides, previously inaccessible through other methods, in sufficient quantities to support research and clinical applications. The synthesis process based upon these microbes is scalable as it avoids expensive starting materials. Most impressive is the high product concentrations (up to 188 g/L) achieved through microbe-catalyzed synthesis. The overall cost for selected molecules has been brought to a reasonable range (estimated $ 30–50/g).Microbial synthesis of oligosaccharides and polysaccharides is a carbon-intensive and energy-intensive process, presenting some unique challenges in metabolic engineering. Unlike nicotinamide cofactors, the required sugar nucleotides are products of multiple interacting pathways, adding significant complexity to the metabolic engineering effort. Besides the challenge of providing the necessary mammalian-originated glycosyltransferases in active form, an adequate uptake of sugar acceptors can be an issue when another sugar is necessary as a carbon and energy source. These challenges are analyzed, and various strategies used to overcome these difficulties are reviewed in this article. Despite the impressive success of the microbial coupling strategy, there is a need to develop a single strain that can achieve at least the same efficiency. Host selection and the manner with which the synthesis interacts with the central metabolism are two important factors in the design of microbial catalysts. Additionally, unlike in vitro enzymatic synthesis, product degradation and byproduct formation are challenges of whole-cell systems that require additional engineering. A systematic approach that accounts for various and often conflicting requirements of the synthesis holds the key to deriving an efficient catalyst.Metabolic engineering strategies applied to selected polysaccharides (hyaluronan, alginate, and exopolysaccharides for food use) are reviewed in this article to highlight the recent progress in this area and similarity to challenges in oligosaccharide synthesis. Many naturally occurring microbes possess highly efficient mechanisms for polysaccharide synthesis. These mechanisms could potentially be engineered into a microbe for oligosaccharide and polysaccharide synthesis with enhanced efficiency.

Advances in preservation methods: keeping biosensor microorganisms alive and active

The ability of bacteria to sense their surroundings can be employed to measure the bioavailability and toxicity of pollutants. However, long-term maintenance of both viability and activity of the sensor bacteria is required for the development of cell-based devices for environmental monitoring. To meet these demands, various techniques to conserve such bacteria have been reported, including freeze drying, vacuum drying, continuous cultivation, and immobilisation in biocompatible polymers of organic or inorganic origin. Much effort has been invested in merging these bacterial preservation schemes with the construction of sensor cell arrays on platforms such as biochips or optic fibres, hopefully leading to effective miniaturised whole-cell biosensor systems. These approaches hold much promise for the future. Nevertheless, their eventual implementation in practical devices calls for significant enhancement of current knowledge on formulation of reporter microorganisms.

Quantitative estimation of channeling from early glycolytic intermediates to CO2 in intact Escherichia coli

A pathway intermediate is said to be 'channeled' when an intermediate just made in a pathway has a higher probability of being a substrate for the next pathway enzyme compared with a molecule of the same species from the aqueous cytoplasm. Channeling is an important phenomenon because it might play a significant role in the regulation of metabolism. Whereas the usual mechanism proposed for channeling is the (often) transient interaction of sequential pathway enzymes, many of the supporting data come from results with pure enzymes and dilute cell extracts. Even when isotope dilution techniques have utilized whole-cell systems, most often only a qualitative assessment of channeling has been reported. Here we develop a method for making a quantitative calculation of the fraction channeled in glycolysis from in vivo isotope dilution experiments. We show that fructose-1,6-bisphosphate, in whole cells of Escherichia coli, was strongly channeled all the way to CO2, whereas fructose-6-phosphate was not. Because the signature of channeling is lost if any downstream intermediate prior to CO2 equilibrates with molecules in the aqueous cytosol, it was not possible to evaluate whether glucose-6-phosphate was channeled in its transformation to fructose-6-phosphate. The data also suggest that, in addition to pathway enzymes being associated with one another, some are free in the aqueous cytosol. How sensitive the degree of channeling is to growth or experimental conditions remains to be determined.

Recent developments in pyridine nucleotide regeneration.

NAD(P)-dependent oxidoreductases are valuable tools for the synthesis of chiral compounds. Due to the high cost of the pyridine cofactors, in situ cofactor regeneration is required for preparative applications. In recent years, existing regeneration methodologies have been improved and new approaches have been devised. These include the use of newly discovered dehydrogenases that are stable in high contents of organic solvent and novel enzymes that can regenerate either the reduced or oxidized forms of the cofactor. The use of electrochemical methods has allowed cofactor regeneration for monooxygenases and natural or engineered whole-cell systems provide alternatives to approaches relying on purified enzymes.