Robert Speight

Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst

BackgroundTrigonopsis variabilisD-amino acid oxidase (Tv DAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of Tv DAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris Tv DAO whole-cell biocatalyst.ResultsAs compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 × 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of ≥ 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used Tv DAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration.ConclusionsStepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell Tv DAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.

A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins.

BACKGROUND Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor kappa B (NF-kappa B) p50 to establish a phenotype-genotype link between the displayed protein and the encoding gene. RESULTS A range of model fusion proteins to either the amino- or carboxy-terminus of NF-kappa B p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-kappa B p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50-DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein-plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:10(8) and to enrich to near homogeneity a single functional protein from a phenotype-genotype linked Escherichia coli genomic library using in vitro functional selections. CONCLUSIONS A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.

Biotechnology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in biotechnology.

Genomic organisation, activity and distribution analysis of the microbial putrescine oxidase degradation pathway.

The catalytic action of putrescine specific amine oxidases acting in tandem with 4-aminobutyraldehyde dehydrogenase is explored as a degradative pathway in Rhodococcus opacus. By limiting the nitrogen source, increased catalytic activity was induced leading to a coordinated response in the oxidative deamination of putrescine to 4-aminobutyraldehyde and subsequent dehydrogenation to 4-aminobutyrate. Isolating the dehydrogenase by ion exchange chromatography and gel filtration revealed that the enzyme acts principally on linear aliphatic aldehydes possessing an amino moiety. Michaelis-Menten kinetic analysis delivered a Michaelis constant (K(M)=0.014 mM) and maximum rate (Vmax=11.2 μmol/min/mg) for the conversion of 4-aminobutyraldehyde to 4-aminobutyrate. The dehydrogenase identified by MALDI-TOF mass spectrometric analysis (E value=0.031, 23% coverage) belongs to a functionally related genomic cluster that includes the amine oxidase, suggesting their association in a directed cell response. Key regulatory, stress and transport encoding genes have been identified, along with candidate dehydrogenases and transaminases for the further conversion of 4-aminobutyrate to succinate. Genomic analysis has revealed highly similar metabolic gene clustering among members of Actinobacteria, providing insight into putrescine degradation notably among Micrococcaceae, Rhodococci and Corynebacterium by a pathway that was previously uncharacterised in bacteria.

Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus

While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH4Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations>1mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis-Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (KM)=190μM and maximum rate (kcat)=21.8s(-1) for the oxidative deamination of putrescine with a lower KM (=60μM) and comparable kcat (=18.2s(-1)) for the copper oxidase. MALDI-TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.

Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris

ABSTRACT Pichia pastoris (syn. Komagataella phaffii) is one of the most common eukaryotic expression systems for heterologous protein production. Expression cassettes are typically integrated in the genome to obtain stable expression strains. In contrast to Saccharomyces cerevisiae, where short overhangs are sufficient to target highly specific integration, long overhangs are more efficient in P. pastoris and ectopic integration of foreign DNA can occur. Here, we aimed to elucidate the influence of ectopic integration by high-throughput screening of >700 transformants and whole-genome sequencing of 27 transformants. Different vector designs and linearization approaches were used to mimic the most common integration events targeted in P. pastoris. Fluorescence of an enhanced green fluorescent protein (eGFP) reporter protein was highly uniform among transformants when the expression cassettes were correctly integrated in the targeted locus. Surprisingly, most nonspecifically integrated transformants showed highly uniform expression that was comparable to specific integration, suggesting that nonspecific integration does not necessarily influence expression. However, a few clones (<10%) harboring ectopically integrated cassettes showed a greater variation spanning a 25-fold range, surpassing specifically integrated reference strains up to 6-fold. High-expression strains showed a correlation between increased gene copy numbers and high reporter protein fluorescence levels. Our results suggest that for comparing expression levels between strains, the integration locus can be neglected as long as a sufficient numbers of transformed strains are compared. For expression optimization of highly expressible proteins, increasing copy number appears to be the dominant positive influence rather than the integration locus, genomic rearrangements, deletions, or single-nucleotide polymorphisms (SNPs). IMPORTANCE Yeasts are commonly used as biotechnological production hosts for proteins and metabolites. In the yeast Saccharomyces cerevisiae, expression cassettes carrying foreign genes integrate highly specifically at the targeted sites in the genome. In contrast, cassettes often integrate at random genomic positions in nonconventional yeasts, such as Pichia pastoris (syn. Komagataella phaffii). Hence, cells from the same transformation event often behave differently, with significant clonal variation necessitating the screening of large numbers of strains. The importance of this study is that we systematically investigated the influence of integration events in more than 700 strains. Our findings provide novel insight into clonal variation in P. pastoris and, thus, how to avoid pitfalls and obtain reliable results. The underlying mechanisms may also play a role in other yeasts and hence could be generally relevant for recombinant yeast protein production strains.

Two Gut-Associated Yeasts in a Tephritid Fruit Fly have Contrasting Effects on Adult Attraction and Larval Survival

Yeast-insect interactions have been well characterized in drosophilid flies, but not in tephritid fruit flies, which include many highly polyphagous pest species that attack ripening fruits. Using the Queensland fruit fly (Bactrocera tryoni) as our model tephritid species, we identified yeast species present in the gut of wild-collected larvae and found two genera, Hanseniaspora and Pichia, were the dominant isolates. In behavioural trials using adult female B. tryoni, a fruit-agar substrate inoculated with Pichia kluyveri resulted in odour emissions that increased the attraction of flies, whereas inoculation with Hanseniaspora uvarum, produced odours that strongly deterred flies, and both yeasts led to decreased oviposition. Larval development trials showed that the fruit-agar substrate inoculated with the ‘deterrent odour’ yeast species, H. uvarum, resulted in significantly faster larval development and a greater number of adult flies, compared to a substrate inoculated with the ‘attractive odour’ yeast species, P. kluyveri, and a yeast free control substrate. GC-MS analysis of volatiles emitted by H. uvarum and P. kluyveri inoculated substrates revealed significant quantitative differences in ethyl-, isoamyl-, isobutyl-, and phenethyl- acetates, which may be responsible for the yeast-specific olfactory responses of adult flies. We discuss how our seemingly counterintuitive finding that female B. tryoni flies avoid a beneficial yeast fits well with our understanding of female choice of oviposition sites, and how the contrasting behavioural effects of H. uvarum and P. kluyveri raises interesting questions regarding the role of yeast-specific volatiles as cues to insect vectors. A better understanding of yeast-tephritid interactions could assist in the future management of tephritid fruit fly pests through the formulation of new “attract and kill” lures, and the development of probiotics for mass rearing of insects in sterile insect control programs.

Improved fermentation efficiency of S . cerevisiae by changing glycolytic metabolic pathways with plasma agitation

Production of ethanol by the yeast Saccharomyces cerevisiae is a process of global importance. In these processes, productivities and yields are pushed to their maximum possible values leading to cellular stress. Transient and lasting enhancements in tolerance and performance have been obtained by genetic engineering, forced evolution, and exposure to moderate levels of chemical and/or physical stimuli, yet the drawbacks of these methods include cost, and multi-step, complex and lengthy treatment protocols. Here, plasma agitation is shown to rapidly induce desirable phenotypic changes in S. cerevisiae after a single treatment, resulting in improved conversion of glucose to ethanol. With a complex environment rich in energetic electrons, highly-reactive chemical species, photons, and gas flow effects, plasma treatment simultaneously mimics exposure to multiple environmental stressors. A single treatment of up to 10 minutes performed using an atmospheric pressure plasma jet was sufficient to induce changes in cell membrane structure, and increased hexokinase 2 activity and secondary metabolite production. These results suggest that plasma treatment is a promising strategy that can contribute to improving metabolic activity in industrial microbial strains, and thus the practicality and economics of industrial fermentations.

Understanding the dynamics of keratin weakening and hydrolysis by proteases

Keratin is the structural protein in hair, nails, feathers and horns. Keratin is recalcitrant, highly disulfide bonded and is generally inaccessible to common proteases. Only certain types of proteases, called keratinases, are able to cleave the peptide bonds within the keratin structure. Due to this outstanding activity, keratinases have potential application in industries such as livestock, cosmetics and pharmaceuticals. Yet, the process of enzymatic keratin degradation is poorly understood, affecting the development of industrial enzyme formulations that may require full or only partial modification or weakening. Here we investigate the dynamics of keratin weakening and hydrolysis, showing that the decrease in hair mechanical strength is associated with cuticle removal and damage to the cortex and complete breakdown is dependent on reducing agents. Proteases with keratinolytic activity were selected and applied to hair with degradation examined by mechanical, biochemical and microscopic techniques. The extent of keratin degradation was highly enhanced by the presence of reducing agents, principally sodium thioglycolate, exceeding 90% degradation within 16 h of enzymatic treatment. Application was extended to feathers showing that the findings are relevant to improving the use of keratinases in a variety of industries. Overall, the outcomes provide valuable insights into the keratin degradation process by enzymes for the optimization of cosmetic and pharmaceutical products and for livestock waste recycling among other important applications.

Biogas, Bioreactors and Bacterial Methane Oxidation

Pure methane is an energy-rich feedstock used to generate electricity, for domestic heating and cooking and as a vehicle fuel. Methane is the second most abundant greenhouse gas and is commonly available as the predominant component of natural gas or biogas. Biogas is viewed as a renewable methane supply, and its production and sources are discussed. Capture of this microbially-derived methane is a significant opportunity not only for heat and energy production, but also for its possible conversion to value-added products from methane-oxidising bacteria. Examples of methanotrophs cultured using methane from biogas are discussed, as well as bioreactor choice and provision of gas to the bacteria. Various bioreactor designs are explained in terms of applicability to methanotroph cultivation. Finally, methanotrophs are discussed in the context of two extremes: their use in methane mitigation and bioremediation versus the synthesis of biological products.