LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae.

Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. 'Turbo' mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37-33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages 'leaky' biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

Chromogenic fusion proteins as alternative textiles dyes.

The widespread adoption of fast fashion has led to a significant waste problem associated with discarded textiles. Using proteins to color textiles can serve as a sustainable alternative to chemical dyes as well as reduce the demand for new raw materials. Here, we explore the use of chromogenic fusion proteins, consisting of a chromoprotein and a carbohydrate-binding module (CBM), as coloring agents for cellulose-based textiles such as cotton. We examined the color properties of chromoproteins AeBlue, SpisPink and Ultramarine alone and fused to CBM under various conditions. AeBlue, SpisPink and Ultramarine exhibited visible color between pH 4-9 and temperatures ranging from 4 to 45℃. Fusing CBM Clos from Clostridium thermocellum and CBM Ch2 from Trichoderma reesei to the chromoproteins had no effect on the chromoprotein color properties. Furthermore, binding assays showed that chromoprotein fusions did not affect binding of CBMs to cellulosic materials. Cotton samples bound with Ultramarine-Clos exhibited visible purple color that faded progressively over time as the samples dried. Applying 10% 8000 polyethylene glycol to cotton samples markedly preserved the color over extended periods. Overall, this work highlights the potential of chromoprotein-CBM fusions for textile dying which could be applied as a color maintenance technology or for reversible coloring of textiles for events or work wear, contributing to sustainable practices and introducing new creative opportunities for the industry.

Asymmetric Ene-Reduction of α,ß-Unsaturated Compounds by F420-Dependent Oxidoreductases A Enzymes from Mycobacterium smegmatis.

The stereoselective reduction of alkenes conjugated to electron-withdrawing groups by ene-reductases has been extensively applied to the commercial preparation of fine chemicals. Although several different enzyme families are known to possess ene-reductase activity, the old yellow enzyme (OYE) family has been the most thoroughly investigated. Recently, it was shown that a subset of ene-reductases belonging to the flavin/deazaflavin oxidoreductase (FDOR) superfamily exhibit enantioselectivity that is generally complementary to that seen in the OYE family. These enzymes belong to one of several FDOR subgroups that use the unusual deazaflavin cofactor F420. Here, we explore several enzymes of the FDOR-A subgroup, characterizing their substrate range and enantioselectivity with 20 different compounds, identifying enzymes (MSMEG_2027 and MSMEG_2850) that could reduce a wide range of compounds stereoselectively. For example, MSMEG_2027 catalyzed the complete conversion of both isomers of citral to (R)-citronellal with 99% ee, while MSMEG_2850 catalyzed complete conversion of ketoisophorone to (S)-levodione with 99% ee. Protein crystallography combined with computational docking has allowed the observed stereoselectivity to be mechanistically rationalized for two enzymes. These findings add further support for the FDOR and OYE families of ene-reductases displaying general stereocomplementarity to each other and highlight their potential value in asymmetric ene-reduction.

Asymmetric Ene-Reduction by F420 -Dependent Oxidoreductases B (FDOR-B) from Mycobacterium smegmatis.

Asymmetric reduction by ene-reductases has received considerable attention in recent decades. While several enzyme families possess ene-reductase activity, the Old Yellow Enzyme (OYE) family has received the most scientific and industrial attention. However, there is a limited substrate range and few stereocomplementary pairs of current ene-reductases, necessitating the development of a complementary class. Flavin/deazaflavin oxidoreductases (FDORs) that use the uncommon cofactor F420 have recently gained attention as ene-reductases for use in biocatalysis due to their stereocomplementarity with OYEs. Although the enzymes of the FDOR-As sub-group have been characterized in this context and reported to catalyse ene-reductions enantioselectively, enzymes from the similarly large, but more diverse, FDOR-B sub-group have not been investigated in this context. In this study, we investigated the activity of eight FDOR-B enzymes distributed across this sub-group, evaluating their specific activity, kinetic properties, and stereoselectivity against α,ß-unsaturated compounds. The stereochemical outcomes of the FDOR-Bs are compared with enzymes of the FDOR-A sub-group and OYE family. Computational modelling and induced-fit docking are used to rationalize the observed catalytic behaviour and proposed a catalytic mechanism.

An enhanced electron transport chain improved astaxanthin production in Phaffia rhodozyma.

Astaxanthin (AX) is a carotenoid pigment with antioxidant properties widely used as a feed supplement. Wild-type strains of Phaffia rhodozyma naturally produce low AX yields, but we increased AX yields 50-fold in previous research using random mutagenesis of P. rhodozyma CBS6938 and fermentation optimization. On that study, genome changes were linked with phenotype, but relevant metabolic changes were not resolved. In this study, the wild-type and the superior P. rhodozyma mutant strains were grown in chemically defined media and instrumented fermenters. Differential kinetic, metabolomics, and transcriptomics data were collected. Our results suggest that carotenoid production was mainly associated with cell growth and had a positive regulation of central carbon metabolism metabolites, amino acids, and fatty acids. In the stationary phase, amino acids associated with the TCA cycle increased, but most of the fatty acids and central carbon metabolism metabolites decreased. TCA cycle metabolites were in abundance and media supplementation of citrate, malate, α-ketoglutarate, succinate, or fumarate increased AX production in the mutant strain. Transcriptomic data correlated with the metabolic and genomic data and found a positive regulation of genes associated with the electron transport chain suggesting this to be the main driver for improved AX production in the mutant strain.

Improving phytase production in Pichia pastoris fermentations through de-repression and methanol induction optimization.

Pichia pastoris (Komagataella phaffii) is a fast-growing methylotrophic yeast with the ability to assimilate several carbon sources such as methanol, glucose, or glycerol. It has been shown to have outstanding secretion capability with a variety of heterologous proteins. In previous studies, we engineered P. pastoris to co-express Escherichia coli AppA phytase and the HAC1 transcriptional activator using a bidirectional promoter. Phytase production was characterized in shake flasks and did not reflect industrial conditions. In the present study, phytase expression was explored and optimized using instrumented fermenters in continuous and fed-batch modes. First, the production of phytase was investigated under glucose de-repression in continuous culture at three dilution factors, 0.5 d-1 , 1 d-1 , and 1.5 d-1 . The fermenter parameters of these cultures were used to inform a kinetic model in batch and fed-batch modes for growth and phytase production. The kinetic model developed aided to design the glucose-feeding profile of a fed-batch culture. Kinetic model simulations under glucose de-repression and fed-batch conditions identified optimal phytase productivity at the specific growth rate of 0.041 h-1 . Validation of the model simulation with experimental data confirmed the feasibility of the model to predict phytase production in our newly engineered strain. Methanol was used only to induce the expression of phytase at high cell densities. Our results showed that high phytase production required two stages, the first stage used glucose under de-repression conditions to generate biomass while expressing phytase, and stage two used methanol to induce phytase expression. The production of phytase was improved 3.5-fold by methanol induction compared to the expression with glucose alone under de-repression conditions to a final phytase activity of 12.65 MU/L. This final volumetric phytase production represented an approximate 36-fold change compared to the flask fermentations. Finally, the phytase protein produced was assayed to confirm its molecular weight, and pH and temperature profiles. This study highlights the importance of optimizing protein production in P. pastoris when using novel promoters and presents a general approach to performing bioprocess optimization in this important production host.

Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris.

Pichia pastoris (Komagataella phaffii) is widely used for industrial production of heterologous proteins due to high secretory capabilities but selection of highly productive engineered strains remains a limiting step. Despite availability of a comprehensive molecular toolbox for construct design and gene integration, there is high clonal variability among transformants due to frequent multi-copy and off-target random integration. Therefore, functional screening of several hundreds of transformant clones is essential to identify the best protein production strains. Screening methods are commonly based on deep-well plate cultures with analysis by immunoblotting or enzyme activity assays of post-induction samples, and each heterologous protein produced may require development of bespoke assays with multiple sample processing steps. In this work, we developed a generic system based on a P. pastoris strain that uses a protein-based biosensor to identify highly productive protein secretion clones from a heterogeneous set of transformants. The biosensor uses a split green fluorescent protein where the large GFP fragment (GFP1-10) is fused to a sequence-specific protease from Tobacco Etch Virus (TEV) and is targeted to the endoplasmic reticulum. Recombinant proteins targeted for secretion are tagged with the small fragment of the split GFP (GFP11). Recombinant protein production can be measured by monitoring GFP fluorescence, which is dependent on interaction between the large and small GFP fragments. The reconstituted GFP is cleaved from the target protein by TEV protease, allowing for secretion of the untagged protein of interest and intracellular retention of the mature GFP. We demonstrate this technology with four recombinant proteins (phytase, laccase, ß-casein and ß-lactoglobulin) and show that the biosensor directly reports protein production levels that correlate with traditional assays. Our results confirm that the split GFP biosensor can be used for facile, generic, and rapid screening of P. pastoris clones to identify those with the highest production levels.

Valorisation of keratin waste: Controlled pretreatment enhances enzymatic production of antioxidant peptides.

Conversion of keratin waste to value-added products not only reduces waste volumes but also creates new revenue streams for the animal production industry. In the present study, combination of alkaline pretreatment of cattle hair with enzymatic hydrolysis was studied to produce keratin hydrolysates with relatively high antioxidant activities. Firstly, the effect of pretreatment conditions at a high solid/liquid mass ratio of 1:2 with different NaOH loadings and temperatures was studied. Increasing NaOH concentration from 1.0% to 2.5% and temperature from room temperature to 110 °C increased hair hydrolysis by keratinase and protein recovery in hydrolysates. Mild pretreatment with 1.5% NaOH at 70 °C for 30 min led to a protein recovery of 30% in the enzymatic hydrolysate. The resulting hydrolysate showed a high antioxidant activity, scavenging 69% of the ABTS radical with a low EC50 of 0.8 mg/mL. Severe pretreatment with 2.5% NaOH at 110 °C for 30 min resulted in a higher protein recovery of 45%, but a lower ABTS radical scavenging activity of 56% and a higher EC50 of 1.3 mg/mL. The reduced antioxidant activity was attributed to the reduced proportion of small peptides (<3 kDa) and the increased extent of amino acid chemical modification. This study demonstrated that controlling alkali pretreatment conditions could lead to the production of enzymatic hydrolysates with higher antioxidant activities for potential value-adding applications. The information generated from this study will aid scale-up and commercialisation of processes with optimised antioxidant peptide production.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris.

BACKGROUND: Pichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains. RESULTS: Potential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter. DISCUSSION: The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

A snapshot of microbial diversity and function in an undisturbed sugarcane bagasse pile.

BACKGROUND: Sugarcane bagasse is a major source of lignocellulosic biomass, yet its economic potential is not fully realised. To add value to bagasse, processing is needed to gain access to the embodied recalcitrant biomaterials. When bagasse is stored in piles in the open for long periods it is colonised by microbes originating from the sugarcane, the soil nearby or spores in the environment. For these microorganisms to proliferate they must digest the bagasse to access carbon for growth. The microbial community in bagasse piles is thus a potential resource for the discovery of useful and novel microbes and industrial enzymes. We used culturing and metabarcoding to understand the diversity of microorganisms found in a uniquely undisturbed bagasse storage pile and screened the cultured organisms for fibre-degrading enzymes. RESULTS: Samples collected from 60 to 80 cm deep in the bagasse pile showed hemicellulose and partial lignin degradation. One hundred and four microbes were cultured from different layers and included a high proportion of oleaginous yeast and biomass-degrading fungi. Overall, 70, 67, 70 and 57% of the microbes showed carboxy-methyl cellulase, xylanase, laccase and peroxidase activity, respectively. These percentages were higher in microbes selectively cultured from deep layers, with all four activities found for 44% of these organisms. Culturing and amplicon sequencing showed that there was less diversity and therefore more selection in the deeper layers, which were dominated by thermophiles and acid tolerant organisms, compared with the top of pile. Amplicon sequencing indicated that novel fungi were present in the pile. CONCLUSIONS: A combination of culture-dependent and independent methods was successful in exploring the diversity in the bagasse pile. The variety of species that was found and that are known for biomass degradation shows that the bagasse pile was a valuable selective environment for the identification of new microbes and enzymes with biotechnological potential. In particular, lignin-modifying activities have not been reported previously for many of the species that were identified, suggesting future studies are warranted.

Yeasts Influence Host Selection and Larval Fitness in Two Frugivorous Carpophilus Beetle Species.

We explored how gut-associated yeasts influence olfactory behaviour and resource use in two pest species of Carpophilus beetle that co-exist in Australian stone fruits. Molecular analysis of yeasts isolated from the gut of C. davidsoni (prefers ripe fruits) and C. hemipterus (prefers overripe and rotting fruits) revealed that the predominant species were Pichia kluyveri and Hanseniaspora guilliermondii. In olfactory attraction and oviposition trials, adult beetles preferred H. guilliermondii over P. kluyveri, and follow up GC-MS analysis revealed unambiguous differences between the odour profiles of these yeasts. In contrast to behavioural trials, larval feeding assays showed that fruit substrates inoculated with P. kluyveri yielded significantly faster development times, higher pupal mass, and a greater number of adult beetles, compared to H. guilliermondii - in other words, the lesser preferred yeast (by foraging adults) was more suitable for larval survival. Moreover, whilst larvae of both species survived to adulthood when fed solely on P. kluyveri (i.e. without a fruit substrate), only larvae of C. davidsoni could develop on H. guilliermondii; and only C. davidsoni reached adulthood feeding on a yeast-free fruit substrate. We discuss how these findings may relate to adaptations towards early colonising of fruits by C. davidsoni, enabling differences in resource use and potentially resource partitioning in the two beetles. More broadly, consideration of microbial interactions might help develop host selection theory. Our results could pave the way to more powerful attractants to mass-trap and monitor Carpophilus pests in fruit orchards.

Enzymatic removal of dags from livestock: an agricultural application of enzyme technology.

The effective removal of dags (manure balls) from cattle, sheep and goats is a significant issue for the livestock industry. Dags are hard recalcitrant deposits composed of materials, such as faeces, hair, soil, urine, feed and straw, and attach to the animal through the hair rather than the skin. Dags build up during wet periods, especially on long haired breeds, and can weigh up to 40 kg per animal for cattle. Dag removal prior to slaughter reduces the risk of microbial meat contamination and damage to the hide during leather processing. Existing removal methods include hair trimming or extensive hose washing that can result in stress to the animal and increased costs. An alternative solution is the application of enzyme formulations that target specific components of the dag so they are more easily removed by washing. Enzymes are already used in other cleaning applications and are proven for the breakdown of materials such as lignocellulose, protein or starch that are found in dags. This mini-review discusses the challenges of current dag removal methods and the state of the art and feasibility of applying enzyme formulations for the effective removal of dags. Although enzyme formulations are yet to be tested in large-scale cattle trials and questions remain regarding how they would be cost-effectively applied to live animals, the results at laboratory scale suggest further research is warranted. Overall, enzymes present an environmentally friendly solution to the high costs and animal welfare issues of current dag removal methods through significant reductions in cleaning time and water use. KEY POINTS: • Dag formation on livestock is a major issue for industry and for animal welfare. • Current methods are costly and challenging for operators and the animal. • Enzymes can degrade dag components to aid release with keratinases showing promise. • Dag removal needs to be field tested, and positive business cases must be generated.

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris.

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.

An improved and general streamlined phylogenetic protocol applied to the fatty acid desaturase family.

Numerous tools to generate phylogenetic estimates are available, but there is no single protocol that will produce an accurate phylogenetic tree for any dataset. Here, we investigated some of those tools, paying particular attention to different alignment algorithms, in order to produce a phylogeny for the integral membrane fatty acid desaturase (FAD) family. Herein, we report a novel streamlined protocol which utilises peptide pattern recognition (PPR). This protocol can theoretically be applied universally to generate accurate multiple sequence alignments and improve downstream phylogenetic analyses. Applied to the desaturases, the protocol generated the first detailed phylogenetic estimates for the family since 2003, which suggested they may have evolved from three functionally distinct desaturases and further, that desaturases evolved first in cyanobacteria. In addition to the phylogenetic outputs, we mapped PPR sequence motifs onto an X-ray protein structure to provide insights into biochemical function and demonstrate the complementarity of PPR and phylogenetics.

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.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

Non-covalent binding tags for batch and flow biocatalysis.

Enzyme immobilization offers considerable advantage for biocatalysis in batch and continuous flow reactions. However, many currently available immobilization methods require that the surface of the carrier is chemically modified to allow site specific interactions with their cognate enzymes, which requires specific processing steps and incurs associated costs. Two carriers (cellulose and silica) were investigated here, initially using fluorescent proteins as models to study binding, followed by assessment of industrially relevant enzyme performance (transaminases and an imine reductase/glucose oxidoreductase fusion). Two previously described binding tags, the 17 amino acid long silica-binding peptide from the Bacillus cereus CotB protein and the cellulose binding domain from the Clostridium thermocellum, were fused to a range of proteins without impairing their heterologous expression. When fused to a fluorescent protein both tags conferred high avidity specific binding with their respective carriers (low nanomolar Kd values). The CotB peptide (CotB1p) induced protein aggregation in the transaminase and imine reductase/glucose oxidoreductase fusions when incubated with the silica carrier. The Clostridium thermocellum cellulose binding domain (CBDclos) allowed immobilization of all the proteins tested, but immobilization led to loss of enzymatic activity in the transaminases (< 2-fold) and imine reductase/glucose oxidoreductase fusion (> 80%). A transaminase-CBDclos fusion was then successfully used to demonstrate the application of the binding tag in repetitive batch and a continuous-flow reactor.

Oxidative stress induced by plasma-activated water stimulates astaxanthin production in Phaffia rhodozyma.

Astaxanthin is used extensively in the nutraceutical, aquaculture, and cosmetic industries. The current market necessitates higher astaxanthin production from Phaffia rhodozyma (P. rhodozyma) due to its higher cost compared to chemical synthesis. In this study, a bubble discharge reactor was developed to generate plasma-activated water (PAW) to produce PAW-made yeast malt (YM) medium. Due to oxidative stress induced by PAW, strains cultured in 15 and 30 min-treated PAW-made medium produced 7.9 ± 1.2 % and 12.6 ± 1.4 % more carotenoids with 15.5 ± 3.3 % and 22.1 ± 1.3 % more astaxanthin, respectively. Reactive oxygen species (ROS) assay results showed that ROS generated by plasma-water interactions elevated intracellular ROS levels. Proteomic analysis revealed increased expression of proteins involved in the cellular response to oxidative stress as well as carotenoid biosynthesis, both of which contribute to higher yields of astaxanthin. Overall, this study supports the potential of PAW to increase astaxanthin yields for industrial-scale production.

A survey of the 2010 quartz crystal microbalance literature.

In 2010 there has again been an increase in the number of papers published involving piezoelectric acoustic sensors, or quartz crystal microbalances (QCM), when compared to the last period reviewed 2006-2009. The average number of QCM publications per annum was 124 in the period 2001-2005, 223 in the period 2006-9, and 273 in 2010. There are trends towards increasing use of QCM in the study of protein adsorption to surfaces (93% increase), homeostasis (67% increase), protein-protein interactions (40% increase), and carbohydrates (43% increase). New commercial systems have been released that are driving the uptake of the technology for characterisation of binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights theoretical and practical aspects of the principals that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells, and membrane interfaces.

Towards commercial levels of astaxanthin production in Phaffia rhodozyma.

Astaxanthin (AX) is a potent antioxidant with increasing biotechnological and commercial potential as a feed supplement, and gives salmonids and crustaceans their attractive characteristic pink color. The red yeast Phaffia rhodozyma naturally produces AX as its main fermentation product but wild-type strains and those previously generated through classical random mutagenesis produce low yields of AX. Existing strains do not meet commercial economic requirements, fundamentally due to a lack of understanding of the underlying mechanisms and genotype-phenotype associations regarding AX production in P. rhodozyma. In the present study, screening of P. rhodozyma CBS 6938 mutant strains generated through chemical and ultra violet radiation mutagenesis delivered increased AX production yields that were then maximized using culture media optimization and fed-batch culture kinetic modeling. The whole genomes of the wild-type and eight increased production strains were sequenced to identify genomic changes. The selected strains produced 50-fold more AX than the wild-type strain with a total biomass of around 100 gDCW/L and a carotenoid production of 1 g/L. Genomic variant analyses found 368 conserved mutations across the selected strains with important mutations found in protein coding regions associated with regulators and catalysts of AX precursors in the mevalonate pathway, the electron transport chain, oxidative stress mechanisms, and carotenogenesis.