Alcohol Oxidase Research Articles

Production of a high purity, C-tagged hepatitis B surface antigen fusion protein VLP vaccine for malaria expressed in Pichia pastoris under cGMP conditions.

Virus‐like particles (VLPs) induce strong humoral and cellular responses and have formed the basis of some currently licensed vaccines. Here, we present the method used for the production of R21, a VLP‐based anti‐sporozoite malaria vaccine, under current Clinical Good Manufacturing Practice regulations (cGMP). Previous preclinical studies in BALB/c mice showed that R21 produced almost complete protection against sporozoite challenge with transgenic Plasmodium berghei parasites. Here, we have modified the preclinical production process to enable the production of sufficient quantities of highly pure, clinical‐grade material for use in human clinical trials. The R21 construct was re‐engineered to include a C‐tag to allow affinity‐based separation from the major contaminant alcohol oxidase 1 (AOX 1, ~74 kDa). To our knowledge, this is the first use of C‐tag technology to purify a VLP vaccine candidate for use in human clinical trials. The R21 vaccine has shown high‐level efficacy in an African Phase IIb trial, and multiple clinical trials are underway to assess the safety and efficacy of the vaccine. Our findings support the future use of C‐tag platform technologies to enable cGMP‐compliant biomanufacturing of high purity yeast‐expressed VLP‐based vaccines for early phase clinical trials when clinical grade material is required in smaller quantities in a quick time frame.

Highly Porous 3D Gold Enhances Sensitivity of Amperometric Biosensors Based on Oxidases and CuCe Nanoparticles.

Metallic nanoparticles potentially have wide practical applications in various fields of science and industry. In biosensorics, they usually act as catalysts or nanozymes (NZs) and as mediators of electron transfer. We describe here the development of amperometric biosensors (ABSs) based on purified oxidases, synthesized nanoparticles of CuCe (nCuCe), and micro/nanoporous gold (pAu), which were electro-deposited on a graphite electrode (GE). As an effective peroxidase (PO)-like NZ, nCuCe was used here as a hydrogen-peroxide-sensing platform in ABSs that were based on glucose oxidase, alcohol oxidase, methylamine oxidase, and L-arginine oxidase. At the same time, nCuCe is an electroactive mediator and has been used in laccase-based ABSs. As a result, the ABSs we constructed and characterized were based on glucose, methanol, methyl amine, L-arginine, and catechol, respectively. The developed nCuCe-based ABSs exhibited improved analytical characteristics in comparison with the corresponding PO-based ABSs. Additionally, the presence of pAu, with its extremely advanced chemo-sensing surface layer, was shown to significantly increase the sensitivities of all constructed ABSs. As an example, the bioelectrodes containing laccase/GE, laccase/nCuCe/GE, and laccase/nCuCe/pAu/GE exhibited sensitivities to catechol at 2300, 5055, and 9280 A·M−1·m−2, respectively. We demonstrate here that pAu is an effective carrier of electroactive nanomaterials coupled with oxidases, which may be promising in biosensors.

Abstract 1892: Expression of bacterial heme-sequestering proteins (HeSPs) in Pichia pastoris for effective treatment of lung cancer

Abstract Heme is a central biosynthetic molecule with important implications in diverse molecular and cellular processes, including oxygen utilization and metabolism. Heme carries out a vital role in mitochondrial energy generation through oxidative phosphorylation (OXPHOS) by acting as a cofactor or prosthetic group for complexes II, II, and IV in the electron transport chain. Lung cancer is the leading cause of cancer related death in the US. Our lab has previously shown that non-small cell lung cancer (NSCLC) cells exhibit an elevated heme flux characterized by elevated heme synthesis and import. This increased heme flux correlates with an enhanced oxygen consumption and ATP production. To target this elevated heme flux, we have engineered recombinant bacterial HasA hemophores as heme-sequestering proteins (HeSPs). HeSPs bind to and sequester heme with an extremely high (pM) affinity, thereby disrupting this elevated heme flux. HeSPs display potent anti-tumor activity in mouse models of NSCLC and triple-negative breast cancer (TNBC). Here, we utilized the Pichia pastoris expression platform to achieve high-yield expression. P. pastoris is a methylotrophic yeast that has minimal endogenous protein secretion and potentially high-yields for heterologous protein expression, due to a tightly regulated alcohol oxidase gene (AOX1). The P. pastoris expression platform is generally easier to handle and requires simpler fermentation conditions than mammalian cell cultures, making it an ideal candidate for expression of recombinant bacterial proteins without endotoxin contamination.We utilized a secretion signal to direct secretion of HeSP2 into the culture medium to allow for easier protein purification. Purification was performed through ion exchange chromatography. We utilized native PAGE and heme-binding spectrum to verify that these yeast-expressed HeSPs adopt a similar conformation to those expressed in bacteria and maintain their heme-binding activity. HeSPs expressed through this P. pastoris expression system demonstrated remarkable anti-cancer efficacy in proliferation assays on NSCLC cells and SCLC (small cell lung cancer) cells. Experiments are underway to test their anti-tumor efficacy in mouse models. These experiments may suggest a novel approach to generate effective anti-cancer agents. Citation Format: Narges Salamat, Adnin Ashrafi, Tianyuan Wang, Zakia Akter, Hanling Gong, Li Zhang. Expression of bacterial heme-sequestering proteins (HeSPs) in Pichia pastoris for effective treatment of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1892.

Heterologous protein expression enhancement of Komagataella phaffii by ammonium formate induction based on transcriptomic analysis

Heterologous protein expression by Komagataella phaffii (K. phaffii) attracts attention of researchers and engineers for high level of heterologous protein, strict pattern of alcohol oxidase 1 promoter (PAOX1), and high cell density fermentation. It is urgent to increase level of heterologous protein by K. phaffii using formate, the only substitute of methanol as inducer, because methanol is toxic and explosive, especially, in industrial scale. A recombinant K. phaffii expressing green fluorescent protein (GFP) was constructed and sequenced by RNA-seq technology based on methanol and ammonium formate (AF) optimization. The transcriptomes were analyzed using COG, GO, KEGG databases with the result of 1172 identical up-regulated genes at both conditions of methanol and AF induction. The transcription levels of 173 genes and 144 genes at AF induction were higher and lower significantly than that of methanol induction respectively within 1172 identical genes. Low energy generation pathway was considered as the reason of low GFP expression level at AF induction based on analysis of 173 and 144 genes. This result was confirmed by nutrients addition obtaining 30.6 % increment of GFP intensity at 2 g/L ATP addition. This result provided a bright future of high-level heterologous protein production by K. phaffii of AF induction.

Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris

BackgroundPichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH).ResultsThe transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells.ConclusionsThis is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris. The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA–protein crosslinking.

Silk-fibroin film as enzyme stabilizing material and optical signal transducer for developing alcohol oxidase-based μPAD methanol biosensor

The commercial success of paper-based microfluidic biosensor devices with enzyme as recognition elements may be achieved greatly by improving the enzyme stability, averting the coffee-ring effect on the detection zone, and making it a stand-alone system. We have addressed these issues here by introducing silk-fibroin (SF) film for immobilizing enzymes and chromogenic reagent (ABTS), and wave-design microfluidic channels in the chromatographic paper for developing a hybrid microfluidic paper-based analytical device (μPAD), for methanol detection through peroxidase reaction. The activity of alcohol oxidase (AOx), used as a biorecognition element, could be wholly retained in a non-dissolvable SF film (pore size <3.5 μm) until 40 days of storage at room temperature (RT) by reducing oxygen permeability to the film. Similarly, ABTS in a dissolvable SF film was protected from air-oxidation even up to two months of storage at RT. The detection approach exploited purple color as a high contrast response signal generated from ABTS di-cation formed from the reaction of SF protein with the ABTS radicals generated from the substrate-dependent peroxidase reaction. The wave-designed microfluidic channels could significantly reduce the coffee ring effect in the detection film. The bi-enzyme SF films prepared by co-immobilizing AOx with peroxidase (HRP) offered high sensitivity to the device with a detection limit (LoD) of 1 ± 0.05 mM and a dynamic range of 1 mM–2 M for methanol. The stand-alone hybrid μPAD biosensor for methanol has a high potential for commercial applications.

Vitreoscilla hemoglobin enhances the catalytic performance of industrial oxidases in vitro.

Oxidases are a group of oxidoreductases and need molecular oxygen in the catalytic process. Vitreoscilla hemoglobin (VHb) can improve the growth and productivity of host cells under hypoxic conditions, rendering it attractive for industrial application. In this work, we demonstrated the addition of immobilized VHb increased the catalytic activity of immobilized D-amino acid oxidase of Trigonopsis variabilis by two-fold when catalyzing cephalosporin C under oxygen-limited conditions. A similar increase of activities was observed in glucose oxidase, alcohol oxidase, and p-hydroxymandelate synthase by adding free VHb or immobilized VHb under hypoxic conditions. When L-glutamate oxidase was used to catalyze L-glutamate to produce α-ketoglutarate, the yield increased from 80.6 to 96.9% by fusing VHb with L-glutamate oxidase. Results demonstrated that the addition of free VHb, immobilized VHb, or fused VHb could increase the catalytic efficiency of oxidases, which was considered by increasing the concentration of the microenvironmental oxygen. Thus, VHb may become a potential additive agent to promote the efficiency of oxidases on industrial scale . KEY POINTS: • First time confirmation of facilitation of VHb on several industrial oxidases in vitro • VHb functions under hypoxic conditions rather than oxygen-enriched conditions • VHb functions in vitro in the form of free, immobilized protein and fusion enzyme.

Breath alcohol sensor based on hydrogel-gated graphene field-effect transistor

The inspection of drunk driving has become an effective measure to reduce the occurrence of traffic accidents. In this work, we constructed a breath alcohol biosensor based on a hydrogel-gated graphene field-effect transistor (HGGT) with chlorella derived layered carbon nanosheets (CNs) and alcohol oxidase (AOx) embedded in the hydrogel. The sensing mechanism of the AOx/CNs functionalized sensor lies in the oxidation reaction of alcohol by AOx and the electrocatalytic oxidation reaction of the generated H2O2. The HGGT based alcohol sensor exhibited an excellent sensitivity with a very low detection limit down to 1 μM (i.e. 0.046 ppm), and has been successfully applied to breath alcohol test after drinking. Compared with normal solution-gated graphene transistors, employment of hydrogel as a source of electrolytes greatly enhances the portability of the sensor, and facilitates functionalization with enzymes and nanomaterials. Due to the advantages of real-time, high portability and accuracy of the functionalized HGGT sensor, it demonstrates a promising platform for constructing biosensors for many other analytes.

Percutaneous implantation of ethanol fueled catalytic hydrogel suppresses tumor growth by triggering ferroptosis

Percutaneous ethanol injection therapy (PEIT) is a nonthermal locoregional treatment modality widely used in the treatment of solid tumors such as small hepatocellular carcinoma (HCC). Restricted by the inhomogeneous diffusion and unwanted leakage of injected ethanol, PEIT that usually requires multiple injections is often associated with a high local recurrence rate and potential severe complications. Herein, we propose an alternative locoregional treatment strategy to effectively ablate tumors by utilizing the reactive oxygen species generated from the percutaneously implanted catalytic couple fueled by orally administrated ethanol. It is shown that the catalytic couple of alcohol oxidase and iron catalyst hemin conjugated chitosan can sequentially convert ethanol to cytotoxic hydrogen peroxides and then hydroxyl radials, thereby result in efficient cancer cell killing by triggering ferroptosis. Upon being fixed inside tumors with in-situ formed alginate hydrogels, such catalytic couple together with multiple ethanol gavages shows great inhibitory effect on murine H22 liver and 4T1 breast tumors, comparable to those treated by PEIT. The efficient therapeutic potency of the combination treatment is further confirmed in a rat orthotopic N1S1 HCC model. This work highlights that our ‘alcohol drinking’ anti-tumor strategy is of great effectiveness, operation convenience, and high safety.

Oxidation of 5-hydroxymethylfurfural with a novel aryl alcohol oxidase from Mycobacterium sp. MS1601.

SummaryBio‐based 5‐hydroxymethylfurfural (HMF) serves as an important platform for several chemicals, among which 2,5‐furan dicarboxylic acid (FDCA) has attracted considerable interest as a monomer for the production of polyethylene furanoate (PEF), a potential alternative for fossil‐based polyethylene terephthalate (PET). This study is based on the HMF oxidizing activity shown by Mycobacterium sp. MS 1601 cells and investigation of the enzyme catalysing the oxidation. The Mycobacterium whole cells oxidized the HMF to FDCA (60% yield) and hydroxymethyl furan carboxylic acid (HMFCA). A gene encoding a novel bacterial aryl alcohol oxidase, hereinafter MycspAAO, was identified in the genome and was cloned and expressed in Escherichia coli Bl21 (DE3). The purified MycspAAO displayed activity against several alcohols and aldehydes; 3,5 dimethoxy benzyl alcohol (veratryl alcohol) was the best substrate among those tested followed by HMF. 5‐Hydroxymethylfurfural was converted to 5‐formyl‐2‐furoic acid (FFCA) via diformyl furan (DFF) with optimal activity at pH 8 and 30–40°C. FDCA formation was observed during long reaction time with low HMF concentration. Mutagenesis of several amino acids shaping the active site and evaluation of the variants showed Y444F to have around 3‐fold higher kcat/Km and ~1.7‐fold lower Km with HMF.

High-Yield Synthesis of Enantiopure 1,2-Amino Alcohols from l-Phenylalanine via Linear and Divergent Enzymatic Cascades.

Enantiomerically pure 1,2-amino alcohols are important compounds due to their biological activities and wide applications in chemical synthesis. In this work, we present two multienzyme pathways for the conversion of l-phenylalanine into either 2-phenylglycinol or phenylethanolamine in the enantiomerically pure form. Both pathways start with the two-pot sequential four-step conversion of l-phenylalanine into styrene via subsequent deamination, decarboxylation, enantioselective epoxidation, and enantioselective hydrolysis. For instance, after optimization, the multienzyme process could convert 507 mg of l-phenylalanine into (R)-1-phenyl-1,2-diol in an overall isolated yield of 75% and >99% ee. The opposite enantiomer, (S)-1-phenyl-1,2-diol, was also obtained in a 70% yield and 98–99% ee following the same approach. At this stage, two divergent routes were developed to convert the chiral diols into either 2-phenylglycinol or phenylethanolamine. The former route consisted of a one-pot concurrent interconnected two-step cascade in which the diol intermediate was oxidized to 2-hydroxy-acetophenone by an alcohol dehydrogenase and then aminated by a transaminase to give enantiomerically pure 2-phenylglycinol. Notably, the addition of an alanine dehydrogenase enabled the connection of the two steps and made the overall process redox-self-sufficient. Thus, (S)-phenylglycinol was isolated in an 81% yield and >99.4% ee starting from ca. 100 mg of the diol intermediate. The second route consisted of a one-pot concurrent two-step cascade in which the oxidative and reductive steps were not interconnected. In this case, the diol intermediate was oxidized to either (S)- or (R)-2-hydroxy-2-phenylacetaldehyde by an alcohol oxidase and then aminated by an amine dehydrogenase to give the enantiomerically pure phenylethanolamine. The addition of a formate dehydrogenase and sodium formate was required to provide the reducing equivalents for the reductive amination step. Thus, (R)-phenylethanolamine was isolated in a 92% yield and >99.9% ee starting from ca. 100 mg of the diol intermediate. In summary, l-phenylalanine was converted into enantiomerically pure 2-phenylglycinol and phenylethanolamine in overall yields of 61% and 69%, respectively. This work exemplifies how linear and divergent enzyme cascades can enable the synthesis of high-value chiral molecules such as amino alcohols from a renewable material such as l-phenylalanine with high atom economy and improved sustainability.

Glucose and Ethanol Detection with an Affinity-Switchable Lateral Flow Assay.

The lateral flow assay (LFA) is one of the most successful analytical platforms for rapid on-site detection of target substances. This type of assay has been used in many rapid diagnoses, for example, pregnancy tests and infectious disease prevention. However, applications of LFAs for very small molecules remain a demanding challenge due to the problem of obtaining the corresponding binding partners to form sandwich complexes. In this paper, we report an affinity-switchable (AS) LFA (ASLFA) for the rapid and selective detection of hydrogen peroxide (H2O2), glucose, and ethanol in blood serum and urine samples. Unlike classical LFAs, which rely on the "always on" interaction between the antigen and the antibody, the working principle of ASLFA is based on the gold nanoparticle-conjugated AS biotin probe Au@H2O2-ASB, which can be activated by H2O2 for binding with the streptavidin (SA) protein. In the presence of glucose and ethanol, glucose oxidase and alcohol oxidase can react with the substrate to generate H2O2 and thereby activate Au@H2O2-ASB for binding with SA. Therefore, this ASLFA approach can be an alternative for classical glucose and ethanol detection methods in a wide variety of samples, where simple and rapid on-site detection is essential.

Mycoremediation of lignocellulosic biorefinery sludge: A reinvigorating approach for organic contaminants remediation with simultaneous production of lignocellulolytic enzyme cocktail

This research work aims to valorize lignocellulosic biorefinery sludge with genetically engineered Trichoderma atroviride for simultaneous removal of organic contaminants, fermentation inhibitors, and lignocellulolytic enzyme cocktail production. Upon analysis, three phenolic compounds (42.6 ± 3.6 μg/g), two polycyclic aromatic hydrocarbons (0.42 ± 0.06 μg/g) and five fermentation inhibitors (2.5 ± 0.3 mg/g) were detected in the sludge. Bioaugmentation of sludge with 72 h-old T. atroviride (5%) results in the production of cellulase (21 U/g), xylanase (84 U/g), laccase (20 U/g), lignin peroxidase (14 U/g) and aryl alcohol oxidase (116 U/g), along with the concomitant removal of organic contaminants (phenol, 2, 4-dinitrophenol, pentchlorophenol, phenanthrene, benzo(a)pyrene) and fermentation inhibitors (furfural, 5-hydroxymethylfurfural, levulinic acid, ferulic acid, and catechol). Subsequently, the enrichment of sludge with nutrients and rhamnolipids enhanced the enzyme production by 5–6-fold and resulted in the removal of 85–95% of organic contaminants and fermentation inhibitors, which constitutes an eco-friendly process.

A novel recombinant chimeric bio-adhesive protein consisting of mussel foot protein 3, 5, gas vesicle protein A, and CsgA curli protein expressed in Pichia pastoris

Despite various efforts to produce potent recombinant bio-adhesive proteins for medical purposes, efficient production of a safe and feasible bio-glue is not yet a commercial reality due to the weak properties or low expression levels. Here, a feasible expression system has been developed to produce strong recombinant fusion bioinspired protein using mussel foot protein 3 and 5 (Mfps) along with gas vesicle protein A (GvpA) of Anabaena flos-aquae, and a curli protein CsgA from E. coli, expressed under the control of alcohol oxidase (AOX1) promoter for high-level production in yeast P. pastoris using pPICZα vector. Purified chimeric proteins were first evaluated using western blotting, and their remaining dihydroxyphenylalanine (DOPA) was measured in the modified proteins by NBT assay. We further elucidated the mechanistic properties of obtained adhesive protein assembly in various pH levels based on its different subunits using atomic force microscopy (AFM) when adsorbed onto the mica surface. We found that both combinational structural features of subunits and post-translational changes during expression in yeast host have led to potent adherence due to higher DOPA residues specially in acidic condition and tetrad complex which is higher than that of earlier reports in prokaryotic systems. We believe that our obtained chimeric protein resulted from the fusion of GvpA and CsgA proteins with DOPA-containing Mfp proteins, expressed in the methylotrophic yeast, P. pastoris, not only presents a candidate for future biomedical applications but also provides novel biological clues used for high-performance bioinspired biomaterial designation.Graphical

Monitoring of alcohol-based hand rubs in SARS-CoV-2 prevention by HS-GC/MS and electrochemical biosensor: A survey of commercial samples

Alcohol-based hand rubs (ABHRs) have found large diffusion during the Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2, thus becoming the most widespread means for hand hygiene. Whereby, it is fundamental to assess the alignment of commercial ABHRs to the indications provided by the principal health agencies regarding alcohol content and possible impurities. In this work, a novel improvement of previous existent methods for the determination of alcohol content in such products was reported. In particular, two alternative sensitive and reproducible methods, such as an electrochemical screen-printed based enzymatic (alcohol oxidase) biosensor and a Headspace Gas Chromatography coupled with Mass Spectrometry (HS-GC/MS) were proposed. The electrochemical device represents a rapid, low-cost and accurate fraud screening method for alcohol-based hand rubs. The second technique confirms, identifies and simultaneously determines ethyl alcohol, isopropyl alcohol, n-propyl alcohol and methyl alcohol, improving their extraction through acidification in the sample pre-treatment step. The developed specific HS-GC/MS method was in-house validated according to ISO/IEC 17025 requirements. Analytical parameters such as limit of detection (LoD 0.13%v/v - 0.17%v/v), limit of quantification (LoQ 0.44% v/v - 0.57% v/v), inter-day repeatability (RSDR 2.1–10.7%) and recovery (80–110%) were assessed. The relative expanded uncertainties range (between 0.1%v/v and 3.4%v/v) for all the analytes were evaluated. Results obtained using the different analytical approaches were compared and indicated that the two data sets were comparable (median; HS-GC/MS, 56%v/v; electrochemical biosensor, 62%v/v) and were not statistically different (one-way ANOVA test; p = 0.062). In addition, a good correlation (95%) was found. This study noticed that only 39% of the tested hand sanitiser products had the recommended average alcohol content, thus highlighting the need for analytical controls on this type of products.

Screening methods for enzyme-mediated alcohol oxidation

Alcohol oxidation for the generation of carbonyl groups, is an essential reaction for the preparation of fine chemicals. Although a number of chemical procedures have been reported, biocatalysis is a promising alternative for more sustainable and selective processes. To speed up the discovery of novel (bio)catalysts for industrial applications, efficient screening approaches need to be established. Here, we report on an enzyme-mediated alcohol oxidation screening platform to rapidly detect the activities and selectivities of three classes of biocatalysts; ketoreductases (KREDs), alcohol oxidases (AlcOXs) and laccase-mediator systems (LMSs) with diverse substrates.

Influence of codon optimization, promoter, and strain selection on the heterologous production of a β-fructofuranosidase from Aspergillus fijiensis ATCC 20611 in Pichia pastoris.

Fructooligosaccharides (FOS) are compounds possessing various health properties and are added to functional foods as prebiotics. The commercial production of FOS is done through the enzymatic transfructolysation of sucrose by β-fructofuranosidases which is found in various organisms of which Aureobasidium pullulans and Aspergillus niger are the most well known. This study overexpressed two differently codon-optimized variations of the Aspergillus fijiensis β-fructofuranosidase-encoding gene (fopA) under the transcriptional control of either the alcohol oxidase (AOX1) or glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters. When cultivated in shake flasks, the two codon-optimized variants displayed similar volumetric enzyme activities when expressed under control of the same promoter with the GAP strains producing 11.7 U/ml and 12.7 U/ml, respectively, and the AOX1 strains 95.8 U/ml and 98.6 U/ml, respectively. However, the highest production levels were achieved for both codon-optimized genes when expressed under control of the AOX1 promoter. The AOX1 promoter was superior to the GAP promoter inbioreactor cultivationsfor both codon-optimized genes with 13,702 U/ml and 2718 U/ml for the AOX1 promoter for ATUM and GeneArt®, respectively, and 6057 U/ml and 1790 U/ml for the GAP promoter for ATUM and GeneArt®, respectively. The ATUM-optimized gene produced higher enzyme activities when compared to the one from GeneArt®, under the control of both promoters.

Methanol oxidase from Hansenula polymorpha shows activity in peroxisome-deficient Pichia pastoris

Pichia pastoris can upgrade the methanol into various of high-valued biochemical products. This biosynthetic method was achieved mostly based on inherent methanol metabolism pathway of yeast. The alcohol oxidase (AOX) from Pichia pastoris plays a crucial role in methanol metabolism pathway. However, when the cells are exposed to excess methanol, the disruption of the peroxisome and inhibition of AOX enzyme activity happens. In our study，the strain Δpex3 derived from P. pastoris GS115 was constructed as initial genetic engineering strain to simulate the damage of the peroxisome which was often happened under high concentrations of methanol. No AOX activity was detected in Δpex3 with peroxisome defected. Subsequently, the methanol oxidase (MOX) from Hansenula polymorpha was employed to make up for activity loss of AOX. Introduction of MOX in the peroxisome-deficient strain Δpex3, thus new recombinant strain was obtained which named as Δpex3-MOX. Δpex3-MOX showed certain activity of alcohol oxidase, and the harvest biomass was higher compared to Δpex3. The ability of utilizing and toleration higher methanol was partially improved when methanol oxidase (MOX) from H. polymorpha was introduced compared to cells with peroxisome defected.

Overview of Mycelial Fungi - Lignin Destructors

In this work, the following genuses of mycelial fungi, capable of producing ligninolytic enzymes of various actions, were considered:Penicillium, Aspergillus, Fusariumand Altermaria. Fungi of the genus Aspergilluswere capable of producing laccase, manganese peroxidase and lignin peroxidase in the medium. Penicillium mostly produced laccase. Fusariumproduced laccase, aryl alcohol oxidase, manganesedependent peroxidase, manganese-independent peroxidase and lignin peroxidase. Alternariaproduced laccase, lignin peroxidase and manganese peroxidase. The results demonstrated the possibility of using specific substrates in the study of enzyme activity, as well as the influence of some factors introduced into the medium on the synthesis of enzymes. The auxiliary influence of these fungi on the synthesis of ligninolytic enzymes in symbiosis with otherswas considered.&#x0D; Keywords: mycelial fungi, ligninolytic enzymes, Penicillium, Aspergillus, Fusarium, Altermaria

Tunable Production of (R)- or (S)-Citronellal from Geraniol via a Bienzymatic Cascade Using a Copper Radical Alcohol Oxidase and Old Yellow Enzyme.

Biocatalytic pathways for the synthesis of (−)-menthol, the most sold flavor worldwide, are highly sought-after. To access the key intermediate (R)-citronellal used in current major industrial production routes, we established a one-pot bienzymatic cascade from inexpensive geraniol, overcoming the problematic biocatalytic reduction of the mixture of (E/Z)-isomers in citral by harnessing a copper radical oxidase (CgrAlcOx) and an old yellow enzyme (OYE). The cascade using OYE2 delivered 95.1% conversion to (R)-citronellal with 95.9% ee, a 62 mg scale-up affording high yield and similar optical purity. An alternative OYE, GluER, gave (S)-citronellal from geraniol with 95.3% conversion and 99.2% ee.