Development of a Combination Fermentation Strategy to Simultaneously Increase Biomass and Enzyme Activity of D-amino Acid Oxidase Expressed in Escherichia coli.

D-amino acid oxidase (DAAO) is widely used in the industrial preparation of L-amino acids, and cultivating Escherichia coli (E. coli) expressing DAAO for the biosynthesis of L-phosphinothricin (L-PPT) is very attractive. At present, the biomass production of DAAO by fermentation is still limited in large-scale industrial applications because the expression of DAAO during the fermentation process inhibits the growth of host cells, which limits higher cell density. In this study, the factors that inhibit the growth of bacterial cells during a 5-L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous D-alanine (D-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation. Under optimal conditions, the DO-STAT feeding mode with DO controlled at 30% ± 5% and the addition of 27.5 g/L lactose mixed with 2 g/L D-Ala during induction at 28 °C resulted in the production of 26.03 g dry cell weight (DCW)/L biomass and 390.0 U/g DCW specific activity of DAAO; an increase of 78% and 84%, respectively, compared with the initial fermentation conditions. The fermentation strategy was successfully scale-up to a 5000-L fermenter.

Overexpression of D-amino acid oxidase prevents retinal neurovascular pathologies in diabetic rats.

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by retinal neurodegeneration and retinal vascular abnormalities, affecting one third of diabetic patients with disease duration of more than 10 years. Accumulated evidence suggests that serine racemase (SR) and D-serine are correlated with the pathogenesis of diabetic retinopathy and the deletion of the Srr gene reverses neurovascular pathologies in diabetic mice. Since D-serine content is balanced by SR synthesis and D-amino acid oxidase (DAAO) degradation, we examined the roles of DAAO in diabetic retinopathy and further explored relevant therapy. METHODS: Rats were used as a model of diabetes by i.p. injection of streptozotocin at the age of 2 months and blood glucose was monitored with a glucometer. Quantitative real-time PCR was used to examine Dao mRNA and western blotting to examine targeted proteins in the retinas. Bisulphite sequencing was used to examine the methylation of Dao mRNA promoter in the retinas. Intravitreal injection of DAAO-expressing adenovirus (AAV8-DAAO) was conducted one week before streptozotocin administration. Brain specific homeobox/POU domain protein 3a (Brn3a) immunofluorescence was conducted to indicate retinal ganglion cells at 3 months after virus injection. The permeability of the blood-retinal barrier was examined by Evans blue leakage from retinal capillaries. Periodic acid-Schiff staining and haematoxylin counterstaining were used to indicate retinal vasculature, which was further examined with double immunostaining at 7 months after virus injection. RESULTS: At the age of 12 months, DAAO mRNA and protein levels in retinas from diabetic animals were reduced to 66.2% and 70.4% of those from normal (control) animals, respectively. The Dao proximal promoter contained higher levels of methylation in diabetic than in normal retinas. Consistent with the observation, DNA methyltransferase 1 was increased in diabetic retinas. Injection of DAAO-expressing virus completely prevented the loss of retinal ganglion cells and the disruption of blood-retinal barrier in diabetic rats. Diabetic retinas contained retinal ganglion cells at a density of 54 ± 4/mm2, which was restored to 68 ± 9/mm2 by DAAO overexpression, similar to the levels in normal retinas. The ratio between the number of endothelial cells and pericytes in diabetic retinas was 6.06 ± 1.93/mm2, which was reduced to 3.42 ± 0.55/mm2 by DAAO overexpression; the number of acellular capillaries in diabetic retinas was 10 ± 5/mm2, which was restored to 6 ± 2/mm2 by DAAO overexpression, similar to the levels in normal retinas. Injection of the DAAO-expressing virus increased the expression of occludin and reduced gliosis, which were examined to probe the mechanism by which the disrupted blood-retinal barrier in diabetic rats was rescued and retinal neurodegeneration was prevented. CONCLUSIONS/INTERPRETATION: Altogether, overexpression of DAAO before the onset of diabetes protects against neurovascular abnormalities in retinas from diabetic rats, which suggests a novel strategy for preventing diabetic retinopathy. Graphical abstract.

TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7.

Azospirillum brasilense is one of the most studied species of diverse agronomic plants worldwide. The benefits conferred to plants inoculated with Azospirillum have been primarily attributed to its capacity to fix atmospheric nitrogen and synthesize phytohormones, especially indole-3-acetic acid (IAA). The principal pathway for IAA synthesis involves the intermediate metabolite indole pyruvic acid. Successful colonization of plants by Azospirillum species is fundamental to the ability of these bacteria to promote the beneficial effects observed in plants. Biofilm formation is an essential step in this process and involves interactions with the host plant. In this study, the tyrR gene was cloned, and the translated product was observed to exhibit homology to TyrR protein, a NtrC/NifA-type activator. Structural studies of TyrR identified three putative domains, including a domain containing binding sites for aromatic amino acids in the N-terminus, a central AAA+ ATPase domain, and a helix-turn-helix DNA binding motif domain in the C-terminus, which binds DNA sequences in promoter-operator regions. In addition, a bioinformatic analysis of promoter sequences in A. brasilense Sp7 genome revealed that putative promoters encompass one to three TyrR boxes in genes predicted to be regulated by TyrR. To gain insight into the phenotypes regulated by TyrR, a tyrR-deficient strain derived from A. brasilense Sp7, named A. brasilense 2116 and a complemented 2116 strain harboring a plasmid carrying the tyrR gene were constructed. The observed phenotypes indicated that the putative transcriptional regulator TyrR is involved in biofilm production and is responsible for regulating the utilization of D-alanine as carbon source. In addition, TyrR was observed to be absolutely required for transcriptional regulation of the gene dadA encoding a D-amino acid dehydrogenase. The data suggested that TyrR may play a major role in the regulation of genes encoding a glucosyl transferase, essential signaling proteins, and amino acids transporters.

Interplay between microbial d-amino acids and host d-amino acid oxidase modifies murine mucosal defence and gut microbiota.

L-Amino acids are the building blocks for proteins synthesized in ribosomes in all kingdoms of life, but d-amino acids (d-aa) have important non-ribosome-based functions(1). Mammals synthesize d-Ser and d-Asp, primarily in the central nervous system, where d-Ser is critical for neurotransmission(2). Bacteria synthesize a largely distinct set of d-aa, which become integral components of the cell wall and are also released as free d-aa(3,4). However, the impact of free microbial d-aa on host physiology at the host-microbial interface has not been explored. Here, we show that the mouse intestine is rich in free d-aa that are derived from the microbiota. Furthermore, the microbiota induces production of d-amino acid oxidase (DAO) by intestinal epithelial cells, including goblet cells, which secrete the enzyme into the lumen. Oxidative deamination of intestinal d-aa by DAO, which yields the antimicrobial product H2O2, protects the mucosal surface in the small intestine from the cholera pathogen. DAO also modifies the composition of the microbiota and is associated with microbial induction of intestinal sIgA. Collectively, these results identify d-aa and DAO as previously unrecognized mediators of microbe-host interplay and homeostasis on the epithelial surface of the small intestine.

A novel pathway for the production of H2 S by DAO in rat jejunum.

BACKGROUND: Hydrogen sulfide (H2 S) is endogenously generated from L-cysteine (L-Cys) by the enzymes cystathionine-ß-synthase (CBS) and cystathionine-γ-Lyase (CSE). Hydrogen sulfide is also produced from D-cysteine (D-Cys) by D-Amino acid oxidase (DAO). METHODS: The H2 S production was measured by the methylene blue assay. The expression of DAO was investigated by Western blotting and immunohistochemistry. The short-circuit current (Isc) was recorded using the Ussing chamber technique. KEY RESULTS: The epithelium in rat jejunum possesses DAO, and generates H2 S. D-cysteine, originally used as a negative control for L-Cys, significantly increases the H2 S release, which is inhibited by I2CA, an inhibitor of DAO. In vitro study by Ussing chamber technique reveals that D-Cys decreases the Isc across the epithelium of the rat jejunum and enhances the Na(+) -coupled L-alanine transport. CONCLUSIONS & INFERENCES: A novel pathway for the production of H2 S by DAO exists in rat jejunum.

High-level soluble and functional expression of Trigonopsis variabilis D-amino acid oxidase in Escherichia coli.

D-Amino acid oxidase is an important biocatalyst used in a variety of fields, and its economically justified level recombinant expression in Escherichia coli has not been established. To accomplish this, after a single Phe54Tyr substitution, fusion proteins of D-amino acid oxidase from Trigonopsis variabilis (TvDAO) with 6 × His-tags were constructed and expressed in E. coli. The effects of his-tags fusing position were revealed. Significant increase in holoenzyme percent and protein solubility made N-terminus tagged TvDAO (termed NHDAO) a suitable choice for TvDAO production. However, reduced cell growth and protein production rates were also observed for the NHDAO bearing strains. To optimize the performance of NHDAO production, changes of culture medium were tested. Finally, a production of 140 U/mL or 3.48 g active enzyme per liter which accounted for 41.4 % of the total protein, and a specific activity of 16.68 U/mg for the crude extract, were achieved in a 3.7 L fermenter in 28.5 h. This indicated a possibility for functional and economical TvDAO expression in E. coli to meet the industrial need.

A rapid in situ immobilization of D-amino acid oxidase based on immobilized metal affinity chromatography.

A rapid in situ immobilization process was developed based on conventional separation technique of immobilized metal affinity chromatography (IMAC) and was studied in the case of D-amino acid oxidase (DAAO) with binding-enhancing Heli-tag (His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly). A recombinant Escherichia coli strain JM105 (Δase)/pGEMK-R-DAAO-Heli was successfully constructed to synthesize chimeric protein DAAO-Heli. Without additional purification procedure, the tagged enzyme DAAO-Heli could be directly immobilized to EP-IDA-Ni(2+) support with purity of 90 % and DAAO activity of over 70 U/g support. Experimental results showed that the immobilized DAAO-Heli was 73 times more thermally stable than free enzyme. Besides, it remained 67 % of initial activity after 100 cycles of batch catalysis and its operational stability was improved 36 times than that of the previously IMAC-immobilized DAAO-His. Furthermore, the epoxy (EP) support could be easily recovered and repeatedly used with simple steps, which could reduce the immobilization costs significantly.

High activity expression of D-amino acid oxidase in Escherichia coli by the protein expression rate optimization.

Two categories of expression systems with different promoters and substitutive ribosome binding site region (RBS) were constructed in order to improve the soluble expression of d-amino acid oxidase (DAAO) basing on the hypothesis that the optimal promoter and suitable RBS would provide the recombinant expression system with better matched expression rate, which served as key factor to the heterogenous synthesis of soluble protein. The results showed that with rational promoter recombination and delicate RBS substitution, significant increase of DAAO activity (20-fold) was obtained for strain JM105/pGEMKT-Tac-R-DAAO over the previously constructed strain BL21(DE3)/pET-DAAO. Furthermore, similar optimization strategy proved feasible in the active expression of other enzymes such as glutaryl-7-aminocephalosporanic acid acylase (GCA) and N-Carbamyl-D-amino acid amidohydrolase (D-Case).

Optimization of media composition for D-amino acid oxidase production by Trigonopsis variabilis using biostatistical analysis.

D-amino acid oxidase (DAAO) is biotechnologically relevant enzyme that is used in various food and pharmaceutical industries. DAAO from the yeast Trigonopsis variabilis is an important agent for use in commercial applications because of its high activity with cephalosporin C and is reasonable resistant to the oxidants O2 and H2O2 byproducts of reaction. In this study, response surface methodology (RSM) in shake flask culture was used to enhance the production of DAAO from T. variabilis by optimization of fermentation media composition. The effects of six factors (DL-alanine, glucose, pH, ZnSO4, (NH4)2SO4 and temperature) were evaluated on DAAO production. Results of Placket-Burman design showed that DL-alanine, pH, glucose and ZnSO4 were significant factors for DAAO production (P<0.05). The optimum values of media components as predicted by the central composite design were inducer (DL-alanine) concentration 3 g/L, pH 7.7, glucose 17 g/L and ZnSO4 34 mg/L. At these optimum values of media composition, maximum production of DAAO was 153 U/g yeast dry weight. Two-fold increase in DAAO production was achieved after optimization of the physical parameters by RSM.

Genetic vulnerability to psychosis and cortical function: epistatic effects between DAAO and G72.

Recent studies have described G72 and DAAO as susceptibility genes for schizophrenia and bipolar disorder. Both genes modulate glutamate neurotransmission, which plays a key role in neurocognitive function and is thought to be altered in these disorders. Moreover, in vitro transcription studies indicate that the two genes interact with each other at the molecular level. However, it is unclear how these genes affect cortical function and whether their effects interact with each other. The aim of this study was therefore to examine the impact of G72 rs746187 and DAAO rs2111902 genotypes on brain function in schizophrenia, bipolar disorder and healthy volunteers. We used functional magnetic resonance imaging and an overt verbal fluency paradigm to examine brain function in a total of 120 subjects comprising 40 patients with schizophrenia, 33 patients with bipolar I disorder and 47 healthy volunteers. A significant 3 way interaction between G72, DAAO and diagnosis was detected in the right middle temporal gyrus (x=60 y=-12 z=-12; z-score: 5.32; p < 0.001 after family-wise error correction), accounting for 8.5% of the individual variance in activation. These data suggest that there is a nonadditive interaction between the effects of variations in the genes implicated in glutamate regulation that affects cortical function. Also, the nature of this interaction is different in patients and healthy controls, providing support for altered glutamate function in psychosis. Future studies could explore the effects of DAAO and G72 in individuals with prodromal symptoms of psychosis, in order to elucidate glutamate dysfunction in this critical phase of the disorder.

D-amino acid oxidase gene therapy sensitizes glioma cells to the antiglycolytic effect of 3-bromopyruvate.

Glioma tumors are refractory to conventional treatment. Glioblastoma multiforme is the most aggressive type of primary brain tumors in humans. In this study, we introduce oxidative stress-energy depletion (OSED) therapy as a new suggested treatment for glioblastoma. OSED utilizes D-amino acid oxidase (DAO), which is a promising therapeutic protein that induces oxidative stress and apoptosis through generating hydrogen peroxide (H2O2). OSED combines DAO with 3-bromopyruvate (3BP), a hexokinase II (HK II) inhibitor that interferes with Warburg effect, a metabolic alteration of most tumor cells that is characterized by enhanced aerobic glycolysis. Our data revealed that 3BP induced depletion of energetic capabilities of glioma cells. 3BP induced H2O2 production as a novel mechanism of its action. C6 glioma transfected with DAO and treated with D-serine together with 3BP-sensitized glioma cells to 3BP and decreased markedly proliferation, clonogenic power and viability in a three-dimensional tumor model with lesser effect on normal astrocytes. DAO gene therapy using atelocollagen as an in vivo transfection agent proved effective in a glioma tumor model in Sprague-Dawley (SD) rats, especially after combination with 3BP. OSED treatment was safe and tolerable in SD rats. OSED therapy may be a promising therapeutic modality for glioma.

[Expression of modified oxidase of D-aminoacids of Trigonopsis variabilis in methylotrophic yeasts Pichia pastoris].

Effective recombinant strains Pichia pastoris that produce functionally active hybrid of Trigonopsis variabilis D-aminoacids bond with chitin-connecting domain of chitinase A1 of Bacillus circulans (DAOcbd) were obtained. The dependence of DAOcbd production levels from production of the number of copies of "expression cassette" integrated in the AOX1 locus of recombinant strains was studied. It was indicated that synthesized DAOcbd may be easily purified and immobilized on chitin sorbents and possessed high specific activity. Produced strains and methods of their cultivation and DAOcbd extraction may be used for development of technologies of obtaining of biocatalyzers in technological processes of obtaining of 7-aminocephalosporane acid.

High-level expression of Rhodotorula gracilis D-amino acid oxidase in Pichia pastoris.

By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

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

BACKGROUND: Trigonopsis variabilis D-amino acid oxidase (TvDAO) 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 TvDAO 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 TvDAO whole-cell biocatalyst. RESULTS: As 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 x 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 >or= 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 TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration. CONCLUSIONS: Stepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO 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.

Spinal D-amino acid oxidase contributes to neuropathic pain in rats.

D-amino acid oxidase (DAO) is an enzyme catalyzing oxidative deamination of neutral and polar d-amino acids and is expressed in the kidneys, liver, and central nervous system (CNS) including the spinal cord. We have previously demonstrated that DAO gene deletion/mutation by using mutant ddY/DAO(-/-) mice and systemic administration of the DAO inhibitor sodium benzoate blocked formalin-induced hyperalgesia in mice. In this study, we further investigated the potential role of DAO in neuropathic pain in a rat model of tight L(5)/L(6) spinal nerve ligation. After L(5)/L(6) spinal nerve ligation, the mRNA expression (measured by real-time quantitative polymerase chain reaction) and enzyme activity (measured by a colorimetric method) of DAO in the lumbar spinal cord were markedly increased, in agreement with the development of neuropathic pain (mechanical allodynia). Intraperitoneal injection of sodium benzoate (400 mg/kg) specifically blocked mechanical allodynia in neuropathic rats and formalin-induced hyperalgesia but did not suppress acute pain responses in the tail-flick test or formalin test. Systemic injection of sodium benzoate also inhibited DAO activity in the lumbar spinal cord of rats. Furthermore, direct intrathecal (spinal cord) injection of benzoate (30 mug/rat) specifically blocked spinal nerve ligation-induced mechanical allodynia in neuropathic rats and formalin-induced hyperalgesia (but not acute pain) in the formalin test. Based on the above results, we conclude that spinal DAO plays a pronociceptive (rather than an antinociceptive) role and might be a target molecule for the treatment of chronic pain of neuropathic origin.

Molecular cloning and expression in Escherichia coli of an active fused Zea mays L. D-amino acid oxidase.

D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717.

Establishment of a cryopreservation method for the industrial use of D-amino acid oxidase-overexpressing Escherichia coli.

A cryopreservation condition for D-amino acid oxidase (DAAO)-overexpressing Escherichia coli (E. coli BL21(DE3)/pET-DAAO) was established. Ten percent was the optimum concentration of glycerol as a cryoprotectant, and its diffusion into stationary phase cells was superior to that into log cells. The results also showed that rather than fast cooling, a slow cooling method was appropriate to our recombinant E. coli. In addition, 15 min was the best equilibration period, at which higher than 90% of recovery rates were maintained at all test points. Most importantly, the relative recovery rates, product yield, and fermentation pattern of the cell banks (CBs) constructed according to our cryopreservation method did not change over 12 months, confirming that our method not only permits exceptional cryopreservation, but offers prolonged productivity. Taken together, our results demonstrating a cryopreservation method for E. coli BL21(DE3)/pET-DAAO provide insight into an improvement in the industrial production of DAAO.

[Fusion expression of D-amino acid oxidase from Trignoposis variabilis with maltose binding protein and Vitreoscilla hemoglobin].

D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.

D-amino acid oxidase: physiological role and applications.

D-Amino acids play a key role in regulation of many processes in living cells. FAD-dependent D-amino acid oxidase (DAAO) is one of the most important enzymes responsible for maintenance proper level of D-amino acids. The most interesting and important data for regulation of the nervous system, hormone secretion, and other processes by D-amino acids as well as development of different diseases under changed DAAO activity are presented. The mechanism of regulation is complex and multi-parametric because the same enzyme simultaneously influences the level of different D-amino acids, which can result in opposing effects. Use of DAAO for diagnostic and therapeutic purposes is also considered.