Identification of novel knockout and up-regulated targets for improving isoprenoid production in E. coli.

Discovery of novel potential genetic targets to increase the supply of isoprenoid precursors, isopentyl/dimethylallyl diphosphate, is of importance for microbial production of isoprenoids. Here, to improve isoprenoid precursor supply, a flux distribution comparison analysis, based on the genome-scale model, was utilized to simultaneously predict the knockout, down- and up-regulated targets in Escherichia coli. 51 targets were in silico discovered. All knockout and up-regulated targets were experimentally tested to enhance lycopene production. Five knockout targets (deoB, yhfw, yahI, pta and eutD) and four up-regulated targets (ompN, ompE, ndk and cmk) led to 10-45% increases of lycopene yield, respectively, which had not been uncovered in previous studies. When engineering of the five most significant targets gdhA, eutD, tpiA, ompE and ompN, were combined the lycopene titer improved by 174% in shake-flask and 81% in bioreactor fermentations with a maximum yield of 454 mg l(-1).

The participation of ferroptosis in fibrosis of the heart and kidney tissues in Dahl salt-sensitive hypertensive rats.

BACKGROUND: Salt-sensitive hypertension is often more prone to induce damage to target organs such as the heart and kidneys. Abundant recent studies have demonstrated a close association between ferroptosis and cardiovascular diseases.Therefore, we hypothesize that ferroptosis may be closely associated with organ damage in salt-sensitive hypertension. This study aimed to investigate whether ferroptosis is involved in the occurrence and development of myocardial fibrosis and renal fibrosis in salt-sensitive hypertensive rats. METHODS: Ten 7-week-old male Dahl salt-sensitive (Dahl-SS) rats were adaptively fed for 1 week, then randomly divided into two groups and fed either a normal diet (0.3% NaCl, NDS group) or a high-salt diet (8% NaCl, HDS group) for 8 weeks. Blood pressure of the rats was observed, and analysis of the hearts and kidneys of Dahl-SS rats was conducted via HE-staining, Masson-staining, Prussian-blue-staining, TEM, tissue iron content detection, MDA content detection, immunofluorescence, and Western blot. RESULTS: Compared to the NDS group, rats in the HDS group increases in systolic blood pressure(SBP) and diastolic blood pressure(DBP)(P<0.05);collagen fiber accumulation was observed in the heart and kidney tissues (P<0.01), accompanied by alterations in mitochondrial ultrastructure,reduced mitochondrial volume, and increased density of the mitochondrial double membrane. Additionally,there were significant increases in both iron content and MDA levels(P<0.05). Immunofluorescence and Western blot results both indicated significant downregulation (P<0.05) of xCT and GPX4 proteins associated with ferroptosis in the HDS group. CONCLUSION: Ferroptosis is involved in the damage and fibrosis of the heart and kidney tissues in salt-sensitive hypertensive rats.

Systems perspectives on erythromycin biosynthesis by comparative genomic and transcriptomic analyses of S. erythraea E3 and NRRL23338 strains.

BACKGROUND: S. erythraea is a Gram-positive filamentous bacterium used for the industrial-scale production of erythromycin A which is of high clinical importance. In this work, we sequenced the whole genome of a high-producing strain (E3) obtained by random mutagenesis and screening from the wild-type strain NRRL23338, and examined time-series expression profiles of both E3 and NRRL23338. Based on the genomic data and transcriptpmic data of these two strains, we carried out comparative analysis of high-producing strain and wild-type strain at both the genomic level and the transcriptomic level. RESULTS: We observed a large number of genetic variants including 60 insertions, 46 deletions and 584 single nucleotide variations (SNV) in E3 in comparison with NRRL23338, and the analysis of time series transcriptomic data indicated that the genes involved in erythromycin biosynthesis and feeder pathways were significantly up-regulated during the 60 hours time-course. According to our data, BldD, a previously identified ery cluster regulator, did not show any positive correlations with the expression of ery cluster, suggesting the existence of alternative regulation mechanisms of erythromycin synthesis in S. erythraea. Several potential regulators were then proposed by integration analysis of genomic and transcriptomic data. CONCLUSION: This is a demonstration of the functional comparative genomics between an industrial S. erythraea strain and the wild-type strain. These findings help to understand the global regulation mechanisms of erythromycin biosynthesis in S. erythraea, providing useful clues for genetic and metabolic engineering in the future.

Real-time fluid dynamics investigation and physiological response for erythromycin fermentation scale-up from 50 L to 132 m3 fermenter.

The physiological response of erythromycin fermentation scale-up from 50 L to 132 m(3) scale was investigated. A relatively high oxygen uptake rate (OUR) in early phase of fermentation was beneficial for erythromycin biosynthesis. Correspondingly, the maximal consistency coefficient (K) reflecting non-Newtonian fluid characteristics in 50 L and 132 m(3) fermenter also appeared in same phase. Fluid dynamics in different scale bioreactor was further investigated by real-time computational fluid dynamics modeling. The results of simulation showed that the impeller combination in 50 L fermenter could provide more modest flow field environment compared with that in 132 m(3) fermenter. The decrease of oxygen transfer rate (OTR) in 132 m(3) fermenter was the main cause for impairing cell physiological metabolism and erythromycin biosynthesis. These results were helpful for understanding the relationship between hydrodynamic environment and physiological response of cells in bioreactor during the scale-up of fermentation process.

Incomplete formation of intramolecular disulfide bond triggers degradation and aggregation of human consensus interferon-alpha mutant by Pichia pastoris.

Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-alpha mutant (cIFN) were serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro conversion assay based on incubation with different concentrations of beta-mercaptoethanol. The results showed that only a wound such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers. Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which result in sharp decrease of bioactivity of cIFN in bioreactor.

Regulation of branched-chain amino acid catabolism: glucose limitation enhances the component of isovalerylspiramycin for the bitespiramycin production.

4''-O-isovalerylspiramycins are the major components of bitespiramycin complex consisting of a group of 4''-O-acylated spiramycins. The availability of isovaleryl group, usually in vivo derived from leucine, one of the branched-chain amino acids, affects the content of isovaleryispiramycin significantly. In this study, the effect of glucose on the activity of branched-chain alpha-keto acid dehydrogenase (BCKDH), which catalyzed the rate-limiting as well as the first irreversible reaction oxidative decarboxylation for branched-chain amino acids degradation, and isovaleryispiramycin biosynthesis was investigated. In the initial glucose concentration experiment, when the residual glucose concentration in the medium declined to 2-4 g/L, the BCKDH activity rose rapidly, and glucose deprivation and the summit of BCKDH activity appeared nearly at the same time. After a delay of about 6 h, the maximal isovalerylspiramycin content was observed. However, the shortage of glucose at the later production phase resulted in the marked decrease in BCKDH activity and isovaleryispiramycin content. In the fermentation in a 50 L fermentor, glucose feeding at the late production phase helped to maintain the residual glucose concentration between 0 and 1 g/L, leading to the high level of BCKDH activity and thus isovalerylspiramycin content. These suggested that glucose concentration could be used as a key parameter to regulate BCKDH activity and isovaleryispiramycin biosynthesis in the bitespiramycin production.

Leucine improves the component of isovalerylspiramycins for the production of bitespiramycin.

Bitespiramycin, a group of 4''-O-acylated spiramycins with 4''-O-isovalerylspiramycins as the major components, is produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4''-O-acyltransferase gene from a carbomycin-producing strain S. mycarofaciens 1748. The effects of leucine feeding on the bitespiramycin fermentation, especially the synthesis of isovalerylspiramycin components, were investigated. The experiment was initially performed in flask culture under the condition of feeding 15.4 mmol/l of leucine at 72 h fermentation, and the culture without leucine feeding was used as control. When 15.4 mmol/l leucine was fed at 72 h, 51.3 +/- 0.33% total isovalerylspiramycins was recorded compared to 40.9 +/- 0.26% under the control condition after 96 h of fermentation. The improvement of total isovalerylspiramycin content was further achieved in 15 l fermentation when 15.4 mmol/l of leucine was supplemented from 65 to 72 h. These results indicated that isovaleryl group derived from leucine catabolism could act as the precursor of the 4'' side chain of bitespiramycin, which profoundly enhanced the synthesis of isovalerylspiramycins in the bitespiramycin complex.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins.

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinant spiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l(-1) Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3% higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5%. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85% and 72.1% of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41%. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9% while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l(-1) leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l(-1) leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1% to 46.9%.

Phosphate limitation increases coenzyme Q10 production in industrial Rhodobacter sphaeroides HY01.

Coenzyme Q10 (CoQ10) is an important component of the respiratory chain in humans and some bacteria. As a high-value-added nutraceutical antioxidant, CoQ10 has excellent capacity to prevent cardiovascular disease. The content of CoQ10 in the industrial Rhodobacter sphaeroides HY01 is hundreds of folds higher than normal physiological levels. In this study, we found that overexpression or optimization of the synthetic pathway failed CoQ10 overproduction in the HY01 strain. Moreover, under phosphate- limited conditions (decreased phosphate or in the absence of inorganic phosphate addition), CoQ10 production increased significantly by 12% to220 mg/L, biomass decreased by 12%, and the CoQ10 productivity of unit cells increased by 27%. In subsequent fed-batch fermentation, CoQ10 production reached 272 mg/L in the shake-flask fermentation and 1.95 g/L in a 100-L bioreactor under phosphate limitation. Furthermore, to understand the mechanism associated with CoQ10 overproduction under phosphate- limited conditions, the comparatve transcriptome analysis was performed. These results indicated that phosphate limitation combined with glucose fed-batch fermentation represented an effective strategy for CoQ10 production in the HY01. Phosphate limitation induced a pleiotropic effect on cell metabolism, and that improved CoQ10 biosynthesis efficiency was possibly related to the disturbance of energy metabolism and redox potential.

Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by P(GAP)-driven Pichia pastoris overexpressing methionine adenosyltransferase.

Methionine adenosyltransferase (MAT) was overexpressed within Pichia pastoris employing the promoter of glyceraldehyde-3-phosphate dehydrogenase gene (P(GAP)), to biosynthesize S-adenosyl-l-methionine (SAM). Effects of five glycerol feeding tactics on MAT activity were first investigated. Strategies A-C were based on limited feeding correlated with dissolved oxygen (DO) at 50.0%, 25.0% and 0.0%, respectively. For strategies D and E, unlimited supplementation was executed by pulsed feeding mode. Gradual decline (2-0%) (w:v) of the residual glycerol level was shown between any two pulses in strategy D, while a nearly stable content (2%) throughout fed-batch cultivation with strategy E. With shifting strategies A-E in alphabetical order, gradual improvements of MAT activities were achieved, with the maximum of 9.05Ug(-1) dried biomass for strategy E, since the specific glycerol consumption rate (F(G)) ascended due to the elevated specific oxygen uptake rate (qO(2)). The success was ascribed to the enhancement of oxygen transfer rate (OTR), because 2% glycerol improved oxygen saturation content in broth (C*) and volumetric oxygen transfer coefficient (k(L)a). Strategy E also led to the highest values of ATP and biomass besides MAT. Consequently, the highest SAM yield and volumetric level were obtained at 0.058gg(-1) and 9.26gl(-1), respectively.

Isolation of soluble proteins from an industrial strain Streptomyces avermitilis in complex culture medium for two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis (2-DE) is a core proteomic technique to study protein expression and function in living organisms. Although 2-DE has been extensively used for the investigation of bacteria, yeast, animal and plant tissue cells, the isolation of proteins from the organisms and elimination of salt, nucleotide, polysaccharide, lipids and other contaminations from the samples often limit its application. In this study, the protocol for protein isolation from cells of Streptomyces avermitilis cultivated in partially insoluble complex medium was investigated. The goal was to make the obtained extraction samples suitable for the two-dimensional electrophoresis, thus make the further proteome analysis possible. Compared to non-denatured procedure, the denatured one, precipitating with 10% TCA in acetone, efficiently eliminated the interference substances from the cell lysate. Thiourea in the rehydration solution enhanced the resolubilization of protein pellets but led to heavy horizontal streaking in the 2-DE gels. High protein loading amount improved the resolution of some low abundance proteins but did not adapt to the high abundance proteins. And it was also important to collect cells at appropriate culture time according to the analysis target. With the optimized protein extraction protocol, the protein expression patterns of S. avermitilis during the onset of avermectin production in complex medium were analyzed.

Inhibition of degradation and aggregation of recombinant human consensus interferon-alpha mutant expressed in Pichia pastoris with complex medium in bioreactor.

The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins. However, limitations such as protein degradation and aggregation became obvious when secreting heterologous protein-recombinant human consensus interferon-alpha mutant. Here, we investigate the effect of induction temperature on the yield and stability of interferon mutant expressed by P. patoris with buffered complex medium. The best results in terms of interferon mutant bioactivity and specific bioactivity were obtained when the microorganism was induced at 15 degrees C, which were 2.91 x 10(8) +/- 0.3 x 10(8) and 2.26 x 10(8 )+/- 0.23 x 10(8) IU mg(-1), respectively. At the same time, the cells grew fast owing to high AOX1-specific activity, and interferon mutant expression level reached 1.23 g l(-1), which was almost 30 times higher than that in the flask. Also, the proteolytic degradation of interferon mutant was inhibited completely because of lower protease bioactivity probably due to a reduced cell death rate at lower temperatures as well as protection of yeast extract and peptone in complex medium. In addition, interferon mutant aggregation was repressed significantly by the addition of Tween-80, and a specific bioactivity of 7.35 x 10(8) +/- 0.56 x 10(8) IU mg(-1) was obtained. These results should be applicable to other low-stability recombinant proteins expressed in P. pastoris.

Real-time viable-cell mass monitoring in high-cell-density fed-batch glutathione fermentation by Saccharomyces cerevisiae T65 in industrial complex medium.

An on-line monitoring of viable-cell mass in high-cell-density fed-batch cultivations of Saccharomyces cerevisiae grown on an industrial complex medium was performed with an in situ capacitance probe fitted to a 50-l fermentor. Conventional off-line biomass determinations of several parameters, including dry cell weight (DCW), optical density at 600 nm wavelength (OD(600)), packed mycelial volume (PMV) and number of colony forming units (CFU), were performed throughout the bioprocess and then compared with on-line viable-cell concentrations measured using a capacitance probe. Capacitance versus viable biomass and all off-line biomass assay values were compared during glutathione fermentation in industrial complex culture media. As a result, the relationship between the number of colony forming units and capacitance with a correlation coefficient (R) of 0.995 was achieved. Simultaneously, compared with those determined by at-line indirect estimation methods including oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER), the specific growth rates estimated by on-line capacitance measurement could be more reliable during glutathione fermentation. Therefore, it is concluded that a capacitance probe is a practical tool for real-time viable biomass monitoring in high-cell-density fed-batch cultivation in a complex medium.

Identification and purification of the main components of cellulases from a mutant strain of Trichoderma viride T 100-14.

A new mutant strain of fungus Trichoderma viride T 100-14 was cultivated on 1% microcrystalline cellulose (Avicel) for 120h and the resulting culture filtrate was prepared for protein identification and purification. To identify the predominant catalytic components, cellulases were separated by an adapted two-dimensional electrophoresis technique. The apparent major spots were identified by high performance liquid chromatography electrospray ionization mass (HPLC-ESI-MS). Seven of the components were previously known, i.e., the endoglucanases Cel7B (EG I), Cel12A (EG III), Cel61A (EG IV), the cellobiohydrolases Cel7A (CBH I), Cel6A (CBH II), Cel6B (CBH IIb) and the beta-glucosidase. The seven major components in the fermentation broth of T. viride T 100-14 probably constitute the essential enzymes for crystalline cellulose hydrolysis and they were further purified to electrophoretic homogeneity by a series of chromatography column. Hydrolysis studies of the purified elements revealed that three of the cellulases were classified as cellobiohydrolases due to their main activities on p-nitrophenyl-beta-d-cellobioside (pNPC). Three of the cellulases, with the abilities of hydrolyzing both carboxymethyl-cellulose (CMC) and Avicel indicate their endoglucanase activities. It deserved noting that the beta-glucosidase from the T 100-14 displayed an extremely high activity on p-nitrophenyl-beta-D-glycopyranoside (pNPG), which suggested it was a good candidate for the conversion of cellobiose to glucose.

Improved large-scale production of vitamin B12 by Pseudomonas denitrificans with betaine feeding.

The strategy of betaine control for vitamin B12 large-scale fermentation by Pseudomonas denitrificans was investigated in this paper. The results obtained in shake-flask experiments demonstrated that betaine could greatly stimulate vitamin B12 biosynthesis but had an inhibition to cell growth. Based on the influence of betaine on the fermentation of P. denitrificans, betaine feeding was a beneficial strategy to solve the inconsistency between cell growth and vitamin B12 production. As a result, an effective and economical strategy of betaine feeding was established for vitamin B12 fermentation in 120-m3 fermenter, in which betaine was continuously fed to maintain betaine concentration of the broth at the range of 5-7g/l during 50-140h of fermentation.

[Study of foot plantar pressure in Chinese diabetic patients].

OBJECTIVE: To investigate the difference and distribution of plantar pressure in normal Chinese population and diabetic patients and their influencing factors. METHODS: 1035 diabetic patients and 1022 normal subjects were evaluated by the "first step approach". Each trial included five acceptable separate barefoot walks across the EMED-AT system for each foot. The whole foot was divided into ten "masks", including the heel, midfoot, the first, second, third, fourth, fifth metatarsals, hallux, second toe and the third-to-fifth toes. The mean of maximum peak pressure (MPP) of each mask as calculated. RESULTS: The MPP were 410 +/- 122 and 427 +/- 128 kPa in left and right foot of diabetics while 415 +/- 113 and 428 +/- 116 kPa in left and right foot of normal subjects with P > 0.05. The top three places with the hight MPP value in the in diabetics and normal subjects were hallux > the second metatarsal > the third metatarsals in both left and right foot. The disease course, sex, age, BMI, WHR and history of foot ulcer had few influence on the plantar pressure in diabetics. The MPP in the diabetics with peripheral neuropathy (DPN) was higher than that of normal subjects, with no significant difference compared to that of non-DPN diabetics. CONCLUSION: The plantar pressure in the diabetics is almost the same as that of the normal subjects, but with different distribution is different. Complication with DPN may lead to the increase of plantar pressure in the diabetic. But factors such as disease course, sex, age, BMI and WHR have few influence on MPP.

[Effects of ammonium on components of biotechspiramycin].

The effects of ammonium ion on the synthesis of biotechspiramycin were investigated. It was proved that the percentage of isovalerylspiramycin III was greatly enhanced in the medium with low concentration of ammonium ion. By greatly reducing ammonium ion concentration in the medium from 62.5mmol/L to 2.5mmol/L, the percentage of isovalerylspiramycin III increased from 5.43% to 28.59% . However, the titer of biotechspiramycin decreased to 107microg/mL in the medium with low concentration of ammonium ion, which was 14.4% lower than that in the medium with high concentration of ammonium ion, due to lack of nitrogen source. The results also showed that the activity of valine dehydrogenase, the key enzyme of leucine catabolic pathway, in the fermentation with high concentration of ammonium ion was lower than that with low ammonium ion concentration. This would lead to the limitation of isovaleryl-CoA, which is the substrate of isovaleryl transfer reaction, and then the decline of percentage of isovalerylspiramycin. The percentage of isovalerylspiramycin III further increased to 37.84% by addition of 0.3mg/mL leucine to the fermentation broth at 36h cultivation.

[Regulatory effects of ammonium ions on the biosynthesis of meilingmycin].

Based on the effects of different ammonium sulfate concentrations on meilingmycin biosynthesis, the results show that lower concentration of ammonium ions stimulates the biosynthesis of meilingmycin, while the concentration of higher than 5mmol/L inhibits the mycelial growth and the biosynthesis of the products. However, the sugar consumption rate increases with the elevating concentration of ammonium sulfate. On this basis, six enzymes, which are greatly related to the meilingmycin biosynthesis and the glucose metabolism, were measured and analyzed during the meilingmycin fermentation process. The results suggest that glucose-6-phosphate dehydrogenase, citrate synthase, succinate dehydrogenase and fatty acid synthase are stimulated by higher concentration of ammonium ions, while valine dehydrogenase and methylmalonyl-CoA carboxyltransferase are inhibited. From the results it follows that ammonium ions favors primary metabolism, that is, the HMP passway and the TCA circle is enhanced, as well as the source of the precursors for the biosynthesis of meilingmycin is restricted, which bring about the lower production of meilingmycin.

[Influence of Mn2+ on the biotechmycin fermentation].

The effect of Mn2+ on the biotechmycin fermentation by Bioengineered strain WSJ-l-195 was studied. In the fermentation process, Mn2+ could improve the biological potency significantly, especially when Mn2+ concentration was 5 mmol/L added at 24 h. The pH profile of fermentation broth decreased gradually after 5 mmol/L Mn2+ supplemented at 24 h, and PMV was lower than that of the control sample. Further research about the influence of Mn2+ on the biosynthesis of biotechmycin was carried out in the aspect of organic acids. The results showed that concentrations of organic acids in a fermentation with 5 mmol/L Mn2+ supplemented at 24 h had been changed greatly, especially the concentration of propionic acid, of which the highest value was about 6 times as that in the control sample at 84 h. In addition, it was found that the yield of biotechmycin could be improved significantly with tiny amount of propionic acid added. Therefore, it can be concluded that Mn2+ has profound influence on the biosynthesis of biotechmycin: it enriches the biotechmycin precursor pool such as propionic acid and thus improves the yield of biotechmycin.

Isolation, characterization, and molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastoris.

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of 60 degrees C.