Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients.

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80°C was extended from 14.81 min to 23.35 min, half-inactivation temperature (T50 30) was increased from 55.12°C to 62.44°C, and Tm value was increased from 48.36°C to 53.18°C. Meanwhile, the specific activity of K189R/K216R at 37°C and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

Production of cleavage-resistant phytase transgenic pigs by handmade cloning.

With the rapid development of intensive animal husbandry in the livestock industry, large quantities of manure waste containing phytate phosphorus are being generated. Phytase can effectively solve the problem of high phosphorus pollution in the feces of monogastric animals. Enviropig, which produces phytase in the salivary glands and secretes the enzyme in the saliva, were first generated in 1999. However, phytase is easily inactivated during digestion. To address this problem, cleavage-resistant phytase transgenic pigs were generated using handmade cloning in this study. Transgene construction was improved and three cell lines carrying Cafp were obtained. In total, 810 blastocysts were generated and 712 good-quality were transferred into six recipients. Fourteen piglets were born, of which six survived after weaning. Polymerase chain reaction and sequencing results showed that seven (three live and four dead) of the fourteen piglets carried Cafp. Phytase activity in the saliva of the six live cloned pigs was tested at four months of age, and only one pig had 0.155 FTU/mL enzyme activity. The other five pigs may not have been activated in the transgenic parotid gland. Among all the transgenic pigs, the highest phosphorus digestion rate was 59.2% of intake, representing a 25.4% decrease in fecal emission compared to the average of controls. Immunohistochemical results on the three Cafp-positive pigs that died after six months of age showed that the transgene was only expressed in parotid glands, confirming tissue-specific gene expression. In conclusion, cleavage-resistant phytase transgenic pigs were successfully produced through handmade cloning. The cloned pigs offer a unique biological approach to managing phosphorus nutrition and environmental pollution in animal husbandry.

Improvement of the recombinant phytase expression by intermittent feeding of glucose during the induction phase of methylotrophic yeast Pichia pastoris.

PURPOSE: For growth of methylotrophic yeast, glycerol is usually used as a carbon source. Glucose is used in some cases, but not widely consumed due to strong repressive effect on AOX1 promoter. However, glucose is still considered as a carbon source of choice since it has low production cost and guarantees growth rate comparable to glycerol. RESULTS: In flask cultivation of the recombinant yeast, Pichia pastoris GS115(pPIC9K-appA38M), while methanol induction point(OD600) and methanol concentration significantly affected the phytase expression, glucose addition in induction phase could enhance phytase expression. The optimal flask cultivation conditions illustrated by Response Surface Methodology were 10.37 OD600 induction point, 2.02 h before methanol feeding, 1.16% methanol concentration and 40.36µL glucose feeding amount(for 20 mL culture volume) in which the expressed phytase activity was 613.4 ± 10.2U/mL, the highest activity in flask cultivation. In bioreactor fermentation, the intermittent glucose feeding showed several advantageous results such as 68 h longer activity increment, 149.2% higher cell density and 200.1% higher activity compared to the sole methanol feeding method. These results implied that remaining glucose at induction point might exhibit a positive effect on the phytase expression. CONCLUSION: Glucose intermittent feeding could be exploited for economic phytase production and the other recombinant protein expression by P. pastoris GS115.

Harnessing alkaline-pH regulatable promoters for efficient methanol-free expression of enzymes of industrial interest in Komagataella Phaffii.

BACKGROUND: The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. RESULTS: We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. CONCLUSIONS: Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.

Characterization of recombinant phytase of Klebsiella sp. and the influence of novel 3-phytase on mineral solubility in broiler diets under an in vitro digestion assay.

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.

Improved Thermal Stability of a Novel Acidophilic Phytase.

Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important enzyme from an environmental perspective because it not only promotes the growth of livestocks but also prevents phosphorus contamination released into the environment. Here we present a novel phytase derived from Turicimonas muris, TmPhy, which has distinctive structure and properties compared to other previously known phytases. TmPhy gene expressed in the Pichia system was confirmed to be 41 kDa in size and was used in purified form to evaluate optimal conditions for maximum activity. TmPhy has a dual optimum pH at pH3 and pH6.8 and exhibited the highest activity at 70°C. However, the heat tolerance of the wildtype was not satisfactory for feed application. Therefore, random mutation, disulfide bond introduction, and N-terminal mutation were performed to improve the thermostability of the TmPhy. Random mutation resulted in TmPhyM with about 45% improvement in stability at 60°C. Through further improvements, a total of three mutants were screened and their heat tolerance was evaluated. As a result, we obtained TmPhyMD1 with 46.5% residual activity, TmPhyMD2 with 74.1%, and TmPhyMD3 with 66.8% at 80°C heat treatment without significant loss of or with increased activity.

CO2-based production of phytase from highly stable expression plasmids in Cupriavidus necator H16.

BACKGROUND: Existing plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2. RESULTS: In this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml. CONCLUSION: The new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Isolation and in-vitro characterization of extracellular phytase producing bacterial isolates for potential application in poultry feed.

BACKGROUND: Phytase catalyses the breakdown of complex organic forms of phosphorous into simpler forms by sequential hydrolysis of phosphate ester bonds to liberate the inorganic phosphate. Supplementation of feeds with bacterial phytase therefore could enhance the bioavailability of phosphorus and micronutrients. Hence, the aim of this study was to isolate and characterize phytase producing bacteria from rhizosphere soil, fresh poultry excreta, and cattle shed to evaluate their potential in improving poultry feeds. Phytase producing bacteria were isolated using wheat bran extract medium. RESULTS: A total of 169 bacterial isolates were purified and screened for phytase activity. Out of these, 36 were confirmed as positive for phytase enzyme activity. The bacterial isolates were identified by cultural, morphological, and biochemical features. The isolates were also identified by using 16 S rRNA gene sequencing. The bacterial isolates (RS1, RS8, RS10 and RS15) were provided with gene bank database accession numbers of MZ407562, MZ407563, MZ407564 and MZ407565 respectively. All isolates increased phytase production when cultured in wheat bran extract medium (pH 6) supplemented with 1% (wt/v) galactose and 1% (wt/v) ammonium sulphate incubated at 50oC for 72 h. Proximate composition analysis after supplementation of phytase showed that phytase supplementation improved bioavailability of phosphorus, calcium, potassium and sodium in poultry feed. CONCLUSIONS: Overall, this study showed that the nutritional value of poultry feed can be improved using microbial phytase enzyme which reduces the cost of supplementation with inorganic phosphate.

Microbial Phytases: Properties and Applications in the Food Industry.

Microbial phytases are enzymes that break down phytic acid, an anti-nutritional compound found in plant-based foods. These enzymes which are derived from bacteria and fungi have diverse properties and can function under different pH and temperature conditions. Their ability to convert phytic acid into inositol and inorganic phosphate makes them valuable in food processing. The application of microbial phytases in the food industry has several advantages. Firstly, adding them to animal feedstuff improves phosphorus availability, leading to improved nutrient utilization and growth in animals. This also reduces environmental pollution by phosphorus from animal waste. Secondly, microbial phytases enhance mineral bioavailability and nutrient assimilation in plant-based food products, counteracting the negative effects of phytic acid on human health. They can also improve the taste and functional properties of food and release bioactive compounds that have beneficial health effects. To effectively use microbial phytases in the food industry, factors like enzyme production, purification, and immobilization techniques are important. Genetic engineering and protein engineering have enabled the development of phytases with improved properties such as enhanced stability, substrate specificity, and resistance to degradation. This review provides an overview of the properties and function of phytases, the microbial strains that produce them, and their industrial applications, focusing on new approaches.

CRISPR/Cas9-Mediated Genome Editing of the Komagataella phaffii to Obtain a Phytase-Producer Markerless Strain.

Using CRISPR/Cas9 system, the recipient strains K. phaffii VKPM Y-5013 (His- phenotype) and K. phaffii VKPM Y-5014 (Leu- phenotype) were derived from the K. phaffii VKPM Y-4287 strain, which has a high expression potential. Based on the developed recipient strains, markerless producers of heterologous proteins could be obtained. Efficiency of the gene inactivation with different variants of sgRNA ranged from 65 to 98% and from 15 to 72% for the HIS4 and LEU2 genes, respectively. The recipient strains retained growth characteristics of the parent strain and exhibited high expression potential, as estimated by the production of heterologous phytase from Citrobacter gillenii. Average productivity of the transformants based on the K. phaffii VKPM Y-5013 and K. phaffii VKPM Y-5014 strains was 2.1 and 2.0 times higher than productivity of the transformants of the commercial K. phaffii GS115 strain. Method for sequential integration of genetic material into genome of the K. phaffii VKPM Y-5013 strain was proposed. A highly effective multicopy markerless strain producing C. gillenii phytase was obtained.

Trait stacking simultaneously enhances provitamin A carotenoid and mineral bioaccessibility in biofortified Sorghum bicolor.

Vitamin A, iron, and zinc deficiencies are major nutritional inadequacies in sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve provitamin A carotenoid or mineral content and/or bioaccessibility in staple crops including sorghum (Sorghum bicolor). However, concurrent carotenoid and mineral enhancement has not been thoroughly assessed and antagonism between these chemical classes has been reported. This work evaluated two genetically engineered constructs containing a suite of heterologous genes to increase carotenoid stability and pathway flux, as well as phytase to catabolize phytate and increase mineral bioaccessibility. Model porridges made from transgenic events were evaluated for carotenoid and mineral content as well as bioaccessibility. Transgenic events produced markedly higher amounts of carotenoids (26.4 µg g-1 DW) compared to null segregants (4.2 µg g-1 DW) and wild-type control (Tx430; 3.7 µg g-1 DW). Phytase activation by pre-steeping flour resulted in significant phytate reduction (9.4 to 4.2 mg g-1 DW), altered the profile of inositol phosphate catabolites, and reduced molar ratios of phytate to iron (16.0 to 4.1), and zinc (19.0 to 4.9) in engineered material, suggesting improved mineral bioaccessibility. Improved phytate : mineral ratios did not significantly affect micellarization and bioaccessible provitamin A carotenoids were over 23 times greater in transgenic events compared to corresponding null segregants and wild-type controls. A 200 g serving of porridge made with these transgenic events provide an estimated 53.7% of a 4-8-year-old child's vitamin A estimated average requirement. These data suggest that combinatorial approaches to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.

Digestion and utilization of plant-based diets by transgenic pigs secreting ß-glucanase, xylanase, and phytase in their salivary glands.

Novel transgenic (TG) pigs co-expressing three microbial enzymes, ß-glucanase, xylanase, and phytase, in their salivary glands were previously generated, which exhibited reduced phosphorus and nitrogen emissions and improved growth performances. In the present study, we attempted to explore the age-related change of the TG enzymic activity, the residual activity of the enzymes in the simulated gastrointestinal tract, and the effect of the transgenes on the digestion of nitrogen and phosphorus content in the fiber-rich, plant-based diets. Results showed that all the three enzymes were stably expressed over the growing and finishing periods in the F2 generation TG pigs. In simulated gastric juice, all the three enzymes exhibited excellent gastrointestinal environment adaptability. The apparent total tract digestibility of phosphorus was increased by 69.05% and 499.64%, while fecal phosphate outputs were decreased by 56.66% and 37.32%, in the TG pigs compared with the wild-type littermates fed with low non-starch polysaccharides diets and high fiber diets, respectively. Over half of available phosphorus and water-soluble phosphorus in fecal phosphorus were reduced. We also found the performance of phosphorus, calcium, and nitrogen retention rates were significantly improved, resulting in faster growth performance in TG pigs. The results indicate that TG pigs can effectively digest the high-fiber diets and exhibit good growth performance compared with wild type pigs.

Functional Metagenomics Approach for the Discovery of Novel Genes Encoding Phosphatase Activity.

Phosphate release from inorganic and organic phosphorus compounds can be enzymatically mediated. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation, and diagnostic analysis. Here, we describe a functional metagenomics approach enabling rapid identification of genes encoding these enzymes. The target genes are detected based on small- and large-insert metagenomic libraries derived from diverse environments. This approach has the potential to unveil entirely new phosphatase families or subfamilies and members of known enzyme classes that hydrolyze phosphomonoester bonds such as phytases. Additionally, we provide a strategy for efficient heterologous expression of phosphatase genes.

Enhancement of activity and thermostability of Aspergillus niger ATCC 10864 phytase A through rational design.

Aspergillus niger ATCC 10864 phytase A was produced in Penicillium verruculosum. The enzyme was found to have two pH optima of 2.5 and 5.0, as well as a T-optimum of 50-55 °C. Two amino acid substitutions, A76M and S265P, were designed for improvement in thermostability, and two more, N300K and D363N, were designed for improvement in enzyme activity. The most thermostable variant, S265P, was characterized by a 3.8-fold increase in time of half-life at 55 °C and a 1.2-fold increase in residual activity at 90 °C compared to the wild-type. The most active variant, D363N, was 1.7-times more active at 40 °C and retained 1.3-times higher residual activity at 90 °C compared to the wild-type. The obtained results revealed the importance of substitutions with proline in α-helixes for the thermostability improvement of phytases. Also, the importance of sequence motif 361HDN363 was demonstrated with relevance to values of catalytic parameters.

Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases.

Among ubiquitous phosphorus (P) reserves in environmental matrices are ribonucleic acid (RNA) and polyphosphate (polyP), which are, respectively, organic and inorganic P-containing biopolymers. Relevant to P recycling from these biopolymers, much remains unknown about the kinetics and mechanisms of different acid phosphatases (APs) secreted by plants and soil microorganisms. Here we investigated RNA and polyP dephosphorylation by two common APs, a plant purple AP (PAP) from sweet potato and a fungal phytase from Aspergillus niger. Trends of δ18O values in released orthophosphate during each enzyme-catalyzed reaction in 18O-water implied a different extent of reactivity. Subsequent enzyme kinetics experiments revealed that A. niger phytase had 10-fold higher maximum rate for polyP dephosphorylation than the sweet potato PAP, whereas the sweet potato PAP dephosphorylated RNA at a 6-fold faster rate than A. niger phytase. Both enzymes had up to 3 orders of magnitude lower reactivity for RNA than for polyP. We determined a combined phosphodiesterase-monoesterase mechanism for RNA and terminal phosphatase mechanism for polyP using high-resolution mass spectrometry and 31P nuclear magnetic resonance, respectively. Molecular modeling with eight plant and fungal AP structures predicted substrate binding interactions consistent with the relative reactivity kinetics. Our findings implied a hierarchy in enzymatic P recycling from P-polymers by phosphatases from different biological origins, thereby influencing the relatively longer residence time of RNA versus polyP in environmental matrices. This research further sheds light on engineering strategies to enhance enzymatic recycling of biopolymer-derived P, in addition to advancing environmental predictions of this P recycling by plants and microorganisms.

Microbial engineering for the production and application of phytases to the treatment of the toxic pollutants: A review.

Phytases are a group of digestive enzymes which are commonly used as feed enzymes. These enzymes are used exogenously in the feeds of monogastric animals thereby it improves the digestibility of phosphorous and thus reduces the negative impact of inorganic P excretion on the environment. Even though these enzymes are widely distributed in many life forms, microorganisms are the most preferred and potential source of phytase. Despite the extensive availability of the phytase-producing microbial consortia, only a few microorganisms have been known to be exploited at industrial level. The high costs of the enzyme along with the incapability to survive high temperatures followed by the poor storage stability are noted to be the bottleneck in the commercialization of enzymes. For this reason, besides the conventional fermentation approaches, the applicability of cloning, expression studies and genetic engineering has been implemented for the past few years to accomplish the abovesaid benefits. The site-directed mutagenesis as well as knocking out have also validated their prominent role in microbe-based phytase production with enhanced levels. The present review provides detailed information on recent insights on the modification of phytases through heterologous expression and protein engineering to make thermostable and protease-resistant phytases.

Expression of B. subtilis Phytase gene driven by fruit specific E8 promoter for enhanced minerals, metabolites and phytonutrient in cucumber fruit.

The fruit nutrigenomics is an interesting and important research area towards nutrition enhancement. The phytic acid is one of the major antinutrient compound, present in seeded fruits and crops. It hinders the absorption of iron (Fe), zinc (Zn), magnesium (Mg), potassium (K) and calcium (Ca), causing mineral deficiencies. In the present study, the BsPhy gene was overexpressed in the cucumber fruits using the tomato fruit specific E8 and constitutive CaMV 35S promoter. The E8 promoter imparted heterologous expression of GUS gene in cucumber fruits, furthermore, the fruit specific expression of E8 promoter with BsPhy gene was confirmed in transgenics (E8::BsPhy) using anti rabbit-phytase antibody. The physio-biochemical analysis of transgenics revealed, maximum phytase activity in E8::BsPhy cucumber fruits at 10 days after anthesis (DAA) compared to 35S::BsPhy and wild-type (WT) fruits. Consequently, E8::BsPhy fruits also showed increased amount of inorganic phosphorus (Pi), total phosphorus (P), minerals (Zn, Fe, Mg, K, Ca), total carotenoid and other macronutrients at 10 DAA compared to 35S::BsPhy fruits. The metabolite profiling of fruits (10 DAA) showed increased sugars, amino acids, sugar acids and polyols, in both E8::BsPhy and 35S::BsPhy transgenics suggesting higher phytate metabolism, compared to WT fruits. Interestingly, both the transgenic fruits showed higher fruit biomass and yield along with improved nutritional quality, which can be attributed to increased P and Zn contents in transgenic fruits, compared to WT fruits. Our findings reveal that the BsPhy gene enhances minerals and macronutrients in transgenic cucumber fruits making it nutritious and healthy.

Structure of a cereal purple acid phytase provides new insights to phytate degradation in plants.

Grain phytate, a mixed metal ion salt of inositol hexakisphosphate, accounts for 60%-80% of stored phosphorus in plants and is a potent antinutrient of non-ruminant animals including humans. Through neofunctionalization of purple acid phytases (PAPhy), some cereals such as wheat and rye have acquired particularly high mature grain phytase activity. As PAPhy activity supplies phosphate, liberates metal ions necessary for seedling emergence, and obviates antinutrient effects of phytate, its manipulation and control are targeted crop traits. Here we show the X-ray crystal structure of the b2 isoform of wheat PAPhy induced during germination. This high-resolution crystal structure suggests a model for phytate recognition that, validated by molecular dynamics simulations, implicates elements of two sequence inserts (termed PAPhy motifs) relative to a canonical metallophosphoesterase (MPE) domain in forming phytate-specific substrate specificity pockets. These motifs are well conserved in PAPhys from monocot cereals, enzymes which are characterized by high specificity for phytate. Tested by mutagenesis, residues His229 in PAPhy motif 4 and Lys410 in the MPE domain, both conserved in PAPhys, are found to strongly influence phytase activity. These results explain the observed phytase activity of cereal PAPhys and open the way to the rational engineering of phytase activity in planta.

Low specific growth rate and temperature in fed-batch cultures of a beta-propeller phytase producing Pichia pastoris strain under GAP promoter trigger increased KAR2 and PSA1-1 gene expression yielding enhanced extracellular productivity.

Previously, we showed that the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) could produce and secrete the beta-propeller phytase FTEII in an active form under the control of the AOX1 promoter and methanol as the inductor. In this work, we engineered P. pastoris strains to construct a constitutive P. pastoris expression system (GAP promoter) and extracellularly produce the phytase FTEII. We optimized the culture conditions to increase the extracellular volumetric phytase productivity (Qp) and evaluated the impact of the optimization process on the physiological response of the host. Moreover, we analyzed the expression levels of the FTEII gene and endogenous genes for P. pastoris cells in cultures with the lowest and highest Qp to understand which processes (from heterologous gene expression to protein secretion) might be responsible for the increase in Qp. The results indicate that a low specific growth rate and temperature in the fed-batch phase increases the Qp, which was correlated with an upregulation of the KAR2 and PSA1-1/MPG1 genes rather than increased heterologous gene transcription.

Enhancing the Thermostability of Phytase to Boiling Point by Evolution-Guided Design.

The good thermostability of enzymes is an important basis for their wide application in industry. In this study, the phytase APPA from Yersinia intermedia was designed by evolution-guided design. Through the collection of homologous sequences in the NCBI database, we obtained a sequence set composed of 5,569 sequences, counted the number and locations of motif N-X-T/S, and selected the sites with high frequency in evolution as candidate sites for experiments. Based on the principle that N-glycosylation modification sites are located on the protein surface, 13 mutants were designed to optimize the number and location of N-glycosylation sites. Through experimental verification, 7 single mutants with improved thermostability were obtained. The best mutant, M14, with equal catalytic efficiency as the wild-type was obtained through combined mutation. The half-life (t1/2) value of mutant M14 was improved from 3.32 min at 65°C to 25 min of at 100°C, allowing it to withstand boiling water treatment, retaining approximately 75% initial activity after a 10-min incubation at 100°C. Differential scanning calorimetry analysis revealed that while the mutants' thermodynamic stability was nearly unchanged, their kinetic stability was greatly improved, and the combined mutant exhibited strong refolding ability. The results of a in vitro digestibility test indicated that the application effect of mutant M14 was about 4.5 times that of wild-type APPA, laying a foundation for its industrial application. IMPORTANCE Due to the harsh reaction conditions of industrial production, the relative instability of enzymes limits their application in industrial production, such as for food, pharmaceuticals, and feed. For example, the pelleting process of feed includes a brief high temperature (80 to 85°C), which requires the enzyme to have excellent thermostability. Therefore, a simple and effective method to improve the thermostability of enzymes has important practical value. In this study, we make full use of the existing homologous sequences (5,569) in the database to statistically analyze the existence frequency of N-X-T/S motifs in this large sequence space to design the phytase APPA with improved thermostability and a high hit rate (~50%). We obtained the best combination mutant, M14, that can tolerate boiling water treatment and greatly improved its kinetic stability without damaging its specific activity. Simultaneously, we proved that its performance improvement is due to its enhanced refolding ability, which comes from N-glycan modification rather than amino acid replacement. Our results provide a feasible and effective method for the modification of enzyme thermostability.