High level expression of a recombinant acid phytase gene in Pichia pastoris

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.     

植酸酶phyAm基因结构延伸突变改善酶的热稳定性

将来源于黑曲霉N25的植酸酶基因phyA^m重组于大肠杆菌表达载体pET-30b（＋）,以重组表达载体pET30b-FphyA^e为模板经PCR扩增获得结构延伸突变植酸酶基因phyA^m（在植酸酶基因C端增加了来源于pET-30b-FphyA^m载体上13氨基酸残基）。含突变基因的重组表达载体pPIC9k-phyA^e在GS115酵母中表达。纯化的突变酶pp-NP^e与野生型酶PP-NP^m-8相比：PP-NPA^e的最适反应温度上升了3气,75℃处理10min,热稳定性提高21％,比活力略有提高。最适反应pH为5．6,有效pH范围pH4,6到pH6．6。比未突变酶扩大了0.4单位。     

植酸酶phyA基因在解脂耶氏酵母po1h中的表达

本实验通过PCR方法从毕赤酵母GS115-phyA中扩增出不含有信号肽及内含子的黑曲霉NRRL3135植酸酶phyA基因,并将其克隆到表达载体pINA1297中,得到表达载体pINA1297-phyA,利用醋酸锂转化法将线性化载体转化到解脂耶氏酵母po1h中,通过YNBcasa和PPB平板筛选出阳性表达菌株,阳性菌株在YM培养基中28℃培养6d后酶活达到最大为636.23U/mL。表达上清经SDS-PAGE分析得到表达植酸酶分子量约为130kDa,但通过去糖基化处理后其分子量变为51kDa,与理论值相符。经过酶学性质分析表明重组植酸酶最适pH为5.5,最适温度为55℃,该酶在pH2.0~8.0处理1h后仍有较高酶活,并且90℃处理10min后还有86.08%的残留酶活,其抵抗胃蛋白酶和胰蛋白酶能力也较强。     

植酸酶基因的多点突变及在毕赤酵母中的高效表达

根据毕赤酵母基因的密码子选择偏爱性,不改变其编码氨基酸序列,对来源于黑曲霉N25植酸酶phyA基因,进行了突变,构建了含有正确突变的酵母表达载体pPIC9k-phyAm-4,电击转化毕赤酵母,获得优化了密码子的重组酵母转化子。经PCR鉴定表明,植酸酶基因已整合到酵母基因组中; 表达产物的SDS-PAGE分析表明,酶蛋白分子大小为70.15KD。Southern blotting结果表明,phyA基因整合到酵母染色体DNA中;转化子酶活测定结果表明,经密码子优化的重组酵母PP-NPm-4-2酶活可达136900U/ml,比Arg没有优化的PP-NPm-8 (47600 Uoml-1)酶活高约2.8倍。     

Role of glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris

Economical and thermostable phytase enzymes are needed to release phytate-phosphorus in plant foods for human and animal nutrition and to reduce phosphorus pollution of animal waste. Our objectives were to determine if a methylotrophic yeast, Pichia pastoris, was able to express a phytase gene (phyA) from Aspergillus niger efficiently and if suppression of glycosylation by tunicamycin affected its functional expression. The gene (1.4 kb) was inserted into an expression vector pPICZalphaA with a signal peptide alpha-factor, under the control of AOX1 promoter. The resulting plasmid was transformed into two P. pastoris strains: KM71 (methanol utilization slow) and X33 (wild-type). Both host strains produced high levels of active phytase (25-65 units/ml of medium) that were largely secreted into the medium. The expressed enzyme was cross-reacted with the polyclonal antibody raised against the wild-type enzyme and showed two pH optima, 2.5 and 5.5, and an optimal temperature at 60 degrees C. Compared with the phyA phytase overexpressed by A. niger, this phytase had identical capacity in hydrolyzing phytate-phosphorus from soybean meal and slightly better thermostability. Deglycosylation of the secreted phytase resulted in reduction in the size from 95 to 55 kDa and in thermostability by 34%. Tunicamycin (20 microg/ml of medium) resulted in significant reductions of both intracellular and extracellular phytase activity expression. Because there was no accumulation of intracellular phytase protein, the impairment did not seem to occur at the level of translocation of phytase. In conclusion, glycosylation was vital to the biosynthesis of the phyA phytase in P. pastoris and the thermostability of the expressed enzyme.     

植酸酶产生菌黑曲霉N14的诱变选育及其基因分析

以植酸酶产生菌黑曲霉03214为出发菌株,经紫外线和亚硝基胍诱变,获得了产酶活性较出发菌株提高了22．3％,达422IU／ml发酵液的突变菌株黑曲霉N14,其最适pH值为2．5,最适温度为50℃。通过对黑曲霉N14植酸酶phyA基因进行PCR扩增,获得了一条长约1．5kb的特异性产物。以pMD18-T为载体,构建了含有目的基因片段的重组质粒。DNA序列测定表明,目的基因片段含有植酸酶phyA基因的完整序列(GenBank Accession：AY426977),phyA基因全长1506bp,其中包含一段长102bp的内含子,编码467个氨基酸,有10个潜在的糖基化位点,5’端有一编码19个氨基酸的信号肽序列。实验结果为植酸酶基因工程菌的构建奠定了基础。     

Site-directed mutagenesis of Aspergillus niger NRRL 3135 phytase at residue 300 to enhance catalysis at pH 4.0

Increased phytase activity for Aspergillus niger NRRL 3135 phytaseA (phyA) at intermediate pH levels (3.0-5.0) was achieved by site-directed mutagenesis of its gene at amino acid residue 300. A single mutation, K300E, resulted in an increase of the hydrolysis of phytic acid of 56% and 19% at pH 4.0 and 5.0, respectively, at 37 degrees C. This amino acid residue has previously been identified as part of the substrate specificity site for phyA and a comparison of the amino acid sequences of other cloned fungal phytases indicated a correlation between a charged residue at this position and high specific activity for phytic acid hydrolysis. The substitution at this residue by either another basic (R), uncharged (T), or acidic amino acid (D) did not yield a recombinant enzyme with the same favorable properties. Therefore, we conclude that this residue is not only important for the catalytic function of phyA, but also essential for imparting a favorable pH environment for catalysis.     

phyA基因新型表达质粒的构建及其在大肠杆菌中的高效表达

利用自行筛选、鉴定的黑曲霉F246,根据植酸酶基因(phyA)成熟肽编码序列设计引物,直接PCR扩增phyA,经酶切分析、DNA测序和氨基酸序列分析证实phyA基因克隆成功。从pMD18T-phyA克隆中获得phyA编码序列,将其与pET30a 质粒连接,构建pET30a -phyA重组质粒,并在大肠杆菌中获得了高效表达。重组质粒经IPTG诱导表达,SDS-PAGE特异区带分子量为50kDa,此重组蛋白占大肠杆菌可溶性蛋白的36.62％,酶活性较天然植酸酶高8倍以上。因此,该phyA基因具有正常的生物学功能,对其进行深入研究,为大量获得高活性植酸酶以及开发新型微生态制剂奠定了基础。     

Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus.

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.     

Biochemical properties of a thermostable phytase from Neurospora crassa

A gene (Ncphy) encoding a putative phytase in Neurospora crassa was cloned and expressed in Pichia pastoris, and the biochemical properties of the recombinant protein were examined in relation to the phytic acid hydrolysis in animal feed. The recombinant phytase (rNcPhy) hydrolyzed phytic acid with a specific activity of 125 U mg-1, Km of 228 micromol L-1, Vmax of 0.31 nmol (phosphate) s-1 mg-1, a temperature optimum of 60 degrees C and a pH optimum of 5.5 and a second pH optimum of 3.5. The enzyme displayed pH stability around pH 3.5-9.5 and showed satisfactory thermostability at 80 degrees C. The phytase from N. crassa has potential for improving animal feed processing at higher temperatures.     

人工合成的黑曲霉NRRL3135菌株植酸酶基因在毕酵母系统中的高效表达

本文以黑曲霉（Aspergillus niger)NRRL3135菌株植酸酶基因为对象,通过基因人工合成的方法去除了该基因的内含子与信号肽编码序列,换用在毕赤酵母（Pichia pastoris）中使用频率较高的密码子以优化其表达。该人工合成植酸酶基因（PhyA-as)以N端融合的方式正确插入到毕赤酵母表达载体pPICZαA。通过电击将重组表达载体整合人酵母染色体DNA中得到重组转化子。SDS－PAGE结果与表达产物酶这性质研究表明植酸酶得到分泌表达,且与天然产物性质基本一致。筛选得若干株高产基因工程菌,其中SPAN－Ⅲ菌株达到了在摇庆培养条件下,每毫升发酵液产生165000u植酸酶的水平,基本满足工业化生产的要求。     

Dephosphorylation of phytate by using the Aspergillus niger phytase with a high affinity for phytate.

A phytase (EC 3.1.3.8) with a high affinity for phytic acid was found in Aspergillus niger SK-57 and purified to homogeneity in four steps by using ion-exchange chromatography (two types), gel filtration, and chromatofocusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme gave a single stained band at a molecular mass of approximately 60 kDa. The Michaelis constant of the enzyme for phytic acid (18.7 +/- 4.6 microM) was statistically analyzed. In regard to the orthophosphate released from phytic acid, a significant difference between a low K(m) phytase from A. niger SK-57 and a high K(m) phytase from Aspergillus ficuum was recognized.     

Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase's activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60 degrees C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.     

Screening, cloning and overexpression of Aspergillus niger phytase (phyA) in Pichia pastoris with favourable characteristics

AIMS: Using gene cloning and overexpression to obtain a potential industrial phytase as a feed additive to upgrade the nutritional quality of phytate-rich seed-based animal feed. METHODS AND RESULTS: A phyA gene from a high extracellular phytase-producing Aspergillus niger sp. was cloned and overexpressed in Pichia pastoris GS115 using the secretive expression vector pPICZalphaA. After cultivation for 4 days in buffered methanol complex medium (BMMY) containing methanol for induction, catalytically active phytase was secreted as a predominantly extracellular protein. The activity of the expressed phytase in fermented broth was 30 000-fold higher than that of native phytase with a specific activity of 503 U mg(-1). The Lineweaver-Burk plot indicated K(m) values of 0.196 mmol l(-1) for sodium phytate and 18.16 mmol l(-1) for p-nitrophenylphosphate (pNPP). Thermostability studies showed that recombinant phytase retained 70% activity after exposure to 90 degrees C for 5 min and 65% activity after 30 min, much higher than for commercial phytase. CONCLUSIONS: The higher activity and high thermostability of recombinant phytase enable it to withstand the temperatures of the feed pelleting process. SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of this recombinant phytase, especially the good thermostability, are likely to render it of potential industrial importance.     

Design and expression of a synthetic phyC gene encoding the neutral phytase in Pichia pastoris

The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed andsynthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering theprotein sequence.The expression vector,pP9K-phyCs,was linearized and transformed in P.pastoris.Theyield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium(BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale,respectively,improving over 90 folds compared with the wild-type isolate.Purified enzyme showed temperature optimumof 70℃ and pH optimum of 7.5.The enzyme activity retained 97% of the relative activity afterincubation at 80℃ for 5 min.Because of the heavy glycosylation the expressed phytase had a molecularsize of approximately 51 kDa.After deglycosylation by endoglycosylase H (EndoH_f),the enzyme had anapparent molecular size of 42 kDa.Its property and thermostability was affected by the glycosylation.     

来源于Escherichia coli的高比活植酸酶基因的高效表达

高效表达高比活植酸酶是进一步提高植酸酶发酵效价、降低植酸酶生产成本的一个有效途径。对源于Escherichiacoli的高比活植酸酶基因appA ,按照毕赤酵母 (Pichiapastoris)密码子的偏爱进行了密码子优化改造。该改造后的基因appA m按正确的阅读框架融合到毕赤酵母表达载体pPIC9上的α 因子信号肽编码序列 3′端 ,通过电击转化得到重组转化子。对重组毕赤酵母的Southernblotting分析证实植酸酶基因已整合到酵母基因组中 ,并确定了整合基因的拷贝数。Northernblotting分析证实植酸酶基因得到了正常转录。SDS PAGE分析和表达产物的研究表明 ,植酸酶得到了高效分泌表达 ,在 5L发酵罐中植酸酶蛋白表达量达到 2 5mg mL发酵液 ,酶活性 (发酵效价 )达到 7 5× 10 6 IU mL发酵液以上 ,大大高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

黑曲霉N-2植酸酶phy4基因的克隆及其重组酵母表达载体的构建

根据已发表的植酸酶phyA基因序列设计并合成1对引物,应用PCR技术,以黑曲霉N-2总DNA为模板,扩增出不包含假定信号肽序列的phyA基因,将其克隆到pMD18-T载体中,测定其核苷酸序列,并推导其氨基酸序列。该基因全长为1350bp,与已发表的黑曲霉NRRL3135的phyA基因的同源性为92．4％(不计内含子),编码1个含449个氨基酸残基的蛋白质,推导的氨基酸序列同源性为95．1％。将该基因与分泌型载体pPIC9K连接,构建了植酸酶基因的重组酵母表达载体pPIC9K／phyA。     

Properties of beta-propeller phytase expressed in transgenic tobacco

Phytases are enzymes that liberate inorganic phosphates from phytate. In a previous study, a beta-propeller phytase (168phyA) from Bacillus subtilis was introduced into transgenic tobacco, which resulted in certain phenotypic changes. In the study described herein, the recombinant phytase (t168phyA) was purified from transgenic tobacco to near homogeneity by a three-step purification scheme. The biochemical properties and kinetic parameters of t168phyA were compared with those of its counterpart from B. subtilis. t168phyA was glycosylated, and it showed a 4 kDa increase in molecular size in SDS-PAGE (44 kDa vs. 40 kDa). Although its thermostability remained unchanged, its temperature optimum shifted from 60 degrees C to 45-50 degrees C and its pH optimum shifted from pH 5.5 to 6.0. Kinetic data showed that the t168phyA had a lower Kcat, but a higher Km than the native enzyme. Despite these changes, t168phyA remained catalytically active and has a specific activity of 2.3 U/mg protein. These results verify the activity of recombinant Bacillus phytase that is expressed in plants.     

黑曲霉N25植酸酶phyA基因的克隆及序列分析

通过对黑曲霉N2 5植酸酶phyA基因PCR扩增 ,获得了一条长约 1 6kb的特异性PCR产物 ,并进行了酶切鉴定。然后在pUC1 8质粒中构建了含有目的基因片段的克隆质粒pFNP 1。DNA序列测定表明 ,目的基因片段含有植酸酶phyA基因的完整序列 ,phyA基因全长1 50 6bp,其中包含一段长 1 0 2bp的内含子 ,编码 467个氨基酸 ,5’端有一段编码 1 9个氨基酸的信号肽序列。黑曲霉N2 5与产植酸酶酶活最高的天然黑曲霉标准菌株NRRL31 35的植酸酶phyA基因 (GenBankAccession :M94550 )相比较 ,其同源性为 96 746% ,编码的氨基酸序列同源性为 97 64%。将黑曲霉N2 5植酸酶phyA基因序列及其相应的氨基酸序列在国际基因库中注册 (注册号分别为 :AF2 1 881 3,AAF2 5481 1 ) ,此基因是目前中国在国际基因库中注册的第一个植酸酶phyA基因。     

Fungal phyA gene expressed in potato leaves produces active and stable phytase

Fungal phyA gene from Aspergillus ficuum (niger) was cloned and expressed in potato leaves. The recombinant enzyme was stable and catalytically active. The expressed protein in the leaves of the dicotyledonous plant retained most physical and catalytic properties of the benchmark A. ficuum phytase. The expressed enzyme was, however, 15% less glycosylated than the native phytase. The usual bi-hump pH optima profile, which is characteristic of the fungal phytase, was altered; however, the pH optimum at 5.0 was unchanged for phytate and at 4.0 for synthetic substrate p-nitrophenyl phosphate. The temperature was, however, unchanged. The expressed phytase was found to be as sensitive as the native enzyme to the inhibitory action of pseudo substrate, myo-inositol hexasulfate, while losing about 90% of the activity at 20 microM inhibitor concentration. Similar to the benchmark phytase, the expressed phytase in leaves was completely inactivated by Arg modifier phenylglyoxal at 60 nM. In addition, the expressed phytase in the leaves was inhibited by antibody raised against a 20-mer internal peptide, which is present on the surface of the molecule as shown by the X-ray deduced 3D structure of fungal phytase. Taken together, the biochemical evidences indicate that fungal phytase when cloned and expressed in potato leaves produces a stable and active biocatalyst. 'Biofarming,' therefore, is an alternative way to produce functional hydrolytic enzymes as exemplified by the expression of A. ficuum (niger) phyA gene in potato leaf.