A Novel Phytase appA from <Emphasis Type="Italic">Citrobacter amalonaticus</Emphasis> CGMCC 1696: Gene Cloning and Overexpression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL⁻¹, and the enzyme activity level reached 15,000 U·mL⁻¹, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg⁻¹. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.     

Heterologous interferons synthesis in yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed. There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features of its metabolism.     

Cloning,expression, and characterization of thermostable Manganese superoxide dismutase from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> var. <Emphasis Type="Italic">levisporus</Emphasis>

A superoxide dismutase (SOD) gene of Thermoascus aurantiacus var. levisporus, a thermophilic fungus, was cloned, sequenced, and expressed in Pichia pastoris and its gene product was characterized. The coding sequence predicted a 231 residues protein with a unique 35 amino acids extension at the N-terminus indicating a mitochondrial-targeting sequence. The content of Mn was 2.46 μg/mg of protein and Fe was not detected in the purified enzyme. The enzyme was found to be inhibited by NaN₃, but not by KCN or H₂O₂. These results suggested that the SOD in Thermoascus aurantiacus var. levisporus was the manganese superoxide dismutase type. In comparison with other MnSODs, all manganese-binding sites were also conserved in the sequence (H88, H136, D222, H226). The molecular mass of a single band of the enzyme was estimated to be 21.7 kDa. The protein was expressed in tetramer form with molecular weight of 68.0 kDa. The activity of purified protein was 2,324 U/mg. The optimum temperature of the enzyme was 55°C and it exhibited maximal activity at pH 7.5. The enzyme was thermostable at 50 and 60°C and the half-life at 80°C was approximately 40 min.     

Isolation of a Novel Lipase Gene from <Emphasis Type="Italic">Serratia liquefaciens</Emphasis> S33 DB-1, Functional Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its Properties

A new lipase gene designated as SlLipA was isolated from Serratia liquefaciens S33 DB-1 by the genomic-walking method. The cloned gene contained an open reading frame (ORF) of 1,845 bp encoding 615 amino acids with a conserved GXSXG motif. Genome sequence analysis showed that an aldo/keto reductase gene closed to the SlLipA gene. The lipase gene was cloned into the expression vector pPICZαA and successfully integrated into the heterologous host, methylotrophic yeast Pichia pastoris GS115. Five transformants could be expressed as secreted recombinant proteins with the high activity on Triglyceride–Agarose plate and as candidates to produce the recombinant enzyme. A C-terminal His tag was used for its purification. The lipase activity of different transformants against substrate para-nitrophenyl laurate (p-NPL) varied from 14 to 16 U ml⁻¹. For the substrates para-nitrophenyl caprate (p-NPC), p-NPL, para-nitrophenyl myristate (p-NPM), para-nitrophenyl palmitate (p-NPP), and para-nitrophenyl stearate (p-NPS), the specific activity was shown to be preferred to long acyl chain length of p-NPS.     

Expression of hepatitis B surface antigen in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> utilizing glyceraldeyhyde-3-phosphate dehydrogenase promoter of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus ‘S’ gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited ∼20–22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.     

Identification of Three <Emphasis Type="Italic">Superoxide dismutase</Emphasis> Genes from a <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

We report the characterization of three Superoxide dismutase (sod) genes isolated from a bacterium in the Geobacillus genus. We isolated the bacterium from high-temperature pond mud and used 16S rRNA gene sequence to confirm its identity in the Geobacillus genus. The three genes Mn-sod, Fe/Mn-sod, and Cu/Zn-sod were cloned and analyzed. Their open reading frames are Mn-sod: 615 bp encoding a 204 amino acid protein; Fe/Mn-sod: 1,236 bp encoding a 411 amino acid protein; Cu/Zn-sod: 522 bp encoding a 173 amino acid protein. When these sod genes were expressed in Escherichia coli, only Mn-SOD was able to be purified. The activity of the purified Mn-SOD we got was about 2,730 U/mg. Studies of this Mn-SOD showed that it was thermostable at 60°, had 70% activity at 80° after 2.5 h, and still had 30% activity at 90° after 2.5 h. Mn-SOD activity required the ion Mn²⁺. Based on gel electrophoresis, we deduced that this Mn-SOD was a homotetramer. No activity was detected after the other two genes (Fe/Mn-sod, Cu/Zn-sod) were expressed in Escherichia coli, but activities were detected when expressed in Pichia pastoris.     

Cloning and functional expression of thermostable β-glucosidase gene from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis>

A thermostable β-glucosidase (BGLI) was purified from Thermoascus aurantiacus IFO9748, and the gene (bgl1) encoding this enzyme was cloned and expressed in yeast Pichia pastoris. The deduced amino acid sequence encoded by bgl1 showed high similarity with the sequence of glycoside hydrolase family 3. The recombinant enzyme was purified and subjected to enzymatic characterization. Recombinant BGLI retained more than 70% of its initial activity after 1 h of incubation at 60°C and was stable in the pH range 3–8. The optimal temperature for enzyme activity was about 70°C and the optimal pH was about 5. P. pastoris expressing recombinant BGLI became able to utilize cellobiose as a carbon source.     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

cDNA cloning,functional expression and antifungal activities of a dimeric plant defensin SPE10 from <Emphasis Type="Italic">Pachyrrhizus erosus</Emphasis> seeds

SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal -mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far.Nucleotide sequence data reported in this paper are available in the DDBJ/EMBL/GenBank database under accession number AY679170     

High production of laccase B from <Emphasis Type="Italic">Trametes</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The coding sequence of a laccase isozyme from Trametes sp. AH28-2 was cloned in pPIC9K vector and heterologously overexpressed in the yeast Pichia pastoris strain GS115. In the minimal medium containing 0.3 mM CuSO₄ and 0.6% alanine, the maximum yield of the recombinant laccase rLacB reached 32,000 U/l (1,012 U/mg), slightly higher than that of the native enzyme nLacB (∼30,000 U/l, 1,356 U/mg). The enzymatic properties of rLacB were different from those of nLacB as well. Regardless of the inferior thermal stability, rLacB had much better stability at both neutral and basic pH range compared to nLacB. In addition, the dye decolorization potential of rLacB was similar to that of nLacB.     

Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2) in methylotrophic yeast Pichia pastoris and its characterizations

To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2) cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris, we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique, which was composed of frequently used codons in the highly expressed Pichia pastoris genes. Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing. The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2. The plasmid was transformed into GS115 cells of the methylotrophic yeast, Pichia pastoris by electroporation, and we got the expression cell through phenotype selection and induction with methanol. Separation, purification, and bioactivity analysis of the expressed products were performed. __________ Translated from Microbiology, 2006, 33(1): 1–6 [译自: 微生物学通报]     

Enhancing survival of <Emphasis Type="Italic">Escherichia coli</Emphasis> by increasing the periplasmic expression of Cu,Zn superoxide dismutase from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The gene for the Cu,Zn superoxide dismutase (Cu,ZnSOD) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli LMG194. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the E. coli periplasmic expression vector pBAD/gIIIA, yielding pBAD-1. E. coli was transformed using the constructed plasmid pBAD-1 and induced by adding 0.02% l-arabinose to express Cu,ZnSOD protein. The results indicated that Cu,ZnSOD enzyme activity in the periplasmic space was about fivefold to sixfold higher in the recombinant E. coli strains bearing the sod gene than in the control strains. The yields of Cu,ZnSOD were about threefold higher at 48 h than at 24 h in the recombinant E. coli cells. Significantly higher survival of strains was obtained in cells bearing the sod gene than in the control cells when the cells were treated by heat shock and superoxide-generating agents, such as paraquat and menadione.     

Recombinant Pichia pastoris overexpressing bioactive phytase

Phytase genephyA2, whose signal peptide encoding sequence and intron sequence had been removed, was modified. The Arg-encoding codons CGG and CAG inphyA2 were mutated into synonymous codon AGA. The modifiedphyA2 was fused behind a-factor signal sequence under the control ofAOX1 promoter in plasmid pPIC9, then introduced into the hostPichia pastoris by electroporation. The results of Southern blotting analysis and Northem blotting analysis demonstrated that thephyA2 gene had integrated into the genome ofP. pastoris and transcribed. The result of SDS-PAGE of the phytase expressed by P.pastoris showed that the modifiedphyA2 had been overexpressed and secreted. The concentration of the phytase expressed by P.pastoris with modifiedphyA2 exceeded 15 000 U/mL, which had a 3 000-fold increase over that of originAspergillus niger 963 and was 37 times higher than that of recombinantP. pastoris with non-modifiedphyA2. Project supported by the “863” program, National Science and Technology Commission of China.     

Translation elongation factor 1-α gene from <Emphasis Type="Italic">Pichia pastoris</Emphasis>: molecular cloning,sequence, and use of its promoter

The gene encoding translation elongation factor 1-α from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P _TEF1 ) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (P _GAP ) by using a bacterial lipase gene as a reporter gene. P _TEF1 demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P _GAP . The sequence of the gene was deposited in GenBank under accession no. EF014948.     

A site-directed integration system for the nonuniversal CUGSer codon usage species Pichia farinosa by electroporation

Halotolerant yeast, Pichia farinosa, is a valuable yeast strain in fermentation industry because it produces high yield of glycerol and xylitol, and can tolerate both contamination and high-density growth during fermentation. However, the lack of genetic manipulation tools makes it less popular as a gene engineering strain. Expression systems commonly used in other yeast systems, such as Saccharomyces cerevisiae and Pichia pastoris cannot be used in P. farinosa because it translates universal Leu codon CUG as Ser. Here we reported a modified expression vector and a transformation system with enhanced efficiency in P. farinosa. The results showed that cells of OD₆₀₀ 0.8–1.0 with DTT treatment can obtain high transformation efficiency. The optimized electroporation condition was 900 V, 25 μF, and 200 Ω. The DNA concentration did not influence the transformation. Our system provides the potential not only for applying P. farinosa as an industrial strain of gene engineering, but also for studying gene function in its native host.     

High level expression of human endostatin in Pichia pastoris using a synthetic gene construct

Endostatin, a 20-kDa C-terminal fragment derived from type XVIII collagen, is a potent angiogenesis inhibitor and an antitumor factor. To improve the production of recombinant human endostatin on increasing demand in clinical practice, we constructed an artificial gene encoding its mature peptide sequence in human collagen XVIII. The synthetic gene consisted of 20 codons in preference in methylotropic yeast—Pichia pastoris and was cloned into expression vector pPICZαA; and the recombinant protein was expressed in P. pastoris strain SMD1168 and purified to near homogeneity using heparin affinity chromatography. The amount of expressed recombinant protein in cultural media using described strategy was 80 mg/l in shake flask cultivation and 435 mg/l in high-density bioreactor fermentation. Methylthiazolium assay demonstrated that human endostatin expressed in P. pastoris using artificial synthetic gene of preference in P. pastoris was able to inhibit the acidic fibroblast growth factor-induced proliferation of endothelial cells in vitro.     

Construction of a <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> glucoamylase gene suitable for expression in distinct host: introns spliced artificially by PCR

Glucoamylase is an industrially extremely important enzyme in the fermentative production of ethanol, used in the enzymatic conversion of starch into high glucose and fructose syrups. The aim of this study is to construct a Rhizopus arrhizus glucoamylase gene (RaGA)—introns artificially spliced by PCR—suitable for expression in S. cerevisiae host and tried expressing in Picha pastoris. In previous work, we failed in amplifying glucoamylase gene from R. arrhizus by RT-PCR, so several primers were designed to splicing the introns by PCR in vitro. Sequence analysis shown that all introns in the RaGA were deleted correctly and no mutant was induced in the extrons compared with the RaGA gene originally cloned. The RaGA gene artificially constructed was transferred into P. pastoris integrative expression vectors pPIC9 (containing а-factor). Consequently, the plasmids pPIC9-RaGA was lineared by SacI and inserted into P. pastoris GS115 (His⁻) genome downstream of the 5′AOX1 promoter by the method of electroporation. Induction by 0.75% methanol for 72 h led to synthesis of secreted glucoamylase. So it is demonstrated that the glucoamylase gene has been expressed in and secreted from P. pastoris.     

Cloning and characterization of a chitinase gene <Emphasis Type="Italic">Lbchi31</Emphasis> from <Emphasis Type="Italic">Limonium bicolor</Emphasis> and identification of its biological activity

Chitinases are digestive enzymes that break down glycosidic bonds in chitin. In the current study, an endochitinase gene Lbchi31 was cloned from Limonium bicolor. The cDNA sequence of Lbchi31 was 1,107 bp in length, encoding 322 amino acid residues with a calculated molecular mass of 31.7 kDa. Clustal analysis showed that there was a highly conserved chitin-binding domains in Lbchi31 protein, containing four sulfide bridges. The Lbchi31 gene was inserted into the pPIC9 vector and transferred into yeast Pichia pastoris GS115 and KM71 for heterologous expression. The transformant harboring the Lbchi31 gene showed a clearly visible protein band with a molecular mass of more than 31 kDa in the SDS-PAGE gel, indicating that it had been translated in P. pastoris. Enzyme characterization showed that the optimal reaction condition for chitinase LbCHI31 activity was: 40°C, pH of 5.0 and 5 mmol l⁻¹ of Mn²⁺. The maximum enzyme activity was 0.88 U ml⁻¹ following exposure to the cell wall chitin of Valsa sordida. The LbCHI31 enzyme can efficiently degrade cell wall chitin of the phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, V. sordida, Septoria tritici and Phytophthora sojae, suggesting that it has the biocontrol function to fungal phytopathogen.     

Modification of a gene encoding hybrid xylanase and its expression in Pichia pastoris

To obtain the protein expression of a hybrid xylanase in yeast, the gene encoding it was modified according to the codon bias of Pichia pastoris and expressed extracellularly in this yeast as an active xylanase, MBtx, exhibited a molecular mass of approximately 35 kDa on SDS–PAGE. The pH behavior of MBtx in terms of both activity and stability was similar to that of Btx, original gene product in Escherichia coli, while a certain difference was observed in optimal temperature for activity and in thermal stability. HPLC analysis revealed the xylan in wheat could be hydrolyzed by MBtx and the major hydrolysis product was xylotriose. These results showed codon usage played a key role in regulating the expression of the hybrid xylanase in P. pastoris and the recombinant hybrid xylanase, MBtx, produced by P. pastoris could be potentially useful in feed industry.