Glucose oxidase (GOD) is an oxygen-consuming dehydrogenase that can catalyze the production of gluconic acid hydrogen peroxide from glucose, and its specific mechanism of action makes it promising for applications, while the low catalytic activity and poor thermostability have become the main factors limiting the industrial application of this enzyme. In this study, we used the glucose oxidase AtGOD reported with the best thermostability as the source sequence for phylogenetic analysis to obtain the GOD with excellent performance. Six genes were screened and successfully synthesized for functional validation. Among them, the glucose oxidase AhGODB derived from Aspergillus heteromorphus was expressed in Pichia pastoris and showed better thermostability and catalytic activity, with an optimal temperature of 40 ℃, a specific activity of 112.2 U/mg, and a relative activity of 47% after 5 min of treatment at 70 ℃. To improve its activity and thermal stability, we constructed several mutants by directed evolution combined with rational design. Compared with the original enzyme, the mutant T72R/A153P showcased the optimum temperature increasing from 40 to 50 ℃, the specific activity increasing from 112.2 U/mg to 166.1 U/mg, and the relative activity after treatment at 70 ℃ for 30 min increasing from 0% to 33%. In conclusion, the glucose oxidase mutants obtained in this study have improved catalytic activity and thermostability, and have potential for application.
Glucose Oxidase/chemistry* ; Enzyme Stability ; Aspergillus/genetics* ; Pichia/metabolism* ; Temperature ; Catalysis ; Fungal Proteins/metabolism* ; Hot Temperature

To prepare antibodies that specifically recognize the conserved domain in the large extracellular loop of the CD63 protein, we expressed anti-CD63 single-chain variable fragment (scFv) antibody in Pichia pastoris in a secreted form. The purified expression product was found to bind specifically with CD63 protein and recognize CD63 on the surface of SK-MEL-28 cells. The variable region of the anti-CD63 monoclonal antibody in an anti-CD63-positive cell line was sequenced. The anti-CD63 scFv consisted of a variable heavy chain and a variable light chain linked by a flexible peptide was then designed. After codon optimization, the gene was synthesized and cloned into the expression plasmid pPICZα-A. The SacI-linearized plasmid was electroporated into P. pastoris X33, and 1% methanol were used to induce the expression of scFv. The fermentation supernatant was purified by Ni column. Anti-CD63 scFv was identified by SDS-PAGE and Western blotting, and its biological activities were analyzed by immunoblotting, immunofluorescence, cell-based ELISA, and flow cytometry. A P. pastoris strain capable of expressing and secreting anti-CD63 scFv was successfully obtained. The antibody had a molecular weight of approximately 30 kDa and specifically recognized CD63 protein. The expression of anti-CD63 scFv in P. pastoris paves the way for the production of anti-CD63 antibodies on a large-scale, which is undoubtedly an economical and effective way of antibody acquisition.
Single-Chain Antibodies/immunology* ; Humans ; Tetraspanin 30/immunology* ; Recombinant Proteins/immunology* ; Pichia/genetics* ; Saccharomycetales/metabolism*

Protein tyrosine phosphatases (PTPs, EC 3.1.3.48) are key regulators of cellular processes, with the catalytic activity attributed to the conserved motif (H/V)CX₅R(S/T), where cysteine and arginine residues are critical. Previous studies revealed that alternative splicing of extracellular phosphatase mRNA precursors in Metarhizium anisopliae generated two distinct transcripts, with the longer sequence containing a novel HCPTPMLS motif resembling PTP signatures but lacking the arginine residue. To identify the novel signature motif and characterize its enzymatic properties, we heterologously expressed and purified both proteins in Pichia pastoris and comprehensively characterized their enzymatic properties. The protein containing the HCPTPMLS motif (designated as L-protein) exhibited the highest activity at pH 5.5 and a strong preference for pTyr substrates. Its phosphatase activity was inhibited by Ag⁺, Zn²⁺, Cu²⁺, molybdate, and tungstate, but enhanced by Ca²⁺ and EDTA. AcP101 (lacking HCPTPMLS) showed the maximal activity at pH 6.5 and a strong preference toward pNPP (P < 0.05), with the activity inhibited by NaF and tartrate, but enhanced by Mg²⁺ and Mn²⁺. Functional analysis confirmed that the L-protein retained the PTP activity despite the absence of arginine in its signature motif, while AcP101 functioned as an acid phosphatase. This study provides the first functional validation of an arginine-deficient PTP motif, expanding the definition of PTP signature motifs and offering new insights for phosphatase classification.
Metarhizium/genetics* ; Protein Tyrosine Phosphatases/chemistry* ; Amino Acid Motifs ; Recombinant Proteins/biosynthesis* ; Amino Acid Sequence ; Pichia/metabolism* ; Fungal Proteins/chemistry* ; Substrate Specificity ; Saccharomycetales

Glycosylation modification is an important post-translational modification of proteins, which participates in regulating protein half-life, biological activity, and immunogenicity, thereby affecting their functions. Glycoproteins expressed in Pichia pastoris predominantly carry high-mannose type glycans, primarily composed of mannose residues, which starkly contrasts with the complex-type glycans synthesized by mammalian cells. This study aims to transform the high mannose glycosylation modification of P. pastoris into a hybrid glycosylation modification similar to that of mammalian cells through genetic engineering technology. We introduced the mannosidase Ⅰ gene (MDSⅠ) from Trichoderma viride and the human β-1,2-N-acetylglucosaminyltransferase I gene (GnTⅠ) into a previously constructed P. pastoris strain (∆och1) capable of producing Man₈GlcNAc₂ glycans. To precisely regulate the expression of MDSⅠ and GnTⅠ, we designed various promoter combinations, including the strong inducible AOX promoter and the constitutive GAP promoter. The receptor-binding domain (RBD, residues 377-588) of the spike protein from the Middle East respiratory syndrome coronavirus (MERS-CoV) was selected as the reporter protein for this investigation (MERS-RBD). The N-glycosylation profile of MERS-RBD was systematically analyzed using PNGase F digestion coupled with mass spectrometry. The results showed that after the knockout of och1 and the introduction of MDSⅠ and GnTⅠ genes with different promoter combinations, P. pastoris strains capable of producing GlcNAcMan₅GlcNAc₂ glycans were successfully generated. When the AOX promoter was used to control the MDSⅠ gene and the GAP promoter was used to control the GnTⅠ gene, the engineered strain exhibited the highest proportion of hybrid-type GlcNAcMan₅GlcNAc₂ glycans, which accounted for 68.38% of the total N-glycosylation. In conclusion, we successfully engineered a P. pastoris strain capable of synthesizing hybrid-type GlcNAcMan₅GlcNAc₂ glycans, establishing a foundation for subsequent research on the biosynthesis of complex-type N-glycans in P. pastoris.
Glycosylation ; Glycoproteins/genetics* ; Polysaccharides/metabolism* ; N-Acetylglucosaminyltransferases/metabolism* ; Pichia/metabolism* ; Humans ; Mannosidases/metabolism* ; Genetic Engineering ; Trichoderma/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales

Human fibroblast growth factor 21 (hFGF21) has become a candidate drug for regulating blood glucose and lipid metabolism. The poor stability and short half-life of hFGF21 resulted in low target tissue availability, which hampers its clinical application. In this study, the hFGF21 was fused with a recombinant human serum albumin (HSA), and the resulted fusion protein rHSA-hFGF21 was expressed in Pichia pastoris. After codon optimization, the recombinant gene fragment rHSA-hFGF21 was inserted into two different vectors (pPIC9k and pPICZαA) and transformed into three different strains (X33, GS115 and SMD1168), respectively. We investigated the rHSA-hFGF21 expression levels in three different strains and screened an engineered strain X33-pPIC9K-rHSA-hFGF21 with the highest expression level. To improve the production efficiency of rHSA-hFGF21, we optimized the shake flask fermentation conditions, such as the OD value, methanol concentration and induction time. After purification by hollow fiber membrane separation, Blue affinity chromatography and Q ion exchange chromatography, the purity of the rHSA-hFGF21 protein obtained was 98.18%. Compared to hFGF21, the biostabilities of rHSA-hFGF21, including their resistance to temperature and trypsinization were significantly enhanced, and its plasma half-life was extended by about 27.6 times. Moreover, the fusion protein rHSA-hFGF21 at medium and high concentration showed a better ability to promote glucose uptake after 24 h of stimulation in vitro. In vivo animal studies showed that rHSA-hFGF21 exhibited a better long-term hypoglycemic effect than hFGF21 in type 2 diabetic mice. Our results demonstrated a small-scale production of rHSA-hFGF21, which is important for large-scale production and clinical application in the future.
Animals ; Blood Glucose/metabolism* ; Diabetes Mellitus, Experimental ; Fibroblast Growth Factors ; Humans ; Hypoglycemic Agents/metabolism* ; Methanol/metabolism* ; Mice ; Pichia/metabolism* ; Recombinant Fusion Proteins ; Recombinant Proteins/metabolism* ; Saccharomycetales ; Serum Albumin/metabolism* ; Serum Albumin, Human/metabolism*

Pichia pastoris is one of the most widely used recombinant protein expression systems. In this study, a novel method for rapid screening of P. pastoris strains capable of efficiently expressing recombinant proteins was developed. Firstly, the ability to express recombinant proteins of the modified strain GS115-E in which a functional Sec63-EGFP (Enhanced green fluorescent protein) fusion protein replaced the endogenous endoplasmic reticulum transmembrane protein Sec63 was tested. Next, the plasmids carrying different copy numbers of phytase (phy) gene or xylanase (xyn) gene were transformed into GS115-E to obtain recombinant strains with different expression levels of phytase or xylanase, and the expression levels of EGFP and recombinant proteins in different strains were tested. Finally, a flow cytometer sorter was used to separate a mixture of cells with different phytase expression levels into sub-populations according to green fluorescence intensity. A good linear correlation was found between the fluorescence intensities of EGFP and the expression levels of the recombinant proteins in the recombinant strains (0.8<|R|<1). By using the flow cytometer, high-yielding P. pastoris cells were efficiently screened from a mixture of cells. The expression level of phytase of the selected high-fluorescence strains was 4.09 times higher than that of the low-fluorescence strains after 120 h of methanol induction. By detecting the EGFP fluorescence intensity instead of detecting the expression level and activity of the recombinant proteins in the recombinant strains, the method developed by the present study possesses the greatly improved performance of convenience and versatility in screening high-yielding P. pastoris strains. Combining the method with high-throughput screening instruments and technologies, such as flow cytometer and droplet microfluidics, the speed and throughput of this method will be further increased. This method will provide a simple and rapid approach for screening and obtaining P. pastoris with high abilities to express recombinant proteins.
6-Phytase/genetics* ; Pichia/genetics* ; Plasmids ; Recombinant Proteins/genetics* ; Saccharomycetales

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.
Gene Editing ; Metabolic Engineering ; Pichia/genetics* ; Synthetic Biology ; Yarrowia/genetics* ; Yeasts

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.
Codon/genetics* ; Humans ; Pichia/genetics* ; Recombinant Proteins/genetics* ; Saccharomycetales ; Vascular Endothelial Growth Factor A/genetics* ; Vascular Endothelial Growth Factors

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.
L-Lactate Dehydrogenase ; genetics ; Lactobacillus casei ; enzymology ; genetics ; Phenylpyruvic Acids ; metabolism ; Pichia ; genetics ; Recombinant Proteins ; genetics ; metabolism

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.
Adaptation, Physiological ; Butanols ; metabolism ; Environment ; Fermentation ; Pichia ; cytology ; metabolism