Expression and characterization of the new antimicrobial peptide AP138L-arg26 anti Staphylococcus aureus.

The low activity and yield of antimicrobial peptides (AMPs) are pressing problems. The improvement of activity and yield through modification and heterologous expression, a potential way to solve the problem, is a research hot-pot. In this work, a new plectasin-derived variant L-type AP138 (AP138L-arg26) was constructed for the study of recombination expression and druggablity. As a result, the total protein concentration of AP138L-arg26 was 3.1 mg/mL in Pichia pastoris X-33 supernatant after 5 days of induction expression in a 5-L fermenter. The recombinant peptide AP138L-arg26 has potential antibacterial activity against selected standard and clinical Gram-positive bacteria (G+, minimum inhibitory concentration (MIC) 2-16 µg/mL) and high stability under different conditions (temperature, pH, ion concentration) and 2 × MIC of AP138L-arg26 could rapidly kill Staphylococcus aureus (S. aureus) (> 99.99%) within 1.5 h. It showed a high safety in vivo and in vivo and a long post-antibiotic effect (PAE, 1.91 h) compared with vancomycin (1.2 h). Furthermore, the bactericidal mechanism was revealed from two dimensions related to its disruption of the cell membrane resulting in intracellular potassium leakage (2.5-fold higher than control), and an increase in intracellular adenosine triphosphate (ATP), and reactive oxygen species (ROS), the decrease of lactate dehydrogenase (LDH) and further intervening metabolism in S. aureus. These results indicate that AP138L-arg26 as a new peptide candidate could be used for more in-depth development in the future. KEY POINTS: • The AP138L-arg26 was expressed in the P. pastoris expression system with high yield • The AP138 L-arg26 showed high stability and safety in vitro and in vivo • The AP138L-arg26 killed S. aureus by affecting cell membranes and metabolism.

Identification a novel Ganoderma FIP gene from Ganoderma capense and its functional expression in Pichia pastoris.

Ganoderma capense is a precious medicinal fungus in China. In this study, a novel fungal immunomodulatory protein gene, named as FIP-gca, was cloned from G. capense by homologous cloning. Sequencing analysis indicated that FIP-gca was composed of 336 bp, which encoded a polypeptide of 110 amino acids. Protein sequence blasting and phylogenetic analysis showed that FIP-gca shared homology with other Ganoderma FIPs. FIP-gca was effectively expressed in Pichia pastoris GS115 at an expression level of 166.8 mg/L and purified using HisTrap™ fast-flow prepack columns. The immunomodulation capacity of rFIP-gca was demonstrated by that rFIP-gca could obviously stimulate cell proliferation and increase IL-2 secretion of murine spleen lymphocytes. Besides, antitumor activity of rFIP-gca towards human stomach cancer AGS cell line was evaluated in vitro. Cell wound scratch assay proved that rFIP-gca could inhibit migration of AGS cells. And flow cytometry assay revealed that rFIP-gca could significantly induce apoptosis of AGS cells. rFIP-gca was able to induce 18.12% and 22.29% cell apoptosis at 0.3 µM and 0.6 µM, respectively. Conclusively, the novel FIP-gca gene from G. capense has been functionally expressed in Pichia and rFIP-gca exhibited ideal immunomodulation and anti-tumour activities, which implies its potential application and study in future.

Food proteins from yeast-based precision fermentation: Simple purification of recombinant ß-lactoglobulin using polyphosphate.

Proteins produced through precision fermentation are often purified through chromatographic methods. Faster and more cost-effective purification methods are desired for food application. Here, we present a simple method for purification of protein produced from yeast, using ß-lactoglobulin secreted from Pichia pastoris as an example. The food-grade salt hexametaphosphate (HMP) was used to precipitate the protein at acidic pH, while the impurities (extracellular polysaccharides; mainly mannan) remained soluble. After re-solubilization of the protein-HMP complex by neutralization, excess HMP was selectively precipitated using calcium chloride. The protein content of the crude sample increased from 26 to 72 wt% (comparable to purification with anion exchange chromatography), containing only residual extracellular polysaccharides (9 wt%) and HMP (1 wt%). The established method had no significant impact on the structural and functional properties (i.e., ability to form emulsions) of the protein. The presented method shows potential for cost-effective purification of recombinant proteins produced through yeast-based expression systems.

Development of a continuous fermentation process for the production of recombinant uricase enzyme by Pichia pastoris.

Recent studies in the biopharmaceutical industry have shown an increase in the productivity and production efficiency of recombinant proteins by continuous culture. In this research, a new upstream fermentation process was developed for the production of recombinant uricase in the methylotrophic yeast Pichia pastoris. Expression of recombinant protein in this system is under the control of the AOX1 promoter and therefore requires methanol as an inducing agent and carbon/energy source. Considering the biphasic growth characteristics of conventional fed-batch fermentation, physical separation of the growth and induction stages for better control of the continuous fermentation process resulted in higher dry-cell weight (DCW) and enhanced recombinant urate oxidase activity. The DCW and recombinant uricase activity enzyme for fed-batch fermentation were 79 g/L and 6.8 u/mL. During the continuous process, in the growth fermenter at a constant dilution rate of 0.025 h-1 , DCW increased to 88.39 g/L. In the induction fermenter, at methanol feeding rates of 30, 60, and 80 mL/h, a recombinant uricase activity was 4.13, 7.2, and 0 u/mL, respectively. The optimum methanol feeding regime in continuous fermentation resulted in a 4.5-fold improvement in productivity compared with fed-batch fermentation from 0.04 u/mL/h (0.0017 mg/mL/h) to 0.18 u/mL/h (0.0078 mg/mL/h).

Selective extraction of recombinant membrane proteins from Hansenula polymorpha by pulsed electric field and lytic enzyme pretreatment.

BACKGROUND: In yeast, recombinant membrane proteins including viral scaffold proteins used for the formation of enveloped Virus-like particles (eVLPs) typically accumulate intracellularly. Their recovery is carried out by mechanical disruption of the cells, often in combination with detergent treatment. Cell permeabilization is an attractive alternative to mechanical lysis because it allows for milder and more selective recovery of different intracellular products. RESULTS: Here, we present a novel approach for extraction of integral membrane proteins from yeast based on cell envelope permeabilization through a combination of pulsed electric field and lytic enzyme pretreatment of the cells. Our primary experiments focused on Hansenula polymorpha strain #25-5 co-expressing the integral membrane small surface protein (dS) of the duck hepatitis B virus and a fusion protein of dS with a trimer of a Human papillomavirus (HPV) L2-peptide (3xL2-dS). Irreversible plasma membrane permeabilization was induced by treating the cell suspension with monopolar rectangular pulses using a continuous flow system. The permeabilized cells were incubated with lyticase and dithiothreitol. This treatment increased the cell wall permeability, resulting in the release of over 50% of the soluble host proteins without causing significant cell lysis. The subsequent incubation with Triton X-100 resulted in the solubilization and release of a significant portion of 3xL2-dS and dS from the cells. By applying two steps: (i) brief heating of the cells before detergent treatment, and (ii) incubation of the extracts with KSCN, an 80% purity on the protein level has been achieved. Experiments performed with H. polymorpha strain T#3-3, co-expressing dS and the fusion protein EDIIIWNV-dS consisting of dS and the antigen from the West Nile virus (WSV), confirmed the applicability of this approach for recovering dS. The treatment, optimal for solubilization of 3xL2-dS and a significant part of dS, was not effective in isolating the fused protein EDIIIWNV-dS from the membranes, resulting in its retention within the cells. CONCLUSIONS: This study presents an alternative approach for the recovery and partial purification of viral membrane proteins expressed in H. polymorpha. The factors influencing the effectiveness of this procedure and its potential use for the recovery of other integral membrane proteins are discussed.

Effect of tandem repeats of antimicrobial peptide CC34 on production of target proteins and activity of Pichia pastoris.

Antimicrobial peptides (AMPs) are attracting attention in the fields of medicine, food, and agriculture because of their broad-spectrum antibacterial properties, low resistance, and low-residue in the body. However, the low yield and instability of the prepared AMP drugs limit their application. In this study, we designed a tetramer of the AMP CC34, constructed and transfected two recombinant expression vectors with pGAPZαA containing a haploid CC34 and tetraploid CC34 (CC34-4js) into Pichia pastoris to explore the effect of biosynthesized peptides. The results showed that CC34 and CC34-4js expression levels were 648.2 and 1105.3 mg/L, respectively, in the fermentation supernatant of P. pastoris. The CC34-4js tetramer showed no antibacterial activity, could be cleaved to the monomer using formic acid, and the hemolytic rate of the polyploid was slightly lower than that of monomeric CC34. The average daily gain, average daily feed intake, feed conversion ratio and immune organ index of rats fed CC34 and CC34-4js showed no differences. In conclusion, CC34-4js exhibited a higher yield and lower hemolysis in P. pastoris than those of CC34. Finally, CC34 and CC34-4js enterokinase lysates showed similar antibacterial activity and both expressed peptides potentially improved the growth performance and organ indices of rats.

Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris.

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris.

Pichia pastoris (Komagataella phaffii) is widely used for industrial production of heterologous proteins due to high secretory capabilities but selection of highly productive engineered strains remains a limiting step. Despite availability of a comprehensive molecular toolbox for construct design and gene integration, there is high clonal variability among transformants due to frequent multi-copy and off-target random integration. Therefore, functional screening of several hundreds of transformant clones is essential to identify the best protein production strains. Screening methods are commonly based on deep-well plate cultures with analysis by immunoblotting or enzyme activity assays of post-induction samples, and each heterologous protein produced may require development of bespoke assays with multiple sample processing steps. In this work, we developed a generic system based on a P. pastoris strain that uses a protein-based biosensor to identify highly productive protein secretion clones from a heterogeneous set of transformants. The biosensor uses a split green fluorescent protein where the large GFP fragment (GFP1-10) is fused to a sequence-specific protease from Tobacco Etch Virus (TEV) and is targeted to the endoplasmic reticulum. Recombinant proteins targeted for secretion are tagged with the small fragment of the split GFP (GFP11). Recombinant protein production can be measured by monitoring GFP fluorescence, which is dependent on interaction between the large and small GFP fragments. The reconstituted GFP is cleaved from the target protein by TEV protease, allowing for secretion of the untagged protein of interest and intracellular retention of the mature GFP. We demonstrate this technology with four recombinant proteins (phytase, laccase, ß-casein and ß-lactoglobulin) and show that the biosensor directly reports protein production levels that correlate with traditional assays. Our results confirm that the split GFP biosensor can be used for facile, generic, and rapid screening of P. pastoris clones to identify those with the highest production levels.

Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein.

Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHß) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHß secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing ß-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHß production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHß. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHß secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC.

Biochemical and biophysical properties of a recombinant serine peptidase from Purpureocillium lilacinum.

The industrial uses of peptidases have already been consolidated; however, their range of applications is increasing. Thus, the biochemical characterization of new peptidases could increase the range of their biotechnological applications. In silico analysis identified a gene encoding a putative serine peptidase from Purpureocillium lilacinum (Pl_SerPep), annotated as a cuticle-degrading enzyme. The Pl_SerPep gene product was expressed as a recombinant in a Komagataella phaffii (previously Pichia pastoris) expression system. The enzyme (rPl_SerPep) showed optimal pH and temperature of 8.0 and 60 °C, respectively. Moreover, rPl_SerPep has a higher thermal stability than the cuticle-degrading enzymes described elsewhere. The structural analysis indicated a conformational change in the rPl_SerPep secondary structure, which would allow an increase in catalytic activity at 60 °C. Komagataella phaffii secretes rPl_SerPep with the pro peptide in its inactive form. Low-resolution small-angle X-ray scattering (SAXS) analysis showed little mobility of the pro peptide portion, which indicates the apparent stability of the inactive form of the enzyme. The presence of 20 mM guanidine in the reaction resulted in the maintenance of activity, which was apparently a consequence of pro peptide structure flexibilization.

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5.

Recombinant human neutrophil elastase (rHNE), a serine protease, was expressed in Pichia pastoris. Glycosylation sites were removed via bioengineering to prevent hyper-glycosylation (a common problem with this system) and the cDNA was codon optimized for translation in Pichia pastoris. The zymogen form of rHNE was secreted as a fusion protein with an N-terminal six histidine tag followed by the heme binding domain of Cytochrome B5 (CytB5) linked to the N-terminus of the rHNE sequence via an enteropeptidase cleavage site. The CytB5 fusion balanced the very basic rHNE (pI = 9.89) to give a colored fusion protein (pI = 6.87), purified via IMAC. Active rHNE was obtained via enteropeptidase cleavage, and purified via cation exchange chromatography, resulting in a single protein band on SDS PAGE (Mr = 25 KDa). Peptide mass fingerprinting analysis confirmed the rHNE amino acid sequence, the absence of glycosylation and the absence of an 8 amino acid C-terminal peptide as opposed to the 20 amino acids usually missing from the C-terminus of native enzyme. The yield of active rHNE was 0.41 mg/L of baffled shaker flask culture medium. Active site titration with alpha-1 antitrypsin, a potent irreversible elastase inhibitor, quantified the concentration of purified active enzyme. The Km of rHNE with methoxy-succinyl-AAPVpNA was identical with that of the native enzyme within the assay's limit of accuracy. This is the first report of full-length rHNE expression at high yields and low cost facilitating further studies on this major human neutrophil enzyme.

N-region of Cry1Ia: A novel fusion tag for Escherichia coli and Pichia pastoris.

Secretory signal peptides (SPs) can increase enhanced green fluorescent protein (eGFP) expression in cytosol. In this study, SPs Iasp (Cry1Ia), Vasp (Vip3A), and their local sequences were used as fusion tags to compare their effects on eGFP expression in Escherichia coli MC4100 and Pichia pastoris GS115. In E coli, the solubility was almost opposite between the proteins encoded by Vegfp and Iegfp. This may be because the overall hydrophobicity of the SPs differed. When the hydrophobic H-region and C-region were removed, the negative effects on eGFP solubility of the N-regions of both SPs (IaN and VN) were significantly reduced without compromise on the expression level. IaN promotes eGFP protein yield 7.1-fold more than Iasp, and using this peptide in tandem (Ia3N) further enhanced fluorescent fusion protein solubility with an efficacy similar to that of a polycationic tag. Furthermore, the GS-IaNeGFP strain produced the highest fluorescent signal intensity when these fusion proteins were expressed in P. pastoris, and the expression was higher than in other strains, including eGFP. In conclusion, we revealed the potential of the N-region of Iasp as a fusion tag in both prokaryotic and eukaryotic cells and further demonstrated the value of the N-regions of abundant SPs.

Cofactor Engineering for Efficient Production of α-Farnesene by Rational Modification of NADPH and ATP Regeneration Pathway in Pichia pastoris.

α-Farnesene, an acyclic volatile sesquiterpene, plays important roles in aircraft fuel, food flavoring, agriculture, pharmaceutical and chemical industries. Here, by re-creating the NADPH and ATP biosynthetic pathways in Pichia pastoris, we increased the production of α-farnesene. First, the native oxiPPP was recreated by overexpressing its essential enzymes or by inactivating glucose-6-phosphate isomerase (PGI). This revealed that the combined over-expression of ZWF1 and SOL3 increases α-farnesene production by improving NADPH supply, whereas inactivating PGI did not do so because it caused a reduction in cell growth. The next step was to introduce heterologous cPOS5 at various expression levels into P. pastoris. It was discovered that a low intensity expression of cPOS5 aided in the production of α-farnesene. Finally, ATP was increased by the overexpression of APRT and inactivation of GPD1. The resultant strain P. pastoris X33-38 produced 3.09 ± 0.37 g/L of α-farnesene in shake flask fermentation, which was 41.7% higher than that of the parent strain. These findings open a new avenue for the development of an industrial-strength α-farnesene producer by rationally modifying the NADPH and ATP regeneration pathways in P. pastoris.

Production of codon-optimized Human papillomavirus type 52 L1 virus-like particles in Pichia pastoris BG10 expression system.

Cervical cancer caused by Human papillomavirus (HPV) is one of the most common causes of cancer death in women worldwide. Even though the disease can be avoided by immunization, the expensive price of HPV vaccines makes it hard to be accessed by women in middle-low-income countries. Thus, the development of generic HPV vaccines is needed to address inequalities in life-saving access. This study aimed to develop the HPV52 L1 VLP-based recombinant vaccine using Pichia pastoris expression system. The l1 gene was codon-optimized based on P. pastoris codon usage resulting CAI value of 0.804. The gene was inserted into the pD902 plasmid under the regulation of the AOX1 promoter. The linear plasmid was transformed into P. pastoris BG10 genome and screened in YPD medium containing zeocin antibiotic. Colony of transformant that grown on highest zeocin concentration was characterized by genomic PCR and sequencing. The positive clone was selected and expressed using BMGY/BMMY medium induced with various methanol concentrations. The SDS-PAGE and Western blot analyses showed that 55 kDa L1 protein was successfully expressed using an optimum concentration of 1% methanol. The self-assembly of HPV52 L1 protein was also proven using TEM analysis. Moreover, we also analyzed the B-cell epitope of HPV52 L1 protein based on several criteria, including antigenicity, surface accessibility, flexibility, and hydrophilicity. We assumed that epitope 476GLQARPKLKRPASSAPRTSTKKKKV500 could be developed as an epitope-based vaccine with a neutralizing antibody response toward HPV52 infection. Finally, our study provided the alternative for developing low-cost HPV vaccines, either VLP or epitope-based.

Biodegradation of Free Gossypol by Helicoverpa armigera Carboxylesterase Expressed in Pichia pastoris.

Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial degradation of gossypol produces metabolites that reduce feed quality. We adopted an enzymatic method to degrade free gossypol safely and effectively. We cloned the gene cce001a encoding carboxylesterase (CarE) into pPICZαA and transformed it into Pichia pastoris GS115. The target protein was successfully obtained, and CarE CCE001a could effectively degrade free gossypol with a degradation rate of 89%. When esterase was added, the exposed toxic groups of gossypol reacted with different amino acids and amines to form bound gossypol, generating substances with (M + H) m/z ratios of 560.15, 600.25, and 713.46. The molecular formula was C27H28O13, C34H36N2O6, and C47H59N3O3. The observed instability of the hydroxyl groups caused the substitution and shedding of the group, forming a substance with m/z of 488.26 and molecular formula C31H36O5. These properties render the CarE CCE001a a valid candidate for the detoxification of cotton meal. Furthermore, the findings help elucidate the degradation process of gossypol in vitro.

Heterologous expression of family GH11 Aspergillus niger xylanase B (AnXylB11) in Pichia pastoris and competitive inhibition by riceXIP: An experimental and simulation study.

The family GH11 Aspergillus niger JL15 xylanase B (AnXylB11) was heterologously expressed in Pichia pastoris X33. The recombinant AnXylB11 (reAnXylB11) was secreted into the culture medium with a molecular weight of approximately 33.0 kDa. The optimal temperature and pH of reAnXylB11 were 40 â„ƒ and 5.0, respectively. reAnXylB11 released xylobiose (X2)-xylohexaose (X6) from beechwood xylan, with xylotriose (X3) as the primary product. The hydrolysates showed significant antioxidant activity. reAnXylB11 was also competitively inhibited by recombinant rice xylanase inhibitory protein (rePriceXIP), with an inhibition constant (Ki) of 106.9 nM. Molecular dynamics (MD) simulations, non-covalent interactions (NCI), and binding free energy calculation and decomposition were conducted to decipher the interactional features between riceXIP and AnXyB11. Representative conformation of riceXIP-AnXylB11 complex showed that a U-shaped long loop between α4 and ß5 (K143-L152) of riceXIP was protruded into the catalytic groove and formed tight interaction with many key residues of AnXylB11. The binding free energy of riceXIP-AnXylB11 was calculated to be - 46.1 ± 10.5 kcal/mol, with Coulomb and van der Waals forces contributing the most. NCI analysis showed that the hydrogen bonding networks such as R148riceXIP-E98AnXyl11B, K143riceXIP-D138AnXyl11B and R148riceXIP-E189AnXyl11B provided terrific contributions to the interface interaction. The Laplacian of electron density values of atom pairs R148riceXIP@ 2HH1-E89AnXylB11@OE2 and N142riceXIP@ 1HD2-D138AnXylB11@OD1 were 0.12190 and 0.16009 a.u., respectively. Exploring the interactional features between xylanase and inhibitor protein may aid in constructing mutant xylanase that is insensitive to xylanase inhibitory proteins (XIs).

Production of biodiesel from Caulerpa racemosa oil using recombinant Pichia pastoris whole cell biocatalyst with double displayed over expression of Candida antartica lipase.

In this study, Caulerpa racemosa oil was used to produce biodiesel by recombinant Pichia pastoris displaying bound (rPp-BL) and secretory lipase (rPp-SL). Collected algae was pre-treated using ultrasonication, microwave and solvent extraction. Defatted C. racemosa was subjected to dilute acid treatment to obtain algal biomass hydrolysate. Both rPp-BL and rPp-SL were cultivated in algal biomass hydrolysate and glycerol. Surfactant treatment was performed on rPp-BL. Screening and optimization of variables were performed for biodiesel production using Plackett Burman design and central composite design, respectively. About 10.64 % (w/w) of algal oil was extracted from C. racemosa. Both rPp-BL and rPp-SL effectively utilized C. racemosa biomass hydrolysate and glycerol. rPp-SL combined with triton X (1.0 % w/v) treated rPp-BL for 3 min improved lipase activity. Methanol to oil ratio, combined whole cell biocatalyst and temperature were significant factors. Under optimum conditions, biodiesel yield reached about 93.64 % after 30 h using developed whole cell biocatalyst.

Engineering and fermenter production of fungi GLA in Pichia pastoris GS115 using oil waste.

γ-Linolenic acid (GLA) is an essential n-6 polyunsaturated fatty acid (PUFA) that has received considerable attention in human and animal feed. GLA is used in many nutritional and medicinal applications, such as the treatment of cancer, inflammatory disorders, and diabetes. Currently, plant seed is the primary dietary source of GLA that is not enough to utilize on an industrial scale. To generate a sustainable novel source of GLA, the gene of delta-6 desaturase, one of the essential enzymes in the GLA production pathway, was isolated from Mucor rouxii DSM1194 and expressed in P. pastoris GS115 by pPICZC vector. The recombinant yeast expressed the GLA up to 19.2% (72 mg/g) of total fatty acids. GLA production of recombinant yeast was studied in a fermenter by oil waste for 5 days, and results detected 6.3 g/l lipid, and 103 mg/g GLA was produced in 72 h. The present study may provide an opportunity to develop an alternative host for manufacturing GLA on an industrial scale.

Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor.

BACKGROUND: Reprogramming in transcriptional regulation provides an effective tool for adjusting cellular metabolic activities. The strong methanol-inducible alcohol oxidase-1 promoter (pAOX1) is commonly used for heterologous gene expression in the yeast Pichia pastoris. Here, we present a novel Pichia pastoris strain engineered to co-express methanol-induced transcription factor 1 (Mit1) and the target protein. Mit1 upregulates pAOX1 in response to methanol. METHODS AND RESULTS: Two model proteins (VEGF and eGFP) have been used as the target proteins under the control of pAOX1. The sequence of Mit1 had obtained from the yeast genome and likewise cloned under the control of pAOX1. The results indicated a 1.9 and 2.2 fold increase in the detected VEGF and eGFP, respectively, when co-expressed with Mit1. Furthermore, the double-recombinant cells, containing Mit-1 and eGFP, produced 1.3 fold more eGFP when the methanol feeding concentration was doubled. The real-time PCR indicated a slight increase in the Mit1 expression, probably due to the negative regulatory feedback loop that exists for the intrinsic yeast Mit1. Overexpression of Mit1 also led to duplication of AOX1 enzyme activity, which may enhance the yeast cells' capacity for methanol detoxification. CONCLUSION: Overexpression of Mit1 could be considered a promising strategy for upregulation of target recombinant proteins in Pichia pastoris. Intracellular overexpression of Mit1 upregulates the heterologous target gene (eGFP) production, which is expressed under the control of pAOX1.

[One-pot synthesis of chondroitin sulfate A by engineered Pichia pastoris].

Chondroitin sulfate (CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue extraction method, microbial synthesis of CS has the advantages of controllability and easiness of scaling-up. In order to achieve an efficient synthesis of chondroitin sulfate A (CSA), we constructed a recombinant Pichia pastoris GS115 strain capable of synthesizing chondroitin (Ch) from glycerol by introducing the Ch synthase coding genes kfoC, kfoA and UDP-glucose dehydrogenase coding gene tuaD into the P. pastoris chromosome. The titer of Ch reached 2.6 g/L in fed-batch cultures upon optimizing the synthesis pathway of Ch. After further expressing the chondroitin-4-O-sulfotransferase (C4ST), we developed a one-pot biosynthesis system for CSA production by directly adding 3'-adenosine-5'-phosphoryl sulfate and C4ST into the high-pressure homogenized recombinant P. pastoris cells. Eventually, controllable synthesis of 0-40% CSA with different sulfation degrees were achieved by optimizing the catalytic conditions. The one-pot biosynthesis system constructed here is easy to operate and easy to scale up for industrial production of CSA. The idea of the present study may also facilitate the biosynthesis of other glycosaminoglycan, for instance, heparin.