Recombinant Pichia Pastoris Research Articles

One-pot synthesis of cellobiose from sucrose using sucrose phosphorylase and cellobiose phosphorylase co-displaying Pichia pastoris as a reusable whole-cell biocatalyst

Cellobiose has received increasing attention in various industrial sectors, ranging from food and feed to cosmetics. The development of large-scale cellobiose applications requires a cost-effective production technology as currently used methods based on cellulose hydrolysis are costly. Here, a one-pot synthesis of cellobiose from sucrose was conducted using a recombinant Pichia pastoris strain as a reusable whole-cell biocatalyst. Thermophilic sucrose phosphorylase from Bifidobacterium longum (BlSP) and cellobiose phosphorylase from Clostridium stercorarium (CsCBP) were co-displayed on the cell surface of P. pastoris via a glycosylphosphatidylinositol-anchoring system. Cells of the BlSP and CsCBP co-displaying P. pastoris strain were used as whole-cell biocatalysts to convert sucrose to cellobiose with commercial thermophilic xylose isomerase. Cellobiose productivity significantly improved with yeast cells grown on glycerol compared to glucose-grown cells. In one-pot bioconversion using glycerol-grown yeast cells, approximately 81.2 g/L of cellobiose was produced from 100 g/L of sucrose, corresponding to 81.2% of the theoretical maximum yield, within 24 h at 60 °C. Moreover, recombinant yeast cells maintained a cellobiose titer > 80 g/L, even after three consecutive cell-recycling one-pot bioconversion cycles. These results indicated that one-pot bioconversion using yeast cells displaying two phosphorylases as whole-cell catalysts is a promising approach for cost-effective cellobiose production.

Oral recombinant Pichia pastoris P-I20H combined with immunopotentiator curcumin remarkably inhibits and clears LMBV in largemouth bass (Micropterus salmoides)

Largemouth bass ranavirus (LMBV) causes massive losses in the largemouth bass industry. A promising therapeutic approach involves the oral administration of novel antimicrobial peptides (AMPs) and immunopotentiators. In this study, the Pichia pastoris expression system was employed for intracellular expression of AMP I20H. After meticulous screening, we successfully developed a strain, P-I20H-G7, demonstrating a high level of expression. Subsequently, we further optimized the induction conditions for this strain, including temperature, pH, and methanol concentration. Through these screening and optimization processes, we achieved an impressive nearly threefold increase in the production of I20H. Transmission electron microscopy, scanning electron microscopy and colony counting assays were utilized to confirm that P. pastoris effectively shielded I20H from the complex gastric and intestinal environments. Afterwards, P-I20H-G7 was lyophilized and co-fed with curcumin to largemouth bass infected by LMBV, the results indicated that oral administration of P-I20H-G7 combined with immunopotentiator curcumin effectively improve the survival rate of largemouth bass, with a 38% increase compared to the control group. qPCR demonstrated that dietary P-I20H-G7 + curcumin significantly inhibited and cleared LMBV within tissues. Additionally, oral administration of P-I20H-G7 + curcumin significantly upregulated the expression of key immune genes (IL-1β, IFN-γ, Mx, and IgM) and enhanced serum enzyme activities (TSOD, TAOC, and C3). Histopathological analysis revealed that P-I20H-G7 + curcumin treatment effectively mitigated tissue damage caused by LMBV infection. In conclusion, we successfully expressed and orally delivered I20H by the P. pastoris system and combined it with curcumin for efficiently curing diseased largemouth bass. These results underscore the potential of P-I20H-G7 + curcumin as a promising antiviral biological agent.

A fungal P450 enzyme from Fusarium equiseti HG18 with 7β-hydroxylase activity in biosynthesis of ursodeoxycholic acid

Cytochrome P450 enzyme with 7β-hydroxylation capacity has attracted widespread attentions due to the vital roles in the biosynthesis of ursodeoxycholic acid (UDCA), a naturally active molecule for the treatment of liver and gallbladder diseases. In this study, a novel P450 hydroxylase (P450FE) was screen out from Fusarium equiseti HG18 and identified by a combination of genome and transcriptome sequencing, as well as heterologous expression in Pichia pastoris. The biotransformation of lithocholic acid (LCA) by whole cells of recombinant Pichia pastoris further confirmed the C7β-hydroxylation with 5.2% UDCA yield. It was firstly identified a fungal P450 enzyme from Fusarium equiseti HG18 with the capacity to catalyze the LCA oxidation producing UDCA. The integration of homology modeling and molecular docking discovered the substrate binding to active pockets, and the key amino acids in active center were validated by site-directed mutagenesis, and revealed that Q112, V362 and L363 were the pivotal residues of P450FE in regulating the activity and selectivity of 7β-hydroxylation. Specifically, V362I mutation exhibited 2.6-fold higher levels of UDCA and higher stereospecificity than wild-type P450FE. This advance provided guidance for improving the catalytic efficiency and selectivity of P450FE in LCA hydroxylation, indicative of the great potential in green synthesis of UDCA from biologically toxic LCA.

Agar plate-based activity assay for easy and fast screening of recombinant Pichia pastoris expressing unspecific peroxygenases.

Unspecific peroxygenases (UPOs) are promising biocatalysts that catalyze oxyfunctionalization reactions without the need for costly cofactors. Pichia pastoris (reclassified as Komagataella phaffii) is considered an attractive host for heterologous expression of UPOs. However, integration of UPO-expression cassettes into the genome via a single cross-over yields recombinant Pichia transformants with different UPO gene copy numbers resulting in different expression levels. Selection of the most productive Pichia transformants by a commonly used screening in liquid medium in 96-well plates is laborious and lasts up to 5 days. In this work, we developed a simple two-step agar plate-based assay to screen P. pastoris transformants for UPO activity with less effort, within shorter time, and without automated screening devices. After cell growth and protein expression on agar plates supplemented with methanol and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), an additional top agar layer supplemented with ABTS and peroxide is added. UPO activity is visualized within 15min by formation of green zones around UPO-secreting P. pastoris transformants. The assay was validated with two UPOs, AbrUPO from Aspergillus brasiliensis and evolved PaDa-I from Agrocybe aegerita. The assay results were confirmed in a quantitative 96-deep well plate screening in liquid medium.

Detection of recombinant Pichia pastoris producing glargine

The global prevalence of diabetes continues to rise. Diabetes management involves the administration of insulin to help regulate blood glucose levels. Currently, two main types of insulin are used for diabetes treatment: human insulin and insulin analogs. One such insulin analog is insulin glargine, categorized as a long-acting insulin. Four prominent organisms used for insulin production are Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha. Pichia pastoris was used to produce insulin glargine in this study. The pPICZαA-G plasmid containing the synthetic glargine gene was inserted into Pichia pastoris. Recombinant Pichia pastoris containing the pPICZαA-G plasmid was detected using selection media with Zeocin, Polymerase Chain Reaction (PCR), and DNA sequencing. Through the implementation of selection media containing Zeocin, PCR, and DNA sequencing techniques, it was known that the recombinant Pichia pastoris contained the synthetic glargine gene. Further research will be carried out testing the expression of glargine in recombinant Pichia pastoris.

Antibacterial peptide PMAP-37(F34-R), expressed in Pichia pastoris, is effective against pathogenic bacteria and preserves plums

BackgroundRecently, researchers have focused on the search for alternatives to conventional antibiotics. Antimicrobial peptides are small bioactive peptides that regulate immune activation and have antibacterial activity with a reduced risk of bacterial resistance. Porcine myeloid antibacterial peptide 37 (PMAP-37) is a small-molecule peptide with broad-spectrum antibacterial activity isolated from pig bone marrow, and PMAP-37(F34-R) is its analogue. In this study, PMAP-37(F34-R) was recombinantly expressed in Pichia pastoris, and the recombinant peptide was further investigated for its antibacterial properties, mechanism and preservative in plums.ResultsTo obtain a Pichia pastoris strain expressing PMAP-37(F34-R), we constructed a plasmid expressing recombinant PMAP-37(F34-R) (pPICZα-PMAP-37(F34-R)-A) and introduced it into Pichia pastoris. Finally, we obtained a highly active recombinant peptide, PMAP-37(F34-R), which inhibited the activity of both Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration is 0.12–0.24 µg/mL, and it can destroy the integrity of the cell membrane, leading to cell lysis. It has good stability and is not easily affected by the external environment. Hemolysis experiments showed that 0.06 µg/mL-0.36 µg/mL PMAP-37(F34-R) had lower hemolysis ability to mammalian cells, and the hemolysis rate was below 1.5%. Additionally, 0.36 µg/mL PMAP-37(F34-R) showed a good preservative effect in plums. The decay and weight loss rates of the treated samples were significantly lower than those of the control group, and the respiratory intensity of the fruit was delayed in the experimental group.ConclusionsIn this study, we constructed a recombinant Pichia pastoris strain, which is a promising candidate for extending the shelf life of fruits and has potential applications in the development of new preservatives.Graphical

Detoxification and decolorization of complex textile effluent in an enzyme membrane reactor: batch and continuous studies.

There is an urgent need to look for bio-based technologies to address the pollution related to textile dyes in waterbodies. The aim of this study was to evaluate an engineered laccase variant, LCC1-62 of Cyathus bulleri, expressed in recombinant Pichia pastoris, for the decolorization and detoxification of real textile effluent. The partially purified laccase effectively (~60-100%) decolorized combined effluent from different dyeing units at a laccase concentration of 500 U/L at a 50-mL level. Decolorization and detoxification of the combined effluents, from a local textile mill, were evaluated at 0.3 L volumetric level in a ray-flow membrane reactor in batch and continuous modes of operation. In batch studies, maximum decolorization of 97% and detoxification of 96% occurred at a hydraulic retention time (HRT) of 6 h without any additional laccase requirement. In continuous studies, the reactor was operated at an HRT of 6 h with a lower enzyme dosage (~120 U/L of the effluent). Decolorization was accompanied by a loss in laccase activity which was restored to ~120 U/L by the addition of laccase in two regimes. The addition of laccase, when the residual laccase activity decreased to 40% (~50 U/L), resulted in high decolorization (~5 ppm residual dye concentration) and low variance (σ2) of 2.77, while laccase addition, when the residual dye concentration decreased to ~8% (~10 U/L), resulted in an average dye concentration of 13 ppm with a high variance of 62.08. The first regime was implemented, and the continuous reactor was operated for over 80 h at an HRT of 3 and 6 h, with the latter resulting in ~95% decolorization and 96% reduction in the mutagenicity of the effluent. Less than 10% membrane fouling was observed over long operations of the reactor. The findings strongly suggest the feasibility of using LCC1-62 in an enzyme membrane reactor for large-scale treatment of textile effluents.

THE OBTAINING OF THE RECOMBINANT CAMEL CHYMOSIN BY SUBMERGE FERMENTATION IN THE PILOT BIOREACTOR

In the cheese making industry, chymosin is used as a milk-clotting enzyme. With its high specific activity against κ-casein, chymosin better than other proteolytic enzymes. Bactrian camel chymosin has a milk-clotting activity higher than calf chymosin. A scheme for obtaining a milk-clotting preparation based on recombinant camel chymosin is proposed. Submerge fermentation of recombinant yeast Pichia pastoris was carried out in a 50-liter bioreactor and recombinant camel chymosin was obtained. The activity of chymosin in the yeast culture was 174.5 U/mL. Chymosin was concentrated 5.6-fold by cross-flow ultrafiltration with 10 kDa cut-off membrane, and chymosin was purified by ion exchange chromatography. The activity of purified chymosin was 4700 U/mL. By sublimation drying with casein peptone, the powder chymosin was obtained with an activity of 36,000 U/g. The proposed scheme for obtaining a milk-clotting drug based on recombinant camel chymosin using submerge fermentation of recombinant yeast has the prospect of being used at biotechnological enterprises.

Ultrathin membranes composed of branched polyethylenimine and poly[(o-cresyl glycidyl ether)-co-formaldehyde] for primary recovery of itaconic acid

Efficient primary recovery of building-block chemicals produced by microbial processes, such as biobased itaconic acid, is a key step for environmentally sustainable and economically feasible manufacturing processes. Ultrathin membranes have the potential to address these challenges. Here, ultrathin membranes composed of branched polyethylenimine and poly[(o-cresyl glycidyl ether)-co-formaldehyde] were fabricated using a facile spin coating procedure, and the composition of the casting solution was adjusted to improve the separation performance. The structural properties of the ultrathin membranes (intrinsic porosity, thickness, surface morphology, chemistry, charge, and wettability) were investigated and linked to the separation performance. By increasing the content of polyethylenimine in the casting solution, the transport rate of itaconic acid across the membranes increased up to 40 times. Membranes with a 4:1 mass ratio of polyethylenimine to poly[(o-cresyl glycidyl ether)-co-formaldehyde] reactive groups were selected, and their ability to recover itaconic acid from synthetic solutions and recombinant Pichia pastoris culture supernatant in diffusion-based mode was demonstrated. Experiments with pure solutions of supernatant components demonstrated that the membranes were equally selective for itaconic acid and potential cultivation byproducts and at least 7-times more selective for itaconic acid than culture medium components. Experiments with synthetic complex solutions and culture supernatant confirmed the high selectivity for itaconic acid: the separation performance was similar for complex and pure itaconic acid solutions, despite the significant difference in the complexity of these solutions. This study is the basis for a new technology to recover microbially produced building-block chemicals.

Simultaneous enzyme production, Levan-type FOS synthesis and sugar by-products elimination using a recombinant Pichia pastoris strain expressing a levansucrase-endolevanase fusion enzyme

BackgroundAlthough Levan-type fructooligosaccharides (L-FOS) have been shown to exhibit prebiotic properties, no efficient methods for their large-scale production have been proposed. One alternative relies on the simultaneous levan synthesis from sucrose, followed by endolevanase hydrolysis. For this purpose, several options have been described, particularly through the synthesis of the corresponding enzymes in recombinant Escherichia coli. Major drawbacks still consist in the requirement of GRAS microorganisms for enzyme production, but mainly, the elimination of glucose and fructose, the reaction by-products.ResultsThe expression of a fusion enzyme between Bacillus licheniformis endolevanase (LevB1) and B. subtilis levansucrase (SacB) in Pichia pastoris cultures, coupled with the simultaneous synthesis of L-FOS from sucrose and the elimination of the residual monosaccharides, in a single one-pot process was developed. The proof of concept at 250 mL flask-level, resulted in 8.62 g of monosaccharide-free L-FOS and 12.83 gDCW of biomass, after 3 successive sucrose additions (30 g in total), that is a 28.7% yield (w L-FOS/w sucrose) over a period of 288 h.At a 1.5 L bioreactor-level, growth considerably increased and, after 59 h and two sucrose additions, 72.9 g of monosaccharide-free L-FOS and 22.77 gDCW of biomass were obtained from a total of 160 g of sucrose fed, corresponding to a 45.5% yield (w L-FOS/w sucrose), 1.6 higher than the flask system. The L-FOS obtained at flask-level had a DP lower than 20 fructose units, while at bioreactor-level smaller oligosaccharides were obtained, with a DP lower than 10, as a consequence of the lower endolevanase activity in the flask-level.ConclusionWe demonstrate here in a novel system, that P. pastoris cultures can simultaneously be used as comprehensive system to produce the enzyme and the enzymatic L-FOS synthesis with growth sustained by sucrose by-products. This system may be now the center of an optimization strategy for an efficient production of glucose and fructose free L-FOS, to make them available for their application as prebiotics. Besides, P. pastoris biomass also constitutes an interesting source of unicellular protein.

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris.

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. β-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.

β-glucosidase production by recombinant Pichia pastoris strain Y1433 under optimal feed profiles of fed-batch cultivation.

Pichia pastoris, a methylotrophic yeast, is known to be an efficient host for heterologous proteins production. In this study, a recombinant P. pastoris Y11430 was found better for β-glucosidase activity in comparison with a wild type P. pastoris Y11430 strain, and thereby, subjected to methanol intermittent feed profiling for β-glucosidase production. The results showed that at 72h of cultivation time, the cultures with 16.67% and 33.33% methanol feeding with constant rate could produce the total dry cell weight of 52.23 and 118.55g/L, respectively, while the total mutant β-glucosidase activities were 1001.59 and 3259.82 units, respectively. The methanol feeding profile was kept at 33% with three methanol feeding strategies such as constant feed rate, linear feed rate, and exponential feed rate which were used in fed-batch fermentation. At 60h of cultivation, the highest total mutant β-glucosidase activity was 2971.85 units for exponential feed rate culture. On the other hand, total mutant β-glucosidase activity of the constant feed rate culture and linear feed rate culture were 1682.25 and 1975.43 units, respectively. The kinetic parameters of exponential feed rate culture were specific growth rate on glycerol 0.228/h, specific growth of methanol 0.061/h, maximum total dry cell weight 196.73g, yield coefficient biomass per methanol ([Formula: see text]) 0.57 gcell/gMeOH, methanol consumption rate ([Formula: see text]) 5.76 gMeOH/h, and enzyme productivity ([Formula: see text]) 75.96 units/h. In conclusion, higher cell mass and β- glucosidase activity were produced under exponential feed rate than constant and linear feed rates.

Efficiency of magnetic immobilization for recombinant Pichia pastoris cells harvesting over consecutive production cycles

ABSTRACT In the current experiment, P. pastoris cells were selected as promising candidate for production of recombinant proteins and were magnetically immobilized by 3-aminopropyltriethoxysilane-coated magnetite nanoparticles (APTES@Fe3O4). The ability of immobilized cells for the production of recombinant human serum albumin (HSA) and the potency of magnetic immobilization for yeast cells recycling was evaluated over three successive batch fermentation cycles. Interestingly, magnetic immobilization resulted in 1.4-fold increase in the HAS production. The productivity was increased from 0.7 mg/mL up to one mg/mL. But, after each production cycle a significant (more than two-fold) reduction in the productivity was recorded in free and immobilized cells. Over consecutive production cycles, efficiency of magnetic immobilization was also reduced. After three production cycles, immobilization efficiency was reduced from 80% down to 64% that was due to appearance of magnetic insensitive cells. Field emission scanning electron microscopy (FESEM) analyses approved that magnetic insensitive cells are nanoparticles free cells. These free cells can be resulted from cell proliferation and detachment of nanoparticles from cells surface. These findings can be considered for development of an efficient immobilization technique to harvest yeast cells over consecutive production cycles.

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.

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.

Application of Ultrafiltration and Ion Exchange Separation Technology for Lysozyme Separation and Extraction

In this study, the fermentation broth of the recombinant Pichia pastoris strain ncy-2 was studied. After pretreatment, separation, and purification, lysozyme was optimized using biofilm and ion exchange separation. Finally, lysozyme dry enzyme powder was prepared by concentrating and vacuum drying. The removal rate of bacterial cells was 99.99% when the fermentation broth was centrifuged at low temperature. The optimum conditions were: transmembrane pressure of 0.20 MPa, pH 6.5, 96.6% yield of lysozyme, enzyme activity of 2612.1 u/mg, which was 1.78 times higher than that of the original enzyme; D152 resin was used for adsorption and elution. Process conditions were optimized: the volume ratio of resin to liquid was 15%; the adsorption time was 4 h; the concentration of NaCl was 1.0 mol/L; the recovery rate of lysozyme activity was 95.67%; the enzyme activity was 3879.6 u/mL; and the purification multiple was 0.5, 3.1 times of the original enzyme activity. The enzyme activity of lysozyme dry enzyme powder was 12,573.6 u/mg, which had an inhibitory effect on microsphere lysozyme. Its enzymatic properties were almost the same as those of natural lysozyme, which demonstrated good application prospects and production potential.

Production, purification, and characterization of recombinant rabies virus glycoprotein expressed in PichiaPink™ yeast

The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/X to 12.9 µg/l and 0.135 µg/g(DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a YP/X of 0.310 µg/g(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.

Boosting expression level of plectasin in recombinant Pichia pastoris via 2A self-processing peptide assembly.

Plectasin is a promising and potent antimicrobial peptide isolated from the fungus Pseudoplectania nigrella which has been heterologously expressed in various hosts. In this study, a four-copy cassette of plectasin was constructed via 2A peptide assembly to further increase its expression level in recombinant Pichia pastoris. The yeast transformant 4Ple-61 harboring four-copy cassette of plectasin could secrete 183.2mg/L total protein containing 60.8% of plectasin at the flask level within 120h, which was 2.3 times higher than that of the yeast transformant Ple-6 carrying one-copy cassette of plectasin. Western blot confirmed the significant peptide expression level in the transformant 4Ple-61. Furthermore, it yielded as high as 426.3mg/L total protein within 120h during a 5-L fermentation. The purified plectasin shows superior stability and good antimicrobial activity against conventional Staphylococcus aureus ATCC 26,001 and some food-borne antibiotic-resistant S. aureus strains with the MICs ranging from 8 to 32μg/mL. Therefore, the strategy based on 2A peptide assembly can enhance the expression of plectasin and further expand its application prospect. KEY POINTS: • A yeast transformant 4Ple-61 with four-copy cassette of plectasin was constructed. • The plectasin level yield by the transformant 4Ple-61 was boosted by 2.3 times. • The plectasin showed good activity against food-borne antibiotic-resistant S. aureus.

Co-culture of Bacillus amyloliquefaciens and recombinant Pichia pastoris for utilizing kitchen waste to produce fengycins

Kitchen waste (KW) is a vast potential source of fermentable substrates. To bio-convert the KW into high-value chemicals, we used KW as substrate for the production of fengycin by an artificial consortium containing Bacillus amyloliquefaciens HM618 producing fengycin and the engineering Pichia pastoris producing amylase, glucosidase, or lipases. The maximal amylase activity of the constructed amylase-producing engineering strain (recombinant P.pastoris GS115-amy98) reached 385.4 U‧mL-1. The engineering strain GS115-α-glu53 producing glucosidase reached an enzyme activity titer of 247.3 U‧mL-1, while the lipase activities of the engineering strains GS115-lip2, GS115-α-lip2, and GS115-lip7 were around 90.0 U‧mL-1, with no significant differences among them. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that the components of fengycin synthesized by B.amyloliquefaciens HM618 were complex, including C14-C18 fengycins A, C13-C14 fengycins B, C16-C18 fengycins B, C16 fengycin B2 and some fengycin homologues with unsaturated fatty acid chains. The levels of fengycin were 15.9mg‧L-1 and 4.6mg‧L-1 under the co-culture with strain HM618 and the recombinant strains producing amylase and lipase, respectively. The maximal titer of fengycin was 21.2mg‧L-1 in the artificial consortia consisting of HM618 and the engineering strains producing glucosidase, amylase and lipase. Taken together, these results show that the co-culture of B.amyloliquefaciens HM618 and engineering strains producing amylase and lipase can promote the conversion of KW into fengycin. The work provides a new strategy for boosting the resource utilization of KW.

Construction of recombinant Pichia pastoris strains for ammonia reduction by the gdhA and glnA regulatory genes in laying hens

Ammonia emissions have become an important environmental challenge for the livestock industry. Probiotics are often used as additives to reduce ammonia, and the ammonia reduction efficiency of common probiotics is approximately 20–40%. In this study, we constructed a gdhA recombinant Pichia pastoris strain, glnA recombinant Pichia pastoris strain and gdhA-glnA Pichia pastoris recombinant strain using the gdhA and glnA genes, which have the potential function of reducing ammonia emissions. The results of in vitro fermentation showed that compared with the control, wild-type Pichia pastoris and pPICZA strains, the gdhA, glnA and gdhA-glnA recombinant strains significantly reduced ammonia emissions in laying hens (P < 0.05), with emission reduction efficiencies of 63.95%, 65.68% and 74.04%, respectively. The reason may be that the recombinant Pichia pastoris strains can convert ammonium nitrogen into amino acids for self-growth through ammonia assimilation, and reduce the pH, uric acid and urea content in the intestinal tract of livestock and poultry, and urease activity. Therefore, the construction of recombinant strains can provide technical support for reducing ammonia pollution in the livestock industry.