Harnessing alkaline-pH regulatable promoters for efficient methanol-free expression of enzymes of industrial interest in Komagataella Phaffii.

BACKGROUND: The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. RESULTS: We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii. Here, we present the utilization of three alkaline pH-responsive promoters (pTSA1, pHSP12 and pPHO89) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference pAOX1-based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. CONCLUSIONS: Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy.

Food-Grade Expression of Two Laccases in Pichia pastoris and Study on Their Enzymatic Degradation Characteristics for Mycotoxins.

Mycotoxin contamination poses substantial health risks to humans and animals. In this study, the two laccases PpLac1 and AoLac2 from Pleurotus pulmonarius and Aspergillus oryzae were selected and heterologously expressed in Pichia pastoris in a food-grade manner to detoxify aflatoxin B1 (AFB1), zearalenone (ZEN), and deoxynivalenol (DON). Both laccases exhibited degradation activity toward these three mycotoxins, while the efficiency of these for DON was relatively low. Therefore, molecular docking between these laccases and DON was conducted to analyze their potential interaction mechanisms. Furthermore, the degradation conditions of AFB1 and ZEN by the two laccases were optimized, and the optimal degradation rates for AFB1 and ZEN by PpLac1 reached 78.51 and 78.90%, while those for AFB1 and ZEN by AoLac2 reached 72.27 and 80.60%, respectively. The laccases PpLac1 and AoLac2 successfully transformed AFB1 and ZEN into the compounds AFQ1 and 15-OH-ZEN, which were 90 and 98% less toxic than the original compounds, respectively. Moreover, the culture supernatants demonstrated effective mycotoxin degradation results for AFB1 and ZEN in contaminated feed samples. The residual levels of AFB1 and ZEN in all samples ranged from 6.61 to 8.72 µg/kg and 3.44 to 98.15 µg/kg, respectively, and these levels were below the limit set by the European Union standards. All of the results in this study indicated that the two laccases have excellent application potential in the feed industry.

Characterization and Hydrolysis Mechanism Analysis of a Cold-Adapted Trypsin-Like Protease from Antarctic Krill.

Cold-adapted proteases are capable of efficient protein hydrolysis at reduced temperatures, which offer significant potential applications in the area of low temperature food processing. In this paper, we attempted to characterize cold-adapted proteases from Antarctic krill. Antarctic krill possesses an extremely active autolytic enzyme system in their bodies, and the production of peptides and free amino acids accompanies the rapid breakdown of muscle proteins following the death. The crucial role of trypsin in this process is recognized. A cold-adapted trypsin named OUC-Pp-20 from Antarctic krill genome was cloned and expressed in Pichia pastoris. Recombinant trypsin is a monomeric protein of 26.8 ± 1.0 kDa with optimum reaction temperature at 25 °C. In addition, the catalytic specificity of OUC-Pp-20 was assessed by identifying its hydrolysis sites through LC-MS/MS. OUC-Pp-20 appeared to prefer Gln and Asn at the P1 position, which is an amino acid with an amide group in its side chain. Hydrolysis reactions on milk and shrimp meat revealed that it can effectively degrade allergenic components in milk and arginine kinase in shrimp meat. These findings update the current knowledge of cold-adapted trypsin and demonstrate the potential application of OUC-Pp-20 in low temperature food processing.

Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.g. Pichia pastoris), the abundance and protein composition of peroxisomes are highly variable, particularly when peroxisome proliferation is induced by specific carbon sources. The intricate subcellular localization of native proteins, the variability of peroxisomal metabolic pathways, and the lack of systematic characterization of peroxisome targeting signals have limited the applications of peroxisomal compartmentalization in P. pastoris. Accordingly, this study established a high-throughput screening method based on ß-carotene biosynthetic pathway to evaluate the targeting efficiency of PTS1s (Peroxisome Targeting Signal Type 1) in P. pastoris. First, 25 putative endogenous PTS1s were characterized and 3 PTS1s with high targeting efficiency were identified. Then, directed evolution of PTS1s was performed by constructing two PTS1 mutant libraries, and a total of 51 PTS1s (29 classical and 22 noncanonical PTS1s) with presumably higher peroxisomal targeting efficiency were identified, part of which were further characterized via confocal microscope. Finally, the newly identified PTS1s were employed for peroxisomal compartmentalization of the geraniol biosynthetic pathway, resulting in more than 30% increase in the titer of monoterpene compared with when the pathway was localized to the cytosol. The present study expands the synthetic biology toolkit and lays a solid foundation for peroxisomal compartmentalization in P. pastoris.

Combined strategies for improving the heterologous expression of a novel xylanase from Fusarium oxysporum Fo47 in Pichia pastoris.

Xylanase, an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat, has been found to improve the organizational structure of dough and thus increase its volume. In our past work, one promising xylanase FXYL derived from Fusarium oxysporum Fo47 and first expressed 779.64 U/mL activity in P. pastoris. It has shown significant potential in improving the quality of whole wheat bread, making it become a candidate for development as a new flour improver. After optimization of expression elements and gene dose, the xylanase activity of FXYL strain carrying three-copies reached 4240.92 U/mL in P. pastoris. In addition, 12 factors associated with the three stages of protein expression pathway were co-expressed individually in order in three-copies strain, and the translation factor Pab1 co-expression increased FXYL activity to 8893.53 U/mL. Nevertheless, combining the most effective or synergistic factors from three stages did not exhibit better results than co-expressing them alone. To further evaluate the industrial potential, the xylanase activity and protein concentration reached 81184.51 U/mL and 11.8 g/L in a 5 L fed-batch fermenter. These engineering strategies improved the expression of xylanase FXYL by more than 104-fold, providing valuable insights for the cost-effective industrial application of FXYL in the baking field.

Optimizing Pichia pastoris Protein Secretion: Role of N-Linked Glycosylation on the α-Mating Factor Secretion Signal Leader.

The methylotrophic yeast, Pichia pastoris (P. pastoris; syn. Komagataella spp.), known for its ability to grow to high cell densities, its strong and tightly regulated promoters, and mammalian liked secretion pathway, has been widely used as a robust system to secrete heterologous proteins. The α-mating factor (MF) secretion signal leader from Saccharomyces cerevisiae (S. cerevisiae) is currently the most successfully used secretion signal sequence in the P. pastoris system. In this study, the secretion efficiency mediated by the α-MF secretion signal leaders from Komagataella pastoris (K. pastoris) and Komagataella phaffii (K. phaffii) was assessed using Enhanced Green Fluorescent Protein (EGFP) as a reporter. The results indicated that the secretion efficiency associated with the α-MF secretion signal leaders from K. pastoris and K. phaffii was notably lower in comparison to the α-MF secretion signal leader from S. cerevisiae. Further research indicated that N-linked glycosylation of the α-MF secretion signal leader enhanced the secretion of EGFP. Disruption of calnexin impaired the secretion of EGFP mediated by the N-linked glycosylated α-MF secretion signal leader, without affecting EGFP secretion mediated by the non-N-linked glycosylation α-MF secretion signal leader. The N-linked glycosylated of the α-MF secretion signal leader reduced the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The enhancement of EGFP secretion by the N-linked glycosylated α-MF secretion signal leader might be achieved through the acceleration of proper folding of glycoproteins by the molecular chaperone calnexin. This study enhances the understanding of protein secretion in P. pastoris, specifically highlighting the influence of N-linked glycosylation on secretion efficiency, and could have implications for the production of recombinant proteins in bioengineering and biotechnological applications in P. pastoris.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection.

BACKGROUND: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. RESULTS: In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. CONCLUSIONS: For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

Production of biliverdin by biotransformation of exogenous heme using recombinant Pichia pastoris cells.

Biliverdin, a bile pigment hydrolyzed from heme by heme oxygenase (HO), serves multiple functions in the human body, including antioxidant, anti-inflammatory, and immune response inhibitory activities. Biliverdin has great potential as a clinical drug; however, no economic and efficient production method is available currently. Therefore, the production of biliverdin by the biotransformation of exogenous heme using recombinant HO-expressing yeast cells was studied in this research. First, the heme oxygenase-1 gene (HO1) encoding the inducible plastidic isozyme from Arabidopsis thaliana, with the plastid transport peptide sequence removed, was recombined into Pichia pastoris GS115 cells. This resulted in the construction of a recombinant P. pastoris GS115-HO1 strain that expressed active HO1 in the cytoplasm. After that, the concentration of the inducer methanol, the induction culture time, the pH of the medium, and the concentration of sorbitol supplied in the medium were optimized, resulting in a significant improvement in the yield of HO1. Subsequently, the whole cells of GS115-HO1 were employed as catalysts to convert heme chloride (hemin) into biliverdin. The results showed that the yield of biliverdin was 132 mg/L when hemin was added to the culture of GS115-HO1 and incubated for 4 h at 30 °C. The findings of this study have laid a good foundation for future applications of this method for the economical production of biliverdin.

Biotransformation of fluorinated drugs and xenobiotics by the model fungus Cunninghamella elegans.

Some species of the genus Cunninghamella (C. elegans, C. echinulata and C. blaskesleeana) produce the same phase I and phase II metabolites when incubated with xenobiotics as mammals, and thus are considered microbial models of mammalian metabolism. This had made these fungi attractive for metabolism studies with drugs, pesticides and environmental pollutants. As a substantial proportion of pharmaceuticals and agrochemicals are fluorinated, their biotransformation has been studied in Cunninghamella fungi and C. elegans in particular. This article details the methods employed for cultivating the fungi in planktonic and biofilm cultures, and extraction and analysis of fluorinated metabolites. Furthermore, protocols for the heterologous expression of Cunninghamella cytochromes P450 (CYPs), which are the enzymes associated with phase I metabolism, are described.

An impact of N-glycosylation on biochemical properties of a recombinant α-amylase from Bacillus licheniformis.

Amylases are enzymes that are known to hydrolyze starch. High efficiency of amylolytic enzymes allows them to compete in the industry with the technology of chemical hydrolysis of starch. A Bacillus licheniformis strain with high amylolytic activity was isolated from soil and designated as T5. The gene encoding α-amylase from B. licheniformis T5 was successfully expressed in both Escherichia coli (rAmyT5-E) and Pichia pastoris (as rAmyT5-P). According to the study, the recombinant α-amylases rAmyT5-E and rAmyT5-P exhibited the highest activity at pH 6.0 and temperatures of 70 and 80 °C, respectively. Over 80% of the rAmyT5-E enzyme activity was preserved following incubation within the pH range of 5-9; the same was true for rAmyT5-P after incubation at pH 6-9. N-glycosylation reduced the thermal and pH stability of the enzyme. The specific activity and catalytic efficiency of the recombinant AmyT5 α-amylase were also diminished by N-glycosylation.

Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in Komagataella phaffii.

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast Komagataella phaffii, a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in K. phaffii. In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).

Glycogen Granules Are Degraded by Non-Selective Autophagy in Nitrogen-Starved Komagataella phaffii.

Autophagy was initially recognized as a bulk degradation process that randomly sequesters and degrades cytoplasmic material in lysosomes (vacuoles in yeast). In recent years, various types of selective autophagy have been discovered. Glycophagy, the selective autophagy of glycogen granules, is one of them. While autophagy of glycogen is an important contributor to Pompe disease, which is characterized by the lysosomal accumulation of glycogen, its selectivity is still a matter of debate. Here, we developed the Komagataella phaffii yeast as a simple model of glycogen autophagy under nitrogen starvation conditions to address the question of its selectivity. For this, we turned the self-glucosylating initiator of glycogen synthesis, Glg1, which is covalently bound to glycogen, into the Glg1-GFP autophagic reporter. Our results revealed that vacuolar delivery of Glg1-GFP and its processing to free GFP were strictly dependent on autophagic machinery and vacuolar proteolysis. Notably, this process was independent of Atg11, the scaffold protein common for many selective autophagy pathways. Importantly, the non-mutated Glg1-GFP (which synthesizes and marks glycogen) and mutated Glg1Y212F-GFP (which does not synthesize glycogen and is degraded by non-selective autophagy as cytosolic Pgk1-GFP) were equally well delivered to the vacuole and had similar levels of released GFP. Therefore, we concluded that glycogen autophagy is a non-selective process in K. phaffii yeast under nitrogen starvation conditions.

A sustainable and efficient strategy for bioconverting naringin to L-rhamnose, 2R-naringenin, and kaempferol.

The development of a sustainable and efficient bioconversion strategy is crucial for the full-component utilization of naringin. In this study, an engineering Pichia pastoris co-culture system was developed to produce L-rhamnose and 2S/2R-naringenin. By optimizing transformation conditions, the co-culture system could completely convert naringin while fully consuming glucose. The production of 2S/2R-naringenin reached 59.5 mM with a molar conversion of 99.2%, and L-rhamnose reached 59.1 mM with a molar conversion of 98.5%. In addition, an engineering Escherichia coli co-culture system was developed to produce 2R-naringenin and kaempferol from 2S/2R-naringenin. Maximal kaempferol production reached 1050 mg/L with a corresponding molar conversion of 99.0%, and 996 mg/L 2R-naringenin was accumulated. Finally, a total of 17.4 g 2R-naringenin, 18.0 g kaempferol, and 26.1 g L-rhamnose were prepared from 100 g naringin. Thus, this study provides a novel strategy for the production of value-added compounds from naringin with an environmentally safe process.

Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.

Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P. pastoris limits its broad applications, particularly when multigene pathways should be manipulated. Here, the CRISPR/Cas9 system is established to efficiently integrate multiple heterologous genes to construct P. pastoris cell factories. In this protocol, with the 2,3-butanediol (BDO) biosynthetic pathway as a representative example, the procedures to construct P. pastoris cell factories are detailed using the established CRISPR-based multiplex genome integration toolkit, including donor plasmid construction, competent cell preparation and transformation, and transformant verification. The application of the CRISPR toolkit is demonstrated by the construction of engineered P. pastoris for converting methanol to BDO. This lays the foundation for the construction of P. pastoris cell factories harboring multi-gene biosynthetic pathways for the production of high-value compounds.

[Analysis of signal peptides and secreted proteins in the whole proteome of Pichia pastoris].

The signal peptide is a key factor that affects the efficiency of protein secretion in Pichia pastoris. Currently, the most used signal peptide is the α-mating factor (MFα) pre-pro leader from Saccharomyces cerevisiae. This exogenous signal peptide has been successfully utilized to express and secret many heterologous proteins. However, MFα is not suitable for the secretory expression of all heterologous proteins. Many typical signal peptides are present in the secretory proteins of P. pastoris, which provides more options besides MFα. Therefore, it is necessary to analyze and identify more efficient endogenous signal peptides that can guide the secretion of heterologous proteins in P. pastoris. In this study, we employed bioinformatics tools such as SignalP, TMHMM, Phobius, WoLF PSORT, and NetGPI to predict endogenous signal peptides from the entire proteome of P. pastoris GS115 (ATCC 20864). Moreover, we analyzed the distribution, length, amino acid composition, and conservation of these signal peptides. Additionally, we screened 69 secreted proteins and their signal peptides, and through secretome validation, we identified 10 endogenous signal peptides that have potential to be used for exogenous protein expression. The endogenous signal peptides obtained in this study may serve as new valuable tools for the expression and secretion of heterologous proteins in P. pastoris.

A superior heterologous prime-boost vaccination strategy against COVID-19: A bivalent vaccine based on yeast-derived RBD proteins followed by a heterologous vaccine.

Various vaccines have been challenged by SARS-CoV-2 variants. Here, we reported a yeast-derived recombinant bivalent vaccine (Bivalent wild-type [Wt]+De) based on the wt and Delta receptor-binding domain (RBD). Yeast derived RBD proteins based on the wt and Delta mutant were used as the prime vaccine. It was found that, in the presence of aluminium hydroxide (Alum) and unmethylated CpG-oligodeoxynucleotides (CpG) adjuvants, more cross-protective immunity against SARS-CoV-2 prototype and variants were elicited by bivalent vaccine than monovalent wtRBD or Delta RBD. Furthermore, a heterologous boosting strategy consisting of two doses of bivalent vaccines followed by one dose adenovirus vectored vaccine exhibited cross-neutralization capacity and specific T cell responses against Delta and Omicron (BA.1 and BA.4/5) variants in mice, superior to a homologous vaccination strategy. This study suggested that heterologous prime-boost vaccination with yeast-derived bivalent protein vaccine could be a potential approach to address the challenge of emerging variants.

Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins.

OBJECTIVE: A convenient strategy was developed to recycle selectable markers using Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins. RESULTS: A plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. phaffii KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recycling of ZeoR and His- markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures. CONCLUSIONS: With easy manipulation, the method was effective in recycling of the selectable markers, and consequently two protein genes were sequential integrated chromosomally and successfully co-expressed in the yeast.

Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant.

A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83 %. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 min, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20 % loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83 % to approximately 92-95 %.

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1 , which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1 . LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

High-Level Expression and Biochemical Characterization of a Maltotetraose Amylase in Pichia pastoris X-33 for Maltotetraose Production.

Maltotetraose amylase, which catalyzes the hydrolysis of amylaceous polysaccharides into maltooligosaccharides with maltotetraose as the main product, is extensively used in the food industry. However, the lack of efficient expression system for maltotetraose amylase has hampered its production and application. In this study, high-level production of a maltotetraose amylase mutant (referred to as Pp-Mta∆CBM) from Pseudomonas saccharophila was achieved in Pichia pastoris X-33. First, the gene of maltotetraose amylase with the carbohydrate-binding module (CBM) removed was codon-optimized and cloned into the pPICZαA vector, followed by transformation into P. pastoris X-33 for expression. Using the promoter PAOX1 and signal peptide α-factor, high-level production of Pp-Mta∆CBM with minimal extracellular impurity proteins was achieved, resulting in an extracellular activity of 367.9 U/mL after 7 days of cultivation in shake flasks. Next, the expressed Pp-Mta∆CBM was purified and characterized. This recombinant enzyme was glycosylated and has maximum activity at 55 ℃ and pH 7.0. Its Km for soluble starch was 4.1 g/L, and its kcat was 3237.6 s-1. Finally, the Pp-Mta∆CBM was found to produce a maximum maltotetraose yield of 57.1% in the presence of 200 g/L of substrate. The findings presented in this study demonstrate the efficient production of Pp-Mta∆CBM in P. pastoris, providing a new expression system for maltotetraose amylase and laying the foundation for its scale-up production and industrial application.