Use of the glyceraldehyde-3-phosphate dehydrogenase promoter from a thermotolerant yeast,Pichia thermomethanolica,for heterologous gene expression,especially at elevated temperature

The glyceraldehyde-3-phosphate dehydrogenase (GAP) gene from the thermotolerant yeast strain Pichia thermomethanolica BCC16875 was characterized. To investigate the efficiency of the GAP promoter for heterologous expression, especially at high temperature in various carbon sources, the promoter was employed for constitutive expression of a phytase reporter gene. The results showed that this promoter was able to drive efficient expression of phytase at 30 °C; the native promoter was highly robust compared with the heterologous GAP promoter from Pichia pastoris. More importantly, the GAP promoter was shown to be able to function at higher temperatures up to 42 °C, which could be useful for large-scale protein production to help reduce cooling costs in the fermenter. Expression in different carbon sources revealed that the GAP promoter was functional in glucose-, glycerol-, and methanol-containing media, with the highest level of expression in YPD medium. This strong promoter will help promote high expression of heterologous protein expression in P. thermomethanolica, especially in large-scale fermentation. In addition, a new tool for heterologous expression in yeast has been gained.     

Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: A review

Pichia pastoris has been recognized as one of the most industrially important hosts for heterologous protein production. Despite its high protein productivity, the optimization of P. pastoris cultivation is still imperative due to strain- and product-specific challenges such as promoter strength, methanol utilization type and oxygen demand. To address the issues, strategies involving genetic and process engineering have been employed. Optimization of codon usage and gene dosage, as well as engineering of promoters, protein secretion pathways and methanol metabolic pathways have proved beneficial to innate protein expression levels. Large-scale production of proteins via high cell density fermentation additionally relies on the optimization of process parameters including methanol feed rate, induction temperature and specific growth rate. Recent progress related to the enhanced production of proteins in P. pastoris via various genetic engineering and cultivation strategies are reviewed. Insight into the regulation of the P. pastoris alcohol oxidase 1 (AOX1) promoter and the development of methanol-free systems are highlighted. Novel cultivation strategies such as mixed substrate feeding are discussed. Recent advances regarding substrate and product monitoring techniques are also summarized. Application of P. pastoris to the production of biodiesel and other value-added products via metabolic engineering are also reviewed. P. pastoris is becoming an indispensable platform through the use of these combined engineering strategies.     

Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50–65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100 % of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.     

An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates

Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47?kDa. Maximum enzyme production was achieved at 96?h, in culture medium supplemented with 1.34?% urea and 1?% yeast extract and upon induction with 1?% methanol. Recombinant enzyme exhibited optimum activity at 60?°C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl ??-d-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.     

以葡萄糖为生长相基质的重组毕赤酵母表达植酸酶发酵

甲醇营养型毕赤酵母表达外源蛋白是在醇氧化酶（alcohol oxidase,AOX）启动子（PAOXI）严格调控下进行的,然而这种启动子在转录水平受到葡萄糖的阻遏。本文研究了毕赤酵母在葡萄糖替代甘油为生长相碳源时表达重组植酸酶蛋白的发酵特征。结果表明：初始葡萄糖浓度为20dL的细胞得率高,为0．39g[DCW]／g。通过基于实时参数（溶氧和呼吸商）调控的葡萄糖补料策略,生长相40h后细胞密度达到100g[DCW]／L,甲醇诱导100h后植酸酶产量达到2200FTUphytase／mL,甲醇得率系数为0．25FTU phytase／gmethnol。因此,在毕赤酵母高表达重组蛋白培养中葡萄糖能够用作生长相基质,并能实现重组蛋白的高效表达。     

Optimization of the production of Aspergillus niger α-glucosidase expressed in Pichia pastoris

The α-glucosidase (AGL) from Aspergillus niger has been applied to produce isomaltooligosaccharides. In the present study, various factors which affect the yield of recombinant AGL, produced by engineered Pichia pastoris, were investigated. The expression level reached 5.5 U ml^?1 in bioreactor after optimization of parameters of initial induction cell density, induction temperature and methanol concentration. In addition, it was found that coexpression of protein disulfide isomerase (PDI) inhibited the growth of the engineered P. pastoris strains and had an adverse effect on the production of AGL, while codon optimization of native A. niger α-glucosidase encoding gene (aglu) resulted in a significant enhancement of enzyme production, which reached 10.1 U ml^?1. We believe that yield of AGL is increased by codon optimization as a result of enhanced translation efficiency as well as more stable mRNA secondary structure. In contrast, PDI coexpression under the control of alcohol oxidase promoter (PAOX1) seems to be less efficient in helping disulfide bond formation in AGL while probably induce unfolded protein response, which further leads to cell apoptosis and increased protein degradation.     

Simplified high-throughput screening of AOX1-expressed laccase enzyme in Pichia pastoris

The heterologous protein expression in Pichia pastoris under the control of alcohol oxidase (AOX1)promoter comprises two steps, the growth and induction phases, which are time-consuming and technically demanding. Here, we describe an alternate method where expression is carried out directly in the methanol-containing medium. Using this method, we were successful in screening high-activity laccase clones from a library of laccase mutants generated by random mutagenesis. This simplified method not only saves time but also is highly efficient and can be used for screening a large number of clones.     

Heterologous protein expression in Pichia thermomethanolica BCC16875, a thermotolerant methylotrophic yeast and characterization of N-linked glycosylation in secreted protein

This study describes Pichia thermomethanolica BCC16875, a new methylotrophic yeast host for heterologous expression. Both methanol-inducible alcohol oxidase (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters from Pichia pastoris were shown to drive efficient gene expression in this host. Recombinant phytase and xylanase were expressed from both promoters as secreted proteins, with the former showing different patterns of N-glycosylation dependent on the promoter used and culture medium. In addition, growth temperature also had an effect on N-glycan modification of cell wall mannoproteins. The major glycoprotein oligosaccharide species produced from P.?thermomethanolica BCC16875 is Man(8-12) GlcNAc(2) , which is similar to that from other methylotrophs. Moreover, mannosylphosphate and α-1,6- and α-1,2-linked mannose modifications of heterologous secreted protein were also detected. The attainably high level of protein production in complement to distinctive thermotolerance rarely found in other industrial yeasts makes this microorganism an attractive host for large-scale fermentation.     

Molecular gene cloning and overexpression of the phytase from Debaryomyces castellii CBS 2923

The ORF encoding the Debaryomyces castellii CBS 2923 phytase was isolated. The deduced 461-amino-acid sequence corresponded to a 51.2 kDa protein and contained the consensus motif (RHGXRXP) which is conserved among phytases. No signal sequence cleavage site was detected. Nine potential N-glycosylation sites have been predicted. The protein shared 21–69% sequence identities with various phytases of yeast or fungal origin. Heterologous expression of the D. castellii CBS 2923 phytase in the methylotrophic yeast Pichia pastoris was tested under both the P. pastoris inducible alcohol oxidase (AOX1) promoter and the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Maximum production levels obtained were 476 U ml⁻¹, with the AOX1 expression system and 16.5 U ml⁻¹ with the GAP one. These productions corresponded to a 320-fold and a 10-fold overexpression of the protein, respectively as compared to the homologous production. The biochemical characteristics of the recombinant phytase were identical to those of the native enzyme.     

Recent advances on the GAP promoter derived expression system of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review. Supported by the National High-tech R&D Program (863 program) (No.2007AA021307).     

Overproduction of a truncated poly (vinyl alcohol) dehydrogenase in recombinant Pichia pastoris by low-temperature induction strategy and related mechanism analysis

Based on the N-terminal sequencing of poly (vinyl alcohol) dehydrogenase (PVADH), a 1,644-bp gene encoding a truncated PVADH (tPVADH) was amplified using the synthetic gene (GenBank accession No. JQ235753) as a template, and was further transformed into Pichia pastoris GS115 with the vector pPIC9K. The maximal tPVADH activity reached 546 U/mL in shake flask. The influence of methanol concentration and induction temperature on tPVADH production was further investigated in 3-L bioreactor. When the methanol concentration and induction temperature were controlled at 15 g/L and 22 °C, respectively, the maximal tPVADH activity reached 8,464 U/mL, which was nearly 10 times that of mature PVADH expressed under the same condition and was the highest level ever reported. The reason responsible for the significant improvement of tPVADH production at low induction temperature was explored in terms of cell viability, extracellular proteases activity, and alcohol oxidase activity.     

Translational Arrest Due to Cytoplasmic Redox Stress Delays Adaptation to Growth on Methanol and Heterologous Protein Expression in a Typical Fed-Batch Culture of Pichia pastoris

Results

We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation.

Conclusion

Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.     

Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

A study of the Mut⁺ phenotype for the expression of recombinant hepatitis B surface antigen (HBsAg) in Pichia pastoris strain GS115 using the pPIC3.5K vector with a two-phase fed-batch protocol in a shake flask system is described. Expression levels of HBsAg protein of 6.74 g L^?1 Dry Cell Weight (DCW) and 26.07 mg L^?1 of HBsAg concentration were achieved 48 h from the induction point which added to a 120 h reduction in production period in comparison with Mut^S expression (168 h). The use of the pPIC3.5K-HBsAg plasmid in the Mut⁺ phenotype enhanced the expression of HBsAg by a nearly 13 times higher volumetric productivity in the first 24 h and 35 times higher at peak production concentration. Comparison of AOX expression cassettes relative to the HBsAg gene in the role of mRNA secondary structure during translation initiation revealed that HBsAg possesses lower folding stability with AOX1 Mut⁺ phenotype. The results from this study demonstrated that expression of HBsAg with Mut⁺ AOX1 promoter is adequate as an alternative for the production of HBsAg. In addition, the AOX promoter of the Mut⁺ phenotype was observed to be better suited for HBsAg expression, which correlates with the ease of translation initiation under shake flask conditions.     

Foreign gene expression in Hansenula polymorpha. A system for the synthesis of small functional peptides

 We describe the synthesis and purification of two functional peptides, namely human insulin-like growth factor II (IGF-II) and Xenopus laevis magainin II in Hansenula polymorpha after their synthesis as hybrid proteins fused to the C terminus of endogenous amine oxidase. The hybrid genes, placed under control of the H. polymorpha alcohol oxidase promoter (P_AOX), were integrated into the genomic alcohol oxidase locus, yielding stable production strains. High-level synthesis of the fusion proteins, exceeding 20% of total cellular protein, was obtained when the transformed strains were grown in methanol-limited chemostat cultures; when expressed by itself, i.e. in the absence of the amine oxidase gene, IGF-II could not be recovered from crude cell extracts, probably as a result of rapid proteolytic degradation. Accumulation in peroxisomes did not significantly affect the IGF-II protein stability when expressed in the absence of the carrier protein. Apparently, fusion to the large (±78 kDa) amine oxidase carrier particularly stabilizes the peptides and prevents them from proteolysis. After partial purification, the fusion partners were readily separated by factor Xa treatment. Received: 16 June 1995 / Accepted: 20 September 1995     

Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm,Bombyx mori

In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5–2.0 and 5.5–6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84 % of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.     

Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.