Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter P_AOX1 and constitutive promoter P_GAP. The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA‐seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol‐inducible promoter P₀₅₄₇ and the constitutive promoter P₀₄₇₂.     

Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris

Pichia pastoris is a well-established cell factory for recombinant protein synthesis. Various optimization strategies of processes based on AOX1 promoter have been investigated, including methanol co-feeding with glycerol or sorbitol during the induction stage. Compared with carbon sources, comparatively little research has been devoted to the effects of nitrogen sources. Several reports have described the benefits of adding casamino acids (CA) to the recombinant protein production medium, however, without considering its effects at the gene expression level. Using enhanced green fluorescent protein as a reporter protein, monitored using flow cytometry, CA was shown to downregulate AOX1 promoter induction. Despite higher growth rates, cultures containing CA exhibited slower transition to the induced state, whereas metabolite analysis revealed that methanol consumption was reduced in the presence of CA compared with its absence. The repressive effect of CA was further confirmed by analysing the synthesis of extracellular recombinant Candida antarctica lipase under control of the AOX1 promoter. These findings highlight nitrogen source selection as an important consideration for AOX1-based protein production.     

Cloning and sequence analysis of the Pichia pastoris TRP1, IPP1 and HIS3 genes

The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trp1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74·1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region. The TRP1 and IPP1 sequences were deposited in the EMBL databank under Accession Number AJ001000. The Accession Number of the HIS3 gene is U69170. © 1998 John Wiley & Sons, Ltd.     

Efficient expression, purification and characterization of mouse salivary alpha-amylase secreted from methylotrophic yeast, Pichia pastoris

We constructed a secretion vector of mouse salivary alpha-amylase, pPAM, using the AOX1 promoter-terminator and the secretion signal of 128 kDa pGKL killer protein, for an alternative yeast, Pichia pastoris. Taking advantage of multicopy insertion of the expression cassette and optimized growth conditions, we succeeded in highly efficient extracellular production (approximately 240 microg/ml) of mouse alpha-amylase in the 10 ml scale by conventional flask culture: this efficiency was about 90-fold higher than that of Saccharomyces cerevisiae. Growth temperature of cells was critical for efficient production of alpha-amylase. P. pastoris transformants secreted both core-glycosylated and non-glycosylated alpha-amylase molecules with a glycosylated:non-glycosylated ratio of about 20:80. Both glycosylated and non-glycosylated alpha-amylases were purified separately to apparent homogeneity. The signal sequence was correctly processed in both species, and the molecular masses of glycosylated and non-glycosylated alpha-amylase were determined to be 58 600 and 56 300, respectively, by mass spectrometry. We further studied the outer chain glycosylation of engineered mouse alpha-amylase secreted by P. pastoris.     

基因拷贝数对重组毕赤酵母的牛乳铁蛋白功能片段表达及细胞存活率的影响

为了提高牛乳铁蛋白功能片段(bovine lactoferrin functional fragment,BlfFf)产量,研究了基因拷贝数对重组毕赤酵母蛋白表达及酵母存活率的影响。将未折叠蛋白响应(unfolded protein response,UPR)激活因子基因HAC1、信号肽切割酶基因Kex2导入重组毕赤酵母BlfFf G01,并以核糖体r DNA非转录基因间隔区同源整合的方法结合PTVA(posttransformational vector amplification)法扩增重组菌株的基因拷贝数。利用SDS-PAGE、Western Blot、ELISA及流式细胞术分析多拷贝重组菌株发酵产物。分析表明,重组蛋白产量随着BlfFf基因拷贝数的增加而增加,但并非线性递增。HAC1拷贝数为3的BlfFf G12菌株产量相比单拷贝的BlfFf G10提高了150%,但更高的HAC1基因拷贝数降低了重组蛋白产量。流式细胞术分析显示,发酵末细胞存活率随着重组菌株中BlfFf基因拷贝数增加而下降,而HAC1基因拷贝数增加可一定程度上提高细胞存活率。摇瓶中BlfFf产量最高的BlfFf...     

Fed‐batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co‐substrate sorbitol

Batch‐wise sorbitol addition as a co‐substrate at the induction phase of methanol fed‐batch fermentation by Pichia pastoris (Mut⁺) was proposed as a beneficial recombinant protein production strategy and the metabolic responses to methanol feeding rate in the presence of sorbitol was systematically investigated. Adding sorbitol batch‐wise to the medium provided the following advantages over growth on methanol alone: (a) eliminating the long lag‐phase for the cells and reaching ‘high cell density production’ at t = 24 h of the process (C_X = 70 g CDW/l); (b) achieving 1.8‐fold higher recombinant human erythropoietin (rHuEPO) (at t = 18 h); (c) reducing specific protease production 1.2‐fold; (d) eliminating the lactic acid build‐up period; (e) lowering the oxygen uptake rate two‐fold; and (f) obtaining 1.4‐fold higher overall yield coefficients. The maximum specific alcohol oxidase activity was not affected in the presence of sorbitol, and it was observed that sorbitol and methanol were utilized simultaneously. Thus, in the presence of sorbitol, 130 mg/l rHuEPO was produced at t = 24 h, compared to 80 mg/l rHuEPO (t = 24 h) on methanol alone. This work demonstrates not only the ease and efficiency of incorporating sorbitol to fermentations by Mut⁺ strains of P. pastoris for the production of any bio‐product, but also provides new insights into the metabolism of the methylotrophic yeast P. pastoris. Copyright © 2009 John Wiley & Sons, Ltd.     

高果糖浆废液杂糖组分分析及其作为毕赤酵母发酵碳源的资源化利用

废杂糖的资源化利用是高果糖浆生产行业迫切需要解决的问题。该研究首先通过高效液相、质谱和红外光谱分析,确定了杂糖成分为葡萄糖、果糖和聚合度为2~16的线性葡聚糖,包括葡萄糖480 g/L,果糖92 g/L,麦芽糖103.6 g/L,麦芽三糖36.8 g/L,总糖含量802.3 g/L。进一步使用2种常用的毕赤酵母宿主(P AOX1型毕赤酵母、P GAP型毕赤酵母)利用杂糖发酵生产内切β-1,3葡聚糖酶并与标准碳源(甘油和葡萄糖)作对比。结果表明,对于P AOX1型毕赤酵母,杂糖做碳源时细胞密度和酶活性与甘油相比均有所下降,最大生物量分别为59.1和82.0 g/L,最高酶活性分别为157.29和199.2 U/mL。对于P GAP型毕赤酵母,杂糖与葡萄糖的发酵效果相当,说明杂糖可以作为P GAP型毕赤酵母生产内切β-1,3葡聚糖酶的优质替代性碳源。     

Identification and characterization of a novel yeast omega3-fatty acid desaturase acting on long-chain n-6 fatty acid substrates from Pichia pastoris

A cDNA sequence putatively encoding a omega(3)-fatty acid desaturase gene was isolated from methylotrophic yeast Pichia pastoris GS115. The deduced amino acid sequence of this cloned cDNA showed high identity to known fungal omega(3)-fatty acid desaturases. Functional identification of this gene heterologously in Saccharomyces cerevisiae strain INVScl indicated that the deduced amino acid sequence exhibited omega(3)-fatty acid desaturase activity. The newly identified omega(3)-fatty acid desaturase, named Pp-FAD3, is novel because it showed broad n-6 fatty acid substrate specificity by its ability to convert all the 18-carbon and 20-carbon n-6 substrates examined to the corresponding n-3 fatty acids, with an approximately equivalent high conversion rate. Pp-FAD3 is the first known yeast omega(3)-fatty acid desaturase to act on long-chain n-6 fatty acid substrates. Heterologous expression of the newly identified omega(3) desaturase in different hosts will be an alternative method to increase the flow of n-6 fatty acid intermediates into their n-3 derivatives.     

Cloning and disruption of the PpURA5 gene and construction of a set of integration vectors for the stable genetic modification of Pichia pastoris

A pair of degenerate primers was used for amplification and cloning of a DNA fragment containing parts of the P. pastoris URA5 and SEC65 genes. Using additional information from a partial genomic sequence of P. pastoris, we cloned and sequenced a 1.9 kb chromosomal fragment containing the complete orotate-phosphoribosyltransferase-encoding URA5 gene. A disruption cassette was constructed by replacing a small part of the open reading frame with a kanamycin-resistance gene. The P. pastoris wild-type strain NRRL Y-11430 was transformed with the disruption cassette and an ura5 auxotrophic strain was identified. To generate marker constructs that can be reused in successive transformations of a single strain, we constructed two lacZ-PpURA3-lacZ and lacZ-PpURA5-lacZ cassettes and used them to disrupt PpOCH1. The PpURA3 and PpURA5 genes in the disruptants were then successfully recycled by selecting for resistance to 5'-fluoro-orotic acid. We also assembled a set of modular plasmids that can be used for the stable genetic modification of P. pastoris via a double cross-over event. The sequence presented here has been submitted to the EMBL data library under Accession No. AY303544.     

Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers

Screening of a partial genomic database of Pichia pastoris allowed us to identify the ARG1, ARG2, ARG3, HIS1, HIS2, HIS5 and HIS6 genes, based on homology to their Saccharomyces cerevisiae counterparts. Based on the cloned sequences, a set of disruption vectors was constructed, using the previously described PpURA5-blaster as a selectable marker, and the cloned genes were individually disrupted. All disruptants exhibited the expected auxotrophic phenotypes, with only the his2 knockouts displaying a bradytroph phenotype. To allow their use as auxotrophic markers, we amplified the open reading frames and respective promoters and terminator regions of PpARG1, PpARG2, PpARG3, PpHIS1, PpHIS2 and PpHIS5. We then designed a set of integration vectors harbouring cassettes of the ARG pathway as selectable markers, to disrupt the genes of the HIS pathway and vice versa. Employing this strategy, we devised a scheme allowing for the rapid and stable introduction of several heterologous genes into the genome of P. pastoris without the need for recyclable markers or strains with multiple auxotrophies. Furthermore, simple replica-plating, instead of cost-consuming and labour-intensive colony PCR or Southern analysis, can be used to identify positive transformants, making this approach amendable for initial high-throughput applications, which can then be followed up by a more careful analysis of the selected transformants.     

Isolation and characterisation of a kallikrein-like enzyme from Agkistrodon halys pallas snake venom

BACKGROUND: Viper snake venoms contain a great variety of toxic proteins. These components mediate their toxicity by either stimulating or inhibiting the haemostatic system of human victims or experimental animals, resulting in common clinical complications of blood clotting or uncontrolled haemorrhage. Therefore it is deemed important to isolate the active component(s) from snake venom with kallikrein‐like activity. RESULTS: A kallikrein‐like proteinase of Agkistrodon halys pallas snake venom, designated AHP‐Ka, was purified by anion exchange chromatography and affinity chromatography. Physicochemical studies showed that the purified enzyme was a 34 kDa monomeric glycoprotein, the molecular weight of which decreased to 26 kDa after deglycosylation with peptide N‐glycosidase F (PNGase F). Sequence studies on the NH₂‐terminal region of the protein indicated that AHP‐Ka shared a high degree of sequence homology with other serine proteinases from snake venoms. AHP‐Ka showed high catalytic activity and kallikrein‐like activity on substrates such as arginine esterase BAEE and chromogenic H‐D‐Pro‐Phe‐Arg‐pNA·2HCl (S‐2302) and was inhibited by protease inhibitor phenylmethylsulfonyl fluoride (PMSF). CONCLUSION: The results showed that AHP‐Ka isolated from A. halys pallas snake venom and purified by anion exchange chromatography and affinity chromatography is in fact a kallikrein‐like enzyme. Copyright © 2011 Society of Chemical Industry     

Mutations in Fks1p affect the cell wall content of beta-1,3- and beta-1,6-glucan in Saccharomyces cerevisiae

Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. Analysis of fks1 delta mutants showed a partial K1 killer toxin-resistant phenotype and a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1 delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. Overexpression of FKS2 suppressed the killer toxin phenotype of fks1 delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1 delta cells. In addition, eight out of 12 fks1ts fks2 delta mutants had altered beta-glucan levels at the permissive temperature: the partial killer resistant FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive allele FKS1T605I M761T increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. We analysed Fks1p and Fks2p in beta-1,6-glucan deficient mutants and found that they were mislocalized and that the mutants had reduced in vitro glucan synthase activity, possibly contributing to the observed beta-1,6-glucan defects.     

Soluble forms of YlCrh1p and YlCrh2p, cell wall proteins of Yarrowia lipolytica, have beta-1,3-glycosidase activity

Crh1p and Crh2p of Saccharomyces cerevisiae are cell wall proteins covalently attached to cell wall glucan and are thought to be putative glycosidases involved in cell wall remodelling. We investigated whether YlCrh1p and YlCrh2p, the Yarrowia lipolytica proteins homologous to ScCrh1p and ScCrh2p, had the required glycosidase activity for cell wall biosynthesis and maintenance. Ylcrh1Delta and Ylcrh2Delta mutants showed sensitivity to compounds that interfere with cell wall construction. Soluble forms of YlCrh1p and YlCrh2p that lacked the C-terminal consensus sequence for GPI anchoring showed glycosidase activity on laminarin, a substrate carrying beta-1,3-glycosidic linkage. Our study suggests that the YlCrh1p and YlCrh2p may participate in cell wall biosynthesis and remodelling through their beta-1,3-glycosidase activity.     

Effect of industrial processing on alfalfa cell walls

Thermal treatment is one of the widely used methods for feedstuff preservation. The aim of this study was to evaluate possible cell wall alterations during the industrial‐scale drying of alfalfa. Alfalfa is mainly dehydrated and pelleted using two drying treatments. First a thermal treatment at 70 °C is applied to the chopped raw material, prior to the main dehydration procedure which involves a temperature of about 600 °C for a restricted time (15 min). The different steps of the dehydration process did not cause significant changes in alfalfa cell wall composition, but the proportion of labile ether‐linked lignin structure (β‐O‐4) increased after the thermal shock. Chemical fractionation of cell wall carbohydrates showed that pectins were less accessible to extraction with ammonium oxalate after alfalfa drying. Moreover, xylanase (endo enzyme) was more effective on cell walls obtained from heated material. These results suggested that heat processing causes some changes in interpolymer bonds (covalent and hydrogen bonds) between pectins and the lignin/hemicellulose matrix, whereby the cell wall network is modified. © 2002 Society of Chemical Industry     

Enzymic degradation and nature of the endosperm cell walls of germinating sorghums and barley

β-(1·3)(1·4)-Glucans are the major components of isolated endosperm cell walls of both sorghum and barley grains and the concentration declines from 75% in unmalted barley walls to 32% in those of the 6-day malts. In sorghums over the same period, the reduction was from 72 and 68% to 54 and 62% in varieties L187 and SK5912, respectively. Following germination wall pentosans, while enhanced in barley, decreased in sorghum. The enzymes which were unable to hydrolyse the walls of sorghum to release sugars (unlike those of barley) did so after the walls had been solubilised in alkali. Cellobiose accounted for 43% of the hydrolysates of the alkali-solubilised walls when incubated with sorghum malt enzymes and 79% when barley enzymes were used. Laminaribiose was not detected in either case which indicates that the polysaccharide of the sorghum walls is largely of a ‘cellulosic’ nature. Preliminary evidence suggests that fuco-xyloglucan may contribute to the resistance of the sorghum endosperm cell walls to enzymic attack during germination.     

A review of malting and malt processing for whisky distillation

This paper encompasses a re‐evaluation of published literature and data regarding wort attenuation in malt distilleries raising questions and discussing how the conventional wisdom has changed over time and what questions still need to be answered. Current knowledge is summarized in the following four points: (a) Under normal malting conditions, starch granules are partially degraded by a combination of α‐amylase and α‐glucosidase. This complex can open up the granule at specific sites on the surface and create characteristic ‘pin‐hole’ lesions, which may be widened by secondary hydrolysis by α‐ and β‐amylase, limit dextrinase and α‐glucosidase (maltase). (b) All of these diastatic enzymes can survive mild kilning, probably by forming heat stable complexes on and within the starch granules and can continue a complete degradation of starch when mashed at ambient temperatures with glucose as the end product. (c) At normal mashing temperatures, starch granules gelatinize and dissolve with a concomitant rapid degradation to glucose, maltose, maltotriose and dextrins ranging from degree of polymerization (DP) 4 to > DP20. If there is immediate wort boiling after run‐off, this is the final composition of starch derived carbohydrates according to the conventional paradigm. (d) All malt worts also contain a small amount of panose, isopanose as well as glucosyl maltodextrins, based on a core of 6₂‐α‐glucosyl maltose (panose) or 6‐α‐maltosyl glucose (isopanose), which are remnants of the α‐amylase/glucosidase degradation of granular starch. These dextrins are resistant to the action of debranching enzymes and their concentration may vary between 4 and 8% of the malt extract, depending on the degree of modification of the host starch granules. They may be created at the active sites of this enzyme complex when the granule is gelatinized. In a conventional mash of unboiled distilling wort, the spectrum of wort dextrins produced from gelatinized starch is reduced to true ‘limit’ dextrins of DP4–8 by continued α‐amylolysis during early fermentation. These dextrins will contain side chains of either maltose or maltotriose residues surrounding the α‐1,6‐glucosidic linkage and can be debranched by limit dextrinase during late fermentation, leaving only the above glucosyl maltodextrins dextrins in the spent wash. Copyright © 2016 The Institute of Brewing & Distilling