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.     

Comparison of expression systems in the yeasts Saccharomyces cerevisiae,Hansenula polymorpha,Klyveromyces lactis,Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.     

Effects of co‐overexpression of secretion helper factors on the secretion of a HSA fusion protein (IL2‐HSA) in pichia pastoris

Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2‐HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% compared with that of HSA (>1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 were involved in protein folding and Sec1 and Sly1 were involved in vesicle trafficking. We evaluated the possible effects of individual overexpression of these secretion helper factors on the secretion of IL2‐HSA in P. pastoris. Constitutive overexpression of the five selected secretion factors did not have an obvious negative effect on cell growth of the IL2‐HSA secreting strain. Individual co‐overexpression of Ero1, Kar2, Pdi1, Sec1 and Sly1 improved the secretion level of IL2‐HSA to ~2.3‐, 1.9‐, 2.2‐, 2.5‐ and 1.9‐fold that in the control strain respectively in shake flasks. We evaluated the changes in mRNA and protein levels of the intracellular IL2‐HSA, as well as the secretion helper factor genes in the co‐overexpressing strains. Our results indicated that manipulating the expression level of ER resident protein Pdi1, Ero1, Kar2 and SM protein Sec1 and Sly1 could improve the secretion level of IL2‐HSA fusion protein in P. pastoris, which provided new candidates for combinatorial engineering in future study. Copyright © 2016 John Wiley & Sons, Ltd.     

Candida albicans TDH3 gene promotes secretion of internal invertase when expressed in Saccharomyces cerevisiae as a glyceraldehyde-3-phosphate dehydrogenase-invertase fusion protein

We have checked the ability of the Candida albicans GAPDH polypeptide, which lacks a conventional N-terminal signal peptide, to reach the cell wall in Saccharomyces cerevisiae by using an intracellular form of the yeast invertase as a reporter protein. A hybrid TDH3-SUC2 gene containing the C. albicans TDH3 promoter sequences and a coding region encoding a fusion protein formed by the C. albicans GAPDH polypeptide, fused at its C-terminus with the yeast internal invertase, was constructed in a centromer derivative plasmid and transformed into a Suc(-) S. cerevisiae strain. Transformants displayed invertase activity measured in intact whole cells, and were able to grow on sucrose as the sole fermentable carbon source. Northern blot analysis with both TDH3 and SUC2 probes detected a single mRNA species of the expected size (about 2.7 kb), and Western immunoblot analysis of cell-free extracts, using a monoclonal antibody (mAb49) against a C. albicans GAPDH epitope, showed the presence of a 90 kDa polypeptide corresponding to the GAPDH-invertase fusion protein. This indicates that the TDH3 gene is able to direct part of the encoded gene product to the cell wall, and that any putative motifs for this targeting should be within the GAPDH amino acid sequence. Further analysis, using the same approach, of a panel of seven N- and C-terminal GAPDH truncates revealed that the region required for the cell wall targeting is located within the N-terminal half of the protein.     

Enhancement of Protein Secretion in Saccharomyces cerevisiae by Overproduction of Sso Protein,a Late-acting Component of the Secretory Machinery

Increased production of secreted proteins in Saccharomyces cerevisiae was achieved by overexpressing the yeast syntaxins, Sso1 or Sso2 protein, the t-SNAREs functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane. Up to four- or six-fold yields of a heterologous secreted protein, Bacillus α-amylase, or an endogenous secreted protein, invertase, were obtained respectively when expressing either one of the SSO genes, SSO1 or SSO2, from the ADH1 promoter on a multicopy plasmid. Direct correlation between the Sso protein level and the amount of secreted α-amylase was demonstrated by modulating the expression level of the SSO2 gene. Quantitation of the α-amylase activity in the culture medium, periplasmic space and cytoplasm suggests that secretion into the periplasmic space is the primary stage at which the SSO genes exert the secretion-enhancing function. Pulse-chase data also support enhanced secretion efficiency obtained by SSO overexpression. Our data suggest that the Sso proteins may be rate-limiting components of the protein secretion machinery at the exocytosis step in yeast. © 1997 John Wiley & Sons, Ltd.     

Investigation of two yeast genes encoding putative isoenzymes of phosphoglycerate mutase

Our previous data indicated that GPM1 encodes the only functional phosphoglycerate mutase in yeast. However, in the course of the yeast genome sequencing project, two homologous sequences, designated GPM2 and GPM3, were detected. They have been further investigated in this work. Key residues in the deduced amino acid sequence, shown to be involved in catalysis for Gpm1 (i.e. His8, Arg59, His181) are conserved in both enzymes. Overexpression of the genes under control of their own promoters in a gpm1 deletion mutant did not complement for any of the phenotypes. This could in part be attributed to a lack of expression due to their weak promoters. Higher level expression under the control of the yeast PFK2 promoter partially complemented the gpm1 defects, without restoring detectable enzymatic activity. Nevertheless, deletion of either GPM2 or GPM3, or the two deletions in concert, did not produce any obvious lesions for growth on a variety of different carbon sources, nor did they change the levels of key intermediary metabolites. We conclude that both genes evolved from duplication events and that they probably constitute non-functional homologues in yeast.     

Isolation and characterisation of invertase inhibitor from sweet potato storage roots

BACKGROUND: Plant invertases play important roles in sucrose metabolism. Cell wall invertase has been reported to participate in phloem loading and unloading. Soluble invertases are involved in hexose level regulation in mature tissues and in utilisation of stored sucrose within vacuoles. Invertase inhibitory proteins have been described as one of the possible components for invertase activity regulation in some plant species. RESULTS: In this work an invertase inhibitor (ITI) coding sequence was cloned by differential display from sweet potato (SP) storage roots. SPITI codes for a protein of 192 amino acids with a predicted molecular mass of 20 624 Da containing a 20‐amino‐acid signal peptide and four cysteines. Computer analysis of the deduced amino acid sequences of the conserved domain revealed that the protein belonged to the plant invertase/pectin methylesterase inhibitor. Both the corresponding mRNA and protein levels were found to be highest in storage roots, followed by veins. Recombinant SPITI protein from the storage root cDNA clone overproduced in Escherichia coli (M15) was purified by affinity chromatography. This protein effectively inhibited the invertase activity in a dose‐dependent manner. The results presented in the Lineweaver‐Burk plots indicated that the invertase inhibitor displayed a mode of competitive inhibition towards the invertase tested, with a K_i of 3.82 × 10^?6 mol L^?1. CONCLUSION: These results suggested that SPITI is a novel member of the ITI family in plants. SPITI genes of sweet potato storage roots display differential gene expression patterns, which may be associated with sucrose metabolism to cope with particular developmental requirements. Copyright © 2008 Society of Chemical Industry     

Development and characterization of a vector set with regulated promoters for systematic metabolic engineering in Saccharomyces cerevisiae

A set of vectors was constructed that enable combined and systematic testing of metabolic pathway genes in Saccharomyces cerevisiae. The vectors are available as CEN/ARS and 2 µ‐based plasmids with a choice of three inducible promoters, P_GAL1, P_CUP1 and P_ADH2. These features offer control over the initiation and level of gene expression. In addition, the vectors can be used as templates to generate PCR fragments for targeted chromosomal integration of gene expression cassettes. Selection markers are flanked by loxP elements to allow efficient CreA‐mediated marker removal and recycling after genomic integration. For each promoter, expression of a bacterial lacZ reporter gene was characterized from plasmid‐based and integrated chromosomal cassettes, and compared to that of the glycolytic P_PGK1 promoter. Plasmid stabilities were also determined. The promoters showed distinct activity profiles useful for modulating expression of metabolic pathway genes. This series of plasmids with inducible promoters extends our previous vector set carrying the constitutive promoters P_PGK1, P_TEF1 and P_HXT7‐391. Copyright © 2012 John Wiley & Sons, Ltd.     

Physiological approach to heterologous human serum albumin production by Kluyveromyces lactis in chemostat culture

Production of recombinant human serum albumin (rHSA) controlled by the constitutive promoter phosphoglycerate kinase was studied in Kluyveromyces lactis. It was governed by both cell concentration and glycolytic flow. The triggering of the fermentation metabolism by unfavourable culture conditions (pH, pO₂, D) caused a decrease in the synthesis of the heterologous protein. The highest productivity (75 mg 1^?1 per h) and rHSA concentration (62 mg 1^?1) were obtained in chemostat culture with a dilution rate of 0·12 h^?1 and with 38 g 1^?1 dry weight.     

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end‐products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast‐based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1·0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA‐1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA‐1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA‐1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1_P‐MFα1_S‐pedA‐ADH1_T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1_P) and terminator (ADH1_T). Secretion of the PED1‐encoded pediocin PA‐1 was directed by the yeast mating pheromone α‐factor's secretion signal (MFα1_S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4·6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria. Copyright © 1999 John Wiley & Sons, Ltd.     

Growth of a tropical marine yeast Yarrowia lipolytica NCIM 3589 on bromoalkanes: relevance of cell size and cell surface properties

Yarrowia lipolytica 3589, a tropical marine yeast, grew aerobically on a broad range of bromoalkanes varying in carbon chain length and differing in degree and position of bromide group. Amongst the bromoalkanes studied, viz. 2‐bromopropane (2‐BP), 1‐bromobutane (1‐BB), 1,5‐dibromopentane (1,5‐DBP) and 1‐bromodecane (1‐BD), the best utilized was 1‐BD, with a maximal growth rate (μ_max) of 0.055 h^?1 and an affinity ratio (μ_max/K_s) of 0.022. Utilization of these bromoalkanes as growth substrates was associated with a concomitant release of bromide (8202.9 µm ) and cell mass (36 × 10⁹ cells/ml), occurring maximally on 1‐BD. Adherence of yeast cells to these hydrophobic bromoalkanes was observed microscopically, with an increase in cell size and surface hydrophobicity. The maximal cell diameter was for 1‐BD (4.66 µm), resulting in an increase in the calculated cell surface area (68.19 µm²) and sedimentation velocity (1.31 µm/s). Cell surface hydrophobicity values by microbial adhesion to solvents (MATS) analysis for yeasts grown on bromoalkanes and glucose were significantly high, i.e. >80%. Similarly, water contact angles also indicate that the cell surface of yeast cells grown in glucose possess a relatively more hydrophilic cell surface (θ = 49.1°), whereas cells grown in 1‐BD possess a more hydrophobic cell surface (θ = 90.7°). No significant change in emulsification activity or surface tension was detected in the cell‐free supernatant. Thus adherence to the bromoalkane droplets by an increase in cell size and surface hydrophobicity leading to debromination of the substrate might be the strategy employed in bromoalkane utilization and growth by Y. lipolytica 3589. Copyright © 2011 John Wiley & Sons, Ltd.     

Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose‐containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose‐induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC–URA plasmid except that gene expression is mediated by constitutive promoters. Copyright © 2010 John Wiley & Sons, Ltd.     

EFFECT OF ADDITION OF PEACH GUM ON PHYSICOCHEMICAL PROPERTIES OF GELATIN-BASED MICROCAPSULE

The physicochemical properties of the microcapsules, which were prepared by spray drying, were evaluated. Gelatin‐sucrose (Gel‐Suc) and gelatin‐peach gum‐sucrose (Gel‐PG‐Suc) were used as wall material, while vitamin A was used as core material. The microencapsulation efficiency of Gel‐Suc and Gel‐PG‐Suc microcapsules significantly varied from 94.77 to 96.70% (P < 0.05). By adding peach gum, the microcapsules exhibited a spherical shape with a smooth surface, as evidenced by scanning electron microscopy, and the volume average diameter (D_4,3) showed significant difference from 73.95 to 68.72 µm (P < 0.05). By employing Fourier transform infrared spectroscopy, it was shown that OH‐stretching vibrations of Gel‐PG‐Suc microcapsules shifted to a lower wave number than that of Gel‐Suc microcapsules (Gel‐Suc microcapsules absorption peak: 3403.80/cm, Gel‐PG‐Suc microcapsules absorption peak: 3395.23/cm), so H‐bonding was strengthened by adding peach gum. Corresponding to the band of C=O in —C=ONH—, Gel‐Suc and Gel‐PG‐Suc microcapsules were 1652.72/cm and 1646.31/cm, respectively. Differential scanning calorimetry results revealed that the glass transition temperature (T_g) increased significantly from 46.103 to 50.448C by the addition of peach gum (P < 0.05).     

The Yarrowia lipolytica PAH1 homologue contributes but is not required for triacylglycerol biosynthesis during growth on glucose

The PAH1-encoded phosphatidate phosphatase (PAP) catalyzes the Mg²⁺-dependent dephosphorylation of phosphatidate to produce diacylglycerol, which can be acylated to form triacylglycerol (TAG). In the model oleaginous yeast Yarrowia lipolytica, TAG is the major lipid produced, and its biosynthesis requires a continuous supply of diacylglycerol, which can be provided by the PAP reaction. However, the regulation of Pah1 has not been studied in detail in Y. lipolytica, and thus its contribution to the biosynthesis of TAG in this yeast is not well understood. In this work, we examined the regulation of the PAH1-mediated PAP activity and Pah1 abundance and localization in cells growing on glucose. We found that Pah1 abundance and localization were regulated in a growth-dependent manner, yet the loss of Pah1 did not have a major effect on PAP activity. We also examined the effects of the Y. lipolytica pah1Δ mutation on cell physiology and lipid biosynthesis. The lack of Pah1 in the pah1Δ mutant resulted in a moderate decrease in TAG levels and an increase in phospholipid levels. These results showed that Pah1 contributed to TAG biosynthesis in Y. lipolytica but also suggested the presence of other activities in the pah1Δ mutant that compensate for the loss of Pah1. Also, the levels of linoleic acid were elevated in pah1Δ cells with a concomitant decrease in the oleic acid levels suggesting that the pah1Δ mutation affected the biosynthesis of fatty acids.     

Characterization of phosphatidic acid phosphatase activity in the oleaginous yeast Yarrowia lipolytica and its role in lipid biosynthesis

Phosphatidic acid phosphatase (PAP) catalyses the committed step of triacylglycerol (TAG) biosynthesis and thus regulates the amounts of TAG produced by the cell. TAG is the target of biotechnological processes developed for the production of food lipids or biofuels. These processes are using oleaginous microorganisms like the yeast Yarrowia lipolytica as the TAG producers. Thus manipulating key enzymatic activities like PAP in Y. lipolytica could drive lipid biosynthesis towards TAG production and increase TAG yields. In this study, PAP activity in Y. lipolytica was characterized in detail and its role in lipid biosynthesis was addressed. PAP activity increased 2.5‐fold with the addition of Mg²⁺ (1 mm ) in the assay mixture, which means that most of the PAP activity was due to Mg²⁺‐dependent PAP enzymes (e.g. Pah1, App1). In contrast, N‐ethylmaleimide (NEM) potently inhibited PAP activity, indicating the presence of NEM‐sensitive PAP enzymes (e.g. App1, Lpp1). Localization studies revealed that the majority of PAP activity resides in the membrane fraction, while the cytosolic fraction harbours only a small amount of activity. PAP activity was regulated in a growth‐dependent manner, being induced at the early exponential phase and declining thereafter. PAP activity did not correlate with TAG synthesis, which increased as cells progressed from the exponential phase to the early stationary phase. In stationary phase, TAG was mobilized with the concomitant synthesis of sterols and sterol esters. These results provide the first insights into the role of PAP in lipid biosynthesis by Y. lipolytica. Copyright © 2016 John Wiley & Sons, Ltd.     

Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus

The bottom‐fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)‐type and two S. bayanus (Sb)‐type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p‐hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G‐418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p‐hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5‐fluoroorotic acid (5‐FOA) plates to generate the desired Ura^– homozygous disruptant (Scura3Δ::TDH3p‐hygro/Scura3Δ::TDH3p‐hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom‐fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1‐overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3? lager strain will be a useful host for constructing strains of relevance to brewing. Copyright © 2012 John Wiley & Sons, Ltd.