不同信号肽对酿酒酵母蔗糖转化酶Suc2分泌表达水平的影响

目前，绝大多数酿酒酵母（Saccharomyces cerevisiae）菌株利用菊糖生产乙醇的能力有限，而蔗糖转化酶Suc2是酿酒酵母水解菊糖的关键酶，其分泌水平直接影响酿酒酵母转化菊糖为乙醇的性能。为提高酿酒酵母中蔗糖转化酶Suc2的分泌表达水平，利用生物信息学的分析方法选择出11种不同的分泌信号肽，包括酿酒酵母内源性、其他菌株来源以及已报道序列优化改造的信号肽，将它们融合至Suc2并构建了相应的酿酒酵母BY4741重组菌。其中，酿酒酵母内源分泌信号肽AGA2能使蔗糖转化酶Suc2更有效的分泌，含有信号肽AGA2的重组菌BY-AG的蔗糖酶酶活和菊糖酶酶活相对于含有天然信号肽的原始菌BY-S分别提高42%和26%，其利用菊糖产乙醇能力较原始菌提高了32%，乙醇产量达到78.11 g/L。在使用毕赤酵母（Pichia pastoris）分泌信号肽MSB2时，蔗糖转化酶Suc2的分泌水平也有提高，含有信号肽MSB2的重组菌BY-MS较原始菌BY-S的蔗糖酶酶活和菊糖酶酶活分别提高了80%和74%，同时，利用菊糖产乙醇能力也提高了56%，产量达到86.31 g/L。最后，对重组菌BY-MS摇瓶发酵过程中的生物量、蔗糖酶酶活、残糖总量和乙醇产量进行了监测，结果表明，重组菌BY-MS的发酵性能较原始菌BY-S有显著提高。本研究为提高蔗糖转化酶Suc2的分泌水平、构建高效菊糖基乙醇生产菌株提供参考。     

Secretory expression of human growth hormone inSaccharomyces cerevisiae using three different leader sequences

A recombinant human growth hormone (hGH) was expressed as a secretory product in the yeastSaccharomyces cerevisiae. Three different leader sequences derived from the mating factor α1 (MFα1), inulinase and invertase were used to direct the secretion of hGH into the extracellular medium. Among three leader sequences tested, the inulinase leader sequence was found to be the most efficient in the secretory expression of hGH. In contrast, no hGH was detected in the extracellular medium with the invertase leader sequence. After 48 h shake-flask culture, the yields of hGH secreted into the medium by the invertase, MFα1, inulinase and invertase leader sequences were approximately 0, 0.3 and 0.9 mg/L, respectively. The secretion efficiencies were also found to be 0, 3.8 and 13% for the invertase, MFα1 and inulinase leader sequences, respectively.     

Yeast secretory expression of insulin precursors

Since the 1980s, recombinant human insulin for the treatment of diabetes mellitus has been produced using either the yeast Saccharomyces cerevisiae or the prokaryote Escherichia coli. Here, development of the insulin secretory expression system in S. cerevisiae and its subsequent optimisation is described. Expression of proinsulin in S. cerevisiae does not result in efficient secretion of proinsulin or insulin. However, expression of a cDNA encoding a proinsulin-like molecule with deletion of threonine^B30 as a fusion protein with the S. cerevisiaeα-factor prepro-peptide (leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g. AAK), or by direct fusion of lysine^B29 to glycine^A1, results in the efficient secretion of folded single-chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor can then be purified and subsequently converted to human insulin by tryptic transpeptidation in organic–aqueous medium in the presence of a threonine ester. The leader confers secretory competence to the insulin precursor, and constructed (synthetic) leaders have been developed for efficient secretory expression of the insulin precursor in the yeasts S. cerevisiae and Pichia pastories. The Kex2 endoprotease, specific for dibasic sites, cleaves the leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptide-insulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide localised between the leader and the insulin precursor has been developed to optimise Kex2 endoprotease processing and insulin precursor fermentation yield. Received: 8 February 2000 / Received revision: 2 May 2000 / Accepted: 2 May 2000     

A novel fusion partner for enhanced secretion of recombinant proteins in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Expressing proteins with fusion partners improves yield and simplifies the purification process. We developed a novel fusion partner to improve the secretion of heterologous proteins that are otherwise poorly excreted in yeast. The VOA1 (YGR106C) gene of Saccharomyces cerevisiae encodes a subunit of vacuolar ATPase. We found that C-terminally truncated Voa1p was highly secreted into the culture medium, even when fused with rarely secreted heterologous proteins such as human interleukin-2 (hIL-2). Deletion mapping of C-terminally truncated Voa1p, identified a hydrophilic 28-amino acid peptide (HL peptide) that was responsible for the enhanced secretion of target protein. A purification tag and a protease cleavage site were added to use HL peptide as a multi-purpose fusion partner. The utility of this system was tested via the expression and purification of various heterologous proteins. In many cases, the yield of target proteins fused with the peptide was significantly increased, and fusion proteins could be directly purified with affinity chromatography. The fusion partner was removed by in vitro processing, and intact proteins were purified by re-application of samples to affinity chromatography.     

Synthetic Leaders with Potential BiP Binding Mediate High-Yield Secretion of Correctly Folded Insulin Precursors fromSaccharomyces cerevisiae

Secretion leaders are essential for expression of many heterologous proteins including insulin in yeast. The function of secretion leaders and their interaction with the secretory pathway is not clear. To determine what constitutes functional pre-pro-leader sequences inSaccharomyces cerevisiae,synthetic leader sequences for secretion of the insulin precursor were developed by a combination of rational design and stepwise systematic optimization. The synthetic leaders efficiently facilitate secretion of the insulin precursor fromS. cerevisiaewhen compared with the α-factor leader, leading to a high yield of correctly folded insulin precursor in the culture supernatant. The synthetic leaders feature two potential N-linked glycosylation sites which are efficiently glycosylated during secretion. Pulse–chase analysis indicates that the synthetic leaders/insulin precursor fusion protein have a prolonged residence in the endoplasmic reticulum compared to the α-factor leader/insulin precursor fusion protein. The longer transition time in the endoplasmic reticulum mediated by the synthetic leaders might provide additional time for correct folding of the insulin precursor and account for the increased fermentation yield.     

Enhanced secretion of<Emphasis Type="Italic"> Bacillus stearothermophilus</Emphasis> L1 lipase in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> by translational fusion to cellulose-binding domain

The secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae was investigated by employing a fusion partner, a cellulose-binding domain (CBD) from Trichoderma harzianum endoglucanase II (THEG). The CBD was connected to the N-terminal of L1 lipase through an endogenous linker peptide from THEG. The expression cassette for the fusion protein in S. cerevisiae was constructed using the -amylase signal peptide and the galactose-inducible GAL10 promoter. Secretion of CBD-linker-L1 lipase by this fusion construct was dramatically 7-fold enhanced, compared with that of the mature L1 lipase without CBD-fusion. The fusion protein was secreted into the culture medium, reaching levels of approximately 1.3 g/l in high-cell-density fed-batch cultures. Insertion of a KEX2 cleavage site into the junction between CBD-linker and L1 lipase resulted in the same level of enhanced secretion, indicating that the CBD-linker fusion probably plays a critical role in secretion from endoplasmic reticulum to Golgi apparatus. Therefore, the CBD from THEG can be used both as an affinity tag and as a secretion enhancer for the secretory production of heterologous proteins in S. cerevisiae, since in vivo breakage at the linker was almost negligible.     

Secretion and localization of invertase and inulinase in recombinant Saccharomyces cerevisiae

Summary Secretion of invertase and inulinase produced by recombinant Saccharomyces cerevisiae cells were investigated under derepression conditions of GALI promoter. Secreted invertase mainly localized in the periplasmic space, but most of inulinase was found in the extracellular culture medium. This high level of extracellular secretion of inulinase was not dependent on the growth phase in which derepression of GALI promoter occurs. Our results indicate that the inulinase polypeptide itself may have a function for the protein secretion into the culture medium.     

The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57–70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.     

Highly efficient secretion of heterologous proteins from Saccharomyces cerevisiae using inulinase signal peptides

The INU genes of Kluyveromyces marxianus encode inulinases which are readily secreted from Saccharomyces cerevisiae into the culture medium. To evaluate the utility of the INU signal peptides for the secretion of heterologous proteins from S. cerevisiae, a variety of expression and secretion vectors were constructed with GAL10 promoter and GAL7 terminator. The coding sequence for human lipocortin-1 (LC1) was inserted in-frame with the INU signal sequences, and then the secretion efficiency and localization of LC1 were investigated in more detail and compared with those when being expressed by the vector with the MFalpha1 leader peptide. The vector systems with INU signal peptides secreted ca. 95% of the total LC1 expressed into the extracellular medium, while the MFalpha1 leader peptide-containing vector resulted in very low secretion efficiency below 10%. In addition, recombinant human interleukin-2 (IL-2) was expressed and secreted with the vector systems with INU signal peptide, and a majority fraction of the human IL-2 expressed was found to be secreted into the extracellular medium as observed in LC1 expression. (c) 1995 John Wiley & Sons, Inc.     

Directed evolution of a secretory leader for the improved expression of heterologous proteins and full‐length antibodies in Saccharomyces cerevisiae

Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in Saccharomyces cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three‐ to tenfold improvements over wild‐type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFα1pp) is screened for the enhanced secretion of a single‐chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to 16‐fold over wild type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a 180‐fold improvement over previous reports in the secretion of full‐length, functional, glycosylated human IgG₁. The production of full‐length IgG₁ at milligram per liter titers in a simple, laboratory‐scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs. Biotechnol. Bioeng. 2009;103: 1192–1201. © 2009 Wiley Periodicals, Inc.     

Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding

High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.     

Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts

Saccharomyces cerevisiae is often used to produce heterologous proteins that are preferentially secreted to increase economic feasibility. We used N-glycosylation as a tool to enhance protein secretion. Secretion of cutinase, a lipase, and llama V_HH antibody fragments by S. cerevisiae or Pichia pastoris improved following the introduction of an N-glycosylation site. When we introduced an N-glycosylation consensus sequence in the N-terminal region of a hydrophobic cutinase, secretion increased fivefold. If an N-glycosylation site was introduced in the C-terminal region, however, secretion increased only 1.8-fold. These results indicate that the use of N glycosylation can significantly enhance heterologous protein secretion.     

Amplification of leader proregions as a mean to increase the secretion of antibody fragments in the Pichia pastoris yeast

The dependence of secretion efficiency in Pichia pastoris cells on the copy number of proregions in leader polypeptides has been studied. The humanized light kappa-chain of the murine H3-1 antibody was used as a reporter protein. The leader pre-pro-polypeptides were composed of the signal peptide (preregion) from the α-factor precursors of Saccharomyces cerevisiae and a variable number of proregions from the prepro-precursors of the α-factor or the Hsp150p protein of S. cerevisiae or Hsp150p of P. pastoris. An increase in the proregion copy number either resulted in an almost 1.5-fold increase or a fivefold decrease in secretion depending on the proregion used. It was concluded that the enhancement of the proregion copy number could be of potential value for the intensification of protein secretion in P. pastoris.     

Protease-Deficient Saccharomyces cerevisiae Strains for the Synthesis of Human-Compatible Glycoproteins

Saccharomyces cerevisiae strains engineered previously to produce proteins with mammalian high mannose structures showed severe growth defects and decreased protein productivity. In strain YAB101, derived from one of these strains by a mutagenesis technique based on the disparity theory of evolution, these undesirable phenotypes were alleviated. Here we describe further engineering of YAB101 with the aim of synthesizing heterologous glycoproteins with Man₅GlcNAc₂, an intermediate for the mammalian hybrid and complex type oligosaccharides. About 60% conversion of Man₈GlcNAc₂ to Man₅GlcNAc₂ was observed after integration of Aspergillus saitoi α-1,2-mannosidase fused to the transmembrane domain of S. cerevisiae Och1. To obtain a higher yield of the target protein, a protease-deficient version of this strain was generated by disruption of PEP4 and PRB1, resulting in YAB101-4. Inactivation of these vacuolar proteases enhanced the secretion of human interferon-β by approximately 10-fold.     

Investigation of Growth Hormone Releasing Hormone Receptor Structure and Activity Using Yeast Expression Technologies

Abstract

Growth hormone releasing hormone (GHRH) is the positive regulator of growth hormone synthesis and secretion in the anterior pituitary. The peptide confers activity by binding to a seven transmembrane domain G protein-coupled receptor. Signal transduction proceeds through subsequent Gas stimulation of adenylyl cyclase. To investigate ligand/receptor and receptor/G protein associations, the human GHRH receptor was expressed in a modified S. cerevisiae strain which allows for facile measurement of receptor activity by cell prototrophy mediated by a reporter gene coupled to the yeast pheromone response pathway. GHRH-dependent signal activation in this system required the substitution of yeast Gα protein with proteins containing C-terminal regions of Gαs. A D60G variant (analogous to the little mouse mutation) of the receptor failed to respond to agonist. In parallel studies, GHRH₂₉ and the N-terminal extracellular region of the receptor were expressed as Gal4 fusion proteins in a 2-hybrid assay. A specific interaction between these proteins was readily observed. The D60G mutation was engineered into the receptor fusion protein. This protein failed to interact with the ligand fusion, confirming the specificity of the association between unmodified proteins. These two yeast expression technologies should prove invaluable in additional structure/activity analyses of this ligand/receptor pair as well as other peptide ligands and receptors.     

Functional production of mammalian concentrative nucleoside transporters in Saccharomyces cerevisiae

The transport of nucleosides and nucleobases in the yeast Saccharomyces cerevisiae is reviewed and the use of this organism to study recombinant mammalian concentrative nucleoside transport (CNT) proteins is described. A selection strategy based on the ability of an expressed nucleoside transporter cDNA to mediate thymidine uptake by yeast under a selective condition that depletes endogenous thymidylate was used to assess the transport capacity of heterologous transporter proteins. The pyrimidine-nucleoside selective concentrative transporters from human (hCNT1) and rat (rCNT1) complemented the imposed thymidylate depletion in S. cerevisiae, as did N-terminally truncated versions of hCNT1 and rCNT1 lacking up to 31 amino acids. Transporter-mediated rescue of S. cerevisiae by both nucleoside transporters was inhibited by cytidine, uridine and adenosine, but not by guanosine or inosine. This work represents the development of a new model system for the functional production of recombinant nucleoside transporters of the CNT family of membrane proteins.     

Simultaneous saccharification of inulin and ethanol fermentation by recombinantSaccharomyces cerevisiae secreting inulinase

VariousSaccharomyces cerevisiae strains were transformed with a 2 μ-based multicopy expression plasmid, pYIGP, carryingKluyveromyces marxianus inulinase gene under the control ofGAPDH promoter. Among them two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinantS. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.     

Recombinant Protein Secretion in Pseudozyma flocculosa and Pseudozyma antarctica with a Novel Signal Peptide

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.     

Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides

Aims: To test seven selected putative signal peptides from Lactobacillus plantarum WCFS1 in terms of their ability to drive secretion of two model proteins in Lact. plantarum, and to compare the functionality of these signal peptides with that of well‐known heterologous signal peptides (Usp45, M6). Methods and Results: Signal peptide functionality was assessed using a series of modular derivatives of the pSIP vectors for peptide pheromone‐controlled high‐level gene expression in lactobacilli. Several of the constructs with homologous signal peptides yielded similar or higher reporter protein activities than constructs with heterologous signal peptides. Two of the homologous signal peptides (Lp_0373 and Lp_0600) appeared as especially promising candidates for directing secretion, as they were among the best performing with both reporter proteins. Conclusions: We have identified homologous signal peptides for high‐level secretion of heterologous proteins in Lact. plantarum. With the model proteins, some of these performed better than commonly used heterologous signal peptides. Significance and Impact of the Study: The homologous signal peptides tested out, in this study, could be useful in food‐grade systems for secretion of interesting proteins in Lact. plantarum. The constructed modular secretion vectors are easily accessible for rapid signal peptide screening.     

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.