The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

AIMS: The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. METHODS AND RESULTS: Two yeast expression/secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase/xylanase gene cassette (pEXS) containing the endo-beta-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-beta-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d'Alexandria grape juices were fermented using the VIN13[pPPK] pectinase- and the VIN13[pEXS] glucanase/xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase/xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase/xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d'Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. CONCLUSIONS: The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d'Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharide-degrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price:quality ratio of wine according to consumer expectations.     

Homologous versus heterologous gene expression in the yeast, Saccharomyces cerevisiae.

DNA sequences normally flanking the highly expressed yeast 3-phosphoglycerate kinase (PGK) gene have been placed adjacent to heterologous mammalian genes on high copy number plasmid vectors and used for expression experiments in yeast. For many genes thus far expressed with this system, expression has been 15-50 times lower than the expression of the natural homologous PGK gene on the same plasmid. We have extensively investigated this dramatic difference and have found that in most cases it is directly proportional to the steady-state levels of mRNAs. We demonstrate this phenomenon and suggest possible causes for this effect on mRNA levels.     

Competitive expression of two heterologous genes inserted into one plasmid in Saccharomyces cerevisiae

Plasmids were constructed which contained two expression units encoding single-chain insulin precursors. Surprisingly, the total amount of insulin precursor produced was similar to that produced from plasmids containing a single expression unit. In this system, therefore, two expression cassettes can be brought to compete for the limited ability of the yeast cell for synthesis and secretion. Using genes encoding B(1-29)-A(1-21) and B(1-29)-Ala-Ala-Lys-A-(1-21), the slightly different precursors could be quantified individually after separation by high-performance liquid chromatography from the culture supernatant. The two-cassette system allowed a sensitive and well controlled comparison of parameters important for optimal expression of a heterologous gene in Saccharomyces cerevisiae. The system was used to compare two promoter constructions and also to evaluate the position of expression cassettes in the plasmid. Finally the codon usage in the gene to be expressed was found to influence its ability to compete for expression.     

酿酒酵母基因组位置效应对外源基因表达的影响

外源基因元件和模块在底盘细胞中发挥特定功能是合成生物学研究的基本过程,而外源元件和模块在基因组中的位置对其功能的实现具有显著影响。为了系统、全面地表征酿酒酵母基因组位置效应对外源基因的表达影响,以绿色荧光蛋白为报告基因,通过双交换同源重组方法,对酿酒酵母单基因敲除库进行高通量转化,构建酿酒酵母基因组单位点荧光标记菌株库。结合流式细胞术和高通量测序技术对单位点荧光标记库菌株进行分析,构建高表达位点库和低表达位点库,共发现促进绿色荧光蛋白表达的位点428个,抑制绿色荧光蛋白表达的位点444个。通过分析高、低表达位点在酵母染色体上的分布,从全基因组尺度上对酿酒酵母基因组整合位置对基因表达的影响进行表征。本研究可为酿酒酵母基因组位置效应的分布规律和产生机理研究提供重要参考,对外源蛋白工业生产和合成生物学中的基因表达精细调控也具有重要的指导意义。     

Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries

Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.     

Cloning and screening of the putative hexokinase genes from Rhizopus oryzae and their heterologous expression in Saccharomyces cerevisiae

Molecular Biology Reports - A filamentous fungus, Rhizopus oryzae (R. oryzae) is one of the ideal candidates for ethanol and lactic acid production due to its ability to grow on renewable carbon...     

Enhanced iron uptake of Saccharomyces cerevisiae by heterologous expression of a tadpole ferritin gene

We genetically engineered Saccharomyces cerevisiae to express ferritin, a ubiquitous iron storage protein, with the major heavy-chain subunit of tadpole ferritin. A 450-kDa ferritin complex can store up to 4,500 iron atoms in its central cavity. We cloned the tadpole ferritin heavy-chain gene (TFH) into the yeast shuttle vector YEp352 under the control of a hybrid alcohol dehydrogenase II and glyceraldehyde-3-phosphate dehydrogenase promoter. We confirmed transformation and expression by Northern blot analysis of the recombinant yeast, by Western blot analysis using an antibody against Escherichia coli-expressed TFH, and with Prussian blue staining that indicated that the yeast-expressed tadpole ferritin was assembled into a complex that could bind iron. The recombinant yeast was more iron tolerant in that 95% of transformed cells, but none of the recipient strain cells, could form colonies on plates containing 30 mM ferric citrate. The cell-associated concentration of iron was 500 microg per gram (dry cell weight) of the recombinant yeast but was 210 microg per gram (dry cell weight) in the wild type. These findings indicate that the iron-carrying capacity of yeast is improved by heterologous expression of tadpole ferritin and suggests that this approach may help relieve dietary iron deficiencies in domesticated animals by the use of the engineered yeast as a feed and food supplement.     

Display of heterologous proteins on the Saccharomyces cerevisiae surface display system using a single constitutive expression vector

In this study, we constructed a novel and simple yeast surface display system with a single expression vector. The newly established system uses a bidirectional expression vector carrying the AGA1 gene driven by the PGK1 promoter in one direction and the AGA2‐expression cassette driven by the TEF1 promoter in the reverse direction, and uses the geneticin, a G418‐resistant gene, as the selection marker for transformants. Because all the display elements are put into one expression vector, the new system is much simpler to use, and there is no need for any genetic modification of the host strains; therefore, the new system can be used in wild type as well as laboratory strains of Saccharomyces cerevisiae. The display efficiency of heterologous proteins using the new system has been confirmed by displaying enhanced green fluorescent protein and Eimeria tenella (a chicken protozoan parasite) microneme protein2 (EtMic2) on several S. cerevisiae strains. We also tested the new system with an aga2 mutant strain of S. cerevisiae. The results indicate that the native expressed Aga2 protein has no effect on the display efficiency of heterologous proteins. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:443–450, 2014     

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae [Anderson, M. S., Muehlbacher, M., Street, I.P., Proffitt, J., & Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175]. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinant IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea [Muehlbacher, M., & Poulter, C. D. (1988) Biochemistry 27, 7315-7328]. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with [4-3H]FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.     

Nodamura virus RNA replication in Saccharomyces cerevisiae: heterologous gene expression allows replication-dependent colony formation

Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 10(4)-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.     

Vectors for the construction of gene banks and the integration of cloned genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae

We have constructed a variety of vectors for use in both budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe). Four of these, pDB262, pWH4, pWH5, and pMAK262, have positive selection for the insertion of cloned DNA, making them convenient for the construction of gene banks. pDB262, pWH4 and pWH5 contain the 2 mu ARS and the LEU2 gene from S. cerevisiae and can be used for gene isolation. They can also be converted into integration vectors for use in the genetic mapping of cloned sequences. pMAK262 contains only the LEU2 gene from budding yeast and can be used to screen for ARS elements or for gene integration. We also describe two other integration vectors, pDAM3 and pDAM6, which have a variety of restriction sites suitable for subcloning.     

Construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae

We report the construction and characterization of a yeast shuttle-expression vector that can be used to express genes in yeast in a modulated form. This vector is centromeric, with a polylinker that optionally can provide an ATG start of translation. Expression features are based on the CUP1 yeast metallothionein gene promoter, which can be tightly modulated by copper.     

Kinetics of beta-glucosidase production by Saccharomyces cerevisiae recombinants harboring heterologous bgl genes

The maximum productivity of -glucosidase by Saccharomyces cerevisiaerecombinants under the control of GALI promoter was 100 IU l^–1 h^–1. The highest productivity of -glucosidase by a S. cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea. The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1 g ethanol g^–1 cellobiose and g ethanol l^–1 h^–1, respectively.