Heterologous production of dihomo-gamma-linolenic acid in Saccharomyces cerevisiae

To make dihomo-gamma-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase, rat Delta6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15 degrees C than in those grown at 20 degrees C, and no DGLA production was observed in the cells grown at 30 degrees C. In NSD at 15 degrees C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15 degrees C, the yield of DGLA reached 2.19 microg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.     

Heterologous production of the epoxycarotenoid violaxanthin in Saccharomyces cerevisiae

Microbial production of carotenoids has mainly focused towards a few products, such as β-carotene, lycopene and astaxanthin. However, other less explored carotenoids, like violaxanthin, have also shown unique properties and promissory applications. Violaxanthin is a plant-derived epoxidated carotenoid with strong antioxidant activity and a key precursor of valuable compounds, such as fucoxanthin and β-damascenone. In this study, we report for the first time the heterologous production of epoxycarotenoids in yeast. We engineered the yeast Saccharomyces cerevisiae following multi-level strategies for the efficient accumulation of violaxanthin. Starting from a β-carotenogenic yeast strain, we first evaluated the performance of several β-carotene hydroxylases (CrtZ), and zeaxanthin epoxidases (ZEP) from different species, together with their respective N-terminal truncated variants. The combined expression of CrtZ from Pantoea ananatis and truncated ZEP of Haematococcus lacustris showed the best performance and led to a yield of 1.6 mg/g_DCW of violaxanthin. Further improvement of the epoxidase activity was achieved by promoting the transfer of reducing equivalents to ZEP by expressing several redox partner systems. The co-expression of the plant truncated ferredoxin-3, and truncated root ferredoxin oxidoreductase-1 resulted in a 2.2-fold increase in violaxanthin yield (3.2 mg/g_DCW). Finally, increasing gene copy number of carotenogenic genes enabled reaching a final production of 7.3 mg/g_DCW in shake flask cultures and batch bioreactors, which is the highest yield of microbially produced violaxanthin reported to date.     

Heterologous protein production from the inducible MET25 promoter in Saccharomyces cerevisiae

Heterologous protein production late in Saccharomyces cerevisiae fermentations is often desirable because it may help avoid the unintentional selection of more rapidly growing, non-protein-expressing cells or allow for the expression of toxic proteins. Here, we describe the use of the MET25 promoter for the production of human serum albumin (HSA) and HSA-fusion proteins in S. cerevisiae. In media lacking methionine, the MET25 promoter yielded high expression levels of HSA and HSA fused to human glucagon, human growth hormone, human interferon alpha, and human interleukin-2. More importantly, we have shown that this system can be used to delay heterologous protein production until late log phase of the growth of the culture and does not require the addition of an exogenous inducer.     

Heterologous biosynthesis of artemisinic acid in Saccharomyces cerevisiae

Artemisinic acid is a precursor of antimalarial compound artemisinin. The titre of biosynthesis of artemisinic acid using Saccharomyces cerevisiae platform has been achieved up to 25 g l^?1; however, the performance of platform cells is still industrial unsatisfied. Many strategies have been proposed to improve the titre of artemisinic acid. The traditional strategies mainly focused on partial target sites, simple up‐regulation key genes or repression competing pathways in the total synthesis route. However, this may result in unbalance of carbon fluxes and dysfunction of metabolism. In this review, the recent advances on the promising methods in silico and in vivo for biosynthesis of artemisinic acid have been discussed. The bioinformatics and omics techniques have brought a great prospect for improving production of artemisinin and other pharmacal compounds in heterologous platform.     

Heterologous expression of syntaxin 6 in Saccharomyces cerevisiae

The molecular mechanisms of vesicular protein transport in eukaryotic cells are highly conserved. Members of the syntaxin family play a pivotal role in the membrane fusion process. We have expressed rat syntaxin 6 and its cytoplasmic domain in wild-type and pep12 mutant strains of Saccharomyces cerevisiae to elucidate the role of the syntaxin 6-dependent vesicular trafficking step in yeast. Immunofluorescence microscopy revealed a punctate, Golgi-like staining pattern for syntaxin 6, which only partially overlapped with Pep12p in wild-type yeast cells. In contrast to Pep12p, syntaxin 6 was not mislocalized to the vacuole upon expression from 2 micron vectors, which might be attributed to conserved sorting and retention signals. Syntaxin 6 was not capable of complementing the sorting and maturation defects of the vacuolar hydrolase CPY in pep12 null mutants. No dominant negative effects of either syntaxin 6 or syntaxin 6 delta C overexpression on CPY sorting and maturation were observed in wild-type yeast cells. We conclude that syntaxin 6 and Pep12p do not act at the same vesicular trafficking step(s) in yeast and higher eukaryotes.     

Heterologous expression of Arabidopsis UDP-glucosyltransferases in Saccharomyces cerevisiae for production of zearalenone-4-O-glucoside

Zearalenone, a secondary metabolite produced by several plant-pathogenic fungi of the genus Fusarium, has high estrogenic activity in vertebrates. We developed a Saccharomyces cerevisiae bioassay strain that we used to identify plant genes encoding UDP-glucosyltransferases that can convert zearalenone into zearalenone-4-O-glucoside (ZON-4-O-Glc). Attachment of the glucose moiety to zearalenone prevented the interaction of the mycotoxin with the human estrogen receptor. We found that two of six clustered, similar UGT73C genes of Arabidopsis thaliana encode glucosyltransferases that can inactivate zearalenone in the yeast bioassay. The formation of glucose conjugates seems to be an important plant mechanism for coping with zearalenone but may result in significant amounts of "masked" zearalenone in Fusarium-infected plant products. Due to the unavailability of an analytical standard, the ZON-4-O-Glc is not measured in routine analytical procedures, even though it can be converted back to active zearalenone in the digestive tracts of animals. Zearalenone added to yeast transformed with UGT73C6 was converted rapidly and efficiently to ZON-4-O-Glc, suggesting that the cloned UDP-glucosyltransferase could be used to produce reference glucosides of zearalenone and its derivatives.     

Heterologous production of five Hepatitis C virus-derived antigens in three Saccharomyces cerevisiae host strains

In this study, the production of recombinant Hepatitis C virus (HCV) derived proteins from transformed Saccharomyces cerevisiae yeast cells is reported. Three different yeast strains (GRF18U, BY4743-4A and CENPK 113-5D) have been transformed for the intracellular expression of five antigens of different dimensions (from 32.8 to 85.2 kDa), all derived from the non-structural (NS) region of different HCV viruses' genotypes and posed under the control of a glycolytic promoter. The putative trans-membrane domains contained in four antigens seem responsible of their accumulation as protein aggregates. Good productions of the smaller and of the bigger antigens (50 and 30 mgl(-1), respectively) have been observed in simple flask batch cultures. Productions are strongly dependent from the genetic background of the yeast host and from the cellular localization of the antigen, while they appear independent from the growth rate of the transformed hosts. For every recombinant antigen tested, the highest production levels were achieved with the CENPK 113-5D-host strain, while the GRF18U strain shows symptoms of a heavily stressed phenotype.     

Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae was genetically modified to assemble a minicellulosome on its cell surface by heterologous expression of a chimeric scaffoldin protein from Clostridium cellulolyticum under the regulation of the phosphoglycerate kinase 1 ( PGK1 ) promoter and terminator regulatory elements, together with the β-xylanase 2 secretion signal of Trichoderma reesei and cell wall protein 2 (Cwp2) of S. cerevisiae . Fluorescent microscopy and Far Western blot analysis confirmed that the Scaf3p is targeted to the yeast cell surface and that the Clostridium thermocellum cohesin domain is functional in yeast. Similarly, functionality of the C. thermocellum dockerin domain in yeast is shown by binding to the Scaf3 protein in Far Western blot analysis. Phenotypic evidence for cohesin–dockerin interaction was also established with the detection of a twofold increase in tethered endoglucanase enzyme activity in S. cerevisiae cells expressing the Scaf3 protein compared with the parent strain. This study highlights the feasibility to future design of enhanced cellulolytic strains of S. cerevisiae through emulation of the cellulosome concept. Potentially, Scaf3p-armed yeast could also be developed into an alternative cell surface display strategy with various tailor-made applications.     

Heterologous expression of cyanobacterial gas vesicle proteins in Saccharomyces cerevisiae

Given the potential applications of gas vesicles (GVs) in multiple fields including antigen-displaying and imaging, heterologous reconstitution of synthetic GVs is an attractive and interesting study that has translational potential. Here, we attempted to express and assemble GV proteins (GVPs) into GVs using the model eukaryotic organism Saccharomyces cerevisiae. We first selected and expressed two core structural proteins, GvpA and GvpC from cyanobacteria Anabaena flos-aquae and Planktothrix rubescens, respectively. We then optimized the protein production conditions and validated GV assembly in the context of GV shapes. We found that when two copies of anaA were integrated into the genome, the chromosomal expression of AnaA resulted in GV production regardless of GvpC expression. Next, we co-expressed chaperone-RFP with the GFP-AnaA to aid the AnaA aggregation. The co-expression of individual chaperones (Hsp42, Sis1, Hsp104, and GvpN) with AnaA led to the formation of larger inclusions and enhanced the sequestration of AnaA into the perivacuolar site. To our knowledge, this represents the first study on reconstitution of GVs in S. cerevisiae. Our results could provide insights into optimizing conditions for heterologous protein production as well as the reconstitution of other synthetic microcompartments in yeast.     

Photoinactivation of peptide transport in Saccharomyces cerevisiae

Oligopeptides and dipeptides are transported into Saccharomyces cerevisiae by a carrier-mediated system. In the dark, leucyl-p-nitroanilide (Leu-p-NA) and leucyl-leucyl-4-azido-2-nitrophenylalanine [Leu-Leu-Phe-(4N3,2NO2)] are competitive inhibitors of peptide transport by S. cerevisiae cells. The photolysis of yeast cells in the presence of Leu-p-NA or Leu-Leu-Phe(4N3,2NO2) at 350 nm results in an irreversible inactivation of peptide transport. Protection against this inactivation is afforded by an excess of trimethionine, a transported peptide. Photolysis with Leu-p-NA or Leu-Leu-Phe(4N3,2NO2) does not affect amino acid or sugar transport, and cell viability is maintained throughout the irradiation procedure. A 5-min irradiation of S. cerevisiae with 2.4 microM Leu-p-NA or 15 microM Leu-Leu-Phe(4N3,2NO2) causes 50% inhibition of trimethionine uptake. p-Nitroaniline, a possible hydrolysis product generated from Leu-p-NA by cellular peptidase activity, has no effect on peptide transport. An exogenous energy source is not required for photoinactivation. The results suggest that a component(s) of the peptide transport system of S. cerevisiae is irreversibly modified by photolysis with Leu-p-NA or Leu-Leu-Phe-(4N3,2NO2) and provide the first example of the use of amino acid p-nitroanilides as photoaffinity labels.     

Heterologous expression of an Entamoeba histolytica chitin synthase in Saccharomyces cerevisiae

Chitin in the cyst wall of Entamoeba histolytica is made by two chitin synthases (Chs), one of which is unique (EhCHS-1) and one of which resembles those of insects and nematodes (EhCHS-2). EhCHS-1 is deposited chitin in the lateral wall of transformed Saccharomyces cerevisiae Chs mutants, independent of accessory proteins (Chs4p to Chs7p) required by yeast Chs3p.     

Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae

Aims: Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. Methods and Results: A dammarenediol‐producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT‐PCR. Fermentation was carried out in a 5‐L GRJY‐bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250 μg l^?1. However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Conclusions: Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. Significance and Impact of the Study: This study showed that the high‐value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene‐derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

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Construction of a Sorbitol-Based Vector for Expression of Heterologous Proteins in Saccharomyces cerevisiae

A new inducible yeast expression vector, pXS7, was constructed by using the promoter and terminator sequences from the Saccharomyces cerevisiae SOR1 gene, which codes for the sorbitol dehydrogenase protein. We cloned the coding sequence of the Saccharomyces YEF3 gene in this vector and demonstrated an increase in YEF3 protein levels when cells were grown in the presence of the sugar sorbitol.