Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae

Manipulation of monoterpene synthases to maximize flux towards targeted products from GPP (geranyl diphosphate) is the main challenge for heterologous monoterpene overproduction, in addition to cell toxicity from compounds themselves. In our study, by manipulation of the key enzymes geraniol synthase (GES) and farnesyl diphosphate synthase (Erg20), geraniol (a valuable acyclic monoterpene alcohol) overproduction was achieved in Saccharomyces cerevisiae with truncated 3-hydroxy-3-methylglutaryl-coenzyme reductase (tHMGR) and isopentenyl diphosphate isomerase (IDI1) overexpressed. The expressions of all above engineered genes were under the control of Gal promoter for alleviating product toxicity. Geraniol production varied from trace amount to 43.19 mg/L (CrGES, GES from Catharanthus roseus) by screening of nine GESs from diverse species. Further through protein structure analysis and site-directed mutation in CrGES, it was firstly demonstrated that among the high-conserved amino acid residues located in active pocket, Y436 and D501 with strong affinity to diphosphate function group, were critical for the dephosphorylation (the core step for geraniol formation). Moreover, the truncation position of the transit peptide from the N-terminus of CrGES was found to influence protein expression and activity significantly, obtaining a titer of 191.61 mg/L geraniol in strain with CrGES truncated at S43 (t3CrGES). Furthermore, directed by surface electrostatics distribution of t3CrGES and Erg20^WW (Erg20^F96W-N127W), co-expression of the reverse fusion of Erg20^ww/t3CrGES and another copy of Erg20^WW promoted the geraniol titer to 523.96 mg/L at shakes flask level, due to enhancing GPP accessibility led by protein interaction of t3CrGES-Erg20^WW and the free Erg20^WW. Eventually, a highest reported titer of 1.68 g/L geraniol in eukaryote cells was achieved in 2.0 L fed-batch fermentation under carbon restriction strategy. Our research opens large opportunities for other microbial production of monoterpenes. It also sets a good reference for desired compounds overproduction in microorganisms in terms of manipulation of key enzymes by protein engineering and metabolic engineering.     

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.     

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

NAD⁺ is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD⁺ pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD⁺ metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD⁺ levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD⁺ homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD⁺ metabolism in higher eukaryotes.     

The imminent role of protein engineering in synthetic biology

Protein engineering has for decades been a powerful tool in biotechnology for generating vast numbers of useful enzymes for industrial applications. Today, protein engineering has a crucial role in advancing the emerging field of synthetic biology, where metabolic engineering efforts alone are insufficient to maximize the full potential of synthetic biology. This article reviews the advancements in protein engineering techniques for improving biocatalytic properties to optimize engineered pathways in host systems, which are instrumental to achieve high titer production of target molecules. We also discuss the specific means by which protein engineering has improved metabolic engineering efforts and provide our assessment on its potential to continue to advance biology engineering as a whole.     

Specificity of yeast (Saccharomyces cerevisiae) in removing carbohydrates by fermentation

The specificity of Saccharomyces cerevisiae yeast on the removal of carbohydrates by fermentation was studied. The common monosaccharides, D-glucose, D-fructose, D-mannose, and D-galactose were completely removed; D-glucuronic acid and D-ribose were partially removed; but D-xylose, D-rhamnose, and L-sorbose were not removed and were completely resistant. Of four glycosides, methyl and phenyl alpha- and beta-D-glucopyranosides, three of the four were partially removed and methyl beta-D-glucopyranoside was not removed. The disaccharides, maltose, sucrose, and turanose were completely removed, while cellobiose, lactose, and melibiose were completely resistant. Isomaltose and alpha,alpha-trehalose were partially removed. Maltotriose and raffinose were partially removed, but isomaltotriose and melezitose were completely resistant. The tetrasaccharides, maltotetraose, isomaltotetraose, and acarbose, were completely resistant. Further, the yeast enzymes did not alter any of the resistant carbohydrates by transglycosylation or condensation reactions or by any other types of reactions.     

Enrichment of threonine content in Saccharomyces cerevisiae by pathway engineering

In a previous work, we have investigated the effect of amplifying individually the genes of the threonine biosynthetic pathway on threonine accumulation by yeast. Here, we present the results of the simultaneous amplification of these genes in strains with different genetic backgrounds. These strains carry a mutant HOM3-R2 allele (coding for a feedback-insensitive aspartate kinase), and/or a mutant cha1 allele that makes it defective in threonine degradation by the catabolic L-serine (L-threonine) deaminase. The results show that the amplification of the clustered genes affects threonine and homoserine accumulation only when it includes the HOM3 gene, or when combined with a HOM3-R2 mutation. Similarly, the cha1 mutation is only effective when a certain amount of threonine is reached. Threonine overproduction affects other cellular functions such as the accumulation of other amino acids, the cell growth and metabolite excretion, probably reflecting a redirection of the carbon flux in the central metabolism.     

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.     

Employing bacterial microcompartment technology to engineer a shell-free enzyme-aggregate for enhanced 1,2-propanediol production in Escherichia coli

Bacterial microcompartments (BMCs) enhance the breakdown of metabolites such as 1,2-propanediol (1,2-PD) to propionic acid. The encapsulation of proteins within the BMC is mediated by the presence of targeting sequences. In an attempt to redesign the Pdu BMC into a 1,2-PD synthesising factory using glycerol as the starting material we added N-terminal targeting peptides to glycerol dehydrogenase, dihydroxyacetone kinase, methylglyoxal synthase and 1,2-propanediol oxidoreductase to allow their inclusion into an empty BMC. 1,2-PD producing strains containing the fused enzymes exhibit a 245% increase in product formation in comparison to un-tagged enzymes, irrespective of the presence of BMCs. Tagging of enzymes with targeting peptides results in the formation of dense protein aggregates within the cell that are shown by immuno-labelling to contain the vast majority of tagged proteins. It can therefore be concluded that these protein inclusions are metabolically active and facilitate the significant increase in product formation.     

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae

Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.     

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.     

Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.     

Identification and characterization of an animal delta(12) fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae

We have cloned a Caenorhabditis elegans cDNA encoding a Delta12 fatty acid desaturase and demonstrated its activity by heterologous expression in Saccharomyces cerevisiae. The predicted protein is highly homologous both to the cloned plant genes with similar function and to the published sequence of the C. elegans omega-3 fatty acid desaturase. In addition, it conforms to the structural constraints expected of a membrane-bound fatty acid desaturase including the canonical histidine-rich regions. This is the first report of a cloned animal Delta(12) desaturase gene. Expression of this cDNA in yeast resulted in the accumulation of 16:2 and 18:2 (linoleic) acids. The increase of membrane fluidity brought about by this change in unsaturation was measured. The production of polyunsaturated fatty acids in yeast cells and the concomitant increase in membrane fluidity was correlated with a modest increase in growth rate at low temperature and with increased resistance to ethanol and oxidative stress.     

Stable continuous constitutive expression of a heterologous protein in Saccharomyces cerevisiae without selection pressure

The stability of heterologous protein expression in Saccharomyces cerevisiae during continuous culture without selection for plasmid-containing cells was investigated. The protein chosen was the leech thrombin inhibitor desulphato-hirudin, which is tolerated well by S. cerevisiae when over-expressed. Expression was from a 2- derived multicopy vector containing a synthetic hirudin gene under control of the constitutive glyceraldehyde-3-phosphate dehydrogenase derived GAPFL promoter. The behaviour of the system was studied at three dilution rates (D) corresponding to approximately 30% (0.06 h^–1), 60% (0.12 h^–1) and 90% (0.17 h^–1) of the estimated maximum D. The level of plasmid loss was low at all Ds, with only 5–10% plasmid-free cells observed at 75 generations. The plasmid was most stably maintained at the intermediate D of 0.12 h^–1, where the rate of loss was comparable to the loss of the native 2- plasmid. Hirudin expression was also highest at D=0.12 h^–1, possibly as a result of cell lysis at D=0.06 h^–1 and D=0.17 h^–1, leading to the release of vacuolar proteases and subsequent proteolysis of hirudin. Differences in expression levels were not a result of changes in plasmid copy number, which was in the range 40–60 throughout all three experiments. The high stability of this system at all Ds investigated shows that heterologous protein expression is not a burden to S. cerevisiae when the protein expressed is tolerated well. Correspondence to: M. Ibba     

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.     

Endoplasmic reticulum-associated degradation (ERAD) and free oligosaccharide generation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, proteins with misfolded lumenal, membrane, and cytoplasmic domains are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L, -M, and -C, respectively. ERAD-L is N-glycan-dependent and is characterized by ER mannosidase (Mns1p) and ER mannosidase-like protein (Mnl1p), which generate Man(7)GlcNAc(2) (d1) N-glycans with non-reducing α1,6-mannosyl residues. Glycoproteins bearing this motif bind Yos9p and are dislocated into the cytoplasm and then deglycosylated by peptide N-glycanase (Png1p) to yield free oligosaccharides (fOS). Here, we examined yeast fOS metabolism as a function of cell growth in order to obtain quantitative and mechanistic insights into ERAD. We demonstrate that both Png1p-dependent generation of Man(7-10)GlcNAc(2) fOS and vacuolar α-mannosidase (Ams1p)-dependent fOS demannosylation to yield Man(1)GlcNAc(2) are strikingly up-regulated during post-diauxic growth which occurs when the culture medium is depleted of glucose. Gene deletions in the ams1Δ background revealed that, as anticipated, Mns1p and Mnl1p are required for efficient generation of the Man(7)GlcNAc(2) (d1) fOS, but for the first time, we demonstrate that small amounts of this fOS are generated in an Mnl1p-independent, Mns1p-dependent pathway and that a Man(8)GlcNAc(2) fOS that is known to bind Yos9p is generated in an Mnl1p-dependent, Mns1p-independent manner. This latter observation adds mechanistic insight into a recently described Mnl1p-dependent, Mns1p-independent ERAD pathway. Finally, we show that 50% of fOS generation is independent of ERAD-L, and because our data indicate that ERAD-M and ERAD-C contribute little to fOS levels, other important processes underlie fOS generation in S. cerevisiae.     

Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.     

Induction of respiration-deficient mutants in Saccharomyces cerevisiae by chelerythrine

Abstract Chelerythrine and sanguinarine, two structurally related benzo/c/phenanthridine alkaloids, prevented growth of yeast cells in medium containing either glucose or non-fermentable carbon sources. At concentrations permitting growth of the yeast Saccharomyces cerevisiae , chelerythrine, but not sanquinarine, induced cytoplasmic respiration-deficient mutants. The petite clones that were analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.     

The effect of a-factor on hybrid formation by protoplast fusion in Saccharomyces cerevisiae

Abstract a_˜-Factor, unlike α-factor, does not significantly enhance hybrid formation by protoplast fusion in the yeast Saccharomyces cerevisiae . When Mat α cells are treated with a-factor prior to being proto-plasted and fused, the frequency of hybrid formation is only slightly increased over unarrested controls.