ERG9 Expression Research Articles

Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.

β-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve β-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for β-carotene production. Point and structural mutations were introduced into β-carotene-producing engineered yeast. The resulting mutants showed higher β-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6mg/L of β-carotene, a value 1.9 times higher than that of the parental strain (20.1mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and β-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with β-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.

Highly Efficient Biosynthesis of γ-Bisabolene with a New Sesquiterpene Synthase AcTPS5 by Dual Cytoplasmic-Peroxisomal Engineering in Saccharomyces cerevisiae

γ-bisabolene is a monocyclic sesquiterpene with various biological activities; it has also been approved as a food additive. Additionally, the hydrogenated form of bisabolene is considered as a potential alternative to D2 diesel. Saccharomyces cerevisiae has the ability to produce a large amount of acetyl-CoA in both cytosol and peroxisomes, which serves as a precursor in terpene biosynthesis. In this study, AcTPS5 was identified as a new γ-bisabolene synthase. By expressing AcTPS5 and the mevalonate pathway in peroxisomes, γ-bisabolene titer was achieved at 125.0 mg/L. Deleting the peroxisome autophagy gene atg36 further improved γ-bisabolene production to 216.9 mg/L. The implementation of dual cytoplasmic–peroxisomal engineering further boosted γ-bisabolene production to 296.4 mg/L. Finally, through increasing the acetyl-CoA supply and down-regulating the expression of ERG9, γ-bisabolene production was achieved at 584.14 mg/L in shake-flask fermentation and 2.69 g/L in fed-batch fermentation, which is the highest reported production of γ-bisabolene to date. The strategy presented in this study provides an efficient approach for terpene production in S. cerevisiae.

Engineering Saccharomyces cerevisiae YPH499 for Overproduction of Geranylgeraniol.

Optimization of supply and conversion efficiency of geranylgeranyl diphosphate (GGPP) is important for enhancing geranylgeraniol (GGOH) production in Saccharomyces cerevisiae. In this study, first, a strain producing 26.92 ± 1.59 mg/g of dry cell weight squalene was constructed with overexpression of all genes of the mevalonate (MVA) pathway, and an engineered strain producing 597.12 mg/L GGOH at the shake flask level was obtained. Second, through additional expression of PaGGPPs-ERG20 and PaGGPPs-DPP1, and downregulating expression of ERG9, the GGOH titer was increased to 1221.96 mg/L. Then, a NADH HMG-CoA reductase from Silicibacter pomeroyi (SpHMGR) was introduced to alleviate the high dependence of the strain upon NADPH, and the GGOH production was further increased to 1271.14 mg/L. Finally, the GGOH titer reached 6.33 g/L after optimizing the fed-batch fermentation method in a 5 L bioreactor, with a 24.9% improvement from the previous report. This study might accelerate the process of developing S. cerevisiae cell factories for diterpenoid and tetraterpenoid production.

Dual regulation of lipid droplet-triacylglycerol metabolism and ERG9 expression for improved \u03b2-carotene production in Saccharomyces cerevisiae

BackgroundThe limitation of storage space, product cytotoxicity and the competition for precursor are the major challenges for efficiently overproducing carotenoid in engineered non-carotenogenic microorganisms. In this work, to improve β-carotene accumulation in Saccharomyces cerevisiae, a strategy that simultaneous increases cell storage capability and strengthens metabolic flux to carotenoid pathway was developed using exogenous oleic acid (OA) combined with metabolic engineering approaches.ResultsThe direct separation of lipid droplets (LDs), quantitative analysis and genes disruption trial indicated that LDs are major storage locations of β-carotene in S. cerevisiae. However, due to the competition for precursor between β-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on β-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in β-carotene content. The transcriptome analysis was adopted to mine OA-repressible promoters and IZH1 promoter was used to replace native ERG9 promoter to dynamically down-regulate ERG9 expression, which diverted the metabolic flux to β-carotene pathway and achieved additional 31.7% increase in β-carotene content without adversely affecting cell growth. By inducing an extra constitutive β-carotene synthesis pathway for further conversion precursor farnesol to β-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of β-carotene, which is 107.3% and 49.5% increase respectively over the parent strain.ConclusionsThis strategy can be applied in the overproduction of other heterogeneous FPP-derived hydrophobic compounds with similar synthesis and storage mechanisms in S. cerevisiae.Graphical

Reconstruction of the Biosynthetic Pathway of Santalols under Control of the GAL Regulatory System in Yeast.

Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/steroid biosynthesis to sesquiterpenoid production, expression of ERG9 (encoding yeast squalene synthase) was depressed by replacing its innate promotor with PHXT1 and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of GAL promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of GAL promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L Z-α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from Santalum album) and WL19 expressing SanSyn (α-santalene synthase from Clausena lansium) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.

Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory

BackgroundAlpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction.Resultsα-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L.ConclusionsAn efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.

Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae.

Production of plant metabolites in microbial hosts represents a promising alternative to traditional chemical-based methods. Diterpenoids are compounds with interesting applications as pharmaceuticals, fragrances and biomaterials. Casbene, in particular, serves as a precursor to many complex diterpenoids found in plants from the Euphorbiaceae family that have shown potential therapeutic effects. Here, we engineered the budding yeast Saccharomyces cerevisiae for improved biosynthesis of the diterpene casbene. We first expressed, in yeast, a geranylgeranyl diphosphate synthase from Phomopsys amygdali in order to boost the geranylgeranyl diphosphate pool inside the cells. The enzyme uses isopentenyl diphosphate and dimethylallyl diphosphate to directly generate geranylgeranyl diphosphate. When co-expressing a casbene synthase from Ricinus communis the yeast was able to produce casbene in the order of 30 mg/L. Redirecting the flux from FPP and sterols, by means of the ergosterol sensitive promoter of ERG1, allowed for plasmid-based casbene production of 81.4 mg/L. Integration of the target genes into the yeast genome, together with the replacement of the promoter regions of ERG20 and ERG9 with combinations of ergosterol- and glucose-sensitive promoters, generated a titer of 108.5 mg/L of casbene. We here succeeded to engineer an improved route for geranylgeranyl diphosphate synthesis in yeast. Furthermore, we showed that the concurrent dynamic control of ERG20 and ERG9 expression, using ergosterol and carbon source regulation mechanisms, could substantially improve diterpene titer. Our approach will pave the way for a more sustainable production of GGPP- and casbene-derived products.

Efficient production of lycopene in Saccharomyces cerevisiae by enzyme engineering and increasing membrane flexibility and NAPDH production.

Lycopene is a red carotenoid pigment with strong antioxidant activity. Saccharomyces cerevisiae is considered a promising host to produce lycopene, but lycopene toxicity is one of the limiting factors for high-level production. In this study, we used heterologous lycopene biosynthesis genes crtE and crtI from Xanthophyllomyces dendrorhous and crtB from Pantoea agglomerans for lycopene production in S. cerevisiae. The crtE, crtB, and crtI genes were integrated into the genome of S. cerevisiae CEN.PK2-1C strain, while deleting DPP1 and LPP1 genes to inhibit a competing pathway producing farnesol. Lycopene production was further improved by inhibiting ergosterol production via downregulation of ERG9 expression and by deleting ROX1 or MOT3 genes encoding transcriptional repressors for mevalonate and sterol biosynthetic pathways. To further increase lycopene production, CrtE and CrtB mutants with improved activities were isolated by directed evolution, and subsequently, the mutated genes were randomly integrated into the engineered lycopene-producing strains via delta-integration. To relieve lycopene toxicity by increasing unsaturated fatty acid content in cell membranes, the OLE1 gene encoding stearoyl-CoA 9-desaturase was overexpressed. In combination with the overexpression of STB5 gene encoding a transcription factor involved in NADPH production, the final strain produced up to 41.8mg/gDCW of lycopene, which is approximately 74.6-fold higher than that produced in the initial strain.

Mannosylinositol phosphorylceramides and ergosterol coodinately maintain cell wall integrity in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group, and breakdown of their normal composition causes several cellular dysfunctions. Previously we found that loss of biosynthesis of mannosylinositol phosphorylceramide (MIPC) causes a defect in cell wall integrity (CWI). In this study, we screened for multicopy suppressor genes that rescue the defect in CWI in cells lacking MIPC synthases (Sur1 and Csh1), and found that the defect is partly suppressed by upregulation of ergosterol biosynthesis. In addition, repression of expression of ERG9, which encodes squalene synthase in the ergosterol biosynthesis pathway, in sur1∆ csh1∆ cells caused a strong growth defect and enhancement of the defect in CWI. The repression of ERG9 and/or the deletion of SUR1 and CSH1 caused an increase in the phosphorylated form of Slt2, a mitogen-activated protein kinase activated through impairment of CWI. Moreover, the deletion of SLT2 or WSC1/2, encoding a sensor protein recognizing CWI, enhanced the growth defect in the ERG9-repressed sur1∆ csh1∆ cells. On the other hand, the ERG9-repressed sur1∆ csh1∆ cells also exhibited an increase in the cell wall chitin level in a Slt2- and Wsc1/2-independent manner. These results suggested that MIPC and ergosterol are coordinately involved in maintenance of CWI, and the activation of Slt2 suppressed the CWI defect caused by these metabolic defects.

Dynamic control of ERG9 expression for improved amorpha-4,11-diene production in Saccharomyces cerevisiae.

BackgroundTo achieve high-level production of non-native isoprenoid products, it requires the metabolic flux to be diverted from the production of sterols to the heterologous metabolic reactions. However, there are limited tools for restricting metabolic flux towards ergosterol synthesis. In the present study, we explored dynamic control of ERG9 expression using different ergosterol-responsive promoters to improve the production of non-native isoprenoids.ResultsSeveral ergosterol-responsive promoters were identified using quantitative real-time PCR (qRT-PCR) analysis in an engineered strain with relatively high mevalonate pathway activity. We found mRNA levels for ERG11, ERG2 and ERG3 expression were significantly lower in the engineered strain over the reference strain BY4742, indicating these genes are transcriptionally down-regulated when ergosterol is in excess. Further replacement of the native ERG9 promoter with these ergosterol-responsive promoters revealed that all engineered strains improved amorpha-4,11-diene by 2 ~ 5-fold over the reference strain with ERG9 under its native promoter. The best engineered strain with ERG9 under the control of PERG1 produced amorpha-4,11-diene to a titer around 350 mg/L after 96 h cultivation in shake-flasks.ConclusionsWe envision dynamic control at the branching step using feedback regulation at transcriptional level could serve as a generalized approach for redirecting the metabolic flux towards product-of-interest.

Dynamic control of gene expression in Saccharomyces cerevisiae engineered for the production of plant sesquitepene α-santalene in a fed-batch mode

Microbial cells engineered for efficient production of plant sesquiterpenes may allow for sustainable and scalable production of these compounds that can be used as e.g. perfumes and pharmaceuticals. Here, for the first time a Saccharomyces cerevisiae strain capable of producing high levels of α-santalene, the precursor of a commercially interesting compound, was constructed through a rationally designed metabolic engineering approach. Optimal sesquiterpene production was obtained by modulating the expression of one of the key metabolic steps of the mevalonate (MVA) pathway, squalene synthase (Erg9). To couple ERG9 expression to glucose concentration its promoter was replaced by the HXT1 promoter. In a second approach, the HXT2 promoter was used to express an ERG9 antisense construct. Using the HXT1 promoter to control ERG9 expression, it was possible to divert the carbon flux from sterol synthesis towards α-santalene improving the productivity by 3.4 fold. Combining this approach together with the overexpression of a truncated form of 3-hydroxyl-3-methyl-glutaryl-CoA reductase (HMGR) and deletion of lipid phosphate phosphatase encoded by LPP1 led to a strain with a productivity of 0.18mg/gDCWh. The titer was further increased by deleting DPP1 encoding a second FPP consuming pyrophosphate phosphatase yielding a final productivity and titer, respectively, of 0.21mg/gDCWh and 92mg/l of α-santalene.

The Candida glabrata putative sterol transporter gene CgAUS1 protects cells against azoles in the presence of serum

The uptake of endogenous sterol from serum may allow Candida glabrata to survive azole treatment. This study aims to determine the contribution of a sterol transporter that alters fluconazole sensitivity in the presence of serum. Bioinformatic analysis predicted CgAUS1 as the C. glabrata orthologue of the Saccharomyces cerevisiae transporters AUS1 and PDR11. To investigate whether the CgAUS1 gene has sterol transporter activity, we investigated the effects of an AUS1 deletion on the growth of a tetracycline-regulatable ERG9 strain (tet-ERG9aus1), wherein ERG9 expression is turned off giving rise to a sterol requirement. Tetracycline-dependent repression of CgAUS1 in the tet-AUS1 strain was used to determine the fluconazole susceptibility of CgAUS1 in the presence and absence of serum. The tetracycline-treated tet-ERG9aus1 strain failed to grow in the presence of serum, whereas the parental tet-ERG9AUS1 strain grew by incorporating sterol from exogenously supplied serum. Serum cholesterol protected cells against the antifungal effects of fluconazole and this protection was lost by repressing CgAUS1 gene expression. Furthermore, such protection was also observed during itraconazole treatment, but not observed in cells treated with non-azole antifungals. CgAUS1 appears to function as a sterol transporter that may contribute to lower azole susceptibility in the presence of serum and to protect C. glabrata against azole toxicity in vivo.

Repression of ergosterol level during oxidative stress by fission yeast F-box protein Pof14 independently of SCF

We describe a new member of the F-box family, Pof14, which forms a canonical, F-box dependent SCF (Skp1, Cullin, F-box protein) ubiquitin ligase complex. The Pof14 protein has intrinsic instability that is abolished by inactivation of its Skp1 interaction motif (the F-box), Skp1 or the proteasome, indicating that Pof14 stability is controlled by an autocatalytic mechanism. Pof14 interacts with the squalene synthase Erg9, a key enzyme in ergosterol metabolism, in a membrane-bound complex that does not contain the core SCF components. pof14 transcription is induced by hydrogen peroxide and requires the Pap1 transcription factor and the Sty1 MAP kinase. Pof14 binds to and decreases Erg9 activity in vitro and a pof14 deletion strain quickly loses viability in the presence of hydrogen peroxide due to its inability to repress ergosterol synthesis. A pof14 mutant lacking the F-box and an skp1-3 ts mutant behave as wild type in the presence of oxidant showing that Pof14 function is independent of SCF. This indicates that modulation of ergosterol level plays a key role in adaptation to oxidative stress.

Positive and negative regulation of squalene synthase ( ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae

To identify regulatory cis-elements in the proximal promoter of the yeast ERG9 squalene synthase gene, promoter deletion analysis was performed. This approach identified two regulatory elements, one an upstream repressing cis-element (URS), and the other an upstream activating cis-element (UAS). Electromobility shift assays (EMSAs) demonstrated that distinct proteins bind each element. Genetic screens were performed to identify yeast mutants that altered expression of ERG9 promoter–reporter gene fusions. Three non-ergosterol biosynthetic pathway genes were identified. A mutation in TPO1( YLL028W) led to a 5.5-fold increase in ERG9 expression while mutations in YER064C and SLK19 ( YOR195W) led to a 3.1- and 5.6-fold decrease, respectively. Deletion analysis of these genes demonstrated that TPO1 and SLK19 specifically regulated ERG9 expression when tested with several different promoter–reporter gene fusions. Additionally, EMSAs demonstrated that extracts derived from the TPO1 deletion strain was unable to shift the repressing cis-element while protein extracts from the SLK19 deletion strain had a reduced shift of the activating cis-element. Furthermore, these two mutants showed quantitative differences in sterols and antifungal drug susceptibilities consistent with their role in regulating ERG9 expression.

Transcriptional regulation of the squalene synthase gene ( ERG9) in the yeast Saccharomyces cerevisiae

The ergosterol biosynthetic pathway is a specific branch of the mevalonate pathway. Since the cells requirement for sterols is greater than for isoprenoids, sterol biosynthesis must be regulated independently of isoprenoid biosynthesis. In this study we explored the transcriptional regulation of squalene synthase ( ERG9) in Saccharomyces cerevisiae, the first enzyme dedicated to the synthesis of sterols. A mutant search was performed to identify genes that were involved in the regulation of the expression of an ERG9- lacZ promoter fusion. Mutants with phenotypes consistent with known sterol biosynthetic mutations ( ERG3, ERG7, ERG24) increased expression of ERG9. In addition, treatment of wild-type cells with the sterol inhibitors zaragozic acid and ketoconazole, which target squalene synthase and the C-14 sterol demethylase respectively, also caused an increase in ERG9 expression. The data also demonstrate that heme mutants increased ERG9 expression while anaerobic conditions decreased expression. Additionally, the heme activator protein transcription factors HAP1 and HAP2/3/4, the yeast activator protein transcription factor yAP-1, and the phospholipid transcription factor complex INO2/4 regulate ERG9 expression. ERG9 expression is decreased in hap1, hap2/3/4, and yap-1 mutants while ino2/4 mutants showed an increase in ERG9 expression. This study demonstrates that ERG9 transcription is regulated by several diverse factors, consistent with the idea that as the first step dedicated to the synthesis of sterols, squalene synthase gene expression and ultimately sterol biosynthesis is highly regulated.