Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase

Saccharomyces cerevisiae utilizes several regulatory mechanisms to maintain tight control over the intracellular level of farnesyl diphosphate (FPP), the central precursor to nearly all yeast isoprenoid products. High-level production of non-native isoprenoid products requires that FPP flux be diverted from production of sterols to the heterologous metabolic reactions. To do so, expression of the gene encoding squalene synthase (ERG9), the first committed step in sterol biosynthesis, was down-regulated by replacing its native promoter with the methionine-repressible MET3 promoter. The intracellular levels of FPP were then assayed by expressing the gene encoding amorphadiene synthase (ADS) and converting the FPP to amorphadiene. Under certain culture conditions amorphadiene production increased fivefold upon ERG9 repression. With increasing flux to amorphadiene, squalene and ergosterol production each decreased. The levels of these three metabolites were dependent not only upon the level of ERG9 repression, but also the timing of its repression relative to the induction of ADS and genes responsible for enhancing flux to FPP.     

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 diverse sesquiterpene profile of patchouli, Pogostemon cablin, is correlated with a limited number of sesquiterpene synthases

Pogostemon cablin (patchouli), like many plants within the Lamiaceae, accumulates large amounts of essential oil. Patchouli oil is unique because it consists of over 24 different sesquiterpenes, rather than a blend of different mono-, sesqui- and di-terpene compounds. To determine if this complex mixture of sesquiterpenes arises from an equal number of unique sesquiterpene synthases, we developed a RT-PCR strategy to isolate and functionally characterize the respective patchouli oil synthase genes. Unexpectedly, only five terpene synthase cDNA genes were isolated. Four of the cDNAs encode for synthases catalyzing the biosynthesis of one major sesquiterpene, including a gamma-curcumene synthase, two germacrene D synthases, and a germacrene A synthase. The fifth cDNA encodes for a patchoulol synthase, which catalyzes the conversion of FPP to patchoulol plus at least 13 additional sesquiterpene products. Equally intriguing, the yield of the different in vitro reaction products resembles quantitatively and qualitatively the profile of sesquiterpenes found in patchouli oil extracted from plants, suggesting that a single terpene synthase is responsible for the bulk and diversity of terpene products produced in planta.     

Progress in terpene synthesis strategies through engineering of Saccharomyces cerevisiae

Terpenes are natural products with a remarkable diversity in their chemical structures and they hold a significant market share commercially owing to their distinct applications. These potential molecules are usually derived from terrestrial plants, marine and microbial sources. In vitro production of terpenes using plant tissue culture and plant metabolic engineering, although receiving some success, the complexity in downstream processing because of the interference of phenolics and product commercialization due to regulations that are significant concerns. Industrial workhorses’ viz., Escherichia coli and Saccharomyces cerevisiae have become microorganisms to produce non-native terpenes in order to address critical issues such as demand-supply imbalance, sustainability and commercial viability. S. cerevisiae enjoys several advantages for synthesizing non-native terpenes with the most significant being the compatibility for expressing cytochrome P450 enzymes from plant origin. Moreover, achievement of high titers such as 40?g/l of amorphadiene, a sesquiterpene, boosts commercial interest and encourages the researchers to envisage both molecular and process strategies for developing yeast cell factories to produce these compounds. This review contains a brief consideration of existing strategies to engineer S. cerevisiae toward the synthesis of terpene molecules. Some of the common targets for synthesis of terpenes in S. cerevisiae are as follows: overexpression of tHMG1, ERG20, upc2-1 in case of all classes of terpenes; repression of ERG9 by replacement of the native promoter with a repressive methionine promoter in case of mono-, di- and sesquiterpenes; overexpression of BTS1 in case of di- and tetraterpenes. Site-directed mutagenesis such as Upc2p (G888A) in case of all classes of terpenes, ERG20p (K197G) in case of monoterpenes, HMG2p (K6R) in case of mono-, di- and sesquiterpenes could be some generic targets. Efforts are made to consolidate various studies (including patents) on this subject to understand the similarities, to identify novel strategies and to contemplate potential possibilities to build a robust yeast cell factory for terpene or terpenoid production. Emphasis is not restricted to metabolic engineering strategies pertaining to sterol and mevalonate pathway, but also other holistic approaches for elsewhere exploitation in the S. cerevisiae genome are discussed. This review also focuses on process considerations and challenges during the mass production of these potential compounds from the engineered strain for commercial exploitation.     

Metabolic engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for linalool production

Objectives

To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool.

Results

Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l^?1 in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l^?1.

Conclusions

Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

     

Detection of farnesyl diphosphate accumulation in yeast ERG9 mutants

A sensitive method was developed for measuring farnesyl diphosphate (FPP) accumulation in a mutant strain of Saccharomyces cerevisiae. The strain was blocked at squalene synthase (ERG9 gene) in the isoprenoid pathway and had the catalytic domain of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene integrated into the chromosome. It required ergosterol for growth and produced E,E-farnesol. The method was based on the isolation of FPP using the anion exchanger Macro Prep High Q and conversion of FPP to E,E-farnesol with alkaline phosphatase. Farnesol was measured using gas chromatography-mass spectrometry. Background farnesol in the cell-free extract was also retained by the anion exchanger, but was removed with repeated washing with methanol. Both 1M NaCl and 40% (v/v) methanol were required in the elution buffer to effectively elute FPP. The preparation of cell-free extract in Bis-Tris propane/HCl, pH 7, buffer containing 0.025% (w/v) Triton X-100 and 15 mM MgCl(2) provided optimum conditions for the stabilization of FPP.     

Farnesol production in Escherichia coli through the construction of a farnesol biosynthesis pathway – application of PgpB and YbjG phosphatases

Farnesol is a sesquiterpenoid alcohol that has important industrial and medical potential. It is usually synthesized from farnesyl diphosphate (FPP) by farnesol synthase in plants. FPP accumulation can cause up‐regulation of phosphatases capable of FPP hydrolysis, resulting in farnesol production in Escherichia coli. We found that PgpB and YbjG, two integral membrane phosphatases, can hydrolyze FPP into farnesol. Overexpression of FPP synthase (IspA) and PgpB, along with a heterologous mevalonate pathway, enabled recombinant E. coli to produce 526.1 mg/L of farnesol. This result indicates that the phosphatases PgpB and YbjG can be used to construct a novel farnesol synthesis pathway for mass production in E. coli.     

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase

The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl₂ at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.     

Growth defects resulting from inhibiting ERG20 and RAM2 in Candida glabrata

Farnesyl pyrophosphate (FPP) is utilized for many cellular processes, including the production of dolichols, ubiquinone (CoQ), sterols, farnesylated heme A and prenylated proteins. This lipid synthesized by FPP synthetase (ERG20) becomes attached to target proteins by the prenyltransferases, CDC43/RAM2 and RAM1/RAM2 complexes after the formation of the C15 and C20 units, respectively. Defects in protein prenylation as a result of inhibiting these enzyme complexes lead to pleiotropic effects in all eukaryotes. In this study, using Candida glabrata conditional mutants, the importance of the ERG20 and RAM2 genes for growth using both in vivo and in vitro assays was assessed by placing the RAM2 and ERG20 genes under the control of a regulatable promoter. Repression of RAM2 gene expression revealed growth defects under both conditions. However, repression of ERG20 gene expression did not impair fungal growth in a mouse host, but did result in growth defects on laboratory media. Thus, FPP synthase is not required for survival in an infected mouse, but the RAM2-encoded prenyltransferase was critical for growth under both conditions. This study strongly suggests that inhibitors of prenyltransferase may be promising antifungals.     

Construction of yeast producing patchoulol by global metabolic engineering strategy

Patchoulol is a sesquiterpene alcohol found in the leaves of the patchouli plant that can be extracted by steam distillation. Notably, patchoulol is an essential natural product frequently used in the chemical industry. However, patchouli produces an insignificant amount of patchoulol, not to mention steam distillation, and requires a lot of energy and time. Recombinant microorganisms that can be cultured in mild conditions and can produce patchoulol from renewable biomass resources may be a promising alternative. We previously developed the global metabolic engineering strategy (GMES), which produces a comprehensive metabolic modification in yeast, using the cocktail δ-integration method. In this study, we aimed to produce patchoulol by modifying engineered yeast. The expression of nine genes involved in patchoulol synthesis was modulated using GMES. Regarding patchoulol production, the resultant strain, YPH499/PAT167/MVA442, showed a concentration of 42.1 mg/L, a production rate of 8.42 mg/L/d, and a yield of 2.05 mg/g-glucose, respectably. These concentration values, production rate, and yield obtained through batch-fermentation in this study were high level when compared to previously reported recombinant microorganism studies. GMES could be used as a potential strategy for producing secondary metabolites from plants in recombinant Saccharomyces cerevisiae.     

The role of ERG20 gene (encoding yeast farnesyl diphosphate synthase) mutation in long dolichol formation. Molecular modeling of FPP synthase

The yeast Saccharomyces cerevisiae strain LB332 bearing a mutation in the ERG20 gene encoding farnesyl diphosphate synthase (FPPS) synthesizes significantly longer dolichols than the wild type strain FL100 (14-31 and 14-19 isoprene units, respectively). The measurement of the short chain prenyl alcohols excreted into the medium shows that increased amounts of geraniol, dimethylallyl and isopentenyl alcohols but not farnesol are synthesized by the mutant strain. The wild type FPPS synthesizes farnesyl diphosphate (FPP) as the only product. The K197E substitution, as opposed to F112A/F113S in avian FPPS, does not change product specificity. Consequently, the possibility that mutated yeast FPPS synthesizes longer polyprenols is unlikely. This is supported by additional evidence such as in vitro analysis of the mutated FPPS products and molecular modeling. We suggest that formation of longer dolichols in vivo is the result of a change in the isopentenyl diphosphate/farnesyl diphosphate ratio caused by the erg20 mutation which in turn affects the activity of cis-prenyltransferase.     

MET3 Promoter: A Tightly Regulated Promoter and Its Application in Construction of Conditional Lethal Strain

The β-galactosidase activity driven by MET3 promoter was assayed in the absence of methionine and in the presence of different concentration of methionine in medium. To compare its activity with GAL1 promoter and the data reported by Mumburg about MET25 promoter, the MET3 promoter was a weak but tightly controlled promoter. Its successful application in the construction of methionine-sensitive tri-mutant (cln1Δcln2Δpho85Δ) demonstrated that the MET3 promoter is a useful promoter in construction of conditional lethal strain and heterologous expression in Saccharomyces cerevisiae. Received: 20 September 2001 / Accepted: 22 October 2001     

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.     

Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes

The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.     

代谢工程改造酿酒酵母提高法尼醇产量

【目的】法尼醇(FOH,C₁₅H₂₆O)是一种具有芳香气味的非环状倍半萜醇,被广泛应用于化妆品和医学药物的工业化生产,也可作为航空燃料的理想替代品。具有食品级安全性的酿酒酵母细胞能够合成内源性法尼醇,但其产量很低,无法满足工业生产的需要。因此,需要采用代谢工程手段,改造法尼醇合成途径,以有效提高法尼醇在酿酒酵母中的产量。【方法】以酿酒酵母工业菌株CEN.PK2-1D为底盘细胞,强化甲羟戊酸途径中关键酶的表达水平和弱化麦角固醇合成分支途径,以提高法尼醇合成所需的直接前体物质法尼基焦磷酸(FPP);并分别表达催化FPP合成法尼醇的五种内源磷酸酶和两种异源合酶,筛选能高效合成法尼醇的磷酸酶或合酶。【结果】通过在CEN.PK2-1D(法尼醇产量<0.1mg/L)中强化表达甲羟戊酸途径中截短形式的HMG-CoA还原酶(tHMGR1)和FPP合酶(ERG20),使法尼醇产量提高约50.8倍,达到5.08 mg/L;使用HXT1启动子替换鲨烯合酶编码基因ERG9启动子以下调其表达水平,使法尼醇产量进一步提升47.1倍,达到239.17 mg/L。在此...     

Farnesol restores wild-type colony morphology to 96% of Candida albicans colony morphology variants recovered following treatment with mutagens.

Candida albicans is a diploid fungus that undergoes a morphological transition between budding yeast, hyphal, and pseudohyphal forms. The morphological transition is strongly correlated with virulence and is regulated in part by quorum sensing. Candida albicans produces and secretes farnesol that regulates the yeast to mycelia morphological transition. Mutants that fail to synthesize or respond to farnesol could be locked in the filamentous mode. To test this hypothesis, a collection of C. albicans mutants were isolated that have altered colony morphologies indicative of the presence of hyphal cells under environmental conditions where C. albicans normally grows only as yeasts. All mutants were characterized for their ability to respond to farnesol. Of these, 95.9% fully or partially reverted to wild-type morphology on yeast malt (YM) agar plates supplemented with farnesol. All mutants that respond to farnesol regained their hyphal morphology when restreaked on YM plates without farnesol. The observation that farnesol remedial mutants are so common (95.9%) relative to mutants that fail to respond to farnesol (4.1%) suggests that farnesol activates and (or) induces a pathway that can override many of the morphogenesis defects in these mutants. Additionally, 9 mutants chosen at random were screened for farnesol production. Two mutants failed to produce detectable levels of farnesol.