Multi-modular engineering of Saccharomyces cerevisiae for high-titre production of tyrosol and salidroside.
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ABSTRACT:
SUBMITTER: Liu H
PROVIDER: S-EPMC8601180 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tyrosol is extensively used in the pharmaceutical industry as an important natural product from plants. In this study, an exogenous pathway involved in catalyzing tyrosine to tyrosol was introduced into Saccharomyces cerevisiae. Furthermore, The pyruvate decarboxylase gene pdc1 was deleted to redirect the flux distribution at the pyruvate node, and a bifunctional NAD+-dependent fused chorismate mutase/prephenate dehydrogenase from E. coli (EcTyrA) and its' tyrosine inhibition resistant mutant (EcTyrAM53I/A354V) were heterologously expression in S. cerevisiae to tuning up the chorismate metabolism effectively directed the metabolic flux toward tyrosol production. Finally, the tyrosol yield of the engineered strain GFT-4 was improved to 126.74 ± 6.70 mg/g DCW at 48 h, increased 440 times compared with that of the control strain GFT-0 (0.28 ± 0.01 mg/g DCW). The new synergetic engineering strategy developed in this study can be further applied to increase the production of high value-added aromatic compounds derived from aromatic amino acid or shikimate in S. cerevisiae.
| S-EPMC6616077 | biostudies-literature
Project description:BACKGROUND:The biological synthesis of high value compounds in industry through metabolically engineered microorganism factories has received increasing attention in recent years. Valencene is a high value ingredient in the flavor and fragrance industry, but the low concentration in nature and high cost of extraction limits its application. Saccharomyces cerevisiae, generally recognized as safe, is one of the most commonly used gene expression hosts. Construction of S. cerevisiae cell factory to achieve high production of valencene will be attractive. RESULTS:Valencene was successfully biosynthesized after introducing valencene synthase into S. cerevisiae BJ5464. A significant increase in valencene yield was observed after down-regulation or knock-out of squalene synthesis and other inhibiting factors (such as erg9, rox1) in mevalonate (MVA) pathway using a recyclable CRISPR/Cas9 system constructed in this study through the introduction of Cre/loxP. To increase the supplement of the precursor farnesyl pyrophosphate (FPP), all the genes of FPP upstream in MVA pathway were overexpressed in yeast genome. Furthermore, valencene expression cassettes containing different promoters and terminators were compared, and PHXT7-VS-TTPI1 was found to have excellent performance in valencene production. Finally, after fed-batch fermentation in 3 L bioreactor, valencene production titer reached 539.3 mg/L with about 160-fold improvement compared to the initial titer, which is the highest reported valencene yield. CONCLUSIONS:This study achieved high production of valencene in S. cerevisiae through metabolic engineering and optimization of expression cassette, providing good example of microbial overproduction of valuable chemical products. The construction of recyclable plasmid was useful for multiple gene editing as well.
| S-EPMC6839068 | biostudies-literature
Project description:Simvastatin is a semisynthetic cholesterol-lowering medication and one of the top-selling statins in the world. Currently, industrial production of simvastatin acid (SVA) is a multistep process starting from the natural product lovastatin. For this reason, there is significant interest in direct production of simvastatin from a microbial host. In this study, six heterologous biosynthetic genes were introduced into Saccharomyces cerevisiae and the acyl-donor dimethylbutyryl-S-methyl mercaptopropionate (DMB-SMMP) was added, resulting in initial production of 0.5?mg/L SVA. Switching the yeast strain from JHY686 to BJ5464-NpgA increased total polyketide production to over 60?mg/L and conversion from dihydromonacolin L acid to monacolin J acid (MJA) was increased from 60% to 90% by tuning the copy number of the P450 lovA. Increasing the media pH to 8.7 led to a further 10-fold increase in SVA production. Optimized chemical lysis of the cell walls in situ after maximum MJA production led to 55?mg/L SVA titer, representing nearly complete conversion from MJA and a 110-fold increase in titer from the initial SVA production strain. The yeast strains developed in this work can be used as an alternative production method for SVA, and the strategies employed can be broadly applied for heterologous production of other fungal polyketides and semisynthetic compounds in yeast.
| S-EPMC6348118 | biostudies-literature
Project description:BACKGROUND:Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. RESULTS:Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively. CONCLUSION:The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.
| S-EPMC6029064 | biostudies-literature
Project description:BackgroundThe natural phenolic glycoside gastrodin is the major bioactive ingredient in the well-known Chinese herb Tianma and is widely used as a neuroprotective medicine in the clinic. Microbial production from sustainable resources is a promising method to replace plant extraction and chemical synthesis which were currently used in industrial gastrodin production. Saccharomyces cerevisiae is considered as an attractive host to produce natural plant products used in the food and pharmaceutical fields. In this work, we intended to explore the potential of S. cerevisiae as the host for high-level production of gastrodin from glucose.ResultsHere, we first identified the plant-derived glucosyltransferase AsUGT to convert 4-hydroxybenzyl alcohol to gastrodin with high catalytic efficiency in yeast. Then, we engineered de novo production of gastrodin by overexpressing codon-optimized AsUGTsyn, the carboxylic acid reductase gene CARsyn from Nocardia species, the phosphopantetheinyl transferase gene PPTcg-1syn from Corynebacterium glutamicum, the chorismate pyruvate-lyase gene UbiCsyn from Escherichia coli, and the mutant ARO4K229L. Finally, we achieved an improved product titer by a chromosomal multiple-copy integration strategy and enhancement of metabolic flux toward the aglycon 4-hydroxybenzyl alcohol. The best optimized strain produced 2.1 g/L gastrodin in mineral medium with glucose as the sole carbon source by flask fermentation, which was 175 times higher than that of the original gastrodin-producing strain.ConclusionsThe de novo high-level production of gastrodin was first achieved. Instead of chemical synthesis or plants extraction, our work provides an alternative strategy for the industrial production of gastrodin by microbial fermentation from a sustainable resource.
| S-EPMC7690157 | biostudies-literature
Project description:24-Methylene-cholesterol is a necessary substrate for the biosynthesis of physalin and withanolide, which show promising anticancer activities. It is difficult and costly to prepare 24-methylene-cholesterol via total chemical synthesis. In this study, we engineered the biosynthesis of 24-methylene-cholesterol in Saccharomyces cerevisiae by disrupting the two enzymes (i.e., ERG4 and ERG5) in the yeast's native ergosterol pathway, with ERG5 being replaced with the DHCR7 (7-dehydrocholesterol reductase) enzyme. Three versions of DHCR7 originating from different organisms-including the DHCR7 from Physalis angulata (PhDHCR7) newly discovered in this study, as well as the previously reported OsDHCR7 from Oryza sativa and XlDHCR7 from Xenopus laevis-were assessed for their ability to produce 24-methylene-cholesterol. XlDHCR7 showed the best performance, producing 178 mg/L of 24-methylene-cholesterol via flask-shake cultivation. The yield could be increased up to 225 mg/L, when one additional copy of the XlDHCR7 expression cassette was integrated into the yeast genome. The 24-methylene-cholesterol-producing strain obtained in this study could serve as a platform for characterizing the downstream enzymes involved in the biosynthesis of physalin or withanolide, given that 24-methylene-cholesterol is a common precursor of these chemicals.
| S-EPMC8615579 | biostudies-literature
Project description:BackgroundGlucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway.ResultsA more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo-inositol, the highest titer that had been ever reported in S. cerevisiae.ConclusionsIn this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae.
| S-EPMC5935971 | biostudies-literature
Project description:Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47?mg?l(-1) with a yield of 67?mg (g glucose)(-1) in shake-flask cultures and a titre of 5.1?g?l(-1) in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals.
| S-EPMC4569842 | biostudies-other
Project description:Genistein, a nutraceutical isoflavone, has various pharmaceutical and biological activities which benefit human health via soy-containing food intake. This study aimed to construct Saccharomyces cerevisiae to produce genistein from sugar via a modular engineering strategy. In the midstream module, various sources of chalcone synthases and chalcone isomerase-like proteins were tested which enhanced the naringenin production from p-coumaric acid by decreasing the formation of the byproduct. The upstream module was reshaped to enhance the metabolic flux to p-coumaric acid from glucose by overexpressing the genes in the tyrosine biosynthetic pathway and deleting the competing genes. The downstream module was rebuilt to produce genistein from naringenin by pairing various isoflavone synthases and cytochrome P450 reductases. The optimal pair was used for the de novo biosynthesis of genistein with a titer of 31.02 mg/L from sucrose at 25 °C. This is the first report on the de novo biosynthesis of genistein in engineered S. cerevisiae to date. This work shows promising potential for producing flavonoids and isoflavonoids by modular metabolic engineering.
| S-EPMC9319343 | biostudies-literature
Project description:Monoterpene indole alkaloids (MIAs) represent a structurally diverse, medicinally essential class of plant derived natural products. The universal MIA building block strictosidine was recently produced in the yeast Saccharomyces cerevisiae, setting the stage for optimization of microbial production. However, the irreversible reduction of pathway intermediates by yeast enzymes results in a non-recoverable loss of carbon, which has a strong negative impact on metabolic flux. In this study, we identified and engineered the determinants of biocatalytic selectivity which control flux towards the iridoid scaffold from which all MIAs are derived. Development of a bioconversion based production platform enabled analysis of the metabolic flux and interference around two critical steps in generating the iridoid scaffold: oxidation of 8-hydroxygeraniol to the dialdehyde 8-oxogeranial followed by reductive cyclization to form nepetalactol. In vitro reconstitution of previously uncharacterized shunt pathways enabled the identification of two distinct routes to a reduced shunt product including endogenous 'ene'-reduction and non-productive reduction by iridoid synthase when interfaced with endogenous alcohol dehydrogenases. Deletion of five genes involved in ?,?-unsaturated carbonyl metabolism resulted in a 5.2-fold increase in biocatalytic selectivity of the desired iridoid over reduced shunt product. We anticipate that our engineering strategies will play an important role in the development of S. cerevisiae for sustainable production of iridoids and MIAs.
| S-EPMC5705256 | biostudies-literature