Efficient production of vindoline from tabersonine by metabolically engineered Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Liu T
PROVIDER: S-EPMC8446080 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Glycyrrhetinic acid (GA) is the most important ingredient in licorice due to its outstanding anti-inflammatory activity and wide application in the medicine and cosmetics industries. Contemporary industrial production of GA by acid hydrolysis of glycyrrhizin which was extracted from Glycyrrhiza plants, is not environment-friendly and devastates farmland since the Glycyrrhiza rhizomes grow up to 10 m underground. RESULTS:In this study, GA was produced through metabolically engineering Saccharomyces cerevisiae by introducing the entire heterogeneous biosynthetic pathway of GA. Codon optimized CYP88D6 and CYP72A154, combined with ?-AS (?-amyrin synthase encoding gene) and the NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana were introduced into S. cerevisiae. The resulting strain (Y1) produced 2.5 mg/L of ?-amyrin and 14 ?g/L of GA. The cytochrome b5 from G. uralensis (GuCYB5) was identified and the introduction of this novel GuCYB5 increased the efficiency of GA production by eightfold. The joint utilization of the GuCYB5 gene along with 10 known MVA pathway genes from S. cerevisiae were overexpressed in a stable chromosome integration to achieve higher GA production. Using the combined strategy, GA concentration improved by 40-fold during batch fermentation. The production was further improved to 8.78 mg/L in fed-batch fermentation, which was increased by a factor of nearly 630. CONCLUSIONS:This study first investigated the influence of carbon flux in the upstream module and the introduction of a newly identified GuCYB5 on GA production. The newly identified GuCYB5 was highly effective in improving GA production. An integrated strategy including enzyme discovery, pathway optimization, and fusion protein construction was provided in improving GA production, achieving a 630 fold increase in GA production. The metabolically engineered yeast cell factories provide an alternative approach to glycyrrhetinic acid production, replacing the traditional method of plant extraction.
| S-EPMC6540369 | biostudies-literature
Project description:BACKGROUND:Diterpenoids are a large class of natural products with complex structures and broad commercial applications as food additives, important medicines, and fragrances. However, their low abundance in plants and high structural complexity limit their applications. Therefore, it is important to create an efficient diterpenoid-producing yeast cell factory of the production of various high-value diterpenoid compounds in a cost-effective manner RESULTS: In this study, 13R-manoyl oxide (13R-MO; 2.31 mg/L) was produced by expressing CfTPS2 and CfTPS3 from Coleus forskohlii in Saccharomyces cerevisiae. The 13R-MO titer was increased by 142-fold to 328.15 mg/L via the stepwise metabolic engineering of the original strain, including the overexpression of the rate-limiting genes (tHMG1 and ERG20) of the mevalonate pathway, transcription and protein level regulation of ERG9, Bts1p and Erg20F96Cp fusion, and the overexpression of tCfTPS2 and tCfTPS3 (excision of the N-terminal plastid transit peptide sequences of CfTPS2 and CfTPS3). The final titer of 13R-MO reached up to 3 g/L by fed-batch fermentation in a 5 L bioreactor. CONCLUSIONS:In this study, an efficient 13R-MO yeast cell factory was constructed, which achieved the de novo production of 3 g/L of 13R-MO from glucose. To the best of our knowledge, this is the highest 13R-MO titer reported to date. Furthermore, the metabolic engineering strategies presented here could be used to produce other valuable diterpenoid compounds in yeast.
| S-EPMC6480505 | biostudies-literature
Project description:We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of the l-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integrated LDH is regulated under the control of the native PDC1 promoter, while PDC1 is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovine LDH. Yeast cells expressing LDH were observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovine LDH under the control of the PDC1 promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologous LDH gene (i.e., either the bovine LDH gene or the Bifidobacterium longum LDH gene): two transgenic strains with 2microm plasmid-based vectors and two genome-integrated strains.
| S-EPMC1082537 | biostudies-literature
Project description:Saccharomyces cerevisiae is a versatile industrial host for chemical production and has been engineered to produce efficiently many valuable compounds. 2-Deoxy-scyllo-inosose (2-DOI) is an important precursor for the biosynthesis of 2-deoxystreptamine-containing aminoglycosides antibiotics and benzenoid metabolites. Bacterial and cyanobacterial strains have been metabolically engineered to generate 2-DOI; nevertheless, the production of 2-DOI using a yeast host has not been reported. Here, we have metabolically engineered a series of CEN.PK yeast strains to produce 2-DOI using a synthetically yeast codon-optimized btrC gene from Bacillus circulans. The expression of the 2-Deoxy-scyllo-inosose synthase (2-DOIS) gene was successfully achieved via an expression vector and through chromosomal integration at a high-expression locus. In addition, the production of 2-DOI was further investigated for the CEN.PK knockout strains of phosphoglucose isomerase (?pgi1), D-glucose-6-phosphate dehydrogenase (?zwf1) and a double mutant (?pgi1, ?zwf1) in a medium consisting of 2% fructose and 0.05% glucose as a carbon source. We have found that all the recombinant strains are capable of producing 2-DOI and reducing it into scyllo-quercitol and (-)-vibo-quercitol. Comparatively, the high production of 2-DOI and its analogs was observed for the recombinant CEN.PK-btrC carrying the multicopy btrC-expression vector. GC/MS analysis of culture filtrates of this strain showed 11 times higher response in EIC for the m/z 479 (methyloxime-tetra-TMS derivative of 2-DOI) than the YP-btrC recombinant that has only a single copy of btrC expression cassette integrated into the genomic DNA of the CEN.PK strain. The knockout strains namely ?pgi1-btrC and ?pgi1?zwf1-btrC, that are transformed with the btrC-expression plasmids, have inactive Pgi1 and produced only traces of the compounds. In contrast, ?zwf1-btrC recombinant which has intact pgi1 yielded relatively higher amount of the carbocyclic compounds. Additionally, 1H-NMR analysis of samples showed slow consumption of fructose and no accumulation of 2-DOI and the quercitols in the culture broth of the recombinant CEN.PK-btrC suggesting that S. cerevisiae is capable of assimilating 2-DOI.
| S-EPMC7348060 | biostudies-literature
Project description:2'-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has potential applications in foods due to its health benefits such as the selective promotion of bifidobacterial growth and the inhibition of pathogenic microbial binding to the human gut. Owing to the limited amounts of 2-FL in human milk, alternative microbial production of 2-FL is considered promising. To date, microbial production of 2-FL has been studied mostly in Escherichia coli. In this study, 2-FL was produced alternatively by using a yeast Saccharomyces cerevisiae, which may have advantages over E. coli.Fucose and lactose were used as the substrates for the salvage pathway which was constructed with fkp coding for a bifunctional enzyme exhibiting L-fucokinase and guanosine 5'-diphosphate-L-fucose phosphorylase activities, fucT2 coding for ?-1,2-fucosyltransferase, and LAC12 coding for lactose permease. Production of 2-FL by the resulting engineered yeast was verified by mass spectrometry. 2-FL titers of 92 and 503 mg/L were achieved from 48-h batch fermentation and 120-h fed-batch fermentation fed with ethanol as a carbon source, respectively.This is the first report on 2-FL production by using yeast S. cerevisiae. These results suggest that S. cerevisiae can be considered as a host engineered for producing 2-FL via the salvage pathway.
| S-EPMC6020385 | biostudies-other
Project description:Biomass, the most abundant carbon source on the planet, may in the future become the primary feedstock for production of fuels and chemicals, replacing fossil feedstocks. This will, however, require development of cell factories that can convert both C6 and C5 sugars present in lignocellulosic biomass into the products of interest. We engineered Saccharomyces cerevisiae for production of 3-hydroxypropionic acid (3HP), a potential building block for acrylates, from glucose and xylose. We introduced the 3HP biosynthetic pathways via malonyl-CoA or β-alanine intermediates into a xylose-consuming yeast. Using controlled fed-batch cultivation, we obtained 7.37±0.17 g 3HP L-1 in 120 hours with an overall yield of 29±1% Cmol 3HP Cmol-1 xylose. This study is the first demonstration of the potential of using S. cerevisiae for production of 3HP from the biomass sugar xylose.
| S-EPMC8193239 | biostudies-literature
Project description:Background2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol.ResultsEndogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C2-compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae. A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h.ConclusionsThe microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.
| S-EPMC6714309 | biostudies-literature
Project description:Monoterpene indole alkaloids (MIAs) from plants encompass a broad class of structurally complex and medicinally valuable natural products. MIAs are biologically derived from the universal precursor strictosidine. Although the strictosidine biosynthetic pathway has been identified and reconstituted, extensive work is required to optimize production of strictosidine and its precursors in yeast. In this study, we engineered a fully integrated and plasmid-free yeast strain with enhanced production of the monoterpene precursor geraniol. The geraniol biosynthetic pathway was targeted to the mitochondria to protect the GPP pool from consumption by the cytosolic ergosterol pathway. The mitochondrial geraniol producer showed a 6-fold increase in geraniol production compared to cytosolic producing strains. We further engineered the monoterpene-producing strain to synthesize the next intermediates in the strictosidine pathway: 8-hydroxygeraniol and nepetalactol. Integration of geraniol hydroxylase (G8H) from Catharanthus roseus led to essentially quantitative conversion of geraniol to 8-hydroxygeraniol at a titer of 227 mg/L in a fed-batch fermentation. Further introduction of geraniol oxidoreductase (GOR) and iridoid synthase (ISY) from C. roseus and tuning of the relative expression levels resulted in the first de novo nepetalactol production. The strategies developed in this work can facilitate future strain engineering for yeast production of later intermediates in the strictosidine biosynthetic pathway.
| S-EPMC6717016 | biostudies-literature
Project description:Biosurfactants are biological tensioactive agents that can be used in the cosmetic and food industries. Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains. These compounds are green alternatives to petrochemical surfactants, but their large-scale production is still in its infancy, hindered due to pathogenicity of natural producer, high substrate and purification costs and low yields and productivities. This study, for the first time, aimed at producing mono-rhamnolipids from sucrose by recombinant GRAS Saccharomyces cerevisiae strains. Six enzymes from P. aeruginosa involved in mono-rhamnolipid biosynthesis were functionally expressed in the yeast. Furthermore, its SUC2 invertase gene was disrupted and a sucrose phosphorylase gene from Pelomonas saccharophila was also expressed to reduce the pathway's overall energy requirement. Two strains were constructed aiming to produce mono-rhamnolipids and the pathway's intermediate dTDP-L-rhamnose. Production of both molecules was analyzed by confocal microscopy and mass spectrometry, respectively. These strains displayed, for the first time as a proof of concept, the potential of production of these molecules by a GRAS eukaryotic microorganism from an inexpensive substrate. These constructs show the potential to further improve rhamnolipids production in a yeast-based industrial bioprocess.
| S-EPMC5811566 | biostudies-literature
Project description:Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6-deoxyerythronolide B (6-dEB) or 3-?-mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60- and 25-fold more erythromycin D (EryD) than the original strain K207-3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.
| S-EPMC3815289 | biostudies-literature