scholarly journals CRISPRi-Guided Metabolic Flux Engineering for Enhanced Protopanaxadiol Production in Saccharomyces cerevisiae

2021 ◽  
Vol 22 (21) ◽  
pp. 11836
Author(s):  
Soo-Hwan Lim ◽  
Jong-In Baek ◽  
Byeong-Min Jeon ◽  
Jung-Woo Seo ◽  
Min-Sung Kim ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Flux ◽  
De Novo ◽  
Carbon Sources ◽  
Biosynthetic Pathways ◽  
High Potency ◽  
Flux System ◽  
High Production ◽  
High Production Cost

Protopanaxadiol (PPD), an aglycon found in several dammarene-type ginsenosides, has high potency as a pharmaceutical. Nevertheless, application of these ginsenosides has been limited because of the high production cost due to the rare content of PPD in Panax ginseng and a long cultivation time (4–6 years). For the biological mass production of the PPD, de novo biosynthetic pathways for PPD were introduced in Saccharomyces cerevisiae and the metabolic flux toward the target molecule was restructured to avoid competition for carbon sources between native metabolic pathways and de novo biosynthetic pathways producing PPD in S. cerevisiae. Here, we report a CRISPRi (clustered regularly interspaced short palindromic repeats interference)-based customized metabolic flux system which downregulates the lanosterol (a competing metabolite of dammarenediol-II (DD-II)) synthase in S. cerevisiae. With the CRISPRi-mediated suppression of lanosterol synthase and diversion of lanosterol to DD-II and PPD in S. cerevisiae, we increased PPD production 14.4-fold in shake-flask fermentation and 5.7-fold in a long-term batch-fed fermentation.

scholarly journals The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis

1999 ◽  
Vol 338 (3) ◽  
pp. 701-708 ◽  
Author(s):  
Evelyne RAUX ◽  
Treasa McVEIGH ◽  
Sarah E. PETERS ◽  
Thomas LEUSTEK ◽  
Martin J. WARREN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Biosynthetic Pathways ◽  
Subtle Difference ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
His Tag ◽  
Cobalamin Biosynthesis

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.

scholarly journals Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of Gastrodin from Glucose

2020 ◽  
Author(s):  
Hua Yin ◽  
Tian Dong Hu ◽  
Yi Bin Zhuang ◽  
Tao Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chemical Synthesis ◽  
Metabolic Flux ◽  
De Novo ◽  
Chinese Herb ◽  
Phenolic Glycoside ◽  
Phosphopantetheinyl Transferase ◽  
High Level ◽  
Level Production ◽  
Hydroxybenzyl Alcohol

Abstract Background: The 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. Results: Here, 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 AsUGT syn , the carboxylic acid reductase gene CAR syn from Nocardia species, the phosphopantetheinyl transferase gene PPTcg-1 syn from Corynebacterium glutamicum , the chorismate pyruvate-lyase gene UbiC syn from Escherichia coli , and the mutant ARO4 K229L . 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. Conclusions: The 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.

scholarly journals Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of Gastrodin from Glucose

2020 ◽  
Author(s):  
Hua Yin ◽  
Tian Dong Hu ◽  
Yi Bin Zhuang ◽  
Tao Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chemical Synthesis ◽  
Metabolic Flux ◽  
De Novo ◽  
Chinese Herb ◽  
Phenolic Glycoside ◽  
Phosphopantetheinyl Transferase ◽  
High Level ◽  
Level Production ◽  
Hydroxybenzyl Alcohol

Abstract Background: The 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.Results: Here, 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.Conclusions: The 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.

scholarly journals Metabolic Engineering of Saccharomyces cerevisiae for High-Level Production of Gastrodin from Glucose

2020 ◽  
Author(s):  
Hua Yin ◽  
Tian Dong Hu ◽  
Yi Bin Zhuang ◽  
Tao Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chemical Synthesis ◽  
Metabolic Flux ◽  
De Novo ◽  
Chinese Herb ◽  
Phenolic Glycoside ◽  
Phosphopantetheinyl Transferase ◽  
High Level ◽  
Level Production ◽  
Hydroxybenzyl Alcohol

Abstract Background: The 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.Results: Here, 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. Conclusions: The 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.

scholarly journals Heterologous Expression of Mycobacterial Proteins in Saccharomyces cerevisiae Reveals Two Physiologically Functional 3-Hydroxyacyl-Thioester Dehydratases, HtdX and HtdY, in Addition to HadABC and HtdZ

2009 ◽  
Vol 191 (8) ◽  
pp. 2683-2690 ◽  
Author(s):  
Aner Gurvitz ◽  
J. Kalervo Hiltunen ◽  
Alexander J. Kastaniotis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lipoic Acid ◽  
Fatty Acid Synthase ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Carbon Sources ◽  
Protective Layer ◽  
Yeast Cells ◽  
Triphenyltetrazolium Chloride ◽  
Mycolic Acids

ABSTRACT We report on Mycobacterium tuberculosis Rv0241c and Rv3389c, representing two physiologically functional 3-hydroxyacyl-thioester dehydratases (Htd). These enzymes are potentially entrained in type 2 fatty acid synthase (FASII). Mycobacterial FASII is involved in the synthesis of mycolic acids, which are the major constituents of the protective layer around the pathogen, shielding it from noxious chemicals and the host's immune system. Mycolic acids are additionally associated with the virulence and resilience of M. tuberculosis. Here, Rv0241c and Rv3389c, which are distinct from the previously identified heterodimers Rv0635-Rv0636 (HadAB) and Rv0636-Rv0637 (HadBC) but also the homodimer Rv0130 (HtdZ), were identified by expressing the corresponding candidate open reading frames in Saccharomyces cerevisiae htd2Δ cells lacking mitochondrial 3-hydroxyacyl-acyl carrier protein dehydratase activity, followed by scoring for phenotype rescue. The htd2Δ mutant fails to produce sufficient levels of lipoic acid and does not respire or grow on nonfermentable carbon sources. Soluble protein extracts made from mutant htd2Δ cells expressing mitochondrially targeted Rv0241c or Rv3389c contained 3-hydroxyacyl-thioester hydratase activity. Moreover, mutant yeast cells expressing Rv0241c or Rv3389c were able to recover their respiratory growth on glycerol medium and efficiently reduce 2,3,5-triphenyltetrazolium chloride. Additionally, expression of mitochondrial Rv0241c or Rv3389c in htd2Δ cells also restored de novo lipoic acid synthesis to 92 and 40% of the level in the wild-type strain, respectively. We propose naming Rv0241c and Rv3389c as HtdX and HtdY, respectively, and discuss the implications of our finding with reference to Rv0098, a candidate mycobacterial FabZ homologue with intrinsic thioesterase and hydratase activities that lacks the eukaryotic-like hydratase-2 motif.

scholarly journals Metabolic engineering of Saccharomyces cerevisiae for high-level production of gastrodin from glucose

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Hua Yin ◽  
Tiandong Hu ◽  
Yibin Zhuang ◽  
Tao Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chemical Synthesis ◽  
Metabolic Flux ◽  
De Novo ◽  
Chinese Herb ◽  
Phenolic Glycoside ◽  
Phosphopantetheinyl Transferase ◽  
High Level ◽  
Level Production ◽  
Hydroxybenzyl Alcohol

Abstract Background The 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. Results Here, 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. Conclusions The 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.

scholarly journals Alternative methodology for isolation of biosurfactant-producing bacteria

2007 ◽  
Vol 67 (1) ◽  
pp. 117-124 ◽  
Author(s):  
N. Krepsky ◽  
FS. Da Silva ◽  
LF. Fontana ◽  
MAC. Crapez
Keyword(s):  
Aqueous Phase ◽  
Growth Curve ◽  
Carbon Sources ◽  
Biosurfactant Production ◽  
Mangrove Sediment ◽  
Light Oil ◽  
Bacterial Growth Curve ◽  
High Production ◽  
High Production Cost ◽  
Petroleum Derivatives

Wide biosurfactant application on biorremediation is limited by its high production cost. The search for cheaper biossurfactant production alternatives has guided our study. The use of selective media containing sucrose (10 g.L-1) and Arabian Light oil (2 g.L-1) as carbon sources showed to be effective to screen and maintain biosurfactant-producing consortia isolated from mangrove hydrocarbon-contaminated sediment. The biosurfactant production was assayed by kerosene, gasoline and Arabian Light Emulsification activity and the bacterial growth curve was determined by bacterial quantification. The parameters analyzed for biosurfactant production were the growth curve, salinity concentration, flask shape and oxygenation. All bacteria consortia screened were able to emulsify the petroleum derivatives tested. Biosurfactant production increased according to the incubation time; however the type of emulsification (non-aqueous phase or aqueous phase) did not change with time but with the compound tested. The methodology was able to isolate biosurfactant-producing consortia from superficial mangrove sediment contaminated by petroleum hydrocarbons and was recommended for selection of biosurfactant producing bacteria in tropical countries with low financial resources.

Effect of salicin on induction and carbon catabolite repression of endoxylanase synthesis in Penicillium janthinellum MTCC 10889

2014 ◽  
Vol 68 (4) ◽  
Author(s):  
Aditi Kundu ◽  
Rina Ray
Keyword(s):  
De Novo ◽  
Carbon Sources ◽  
Fold Increase ◽  
Enzyme Synthesis ◽  
Submerged Cultivation ◽  
Penicillium Janthinellum ◽  
Inducer Exclusion ◽  
Repressive Effect ◽  
High Production ◽  
Exclusion Mechanism

AbstractAmongst various carbon sources, xylan was found to be the sole inducer of endoxylanase production by Penicillium janthinellum MTCC 10889 in submerged cultivation. Endoxylanase synthesis by a xylan induced culture was initially repressed after a simultaneous addition of xylose, probably by the inducer exclusion mechanism, but it was resumed and achieved its highest level at a much later stage of growth (at 120 h). Xylose added after 30 h of growth cannot exert its full repressive effect. Although glucose was proved to be a more potent repressor than xylose, supplementation of salicin, an alcoholic β-glycoside containing d-glucose, with pure xylan resulted in an about 3.22 fold increase in the enzyme synthesis at 72 h followed by constant high production of the enzyme at least until the 144th h of growth. Inducing capacity of salicin in a xylan induced culture was significantly reduced when it was added after 30 h of growth. Addition of salicin and xylan help to partially overcome the repressive effect of xylose and glucose. Failure of salicin in recovering the endoxylanase synthesis in actinomycin D and cyclohexamide inhibited the xylan induced culture indicating that salicin cannot initiate the de novo synthesis of the enzyme.

Is 3 years adequate for tracking completely occluded coiled aneurysms?

2020 ◽  
Vol 133 (3) ◽  
pp. 758-764
Author(s):  
Eung Koo Yeon ◽  
Young Dae Cho ◽  
Dong Hyun Yoo ◽  
Su Hwan Lee ◽  
Hyun-Seung Kang ◽  
...  
Keyword(s):  
De Novo ◽  
Careful Monitoring ◽  
Surveillance Period ◽  
Radiological Data ◽  
De Novo Aneurysm ◽  
Low Probability ◽  
Almost All ◽  
Annual Risk

OBJECTIVEThe authors conducted a study to ascertain the long-term durability of coiled aneurysms completely occluded at 36 months’ follow-up given the potential for delayed recanalization.METHODSIn this retrospective review, the authors examined 299 patients with 339 aneurysms, all shown to be completely occluded at 36 months on follow-up images obtained between 2011 and 2013. Medical records and radiological data acquired during the extended monitoring period (mean 74.3 ± 22.5 months) were retrieved, and the authors analyzed the incidence of (including mean annual risk) and risk factors for delayed recanalization.RESULTSA total of 5 coiled aneurysms (1.5%) occluded completely at 36 months showed recanalization (0.46% per aneurysm-year) during the long-term surveillance period (1081.9 aneurysm-years), 2 surfacing within 60 months and 3 developing thereafter. Four showed minor recanalization, with only one instance of major recanalization. The latter involved the posterior communicating artery as an apparent de novo lesion, arising at the neck of a firmly coiled sac, and was unrelated to coil compaction or growth. Additional embolization was undertaken. In a multivariate analysis, a second embolization for a recurrent aneurysm (HR = 22.088, p = 0.003) independently correlated with delayed recanalization.CONCLUSIONSAlmost all coiled aneurysms (98.5%) showing complete occlusion at 36 months postembolization proved to be stable during extended observation. However, recurrent aneurysms were predisposed to delayed recanalization. Given the low probability yet seriousness of delayed recanalization and the possibility of de novo aneurysm formation, careful monitoring may be still considered in this setting but at less frequent intervals beyond 36 months.

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