Complementation of thefol2Deletion inSaccharomyces cerevisiaeby Human andEscherichia coliGenes Encoding GTP Cyclohydrolase I

Mancini, R. C.; Saracino, Francesca; Buscemi, Giacomo; Fischer, Markus; Schramek, Nicholas; Bracher, A.; Bacher, Adelbert; Gütlich, Markus; Carbone, M.

doi:10.1006/bbrc.1998.9951

Cited by 10 publications

(14 citation statements)

References 16 publications

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“…FOL2 encodes GTP cyclohydrolase I, which catalyzes the initial step in the biosynthesis of the pteridine moiety of folate ( Fig. 1) [22,23]. In tomatoes and Arabidopsis, overexpression of the homologous gene results in a two-to four-fold increase in folate levels [24][25][26].…”

Section: Resultsmentioning

confidence: 99%

Bioengineering yeast to enhance folate levels in wine

Walkey

Kitts

Liu

et al. 2015

Process Biochemistry

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Section: Resultsmentioning

confidence: 99%

Bioengineering yeast to enhance folate levels in wine

Walkey

Kitts

Liu

et al. 2015

Process Biochemistry

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“…Yeast strains and media: All the S. cerevisiae strains used in this study derived from the haploid strain 971/6a (MATa ade2-1 his3-11,15 leu2-3,112 ura3-1 can1 fol2D), which shows auxotrophy for folinic acid, and the isogenic strain 971/6c (FOL2) as wild-type control. Yeast cells were grown in SD medium (0.67% DIFCO yeast nitrogen base without amino acids, 2% glucose or galactose) plus appropriate supplements, as previously reported (Mancini et al 1999). When needed, folinic acid (Sigma, Italy) was added at a ¢nal concentration of 50 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

“…The mutations ÁG693 (patient D35), 614T>G (patient D37) and 595C>G (patients D89/90) were introduced by PCR site-directed mutagenesis into the human GCH1 coding region, cloned under control of the yeast GAL1 promoter in plasmid pLA732, carrying the selection marker URA3 (described in Mancini et al 1999). To this purpose, the entire GCH1 coding region of plasmid pLA732 was ampli¢ed by Pfu polymerase (Stratagene, M-Medical, Italy), using the speci¢c primer (P2) carrying the desired mutation (OLP100, OLP102 and OLP114, respectively) (Table 1), in association with the OLP47 primer (below).…”

Section: Pcr Site-directed Mutagenesismentioning

confidence: 99%

“…The resulting PCR products, carrying the GCH1 coding region, with the desired mutation, £anked by short regions of the 5 0 and 3 0 untranslated FOL2 sequences, were used to substitute the wild-type with the mutant GCH1 sequence in plasmid pLA732 by in vivo recombination in strain 971/6a, by gap-repair, as previously described (Mancini et al 1999). The expected mutation, as well as the absence of additional mutations, was assessed by DNA sequencing of the entire GCH1 coding region.…”

Section: Pcr Site-directed Mutagenesismentioning

confidence: 99%

“…The budding yeast Saccharomyces cerevisiae is so far the only organism in which a knockout mutant (fol2D) in the gene encoding GTP-CH1 (FOL2) has been obtained. In yeast, the enzyme mediates the ¢rst and committing step of the folate-synthesis pathway, and the fol2D allele causes auxotrophy for folinic acid (Mancini et al 1999). The yeast fol2D mutant was shown to be a suitable system for the in vivo functional characterization of heterologous GTP-CH1 enzyme activity (Basset et al 2002;Mancini et al 1999).…”

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GTP‐cyclohydrolase I gene mutations in patients with autosomal dominant and recessive GTP‐CH1 deficiency: Identification and functional characterization of four novel mutations

Garavaglia

Invernizzi

Carbone

et al. 2004

J of Inher Metab Disea

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GTP-cyclohydrolase I (GTP-CH1, EC 3.5.4.16) is encoded by the GCH1 gene. Mutations in the GCH1 gene cause both dopa-responsive dystonia (McKusick 128230) and recessive GTP-CH1 deficiency (McKusick 600225). The exact molecular mechanism resulting in decreased GTP-CH1 activity in the patients is still obscure. We report the clinical features and molecular and functional study of the GCH1 gene in eight Italian patients affected by dominant and recessive GTP-CH1 deficiency. All the studied patients had mutations in the GCH1 gene. Three missense mutations (V205G, K224R, P199A), a frameshift mutation (Delta G693), and a splice-site mutation (ivs5 + 1g > c) were found. Except for K224R these are all novel mutations. To analyse the defect caused by the novel mutations, an in vivo functional assay in a Saccharomyces cerevisiae strain lacking the endogenous gene encoding GTP-CH1 ( FOL2 ) was performed. Complementation analysis showed that the Delta G693 and V205G mutations abolish the enzymatic function, while the P199A mutation causes a conditional defect. In conclusion, the clinical phenotypes displayed by our patients confirm the wide clinical spectrum of the disease and further support the lack of correlation between a given mutation and a clinical phenotype. Complementation analysis in yeast is a useful tool for confirming the pathogenetic effect of GCH1 mutations.

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Systems metabolic engineering of Bacillus subtilis for efficient biosynthesis of 5‐methyltetrahydrofolate

Yang

Liu

et al. 2020

Biotech & Bioengineering

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5‐Methyltetrahydrofolate (5‐MTHF) is the major form of folate in human plasma and is the only folate form that can penetrate the blood–brain barrier. It has been widely used for the prevention and treatment of various diseases. It is mainly produced by chemical synthesis. However, the low production rate cannot meet the increasing demand. In addition, chemical synthesis is potentially detrimental to the environment. Despite various microorganisms synthetizing 5‐MTHF, an efficient 5‐MTHF bioproduction approach is lacking because of the tight regulation of the 5‐MTHF pathway and limited metabolic flux toward the folic acid pathway. In this study, the 5‐MTHF synthetic pathway in Bacillus subtilis was systematically engineered to realize 5‐MTHF accumulation and further improve 5‐MTHF production. Specifically, the 5‐MTHF synthesis pathway with dihydrofolate (DHF) as the precursor was strengthened to shift the metabolic flux to 5‐MTHF biosynthesis by replacing the native yitJ gene with Escherichia coli metF, knockout of purU, and overexpressing dfrA. The intracellular level of 5‐MTHF increased 26.4‐fold, reaching 271.64 µg/L. Next, the 5‐MTHF precursor supply pathway was strengthened by co‐overexpression of folC, pabB, folE, and yciA. This resulted in a 93.2‐fold improvement of the 5‐MTHF titer, which reached 960.27 µg/L. Finally, the clustered regularly interspaced short palindromic repeats interference system was used to identify key genes in the competitive and catabolic pathways for repression to further shift the metabolic flux toward 5‐MTHF biosynthesis. The repression of genes thyA (existing in the purine metabolic pathway), pheA (existing in the competitive metabolic pathway), trpE (existing in the competitive metabolic pathway), and panB (existing in the pantoate synthesis pathway) significantly increased the titer of 5‐MTHF. By repressing the pheA gene, the 5‐MTHF titer reached 1.58 mg/L, which was 153.8‐fold that of the wild‐type strain of B. subtilis 168. Through medium optimization, the 5‐MTHF titer reached 1.78 mg/L, which was currently the highest titer of 5‐MTHF in B. subtilis. Apart from the highest titer of 5‐MTHF, the highest titer of total folates including 5‐MTHF, 5‐FTHF, folic acid, and THF could reach 3.31 mg/L, which was 8.5‐fold that in B. subtilis. To the best of our knowledge, the 5‐MTHF and total folate titers reported here are the highest using a Generally regarded as safe (GRAS) bacterium as the production host. Overall, this study provides a good starting point for further metabolic engineering to achieve efficient biosynthesis of 5‐MTHF by GRAS bacteria.

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Complementation of thefol2Deletion inSaccharomyces cerevisiaeby Human andEscherichia coliGenes Encoding GTP Cyclohydrolase I

Cited by 10 publications

References 16 publications

Bioengineering yeast to enhance folate levels in wine

Bioengineering yeast to enhance folate levels in wine

GTP‐cyclohydrolase I gene mutations in patients with autosomal dominant and recessive GTP‐CH1 deficiency: Identification and functional characterization of four novel mutations

Systems metabolic engineering of Bacillus subtilis for efficient biosynthesis of 5‐methyltetrahydrofolate

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