The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C. boidinii genome (fdh1 delta) by one-step gene disruption. In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium. In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h[-1]), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain. Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source). Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level. Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant. Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol. As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.

Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline.

In methylotrophic yeasts, formaldehyde is the key intermediate in methanol metabolism since it stands at the branchpoint of pathways for methanol assimilation and dissimilation. Since formaldehyde is an extremely toxic compound, its intracellular level should be strictly regulated.

Dihydroxyacetone synthase (DHAS) (EC 2.2.1.3) catalyzes the first reaction in the assimilation pathway by fixing formaldehyde to d-xylulose 5-phosphate, after formaldehyde is generated from methanol via alcohol oxidase (AOD) (EC 1.1.3.13) (3, 11). Otherwise, in a formaldehyde oxidation pathway, formaldehyde is dissimilated to CO2 through enzymes, e.g., glutathione-dependent formaldehyde dehydrogenase (FLD) (EC 1.2.1.1) and formate dehydrogenase (FDH) (EC 1.2.1.2).

So far, the physiological significance of these methanol-metabolizing enzymes has been estimated mainly from the enzymatic properties of purified enzyme or from phenotypes of mutants deficient in the specific enzyme obtained via random mutagenesis. Through such analyses, for example, FDH had been considered to be essential to the energy supply for methylotrophic growth (2, 34). However, through gene disruption analysis with Candida boidinii (23), the physiological role of FDH was revealed to be mainly detoxification of formate rather than stimulated energy generation. Such unexpected results with FDH led us to reevaluate the physiological roles of a key enzyme in formaldehyde assimilation, DHAS, using a gene-disrupted strain.

Similar to other cases, the physiological function of DHAS as a methanol assimilation enzyme has been estimated from its enzymatic properties and by analysis of a mutant obtained by random mutagenesis (13, 14). However, our previous study showed that loss of a peroxisome membrane protein, Pmp47, not only inhibits transport of DHAS into peroxisomes but also leads to loss of DHAS activity (26), raising the possibility that a previously derived DHAS-deficient mutant strain (13, 14) did not represent the specific mutation in the DHAS structural gene. In addition, we needed to clone and disrupt the gene for DHAS in C. boidinii to further investigate the molecular mechanism of peroxisomal transport of DHAS in relation to the function of Pmp47. Furthermore, although the strong and methanol-inducible promoter of the DHAS-encoding gene is expected to be applicable to expression of various heterologous genes in methylotrophic yeasts (37), the regulation of DHAS has not been studied in detail.

This study was conducted (i) to see whether DHAS is involved in detoxification of formaldehyde and (ii) to reveal how DHAS synthesis is regulated by various carbon and nitrogen sources. First, C. boidinii DAS1, encoding DHAS, was cloned from the C. boidinii genome and its primary structure was determined. Next, the regulation of DAS1 expression by various carbon and nitrogen sources was investigated and compared with that of other methanol-metabolizing enzymes. Lastly, the das1Δ strain, a mutant of C. boidinii harboring disrupted DAS1, was constructed and used to study the physiological importance of the enzyme in growth on various carbon and nitrogen sources. Our results suggest that DHAS is involved mainly in the assimilation of formaldehyde and that the physiological significance of DHAS for formaldehyde detoxification is minor.

Regulation and Physiological Role of the DAS1 Gene, Encoding Dihydroxyacetone Synthase, in the Methylotrophic Yeast Candida boidinii

In this paper, we describe molecular characterization of the FLD1 gene, which encodes glutathione-dependent formaldehyde dehydrogenase (FLD), from the methylotrophic yeast Pichia methanolica The P methanolica FLD1 gene contains two exons corresponding to a gene product of 380 amino acid residues and a 225 bp intron, respectively, and its deduced amino acid sequence shows high similarity to those of Fld1ps from other methylotrophic yeasts (80–88%) In P methanolica, FLD activity is mainly induced by methanol, and this induction is not completely repressed by glucose Moreover, the expression of the PmFLD1 is strictly regulated, mainly at the mRNA level, its expression increasing with increasing methanol concentrations in the medium These results suggest that FLD1 is involved in the detoxification of formaldehyde in methanol metabolism, and Fld1p coordinates the formaldehyde level in methanol-grown cells according to the methanol concentration on growth The sequence has been submitted to the DDBJ/EMBL/GenBank databases under Accession No AB112935 Copyright © 2004 John Wiley & Sons, Ltd

Molecular characterization of the glutathione-dependent formaldehyde dehydrogenase gene FLD1 from the methylotrophic yeast Pichia methanolica

Trimethylamine (TMA) found in some leafy vegetables, such as spinach, cabbage, and lettuce, at alkaline pH was identified and quantified using headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME and GC-MS). HS-SPME conditions were optimized at an adsorption temperature of 50 °C, equilibration time of 5 min, and adsorption time of 5 min with 65 μm of polydimethylsiloxane/divinylbenzene fiber. The TMA that was formed from spinach, cabbage, and lettuce was assayed at pH 7 to 11 for 0 to 4 h at 50 °C using HS-SPME. The results showed that the amount of TMA formed was dependent on pH. The amount of TMA formed increased dramatically at a pH greater than 9. TMA was not formed at a pH lower than 7. Spinach produced a higher amount of TMA than cabbage or lettuce. TMA was formed at alkaline pH from choline, betaine, and carnitine, which are TMA precursors. To confirm the SPME results, TMA was quantitated using the AOAC official method. Data obtained from chemical analysis were in good agreement with the SPME data. The formation mechanism of TMA is thought to be the Hofmann elimination reaction, which generates amine compounds at alkaline pH.



Practical Application:  Fishy off-flavor in foods is associated with trimethylamine (TMA), which is frequently found in fish and seafood. In this study, TMA was identified for the first time in some leafy vegetables, such as spinach, cabbage, and lettuce, at alkaline pH. The presence of TMA in leafy vegetables under certain circumstances such as high pH and temperature may affect the sensory properties of foods containing these vegetables.

Determination of trimethylamine in spinach, cabbage, and lettuce at alkaline pH by headspace solid-phase microextraction.

During pregnancy, dysbiosis in the vaginal microbiota directly affects the metabolic profiles, which might impact preterm birth (PTB). In this study, we performed cervicovaginal fluid (CVF) metabolic profiling using nuclear magnetic resonance (NMR) spectroscopy and identified the metabolic markers for predicting PTB. In this nested case-control study, 43 South Korean pregnant women with PTB (n = 22), and term birth (TB; n = 21) were enrolled with their demographic profiles, and CVF samples were collected by vaginal swabs. The PTB group had two subgroups based on post-CVF sampling birth: PTB less than (PTB < 7 d) and more than 7 days (PTB ≥ 7 d). We observed significant differences in the gestational age at birth (GAB), cervical length (CL), and neonatal birth weight among the groups. The principal component analysis (PCA), and partial least square discriminant analysis (PLS-DA) scatter plot showed the separation between the PTB < 7 d group, and the TB group. Out of 28 identified metabolites, acetone, ethanol, ethylene glycol, formate, glycolate, isopropanol, methanol, and trimethylamine N-oxide (TMAO) were significantly increased in the PTB group compared with the TB group. The ROC curve analysis revealed that the acetone, ethylene glycol, formate, glycolate, isopropanol, methanol, and TMAO had the best predictive values for PTB. Additionally, the correlation analysis of these metabolites showed a strong negative correlation with GAB and CL. These metabolites could be beneficial markers for the clinical application of PTB prediction.

https://www.mdpi.com/2218-1989/10/9/349/pdf

Identification of Potential Biomarkers in the Cervicovaginal Fluid by Metabolic Profiling for Preterm Birth.

The degradation of choline by Candida tropicalis cells grown in a medium containing choline as a nitrogen source was examined. The degradation of choline by resting cells was stimulated by the addition of Cu2+ or glutathione, and inhibited by 2-mercaptoethanol or potassium cyanide. With feeding of [1,2-14C]choline in the resting cell reaction, the release of 14C-labelled ethylene glycol was observed on radio-gas-liquid chromatography. Ethylene glycol, as one of the degradation products, was also observed on thin-layer and gas-liquid chromatographies, and mass spectrometry. Thus, it is suggested that choline is converted to ethylene glycol and trimethylamine by C. tropicalis.

Ken-saku Uzura

Papers

Formation of ethylene glycol and trimethylamine from choline by Candida tropicalis