Depletion of L-Methionine in Foods with an Engineered Thermophilic Methionine γ-lyase Efficiently Inhibits Tumor Growth.

Dietary restriction of l-methionine, an essential amino acid, exerts potent antitumor effects on l-methionine-dependent cancers. However, dietary restriction of l-methionine has not been practical for human therapy because of the problem with the administration of l-methionine concentration in foods. Here, a thermophilic methionine γ-lyase (MGL), that catalyzes the cleavage of the C-S bond in l-methionine to produce α-ketobutyric acid, methanethiol, and ammonia was engineered from human cystathionine γ-lyase and almost completely depleted l-methionine at 65 °C, a temperature that accelerates the volatilization of methanethiol and its oxidation products. The high efficiency of l-methionine lysis may be attributed to the cooperative fluctuation and moderate the structural rigidity of 4 monomers in the thermophilic MGL, which facilitates l-methionine access to the entrance of the active site. Experimental diets treated with thermophilic MGL markedly inhibited prostate tumor growth in mice, and in parallel, the in vivo concentrations of l-methionine, its transformation product l-cysteine, and the oxidative stress indicator malondialdehyde significantly decreased. These findings provide a technology for the depletion of l-methionine in foods with an engineered thermophilic MGL, which efficiently inhibits tumor growth in mice.

YALI0C22088g from Yarrowia lipolytica catalyses the conversion of l-methionine into volatile organic sulfur-containing compounds.

The enzymatic conversion of l-methionine (l-Met) into volatile organic sulfur-containing compounds (VOSCs) plays an important role in developing the characteristic aroma of foods. However, the mechanism for the direct conversion of l-Met into VOSCs is still unclear in yeast cells used to make food products. Here, we show that the transcription profile of YALI0C22088g from Yarrowia lipolytica correlates positively with l-Met addition. YALI0C22088g catalyses the γ-elimination of l-Met, directly converting l-Met into VOSCs. YALI0C22088g also exhibits strong C-S lysis activities towards l-cystathionine and the other sulfur-containing compounds and forms a distinct cystathionine-γ-lyase subgroup. We identified eight key amino acid residues in YALI0C22088g, and we inferred that the size of the tunnel and the charges carried by the entrance amino acid residue are the determinants for the enzymatic conversion of l-Met into VOSCs. These findings reveal the formation mechanism of VOSCs produced directly from l-Met via the demethiolation pathway in Yarrowia lipolytica, which provides a rationale for engineering the enzymatic conversion of l-Met into VOSCs and thus stimulates the enzymatic production of aroma compounds.

Enzymatic O-Glycosylation of Etoposide Aglycone by Exploration of the Substrate Promiscuity for Glycosyltransferases.

The 4-O-ß-d-glucopyranoside of DMEP ((-)-4'-desmethylepipodophyllotoxin) (GDMEP), a natural product from Podophyllum hexandrum, is the direct precursor to the topoisomerase inhibitor etoposide, used in dozens of chemotherapy regimens for various malignancies. The biosynthesis pathway for DMEP has been completed, while the enzyme for biosynthesizing GDMEP is still unclear. Here, we report the enzymatic O-glycosylation of DMEP with 53% conversion by exploring the substrate promiscuity and entrances of glycosyltransferases. Notably, we found 6 essential amino acid residues surrounding the putative substrate entrances exposed to the protein surface in UGT78D2, CsUGT78D2, and CsUGT78D2-like, and these residues may determine substrate specificity and high O-glycosylation activity toward DMEP. Our results provide an effective route for one-step synthesis of GDMEP. Identification of the key residues and entrances of glycosyltransferases will promote precise identification of glycosyltransferase biocatalysts for novel substrates and provide a rational basis for glycosyltransferase engineering.

Clonostachys rosea demethiolase STR3 controls the conversion of methionine into methanethiol.

Eukaryote-derived methioninase, catalyzing the one-step degradation of methionine (Met) to methanethiol (MTL), has received much attention for its low immunogenic potential and use as a therapeutic agent against Met-dependent tumors. Although biological and chemical degradation pathways for Met-MTL conversion are proposed, the concrete molecular mechanism for Met-MTL conversion in eukaryotes is still unclear. Previous studies demonstrated that α-keto-methylthiobutyric acid (KMBA), the intermediate for Met-MTL conversion, was located extracellularly and the demethiolase STR3 possessed no activities towards Met, which rule out the possibility of intracellular Met-MTL conversion pathway inside eukaryotes. We report here that degradation of Met resulted in intracellular accumulation of KMBA in Clonostachys rosea. Addition of Met to culture media led to the production of MTL and downregulation of STR3, while incubation of Met with surrogate substrate α-ketoglutaric acid enhanced the synthesis of MTL and triggered the upregulation of STR3. Subsequent biochemical analysis with recombinant STR3 showed that STR3 directly converted both Met and its transamination product KMBA to MTL. These results indicated that STR3 as rate-limiting enzyme degrades Met and KMBA into MTL. Our findings suggest STR3 is a potential target for therapeutic agents against Met-dependent tumors and aging.

[Construction of a versatile degrading bacteria Pseudomonas putida KT2440-DOP and its degrading characteristics].

Triazophos is a kind of organophosphorous pesticide which was widely used by farmers all over the world in 1990's. It is effective in controlling pesticide but harmful to human beings. Bioremediation is an effective and economic method to treat environment that has been polluted by hazardous organic compounds, so researchers paid much attention in this area. Most of which focused on isolating functional bacteria, studying its degrading mechanism, and cloning degradation-related genes. MP-4 was isolated from soil polluted by Triazophos for a long time and identified as Ochrobactrum sp.. The triazophos hydrolase (tpd) gene was cloned by the method of shotgun cloning, and the sequences were determined and analyzed. In the former tests it was found that there was only 18 base pairs different in tpd gene from mpd gene, which was isolated from methyl parathion degrading strain Pseudomonas putida DLL-1. Enzyme TPD can hydrolyze triazophos and methyl parathion while MPD cannot hydrolyze triazophos. Pseudomonas putida KT2440 is a metabolically versatile saprophytic soil bacterium that has been certified as a biosafety host for the cloning of foreign genes. This bacterium is known for its diverse metabolism and potential for development of biopesticides and plant growth promoters because of its ability to colonize rhizosphere of crop plants. Tpd gene was isolated from the genomic DNA of Ochrobactrum sp. MP-4 by PCR amplification. Recombinant plasmids pTPD was constructed by ligating tpd gene into broad host vector pBBRMCS-5. With the help of plasmid pRK2013, pTPD was transferred into Pseudomonas putida KT2440 to construct KT2440-DOP. KT2440-DOP can degrade many organophosphate pesticides and aromatics compounds. The specific activity of organophosphate hydrolase of KT2440-DOP was approximately 2 times of MP-4. Later, parameters affecting bioremediation of Organophosphate pesticide in soil using KT2440-DOP will be studied.

[Glycine betaine supplied exogenously enhance salinity tolerance of Pseudomonas putida DLL-1].

The salinity tolerance characteristic of Pseudomonas putida DLL-1 and the environmental factors affecting the salinity tolerance of DLL-1 were investigated. The result showed that the enrichment of culture medium influenced the salt tolerance of DLL-1, in complete medium DLL-1 could survive higher salinities than in chemically defined medium. When inoculated to complete medium with 1mol/L NaCl, the least initial biomass guarantee DLL-1 surviving was 1/100(V/V) of the medium volumes. But when inoculated to chemically defined medium with 1mol/L NaCl, the least initial biomass guarantee DLL-1 surviving was 1/10(V/V) of the medium volumes. The effects of glycine betaine exogenously supplied on the salinity tolerance of DLL-1 and its osmo protection mechanisms were also studied. The results indicated that the glycine betaine present externally could promote the growth of DLL-1 under high salinity. 10mg/L of exogenous glycine betaine was sufficient to promote the growth condition of DLL-1 cells under high salinities. 150mg/L glycine betaine could support DLL-1 cells grow in chemically defined medium with 1.2mol/L NaCl. The presence of glycine betaine in chemically defined medium could reduce significantly the lag time and generation time and increase the final biomass of DLL-1 under salt stress. Compared with the control without exogenous glycine betaine, the lag time of the treatment with exogenous glycine betaine could be reduced from 24h to 6h, and the generation time from 60min to 35.7min, the final OD610 value of culture increased from 1.29 to 1.57. Under osmotic stress, DLL-1 cells could synthesis glycine betaine, trehalose and free amino acid as the main compatible solute. When exogenously supplied, DLL-1 cells accumulated exogenous glycine betaine rapidly from medium to balance the extracellular osmolality instead of the synthesis of compatible solutes.