top of page

Hello

Public·9 members
Jameson Cook
Jameson Cook

Phylogenetic Analysis Of Methionine Synthesis G... _HOT_



Methionine synthase encoded by the MTR gene is one of the key enzymes involved in the SAM (S- Adenosyl Methionine) cycle catalyzing the conversion of homocysteine to methionine. Methionine plays an important role in the DNA, RNA, protein, phospholipids, and neurotransmitters methylation. It also maintains serum homocysteine level and indirectly regulates de novo nucleotide synthesis and repair. The current study predicted the functional consequences of nsSNPs in human MTR gene using SIFT, PolyPhen2, PROVEAN, SNAP2, PMut, nsSNPAnalyzer, PhD-SNP, SNPs&GO, I-Mutant, MuPro, and iPTREE-STAB. The PTM sites within the protein were predicted using ModPred and the phylogenetic conservations of amino acids & conserved domains of protein were predicted using ConSurf and NCBI conserved domain search tool respectively. The protein 3D structure was generated using SPARKS-X and analyzed using RAMPAGE. Structural deviation was analyzed using TM-Score. STRING analysis was preformed to predict protein-protein interactions. D621G, G682D, V744L, V766E, and R1027W were predicted to be the most deleterious nsSNPs in MTR. R1027 was predicted to having the three PTM sites and G682 & V744 were predicted as highly conserved residues. D621G, G682D, V744L, V776E, and R1027W were predicted to be within conserved domains of methionine synthase. The G682D, V744L, V776E, and R1027W were predicted to alter protein 3D structure. STRING predicted that methionine synthase interacting with 10 different proteins. The present study predicted D621G, G682D, V744L, V766E, and R1027W as functionally and structurally significant nsSNPs in human MTR gene. The present study can provide the significant information for further experimental analysis. Abbreviations: cblG: methylcobalamin deficiency G; MTR: 5-methyl tetrahydrofolate-homocysteine methyl transferase; MS: methionine synthase; SAM: S-adenosyl methionine; nsSNPs: non-synonymous single nucleotide polymorphisms; OMIM: online mendelian inheritance in man; NCBI: national center for biological information; SIFT: sorting intolerant from tolerant; PolyPhen2: polymorphism phenotyping 2; PROVEAN: protein variation effect analyzer; SNPs&GO: single nucleotide polymorphisms and gene ontology; PhD-SNP: predictor of human deleterious single nucleotide polymorphisms; RI: reliability index; PTM: post translational modification; SPDBV: Swiss PDB viewer; PDB: protein data bank; RMSD: root mean square deviation; STRING: search tool for the retrieval of interacting proteins.




Phylogenetic analysis of methionine synthesis g...



To further address the reason, the intracellular SAM concentration of AcsamsOE was measured after fermentation in the MDFA medium with or without addition of exogenous methionine. As expected, the intracellular SAM concentration of AcsamsOE was enhanced significantly in both situations, but addition of exogenous methionine in AcsamsOE did not further increase the intracellular SAM concentration (Fig. 3b), indicating that the endogenous methionine was either enough for the synthesis of SAM or the extra methionine could suppress the synthesis of SAM in AcsamsOE, this could also be the reason that the concentration of SAM was very low in AcsamsOE at 3rd day of fermentation when the exogenous methionine was added. On the other hand, it was also validated the importance of the endogenous methionine for CPC production as reported previously [27]. Not like WT in which the SAM concentration declined dramatically after 4 days fermentation, AcsamsOE almost remained the same level of SAM concentration even after 5 days fermentation (Fig. 3b). The increment of CPC production in AcsamsOE could be due to the accumulation of intracellular SAM, especially at the late stage of fermentation. These results indicated the dependence of methionine stimulation during CPC production could be significantly reduced by enhancing the intracellular SAM in the metabolic engineered strain.


The concentration of intracellular SAM was also quantified and discovered that the SAM concentration in Acppm1DM was twofold higher than that in WT when 0.32 g/L methionine was added in the MDFA medium. With 0.32 g/L methionine in the fermentation medium, the SAM concentration in Acppm1DM reached to a maximum level after 4 days fermentation and retained high level even after 5 days fermentation. While, the SAM concentration of WT was decreased to a very low level after 5 days fermentation, it may be caused by the deficiency of methionine (Fig. 5c). When 3.2 g/L methionine was added in the MDFA medium, the intracellular SAM decreased sharply in Acppm1DM (Fig. 5c). To assess the possible reason for this phenomenon, we detected the transcriptional level of the AcsamS in WT and Acppm1DM. The result showed that addition of exogenous methionine led to a reduction of AcsamS transcription (Additional file 1: Fig. S13a, b), the same as reported before in human hepatocarcinoma cells [31]. Furthermore, the transcriptional level of AcsamS in Acppm1DM was obviously lower than that in WT with 3.2 g/L methionine in the fermentation medium (Additional file 1: Fig. S13c). Thus it was possible that inhibition of SAM biosynthesis by adding 3.2 g/L methionine caused a decrease of SAM concentration in Acppm1DM. These results indicated that the main reason for CPC increment in Acppm1DM was the intracellular SAM accumulation.


Furthermore, the plasmid pAg1PT-G418-AcsamS-ble was constructed and transformed into Acppm1DM-mecBOE to obtain the AcsamS overexpressed strain Acppm1DM-mecBOE-AcsamsOE. After verified by real-time RT-PCR (Additional file 1: Fig. S20), one transformant was randomly selected and used for fermentation. For fermentation, the Acppm1DM-mecBOE-AcsamsOE was cultured in the MDFA medium with addition of varying concentrations of methionine (0, 0.32 and 3.2 g/L, respectively). During fermentation in the MDFA medium without methionine, the CPC production of Acppm1DM-mecBOE-AcsamsOE was almost the same as that of Acppm1DM-mecBOE (Additional file 1: Fig. S21), indicating that the expression of mecB is the rate-limiting step of the CPC production in Acppm1DM and Acppm1DM-AcsamsOE. Like in Acppm1DM-mecBOE, the CPC production in Acppm1DM-mecBOE-AcsamsOE was decreased in the MDFA medium with addition of methionine (Additional file 1: Fig. S21). Since the regulation of CPC biosynthesis is complicated and the intracellular SAM accumulation could inhibit expression of AcsamS or affect the activity of AcSAMS, Acppm1DM-AcsamsOE and Acppm1DM-mecBOE-AcsamsOE did not increased the CPC production further compared with Acppm1DM and Acppm1DM-mecBOE.


Our results revealed that methionine stimulated CPC production through enhancing the intracellular SAM of A. chrysogenum and the CPC production could be improved by accumulating endogenous SAM during fermentation. Two strategies, including enhancement of SAM synthesis and blockage of SAM consumption, were used to overcome the dependence of methionine stimulation during CPC production. Finally, an optimum recombinant strain Acppm1DM-mecBOE was constructed through engineering the methionine cycle. In this strain, CPC production was increased 5.5-fold and its improvement was totally independent of methionine stimulation. This study provides a novel insight for improving CPC production independent of methionine stimulation.


Strains and plasmids used in this study. Table S2. Primers used in this study. Fig. S1. Cephalosporin C production of WT detected by UPLC/MS in the MDFA medium with or without addition of 3.2 g/L methionine. Fig. S2. Mycelium dry weight of A. chrysogenum in the MDFA medium with or without addition of 3.2 g/L methionine. Fig. S3. Sequence alignment and phylogenetic analysis of the SAM synthetase family proteins. Fig. S4. Cephalosporin C production of WT and WT/pAg1PT-G418 in the MDFA medium with or without addition of 3.2 g/L methionine. Fig. S5. Construction and validation of the AcsamS overexpressed strain (AcsamsOE). Fig. S6. Cephalosporin C production of WT and AcsamsOE was detected by UPLC/MS in MDFA medium. Fig. S7. Mycelium dry weight of AcsamsOE in the MDFA medium with or without addition of 3.2 g/L methionine. Fig. S8. Cephalosporin C production of AcsamsOE in the MDFA medium supplemented with different concentration of SAM. Fig. S9. Sequence alignment of the leucine carboxyl methyltransferase superfamily proteins. Fig. S10. Construction and validation of the Acppm1 disruption mutant (Acppm1DM). Fig. S11. Cephalosporin C production of WT and Acppm1DM was detected by UPLC/MS. Fig. S12. Mycelium dry weight of Acppm1DM, Acppm1CM, Acppm1OE in the MDFA medium with or without addition of 0.32 g/L methionine. Fig. S13. The relative transcriptional level of AcsamS in WT and Acppm1DM. Fig. S14. The relative transcriptional level of AcmetH, AccysD, AcmecA and mecB of WT in the MDFA medium with or without addition of 3.2 g/L methionine. Fig. S15. Cephalosporin C production of Acppm1DM and Acppm1DM-AcsamsOE. Fig. S16. The relative transcriptional level of mecB in Acppm1DM. Fig. S17. Construction and validation of the mecB overexpressed strain (Acppm1DM-mecBOE). Fig. S18. Cephalosporin C production of WT and Acppm1DM-mecBOE was detected by UPLC/MS in MDFA medium. Fig. S19. Mycelium dry weight of WT and Acppm1DM-mecBOE in the MDFA medium supplemented with 0, 0.32 g/L and 3.2 g/L of methionine respectively. Fig. S20. Construction and validation of Acppm1DM-mecBOE-AcsamsOE. Fig. S21. Cephalosporin C production of Acppm1DM-mecBOE-AcsamsOE.


Three different analyses were performed to validate previous phylogenetic placement of Ca. G. gigasporarum (Lumini et al., 2006; Castillo and Pawlowska, 2010). First, 21 structural genes retrieved from 67 completely sequenced bacterial species were used for a large multigene phylogenetic analysis (Supplementary Table S1). As a further step, the analysis was restricted to β-proteobacteria, whose both 16S and 23S were available (Supplementary Table S2 and Supplementary Text S1). 041b061a72


About

Welcome to the group! You can connect with other members, ge...

Members

  • Ife Yoga
  • Miller Torbert
    Miller Torbert
  • Josie Allison
    Josie Allison
  • Arkady Kolesnikov
    Arkady Kolesnikov
  • Lawrence Jean
    Lawrence Jean
bottom of page