Molecular characterization of two alkylresorcylic acid synthases from <i>Sordariomycetes</i> fungi

Ramakrishnan, Dhivya,Tiwari, Manish K.,Manoharan, Gomathi,Sairam, Thiagarajan,Thangamani, Rajesh,Lee, Jung-Kul,Marimuthu, Jeya Elsevier 2018 Enzyme and microbial technology Vol.115 No.-

<P><B>Abstract</B></P> <P>Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from <I>Sordaria macrospora</I> (SmPKS) and <I>Chaetomium thermophilum</I> (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, <I>Escherichia Coli</I> BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C<SUB>4</SUB>–C<SUB>20</SUB>) as starter units. The truncated <I>S. macrospora</I> polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>−1</SUP> catalytic efficiency towards stearoyl-CoA.Whereas, truncated <I>C. thermophilum</I> polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>−1</SUP>. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium–thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. <I>macrospora</I> and C. <I>thermophilum</I> respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency. </LI> <LI> Both the recombinant PKSs efficiently synthesize pyrones, resorcylic acids and resorcinols using various acyl- CoA (C<SUB>4</SUB>–C<SUB>20</SUB>) as starter units. </LI> <LI> TrCtPKS produces pentaketide and hexaketide resorcinols from arachidoyl-CoA with a high catalytic efficiency of 6.2 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>−1</SUP>. </LI> <LI> Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity. </LI> </UL> </P>

Psychiatric, Somatic and Other Functional Gastrointestinal Disorders in Patients With Irritable Bowel Syndrome at a Tertiary Care Center

( Prashant Singh ),( Abhishek Agnihotri ),( Manish K Pathak ),( Asef Shirazi ),( Rajeew P Tiwari ),( V Sreenivas ),( Rajesh Sagar ),( Govind K Makharia ) 대한소화기기능성질환·운동학회 2012 Journal of Neurogastroenterology and Motility (JNM Vol.18 No.3

Background/Aims To study the prevalence of somatic and psychiatric co-morbidities in the patients of irritable bowel syndrome (IBS) and to assess the quality of life (QOL) of these patients. Methods One hundred and eighty-four IBS patients and 198 controls were included. Diagnosis of IBS, its sub-classification and assessment of other functional gastrointestinal disorders (FGIDs) was made on basis of Rome III criteria. Severity of IBS was assessed using IBS severity scoring system. Psychiatric evaluation was done using Patient Heath Questionnaire. QOL was evaluated using WHO QOL-BREF. Results One hundred and forty-seven (79.9%) and 158 (85.9%) patients with IBS had at least one other FGID or at least one somatic co-morbidity, respectively. Higher number of patients had at least one psychiatric co-morbidity compared to controls (79.9% vs 34.3%; P < 0.001). Major depressive syndrome (47.3% vs 5.1%; P < 0.001), somatoform disorder (50% vs 14.6%; P < 0.001) and panic syndrome (44% vs 11.6%; P < 0.001) were more common in IBS than controls. Only 14 (7.6%) patients were receiving drug treatment for their psychiatric illness. Severe IBS symptoms were present in significantly higher number of patients with constipation predominant IBS than diarrhea predominant IBS. Those with severe disease had higher prevalence of psychiatric (95.1%) and somatic (96.7%) co-morbidities compared with mild disease. QOL of IBS patients was significantly lower in all four domains compared to controls. Presence of at least one other FGID was significantly associated with presence of one or more psychiatric co-morbidity (P < 0.001). Conclusions Majority of IBS patients presenting to a tertiary care center had associated psychiatric, somatic co-morbidities and reduced QOL. Very few of them received specific psychiatric treatment. (J Neurogastroenterol Motil 2012;18:324-331)

Identification and characterization of type III polyketide synthase genes from culturable endophytes of ethnomedicinal plants

Manoharan, Gomathi,Sairam, Thiagarajan,Thangamani, Rajesh,Ramakrishnan, Dhivya,K.Tiwari, Manish,Lee, Jung-Kul,Marimuthu, Jeya IPC Science and Technology Press 2019 Enzyme and microbial technology Vol.131 No.-

<P><B>Abstract</B></P> <P>Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including <I>Arbus precatorius</I>, <I>Bacopa monnieri,Citrus aurantifolia</I> and <I>Datura metel</I> to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60–99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from <I>Fusarium incarnatum</I> BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in <I>E. coli</I> Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 10<SUP>4</SUP> s<SUP>−1</SUP> M<SUP>-1</SUP> with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A CODEHOP PCR based screening method was employed for type III polyketide synthase gene identification in fungal endophytes. </LI> <LI> By this approach, partial type III PKS genes from eight fungal endophytes were amplified and sequenced. </LI> <LI> FiPKS gene from Fusarium incarnatum BMER1, an endophyte of Bacopa monnieri was cloned and functionally characterized. </LI> <LI> FiPKS produced pyrones and resorcinols with the highest catalytic efficiency of 7.6 x 104 s-1 M-1towards stearoyl CoA. </LI> </UL> </P>