Biology subjects

Dastmalchi, M.

Publications and source records attributed to Dastmalchi, M..

3 recordsLinked to original sources

bioRxiv2025

Chalcone isomerase-like impedes the lactone shunt and enhances flux partitioning in a bifurcated pathway towards isoflavonoid biosynthesis

The reconstitution of biosynthetic pathways in heterologous hosts is often challenged by the switch to a foreign cellular environment, lacking compatible structural or regulatory features. Auxiliary or non-catalytic proteins can play a critical role in modulating metabolic flux and pathway efficiency. Chalcone isomerase-like (CHIL) is a non-catalytic protein known to serve as a partner to chalcone synthase (CHS) in flavonoid biosynthesis, rectifying its promiscuous activity and preventing by-product formation, such as the aberrant p-coumaroyltriacetic acid lactone (CTAL). Here, we extended the characterization of CHILs to the legume-characteristic isoflavonoid pathway. We assessed four CHIL orthologs from diverse plant lineages: Glycine max (GmCHIL), Oryza sativa (OsCHIL), Selaginella moellendorffii (SmCHIL), and Marchantia polymorpha (MpCHIL). Structural modelling suggested that naringenin (flavanone) entry into the CHIL binding cleft may be sterically hindered compared to catalytic CHIs. Moreover, legume CHIL isoforms possess an additional bulky residue, Tyr48, that is expected to impose further constraints on ligand binding. In vitro, CHS produced up to 60% lactone CTAL instead of its desired output; however, CHIL suppressed this aberrant activity to 10%, concomitantly increasing target compound titers. Combinatorial enzyme and yeast biotransformation assays revealed a critical role for CHIL in conducting flux through chalcone, flavanone, and isoflavone biosynthesis. The inclusion of CHIL in our engineered yeast strains enhanced overall titers and, unexpectedly, promoted carbon flux toward the so-called deoxy-branch (isoliquiritigenin, liquiritigenin, and daidzein) by up to 67%, with a 33% increase in final daidzein titers. By extending CHIL characterization to the isoflavonoid pathway, we have revealed an expanded role for this auxiliary protein and underscored its utility in engineered metabolic contexts. Our findings reiterate the often-overlooked impact of non-catalytic proteins in shaping specialized metabolism. HIGHLIGHTSO_LICombinatorial enzyme assays reveal a species-dependent preference for CHILs from more closely related plant phyla by soybean CHS, which improved chalcone and downstream flavanone output by suppressing aberrant lactone formation. C_LIO_LIYeast co-expressing CHIL with the components of the isoflavonoid metabolon exhibited a 67% increase in flux through a legume-characteristic branch of the pathway, resulting in a 33% increase in titers of the major isoflavone, daidzein. C_LIO_LICHIL proteins can be included to engineer the output of phenylpropanoid-derived intermediates preferentially toward isoflavonoid biosynthesis. C_LIO_LIAuxiliary components, such as CHIL, can be designed to refine metabolic composition in bifurcated pathways, as well as enhancing general flux through pathways. C_LI

synthetic biology↗

bioRxiv2024

Serum from patients with Idiopathic inflammatory myopathy induces skeletal muscle weakness

Idiopathic inflammatory myopathies (IIM) are a group of systemic autoimmune inflammatory disorders that primarily affect striated muscles leading to weakness and accelerated fatigue. The disseminated muscle phenotype points to systemic humoral factors as mediators of the disease. Autoantibodies are important biomarkers for disease classification; it is not known if they play a direct role in IIM disease development. This study aims to investigate if IIM patient serum or isolated IgG could directly impair contractile function in muscle. Isolated flexor digitorum brevis (FDB) muscles from healthy mice were exposed to serum (10-50%) from healthy controls or patients with recent onset IIM. Some muscles were exposed to isolated total IgG (50 or 150g/ml) from patients with IIM. Muscle force in whole muscles was measured before and after exposure to sera or IgG. Muscle force and intracellular [Ca2+] in single muscle fibers were measured after exposure to serum. FDB muscles exposed to serum from patients with IIM displayed a marked reduction in force production in both 10% and 50% serum. Moreover, single myofibers dissected from FDB muscles exposed to IIM sera displayed lower force but unaffected Ca2+ release during contractions, which indicates myofibrillar dysfunction, but not intracellular Ca2+ release, as the cause to weakness. FDB muscles exposed to total IgG from IIM patients did not display any reduction in muscle force. In conclusion IIM patient serum, but not total IgG, impairs force production in skeletal muscle fibers. As the experiments were performed in isolated muscle, our results cannot be explained by infiltrating immune cells, impaired neuronal or vascular functions. This suggests that humoral factors play a direct role in the pathogenesis of muscle weakness in recent onset IIM.

physiology↗

bioRxiv2023

In vivo characterization of a secologanin transporter from Catharanthus roseus

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have fully characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1/SLTr. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. A full panel of in vivo biochemical characterization using the Xenopus laevis oocyte expression system was used to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, rapidly transporting 1 mM of secologanin within 25 min. Notably, the absence of CrMATE1 leads to a transport bottleneck, resulting in the conversion of secologanin to its reduced form, secologanol, both in planta and in the X. laevis system. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of metabolic flux, mediating the balance between anti-herbivory potency and cell homeostasis in planta. MIA and secoiridoid transporters could also be deployed in heterologous systems to guide biosynthetic pathways and improve titers of valuable and life-saving MIAs. SIGNIFICANCEWe have fully characterized CrMATE1, a multidrug and toxic compound extrusion (MATE) family transporter in Catharanthus roseus, as a vacuolar importer of secologanin. The translocation of secologanin into the vacuole is necessary for the first committed step of monoterpenoid indole alkaloid (MIA) biosynthesis.

plant biology↗