Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches

Abstract

Thiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for c. 20% of global primary production. They have been predicted to contain TPP aptamers in the 3 0 UTR of some thiamine metabolism-related genes, but little information is known about their function and regulation.We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum.We found that thiamine-related genes with putative TPP aptamers are not responsive to supplementation with thiamine or its precursor 4-amino-5-hydroxymethyl-2methylpyrimidine (HMP), and targeted mutation of the TPP aptamer in the THIC gene encoding HMP-P synthase does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtTHIC is essential for thiamine biosynthesis and another gene, PtSSSP, is necessary for thiamine uptake.Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

Thiamine-Mediated Microbial Interaction between Auxotrophic Rhodococcus ruber ZM07 and Prototrophic Cooperators in the Tetrahydrofuran-Degrading Microbial Community H-1

“…Similar results were found in a study of the microbial community in the North Atlantic Ocean, and thiamine-related compounds were positively correlated with bacterial production ( 37 ). However, a recent study showed that thiamine metabolism genes in diatoms cannot be regulated by exogenous thiamine ( 38 ). Herein, the thiamine metabolic genes were significantly downregulated in some genera with exogenous thiamine ( Fig.…”

Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-Associated Protein and Its Utility All at Sea: Status, Challenges, and Prospects

“…The result showed that EukCatA played critical roles in voltage-regulated Ca 2+ signaling and Ca 2+ -dependent gliding motility and potentially served as an alternative mechanism of 4D-Ca v /Na v channels in pennate diatoms [ 214 ]. The CRISPR/Cas9 method was employed to investigate the thiamine metabolic process in P. tricornutum [ 215 ]. The knockout of the HMP-P synthase ( THIC ) gene ( PtTHIC ) and thiamine-related proteins SSSP gene ( PtSSSP ) indicated that the PtTHIC was essential for thiamine biosynthesis while PtSSSP served in thiamine uptake [ 215 ].…”

“…The CRISPR/Cas9 method was employed to investigate the thiamine metabolic process in P. tricornutum [ 215 ]. The knockout of the HMP-P synthase ( THIC ) gene ( PtTHIC ) and thiamine-related proteins SSSP gene ( PtSSSP ) indicated that the PtTHIC was essential for thiamine biosynthesis while PtSSSP served in thiamine uptake [ 215 ].…”

Genetic engineering to enhance microalgal-based produced water treatment with emphasis on CRISPR/Cas9: A review

“…Frontiers in Bioengineering and Biotechnology frontiersin.org degradation could induce increased lipid yields in different microalgal species (Nguyen A. D. et al, 2020;Chang et al, 2020). Several other studies were also successful in implementing CRISPR/Cas9 to increase the production of different carotenoids in Dunaliella salina (Hu et al, 2021); and in Chlamydomonas reinhardtii (Baek et al, 2016), to improve the thermal tolerance of Tetraselmis suecica (Xu J. et al, 2020), and to investigate gene function in Phaeodactylum tricornutum (Hao et al, 2022;Llavero-Pasquina et al, 2022). Overall, CRISPR/ Cas9 has been successfully applied on many other algae species to improve their biomass productivities, tolerances to abiotic stressors, or increase lipid content, or that of other biomolecules and value-added products (Kumar et al, 2020;Jeon et al, 2021;Wang et al, 2021).…”