Saccharomyces cerevisiae,a widely utilized model organism,has seen continuous updates to its genome-scale metabolic model(GEM)to enhance the prediction performance for metabolic engineering and systems biology.This st...Saccharomyces cerevisiae,a widely utilized model organism,has seen continuous updates to its genome-scale metabolic model(GEM)to enhance the prediction performance for metabolic engineering and systems biology.This study presents an auxotrophy-based curation of the yeast GEM,enabling facile upgrades to yeast GEMs in future endeavors.We illustrated that the curation bolstered the predictive capability of the yeast GEM particularly in predicting auxotrophs without compromising accuracy in other simulations,and thus could be an effective manner for GEM refinement.Last,we leveraged the curated yeast GEM to systematically predict auxotrophs,thereby furnishing a valuable reference for the design of nutrient-dependent cell factories and synthetic yeast consortia.展开更多
Dinoflagellates are responsible for most marine harmful algal blooms (HABs) and play vital roles in many ocean processes.More than 90% of dinoflagellates are vitamin B_(12) auxotrophs and that B_(12) availability can ...Dinoflagellates are responsible for most marine harmful algal blooms (HABs) and play vital roles in many ocean processes.More than 90% of dinoflagellates are vitamin B_(12) auxotrophs and that B_(12) availability can control dinoflagellate HABs,yet the genetic basis of B_(12) auxotrophy in dinoflagellates in the framework of the ecology of dinoflagellates and particularly HABs,which was the objective of this work.Here,we investigated the presence,phylogeny,and transcription of two methionine synthase genes(B_(12)-dependent metH and B_(12)-independent metE)via searching and assembling transcripts and genes from transcriptomic and genomic databases,cloning 38 cDNA isoforms of the two genes from 14 strains of dinoflagellates,measuring the expression at different scenarios of B_(12),and comprehensive phylogenetic analyses of more than 100 organisms.We found that 1)metH was present in all 58 dinoflagellates accessible and metE was present in 40 of 58 species,2)all metE genes lacked N-terminal domains,3)metE of dinoflagellates were phylogenetically distinct from other known metE genes,and 4)expression of metH in dinoflagellates was responsive to exogenous B_(12) levels while expression of metE was not responding as that of genuine metE genes.We conclude that most,hypothetically all,dinoflagellates have either non-functional metE genes lacking N-terminal domain for most species,or do not possess metE for other species,which provides the genetic basis for the widespread nature of B_(12) auxotrophy in dinoflagellates.The work elucidated a fundamental aspect of the nutritional ecology of dinoflagellates.展开更多
Agrobacterium mediated plant transformation largely depends on two distinct strain lineages-C58 and Ach5.To better serve the plant transformation community,we have created a suite of auxotrophic and auxotrophic re-com...Agrobacterium mediated plant transformation largely depends on two distinct strain lineages-C58 and Ach5.To better serve the plant transformation community,we have created a suite of auxotrophic and auxotrophic re-combinant deficient mutants of C58 derivatives EHA105,GV3101::pMP90,and Ach5 derivative LBA4404.While these derivatives are useful,having additional strain backgrounds available would help expand the repertoire for plant transformation even further.Toward that end,two underutilized hypervirulent strains are K599(NCPPB 2659),and Chry5—but disarmed variants are not easily accessible.To improve availability,we produced dis-armed versions of A.rhizogenes strain K599 and A.tumefaciens strain Chry5 and introduced the same desirable mutations as with the other lineages.Each thymidine auxotrophy and recombination deficiency were introduced to existing and newly disarmed Agrobacterium strains via loss of function mutations conferred to thyA and recA,respectively,through CRISPR-mediated base-editing of codons amenable to nonsense mutation.To streamline the editing process,we created a series of visually marked single component base-editor vectors and a corresponding guide-filtering Geneious Prime wrapper plugin for expedited guide filtering.These new strains,the simplified CRISPR-mediated base-editor plasmids,and streamlined workflow will improve the ease with which future Agrobacterium strain derivatives are created while also supporting plant transformation at large.展开更多
基金supported by the National Key R&D Program of China(2023YFA0913900)the Shenzhen Medical Research Fund(A2303026)the Shenzhen Science and Technology Program(KJZD20230923114415032).
文摘Saccharomyces cerevisiae,a widely utilized model organism,has seen continuous updates to its genome-scale metabolic model(GEM)to enhance the prediction performance for metabolic engineering and systems biology.This study presents an auxotrophy-based curation of the yeast GEM,enabling facile upgrades to yeast GEMs in future endeavors.We illustrated that the curation bolstered the predictive capability of the yeast GEM particularly in predicting auxotrophs without compromising accuracy in other simulations,and thus could be an effective manner for GEM refinement.Last,we leveraged the curated yeast GEM to systematically predict auxotrophs,thereby furnishing a valuable reference for the design of nutrient-dependent cell factories and synthetic yeast consortia.
基金edge the financial support from the National Natural Science Foundation of China(Grant No.41776125)the Key Deployment Project of Centre for Ocean Mega-Research of Science,Chinese Academy of Sciences(Grant No.COMS2019Q09)+3 种基金the Science&Technology Basic Resources Investigation Program of China(Grant No.2018FY100200)National Natural Science Foundation of China(Grants Nos.41976134 and 61533011)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(Grant No.2018SDKJ0504-2)supported by the Chicago Community Trust.
文摘Dinoflagellates are responsible for most marine harmful algal blooms (HABs) and play vital roles in many ocean processes.More than 90% of dinoflagellates are vitamin B_(12) auxotrophs and that B_(12) availability can control dinoflagellate HABs,yet the genetic basis of B_(12) auxotrophy in dinoflagellates in the framework of the ecology of dinoflagellates and particularly HABs,which was the objective of this work.Here,we investigated the presence,phylogeny,and transcription of two methionine synthase genes(B_(12)-dependent metH and B_(12)-independent metE)via searching and assembling transcripts and genes from transcriptomic and genomic databases,cloning 38 cDNA isoforms of the two genes from 14 strains of dinoflagellates,measuring the expression at different scenarios of B_(12),and comprehensive phylogenetic analyses of more than 100 organisms.We found that 1)metH was present in all 58 dinoflagellates accessible and metE was present in 40 of 58 species,2)all metE genes lacked N-terminal domains,3)metE of dinoflagellates were phylogenetically distinct from other known metE genes,and 4)expression of metH in dinoflagellates was responsive to exogenous B_(12) levels while expression of metE was not responding as that of genuine metE genes.We conclude that most,hypothetically all,dinoflagellates have either non-functional metE genes lacking N-terminal domain for most species,or do not possess metE for other species,which provides the genetic basis for the widespread nature of B_(12) auxotrophy in dinoflagellates.The work elucidated a fundamental aspect of the nutritional ecology of dinoflagellates.
基金supported by the Center for Bioenergy Innovation(CBI),U.S.Department of Energy,Office of Science,Biological and Environmental Research Program under Award Number ERKP886.
文摘Agrobacterium mediated plant transformation largely depends on two distinct strain lineages-C58 and Ach5.To better serve the plant transformation community,we have created a suite of auxotrophic and auxotrophic re-combinant deficient mutants of C58 derivatives EHA105,GV3101::pMP90,and Ach5 derivative LBA4404.While these derivatives are useful,having additional strain backgrounds available would help expand the repertoire for plant transformation even further.Toward that end,two underutilized hypervirulent strains are K599(NCPPB 2659),and Chry5—but disarmed variants are not easily accessible.To improve availability,we produced dis-armed versions of A.rhizogenes strain K599 and A.tumefaciens strain Chry5 and introduced the same desirable mutations as with the other lineages.Each thymidine auxotrophy and recombination deficiency were introduced to existing and newly disarmed Agrobacterium strains via loss of function mutations conferred to thyA and recA,respectively,through CRISPR-mediated base-editing of codons amenable to nonsense mutation.To streamline the editing process,we created a series of visually marked single component base-editor vectors and a corresponding guide-filtering Geneious Prime wrapper plugin for expedited guide filtering.These new strains,the simplified CRISPR-mediated base-editor plasmids,and streamlined workflow will improve the ease with which future Agrobacterium strain derivatives are created while also supporting plant transformation at large.
