Erwinia persicina is a bacterium that has been known to produce secondary metabolites,such as andrimid,pink pigment,and exopolysaccharides,and to infect more than twenty plant species.However,traditional gene manipula...Erwinia persicina is a bacterium that has been known to produce secondary metabolites,such as andrimid,pink pigment,and exopolysaccharides,and to infect more than twenty plant species.However,traditional gene manipulation methods have been hindered by the inefficient of suicide plasmid-mediated genome editing.In this study,we describe the successful application of the CRISPR-Cas9 system in E.persicina.Efficient genome editing was achieved by substituting the native gRNA promoter with J23119 in a single-plasmid system(pRed_Cas9_ΔpoxB)and optimizing the gRNA design.The use of double gRNAs led to the deletion of a 42 kb genomic fragment,and the incorporation of a sacB screening marker facilitated iterative knockouts.Additionally,a 22 kb plasmid containing a self-resistance gene was conjugally transferred into E.persicina,resulting in the insertion of a 6.4 kb fragment with 100%efficiency.Furthermore,we demonstrated the expression of shinorine,an anti-UV compound,within the E.persicina chassis.This study establishes E.persicina as a promising chassis for synthetic biology and provides a model for gene-editing systems in non-model microorganisms.展开更多
Monoethanolamine(MEA)is a frequently utilized absorbent for CO_(2)capture in many settings,enabling biomanufacturing using carbon as the resource.Nevertheless,evidence indicates that MEA is toxic to biological systems...Monoethanolamine(MEA)is a frequently utilized absorbent for CO_(2)capture in many settings,enabling biomanufacturing using carbon as the resource.Nevertheless,evidence indicates that MEA is toxic to biological systems,and its emissions can exacerbate ecosystem pollution.Therefore,it is imperative that disposal or valorization measures be implemented for effective green biomanufacturing with MEA as the absorbent.This study examined the removal of MEA by Haematococcus pluvialis(H.pluvialis),an astaxanthin-rich microalgae,and its effects on microalgal cells and related mechanisms.Approximately half of the initial MEA was metabolized by H.pluvialis,with the resulting metabolic intermediates including acetyl-CoA.The genes involved in MEA utilization exhibited a significant increase in expression,signifying a pivotal advancement in our understanding of its potential as a nutrient for microalgae.Moreover,the exposure of H.pluvialis to MEA resulted in notable alterations in cellular components,including a 21.7%increase in lipid content and a 27.8% increase in carbohydrate content.Notably,there was a 1.49-fold increase in astaxanthin content,which was accompanied by notable changes in cell morphology.In addition to the increase in astaxanthin production,the antioxidant system was activated to counteract the adverse effects of MEA-induced oxidative stress.Furthermore,enhanced biosynthesis of both carotenoids and fatty acids directly contributed to the elevated cellular astaxanthin levels achieved through MEA metabolism by H.pluvialis.These findings offer valuable insights into the treatment of CO_(2)absorbents using microalgae while simultaneously producing high-value and healthy products,which may prove beneficial for the development of sustainable solutions for green biomanufacturing.展开更多
基金funding from the Hundred Talents Program of the Chinese Academy of Sciences to LZ(E3J56201).
文摘Erwinia persicina is a bacterium that has been known to produce secondary metabolites,such as andrimid,pink pigment,and exopolysaccharides,and to infect more than twenty plant species.However,traditional gene manipulation methods have been hindered by the inefficient of suicide plasmid-mediated genome editing.In this study,we describe the successful application of the CRISPR-Cas9 system in E.persicina.Efficient genome editing was achieved by substituting the native gRNA promoter with J23119 in a single-plasmid system(pRed_Cas9_ΔpoxB)and optimizing the gRNA design.The use of double gRNAs led to the deletion of a 42 kb genomic fragment,and the incorporation of a sacB screening marker facilitated iterative knockouts.Additionally,a 22 kb plasmid containing a self-resistance gene was conjugally transferred into E.persicina,resulting in the insertion of a 6.4 kb fragment with 100%efficiency.Furthermore,we demonstrated the expression of shinorine,an anti-UV compound,within the E.persicina chassis.This study establishes E.persicina as a promising chassis for synthetic biology and provides a model for gene-editing systems in non-model microorganisms.
基金supported by the National Key R&D Program of China(2022YFC3401800)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0120301)+5 种基金the National Natural Science Foundation of China(32470253)the Hundred Talents Program of the Chinese Academy of Sciences(E3J56201)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-IJCP-001TSBICIP-IJCP-002TSBICIP-CXRC-027)the Tangshan Science and Technology Program(24150214C).
文摘Monoethanolamine(MEA)is a frequently utilized absorbent for CO_(2)capture in many settings,enabling biomanufacturing using carbon as the resource.Nevertheless,evidence indicates that MEA is toxic to biological systems,and its emissions can exacerbate ecosystem pollution.Therefore,it is imperative that disposal or valorization measures be implemented for effective green biomanufacturing with MEA as the absorbent.This study examined the removal of MEA by Haematococcus pluvialis(H.pluvialis),an astaxanthin-rich microalgae,and its effects on microalgal cells and related mechanisms.Approximately half of the initial MEA was metabolized by H.pluvialis,with the resulting metabolic intermediates including acetyl-CoA.The genes involved in MEA utilization exhibited a significant increase in expression,signifying a pivotal advancement in our understanding of its potential as a nutrient for microalgae.Moreover,the exposure of H.pluvialis to MEA resulted in notable alterations in cellular components,including a 21.7%increase in lipid content and a 27.8% increase in carbohydrate content.Notably,there was a 1.49-fold increase in astaxanthin content,which was accompanied by notable changes in cell morphology.In addition to the increase in astaxanthin production,the antioxidant system was activated to counteract the adverse effects of MEA-induced oxidative stress.Furthermore,enhanced biosynthesis of both carotenoids and fatty acids directly contributed to the elevated cellular astaxanthin levels achieved through MEA metabolism by H.pluvialis.These findings offer valuable insights into the treatment of CO_(2)absorbents using microalgae while simultaneously producing high-value and healthy products,which may prove beneficial for the development of sustainable solutions for green biomanufacturing.
