Halomonas bluephagenesis TD serves as an exceptional chassis for next generation industrial biotechnology to produce various products.However,the simultaneous editing of multiple loci in H.bluephagenesis TD remains a ...Halomonas bluephagenesis TD serves as an exceptional chassis for next generation industrial biotechnology to produce various products.However,the simultaneous editing of multiple loci in H.bluephagenesis TD remains a significant challenge.Herein,we report the development of a multiple loci genome editing system,named CRISPR-deaminase-assisted base editor(CRISPR-BE)in H.bluephagenesis TD.This system comprises two components:a cytidine(CRISPR-cBE)and an adenosine(CRISPR-aBE)deaminase-based base editor.CRISPR-cBE can introduce a cytidine to thymidine mutation with an efficiency of up to 100%within a 7-nt editing window in H.bluephagenesis TD.Similarly,CRISPR-aBE demonstrates an efficiency of up to 100%in converting adenosine to guanosine mutation within a 7-nt editing window.CRISPR-cBE has been further validated and successfully employed for simultaneous multiplexed editing in H.bluephagenesis TD.Our findings reveal that CRISPR-cBE efficiently inactivated all six copies of the IS1086 gene simultaneously by introducing stop codon.This system achieved an editing efficiency of 100%and 41.67%in inactivating two genes and three genes,respectively.By substituting the Pcas promoter with the inducible promoter PMmp1,we optimized CRISPR-cBE system and ultimately achieved 100%editing efficiency in inactivating three genes.In conclusion,our research offers a robust and efficient method for concurrently modifying multiple loci in H.bluephagenesis TD,opening up vast possibilities for industrial applications in the future.展开更多
Polyhydroxyalkanoates(PHA)have been produced by some bacteria as bioplastics for many years.Yet their commercialization is still on the way.A few issues are related to the difficulty of PHA commercialization:namely,hi...Polyhydroxyalkanoates(PHA)have been produced by some bacteria as bioplastics for many years.Yet their commercialization is still on the way.A few issues are related to the difficulty of PHA commercialization:namely,high cost and instabilities on molecular weights(Mw)and structures,thus instability on thermo-mechanical properties.The high cost is the result of complicated bioprocessing associated with sterilization,low conversion of carbon substrates to PHA products,and slow growth of microorganisms as well as difficulty of downstream separation.Future engineering on PHA producing microorganisms should be focused on contamination resistant bacteria especially extremophiles,developments of engineering approaches for the extremophiles,increase on carbon substrates to PHA conversion and controlling Mw of PHA.The concept proof studies could still be conducted on E.coli or Pseudomonas spp.that are easily used for molecular manipulations.In this review,we will use E.coli and halophiles as examples to show how to engineer bacteria for enhanced PHA biosynthesis and for increasing PHA competitiveness.展开更多
Microbial polyhydroxyalkanoates(PHA)have been produced as bioplastics for various purposes.Under the support of China National Basic Research 973 Project,we developed synthetic biology methods to diversify the PHA str...Microbial polyhydroxyalkanoates(PHA)have been produced as bioplastics for various purposes.Under the support of China National Basic Research 973 Project,we developed synthetic biology methods to diversify the PHA structures into homo-,random,block polymers with improved properties to better meet various application requirements.At the same time,various pathways were assembled to produce various PHA from glucose as a simple carbon source.At the end,Halomonas bacteria were reconstructed to produce PHA in changing morphology for low cost production under unsterile and continuous conditions.The synthetic biology will advance the PHA into a bio-and material industry.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.32171415)the Chongqing Talents Top Youth Talent Program(No.CQYC202105065).
文摘Halomonas bluephagenesis TD serves as an exceptional chassis for next generation industrial biotechnology to produce various products.However,the simultaneous editing of multiple loci in H.bluephagenesis TD remains a significant challenge.Herein,we report the development of a multiple loci genome editing system,named CRISPR-deaminase-assisted base editor(CRISPR-BE)in H.bluephagenesis TD.This system comprises two components:a cytidine(CRISPR-cBE)and an adenosine(CRISPR-aBE)deaminase-based base editor.CRISPR-cBE can introduce a cytidine to thymidine mutation with an efficiency of up to 100%within a 7-nt editing window in H.bluephagenesis TD.Similarly,CRISPR-aBE demonstrates an efficiency of up to 100%in converting adenosine to guanosine mutation within a 7-nt editing window.CRISPR-cBE has been further validated and successfully employed for simultaneous multiplexed editing in H.bluephagenesis TD.Our findings reveal that CRISPR-cBE efficiently inactivated all six copies of the IS1086 gene simultaneously by introducing stop codon.This system achieved an editing efficiency of 100%and 41.67%in inactivating two genes and three genes,respectively.By substituting the Pcas promoter with the inducible promoter PMmp1,we optimized CRISPR-cBE system and ultimately achieved 100%editing efficiency in inactivating three genes.In conclusion,our research offers a robust and efficient method for concurrently modifying multiple loci in H.bluephagenesis TD,opening up vast possibilities for industrial applications in the future.
基金This research was financially supported by a grant from Ministry of Sciences and Technology(Grant No.2016YFB0302504)grants from National Natural Science Foundation of China(Grant No.31430003)Tsinghua President Fund also supported this project(Grant No.2015THZ10).
文摘Polyhydroxyalkanoates(PHA)have been produced by some bacteria as bioplastics for many years.Yet their commercialization is still on the way.A few issues are related to the difficulty of PHA commercialization:namely,high cost and instabilities on molecular weights(Mw)and structures,thus instability on thermo-mechanical properties.The high cost is the result of complicated bioprocessing associated with sterilization,low conversion of carbon substrates to PHA products,and slow growth of microorganisms as well as difficulty of downstream separation.Future engineering on PHA producing microorganisms should be focused on contamination resistant bacteria especially extremophiles,developments of engineering approaches for the extremophiles,increase on carbon substrates to PHA conversion and controlling Mw of PHA.The concept proof studies could still be conducted on E.coli or Pseudomonas spp.that are easily used for molecular manipulations.In this review,we will use E.coli and halophiles as examples to show how to engineer bacteria for enhanced PHA biosynthesis and for increasing PHA competitiveness.
基金the State Basic Science Foundation 973(Grant no.2012CB725201)National Natural Science Foundation of China(Grant no.31430003 and 31270146).
文摘Microbial polyhydroxyalkanoates(PHA)have been produced as bioplastics for various purposes.Under the support of China National Basic Research 973 Project,we developed synthetic biology methods to diversify the PHA structures into homo-,random,block polymers with improved properties to better meet various application requirements.At the same time,various pathways were assembled to produce various PHA from glucose as a simple carbon source.At the end,Halomonas bacteria were reconstructed to produce PHA in changing morphology for low cost production under unsterile and continuous conditions.The synthetic biology will advance the PHA into a bio-and material industry.
