Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production.However,the direct application of this strain suffers from the transformation inefficiency and plasmid inst...Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production.However,the direct application of this strain suffers from the transformation inefficiency and plasmid instability.Herein,we conducted genetic modifications at a serial of loci of MG1655 genome,generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways.Using JW128 as the host,the titer of isobutanol reached 5.76 g/L in shake-flask fermentation,and the titer of lycopene reached 1.91 g/L in test-tube fermentation,40-fold and 5-fold higher than that of original MG1655,respectively.These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.展开更多
Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these me...Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these methods still face some problems such as low yield and high cost.The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E.coli K12 MG1655.The lgtA and lgtB were first expressed by a plasmid,resulting in a LNnT titer of 0.04 g/L.To improve the yield of LNnT on substrate lactose,lacZ and lacI were knocked out,and lacY was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,the pathway was regulated using the titer of Lacto-N-triose II(LNTII)as a measure,and obtained a high titer strain of LNnT for 1.04 g/L.Finally,the gene expressions were fine-tuned,the titer of LNnT reached 1.2 g/L,which was 93%higher than the control strain,and the yield on lactose reached 0.28 mol/mol.The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E.coli.展开更多
基金The work finished in Beijing Institute of Technology was supported by the National Key R&D Program of China(grant No.2019YFA0904104)the National Natural Science Foundation of China(Grant No.21676026)the Fundamental Research Funds for the Central Universities.
文摘Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production.However,the direct application of this strain suffers from the transformation inefficiency and plasmid instability.Herein,we conducted genetic modifications at a serial of loci of MG1655 genome,generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways.Using JW128 as the host,the titer of isobutanol reached 5.76 g/L in shake-flask fermentation,and the titer of lycopene reached 1.91 g/L in test-tube fermentation,40-fold and 5-fold higher than that of original MG1655,respectively.These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.
基金This work was supported by the National Natural Science Foundation of China(31930085,32021005)the key research and development program of China(2018YFA0900300,2020YFA0908300).
文摘Lacto-N-neotetraose(LNnT),one of the most important human milk oligosaccharides,can be used as infants’food addi-tives.Nowadays,extraction,chemical and biological synthesis were utilized to obtain LNnT,while these methods still face some problems such as low yield and high cost.The aim of current work is to construct a de novo biosynthesis pathway of LNnT in E.coli K12 MG1655.The lgtA and lgtB were first expressed by a plasmid,resulting in a LNnT titer of 0.04 g/L.To improve the yield of LNnT on substrate lactose,lacZ and lacI were knocked out,and lacY was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,the pathway was regulated using the titer of Lacto-N-triose II(LNTII)as a measure,and obtained a high titer strain of LNnT for 1.04 g/L.Finally,the gene expressions were fine-tuned,the titer of LNnT reached 1.2 g/L,which was 93%higher than the control strain,and the yield on lactose reached 0.28 mol/mol.The engineering strategy of pathway construction and modulation used in this study is applicable to facilitate the microbial production of other metabolites in E.coli.
