[Effects of knockout genes related to outer membrane on extracellular secretion of recombinant proteins in Escherichia coli BL21 (DE3)].
OBJECTIVE
We knocked out the genes related to lipopolysaccharide in outer membrane of Escherichia coli BL21 (DE3) to study the effects on extracellular secretion of recombinant proteins.


METHODS
We generated waaF or msbB knockout mutants [E. coli BL21 (ΔwaaF) or E. coli BL21 (ΔmsbB) ] of E. coli BL21 (DE3) by using lambda-Red recombination system. Then, we transformed recombinant plasmids pET-ffase or pET-pga into E. coli BL21 (AmsbB) , E. coli BL21 (ΔwaaF) and E. coli BL21 (DE3) respectively, to generate the engineering strains E. coli BL21 (ΔmsbB)/ pET-ffase, E. coli BL21 (ΔwaaF)/pET-ffase, E. coli BL21 (DE3)/pET-ffase, E. coli BL21 (ΔmsbB)/pET-pga, E. coli BL21 (ΔwaaF)/pET-pga and E. coli BL21 (DE3)/pET-pga. Finally, we studied the effects of mutants on extracellular secretion of beta-fructofuranosidase (EC 3. 2. 1. 26, beta-FFase) and penicillin G acylase (EC 3. 5. 1. 11) in shaking flask fermentation.


RESULTS
After induced expression for 4 hours, up to 19.7% of the beta-FFase activity was found in the culture medium with the msbB deletion mutant, and 50.9% with the waaF deletion mutant, compared to the original 2.6%. Besides, after induced expression for 24 hours, up to 1708 U/L extracellular activity of penicillin G acylase was found in the culture medium with the waaF deletion mutant, which was 4.1 times of the original.


CONCLUSIONS
Knockout mutants (ΔmsbB and ΔwaaF) had significantly higher excretion of beta-FFase and the waaF deletion mutant had higher excretion of penicillin G acylase.
Yun
Y
Zhu
Youzhi
Y
Zhou
Jianlin
J
Chu
Bingfang
B
He
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D058947:Bacterial Secretion Systems; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014166:Transferases / Q000235:genetics / Q000378:metabolism
D058947:Bacterial Secretion Systems; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014166:Transferases; D000217:Acyltransferases; C097895:MsbB protein, E coli; C019917:3-deoxy-D-manno-octulosonate transferase