Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3).
Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-beta- D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7,000 and 21,000 U/(g cell dry weight)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2-3 h into the post-induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l(-1)), as well as in induced cultivations employing metabolite-supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U/(g cell dry weight)] remained relatively unaffected in all cases. The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed.
Ramchuran
S O
SO
Dept. Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O.Box 124, Sweden. santosh.ramchuran@biotek.lu.se
Nordberg Karlsson
E
E
Velut
S
S
De Maré
L
L
Hagander
P
P
Holst
O
O
D001431:Bacteriological Techniques; D003470:Culture Media / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D005246:Feedback; D005285:Fermentation; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis
D003470:Culture Media; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D010450:Endopeptidases; D005947:Glucose