Effect of postinduction nutrient feed composition and use of lactose as inducer during production of thermostable xylanase in Escherichia coli glucose-limited fed-batch cultivations.
Escherichia coli is a microorganism routinely used in the production of heterologous proteins. The overexpression of a xylanase (Xyn 10 A Delta NC), which originated from the thermophile Rhodothermus marinus cloned under the control of the strong T7/lac promoter in a defined medium (mAT) using a substrate-limited feed strategy, was however shown to impose a significant metabolic burden on host cells. This resulted in a decreased cell growth rate and ultimately also a decreased target protein production. The investigation hence centers on the effect of some selected nutrient feed additives (amino acid [Cys] or TCA-intermediates [citrate, succinate, malate]) used to relieve the metabolic burden imposed during the feeding and postinduction phases of these glucose-limited fed-batch cultivations. The use of either succinic acid or malic acid as feed-additives resulted in an increase in production of approximately 40% of the heterologous thermostable xylanase. Furthermore, use of lactose as an alternative inducer of the T7/lac promoter was also proven to be a suitable strategy that significantly prolonged the heterologous protein production phase as compared with induction using isopropyl beta-D-thiogalactopyranoside (IPTG).
Ramchuran
Santosh O
SO
Department of Biotechnology, Centre for Chemistry and Chemical Engineering, Lund University, P.O.Box 124, SE-22100 Lund, Sweden.
Holst
Olle
O
Karlsson
Eva Nordberg
EN
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005947:Glucose / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases; D005947:Glucose; D007785:Lactose