TY - JOUR
T1 - Decoupling of recombinant protein production from Escherichia coli cell growth enhances functional expression of plant Leloir glycosyltransferases
AU - Lemmerer, Martin
AU - Mairhofer, Juergen
AU - Lepak, Alexander
AU - Longus, Karin
AU - Hahn, Rainer
AU - Nidetzky, Bernd
PY - 2019/6/1
Y1 - 2019/6/1
N2 - Sugar nucleotide-dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology-based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l-arabinose-inducible expression of the T7 phage-derived Gp2 protein from a genome-integrated site. The glycosyltransferase genes are encoded on conventional pET-based expression plasmids that allow T7 RNA polymerase-driven inducible expression by isopropyl-β- d-galactoside. Laboratory batch and scaled-up (20 L) fed-batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12-fold as a result of production under growth-decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ∼5 and ∼1% of total intracellular protein. Fed-batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3-fold) and quality (2.3-fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth-arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.
AB - Sugar nucleotide-dependent (Leloir) glycosyltransferases from plants are important catalysts for the glycosylation of small molecules and natural products. Limitations on their applicability for biocatalytic synthesis arise because of low protein expression (≤10 mg/L culture) in standard microbial hosts. Here, we showed two representative glycosyltransferases: sucrose synthase from soybean and UGT71A15 from apple. A synthetic biology-based strategy of decoupling the enzyme expression from the Escherichia coli BL21(DE3) cell growth was effective in enhancing their individual (approximately fivefold) or combined (approximately twofold) production as correctly folded, biologically active proteins. The approach entails a synthetic host cell, which is able to shut down the production of host messenger RNA by inhibition of the E. coli RNA polymerase. Overexpression of the enzyme(s) of interest is induced by the orthogonal T7 RNA polymerase. Shutting down of the host RNA polymerase is achieved by l-arabinose-inducible expression of the T7 phage-derived Gp2 protein from a genome-integrated site. The glycosyltransferase genes are encoded on conventional pET-based expression plasmids that allow T7 RNA polymerase-driven inducible expression by isopropyl-β- d-galactoside. Laboratory batch and scaled-up (20 L) fed-batch bioreactor cultivations demonstrated improvements in an overall yield of active enzyme by up to 12-fold as a result of production under growth-decoupled conditions. In batch culture, sucrose synthase and UGT71A15 were obtained, respectively, at 115 and 2.30 U/g cell dry weight, corresponding to ∼5 and ∼1% of total intracellular protein. Fed-batch production gave sucrose synthase in a yield of 2,300 U/L of culture (830 mg protein/L). Analyzing the isolated glycosyltransferase, we showed that the improvement in the enzyme production was due to the enhancement of both yield (5.3-fold) and quality (2.3-fold) of the soluble sucrose synthase. Enzyme preparation from the decoupled production comprised an increased portion (61% compared with 26%) of the active sucrose synthase homotetramer. In summary, therefore, we showed that the expression in growth-arrested E. coli is promising for recombinant production of plant Leloir glycosyltransferases.
KW - growth-arrested E. coli
KW - Leloir glycosyltransferase
KW - protein quality
KW - recombinant protein production
KW - small-molecule glycosylation
KW - synthetic biology
UR - http://www.scopus.com/inward/record.url?scp=85061056492&partnerID=8YFLogxK
U2 - 10.1002/bit.26934
DO - 10.1002/bit.26934
M3 - Article
C2 - 30659592
AN - SCOPUS:85061056492
SN - 0006-3592
VL - 116
SP - 1259
EP - 1268
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
IS - 6
ER -