TY - JOUR
T1 - 2′-Fucosyllactose production in engineered Escherichia coli with deletion of waaF and wcaJ and overexpression of FucT2
AU - Lee, Jae Won
AU - Kwak, Suryang
AU - Liu, Jing Jing
AU - Yun, Eun Ju
AU - Jin, Yong Su
N1 - Funding Information:
This study was supported by the USDA National Institute of Food and Agriculture (NIFA) through the Hatch/Multistate project NC1202 ( ILLU-698-928 ). The authors thank Cassie Liu for her diligent proofreading of this paper.
Publisher Copyright:
© 2021 Elsevier B.V.
PY - 2021/11/10
Y1 - 2021/11/10
N2 - 2′-Fucosyllactose (2′-FL), a major oligosaccharide of human breast milk, and is currently supplemented into infant formula. For the overproduction of 2′-FL via fucosylation of lactose, conventional approaches have focused on the episomal overexpression of de novo or salvage GDP-L-fucose biosynthetic pathway and α-1,2-fucosyltransferase (FucT2) through T7 RNA polymerase expression system in engineered E. coli. However, these approaches have drawbacks of metabolic burden, plasmid instability, and inclusion body formation. In this study, a deletion mutant of waaF coding for ADP-heptose:LPS heptosyltransferase II was employed for 2′-FL production. As the waaF deletion induces accumulation of colanic acid, additional deletion of wcaJ coding for UDP-glucose-1-phosphate transferase in the waaF deletion mutant resulted in enhanced accumulation of GDP-L-fucose. Besides, 2′-FL yields and titers were drastically improved when T7 promoter was replaced with Trc promoter for α-1,2 fucosyltransferase expressions in the waaF and wcaJ deleted strain. As a result, when FucT2 was expressed under Trc promoter in the E. coli JM109(DE3) ΔwaaFΔwcaJ, 14.7 g/L of 2′-FL was produced with a productivity of 0.31 g/L/h in a fed-batch fermentation. We envision that the deletion-based metabolic design and decreased promoter strength for fucosyltransferase expression can resolve the drawbacks of T7 RNA polymerase-based expression design for 2′-FL production in E. coli.
AB - 2′-Fucosyllactose (2′-FL), a major oligosaccharide of human breast milk, and is currently supplemented into infant formula. For the overproduction of 2′-FL via fucosylation of lactose, conventional approaches have focused on the episomal overexpression of de novo or salvage GDP-L-fucose biosynthetic pathway and α-1,2-fucosyltransferase (FucT2) through T7 RNA polymerase expression system in engineered E. coli. However, these approaches have drawbacks of metabolic burden, plasmid instability, and inclusion body formation. In this study, a deletion mutant of waaF coding for ADP-heptose:LPS heptosyltransferase II was employed for 2′-FL production. As the waaF deletion induces accumulation of colanic acid, additional deletion of wcaJ coding for UDP-glucose-1-phosphate transferase in the waaF deletion mutant resulted in enhanced accumulation of GDP-L-fucose. Besides, 2′-FL yields and titers were drastically improved when T7 promoter was replaced with Trc promoter for α-1,2 fucosyltransferase expressions in the waaF and wcaJ deleted strain. As a result, when FucT2 was expressed under Trc promoter in the E. coli JM109(DE3) ΔwaaFΔwcaJ, 14.7 g/L of 2′-FL was produced with a productivity of 0.31 g/L/h in a fed-batch fermentation. We envision that the deletion-based metabolic design and decreased promoter strength for fucosyltransferase expression can resolve the drawbacks of T7 RNA polymerase-based expression design for 2′-FL production in E. coli.
KW - 2′-Fucosyllactose
KW - Escherichia coli
KW - GDP-L-fucose
KW - WaaF
KW - WcaJ
UR - http://www.scopus.com/inward/record.url?scp=85114324494&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85114324494&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2021.08.007
DO - 10.1016/j.jbiotec.2021.08.007
M3 - Article
C2 - 34450187
AN - SCOPUS:85114324494
SN - 0168-1656
VL - 340
SP - 30
EP - 38
JO - Journal of Biotechnology
JF - Journal of Biotechnology
ER -