Insights into the biosynthesis of septacidin l-heptosamine moiety unveils a VOC family sugar epimerase

Reconstitution of Septacidin Biosynthesis in Escherichia coli by Redirecting an ADP-Heptose Precursor from Primary Metabolism

“…1,2,12 In the previous works, we delineated the biosynthetic pathway of SEPs clearly and found that E. coli is able to supply almost all the precursors for SEP biosynthesis. 14,16,17 Specifically, a key intermediate of SEP, ADP-heptose, is a precursor involved in the inner core assembly of LPSs, which are essential components of Gram-negative bacteria including E. coli. 21 In light of this discovery, we successfully reconstituted SEP biosynthesis in E. coli by redirecting ADP-heptose flow from LPS metabolism to SEP core SEP-327 production, which significantly reduced the difficulty of SEP heterologous expression by shortening its biosynthetic pathway into eight steps.…”

“…SEPs have attracted much attention among nucleoside antibiotics due to their unique structures and antifungal, antitumor, and pain relief activities. ,, In the previous works, we delineated the biosynthetic pathway of SEPs clearly and found that E. coli is able to supply almost all the precursors for SEP biosynthesis. ,, Specifically, a key intermediate of SEP, ADP-heptose, is a precursor involved in the inner core assembly of LPSs, which are essential components of Gram-negative bacteria including E. coli .…”

“… The ADP activated l - glycero -β- d - manno -heptose ( l,d - manno -heptose) is transferred to the N 6 -position of adenine monophosphate (AMP) by SepE (glycosyltransferase) to form SEP-540, which is then converted to SEP-328 by detaching the ribosyl-5-phosphate by SepF (glycosidase) . Subsequently, the l,d - manno -heptose moiety of SEP-328 is converted to 4′-amino-4′-deoxy- l - glycero -β- d - gluco -heptose ( l,l - gluco -heptosamine) by SepI (C-4′ oxidase), SepJ (C-5′ epimerase), SepA (C-3′ epimerase), and SepG (C-4′ aminotransferase using l -glutamate (l -Glu) as an amino donor) sequentially to generate SEP-327, the N 6 -glycosylated l -heptosamine-adenine core of SEPs . SEPs are formed finally by SepH and SepD, two acyl-transferases that add a N 4′ -glycyl group and a fatty acyl chain, respectively (Figure S1).…”

“…Recently, we have delineated the complete biosynthetic pathway of SEPs, which contains 13 steps that are catalyzed by 11 enzymes (including a trifunctional protein, SepB) (Figure S1). 14,16,17 Furthermore, most of the Sep enzymes were successfully expressed in Escherichia coli and characterized in vitro during the biosynthesis studies, which set a solid stage for the reconstitution of SEP biosynthesis in E. coli, a well-developed chassis cell with fast growthrate, expedient genetic toolbox, and mature scale-up fermentation process. 18 In brief, the biosynthesis of SEPs is started from Dsedoheptulose 7-phosphate (S7P), a sugar from the pentose phosphate pathway.…”

“…16 Subsequently, the L,D-manno-heptose moiety of SEP-328 is converted to 4′amino-4′-deoxy-L-glycero-β-D-gluco-heptose (L,L-gluco-heptosamine) by SepI (C-4′ oxidase), SepJ (C-5′ epimerase), SepA (C-3′ epimerase), and SepG (C-4′ aminotransferase using Lglutamate (L-Glu) as an amino donor) sequentially to generate SEP-327, the N 6 -glycosylated L-heptosamine-adenine core of SEPs. 17 SEPs are formed finally by SepH and SepD, two acyltransferases that add a N 4 ′-glycyl group and a fatty acyl chain, respectively (Figure S1). 14 Fortunately, SEP and its analogues exhibit no inhibitory activity against bacteria including E. coli, and the primary metabolism of E. coli can supply almost all the necessary precursors for SEP synthesis, except for the branched fatty groups.…”