Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 6 (1), 95-100

Recombinant E. Coli Prototype Strains for in Vivo Glycorandomization

Recombinant E. Coli Prototype Strains for in Vivo Glycorandomization

Gavin J Williams et al. ACS Chem Biol.

Abstract

In vitro glycorandomization is a powerful strategy to alter the glycosylation patterns of natural products and small molecule therapeutics. Yet, such in vitro methods are often difficult to scale and can be costly given the requirement to provide various nucleotides and cofactors. Here, we report the construction of several recombinant E. coli prototype strains that allow the facile production of a range of small molecule glycosides. This strategy relies on the engineered promiscuity of three key enzymes, an anomeric kinase, a sugar-1-phosphate nucleotidyltransferase, and a glycosyltransferase, as well as the ability of diverse small molecules to freely enter E. coli. Subsequently, this work is the first demonstration of "in vivo glycorandomization" and offers vast combinatorial potential by simple fermentation.

Figures

Figure 1
Figure 1
Comparison of methods for glycodiversification of natural products. (a) In vitro glycorandomization. Reducing sugars are converted to sugar-1-phosphates by E1, a flexible anomeric kinase. E2, A suitably flexible sugar-1-phosphate nucleotidyltransferase activates each sugar phosphate to the corresponding nucleotide sugar. Large panels of NDP-donors are used to probe the specificity of natural product GTs. Grey oval represents diverse natural product or natural product-like aglycons (X = O, S, or NH). (b) In vivo glycodiversification via a ‘non-natural glycoside host’ strain. Reducing sugars and aglycons are fed to a bacterial host engineered to express E1, E2, and a promiscuous GT. The endogenous biosynthetic machinery ensures recycling of necessary cofactors and aglycons decorated with non-natural sugars are collected from the culture media. (c) In vivo glucoside host. Aglycons are fed into a bacterial host engineered to express a GT which uses endogenous dTDP/UDPGlc as the glycosyl donor.
Figure 2
Figure 2
Structures of substrates used in this study.
Figure 3
Figure 3
Activity of prototype glycoside producing strains. (a) Yields (% conversion from acceptor) of glucosides using the TDP16-, WT-, and 1C9-based glucoside host with a small panel of diverse acceptors. (b) Yields (% conversion from 3) of glycosides using the TDP16- and WT-based non-natural glycoside host using acceptor 3 and a panel of free sugars. ‘w/o GalK/RmlA’ refers to the TDP16-based host but which lacks the pDuet-GalK/RmlA vector. See Supplementary Materials for full description of the strains used, details of bioconversion conditions and detection. The standard deviation of the % conversions using data from three independent determinations was less than 20%.

Comment in

Similar articles

See all similar articles

Cited by 22 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback