Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3'-phosphoadenosine-5'-phosphosulfate

Abstract

Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.

Probing Structural Selectivity of Synthetic Heparin Binding to Stabilin Protein Receptors

“…35 S]PAPS, UDP-GlcNTFA, and disaccharide (GlcAAnMan) starting materials are described elsewhere (22,23,27). The enzymes used for the synthesis, including KfiA, pmHS2, NST, C 5 -epimerase, 2-OST, 6-OST-1, 6-OST-3, and 3-OST-1, were expressed in E. coli as described previously (24).…”

Uncovering Biphasic Catalytic Mode of C5-epimerase in Heparan Sulfate Biosynthesis

“…The UDP-GlcNTFA synthesis was completed by transforming GlcNTFA-1-phosphate using glucosamine-1-phosphate acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase as described (16). The protocols for the expression of other HS biosynthetic enzymes and the preparation of PAPS were described elsewhere (16,18). Additional methods are presented under supplemental "Methods.…”

Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering

“…Treatment of N -sulfo, N -acetyl heparosan with recombinant O -sulfotransferases and C5-epimerase in the presence of a PAPS cofactor recycling system results in a bioengineered heparin that closely resembles the chemical and biological properties of heparin. Key elements for the commercialization include: process control, scale-up, and a reduction in the costs of CPS, recombinantly expressed biosynthetic enzymes, and PAPS cofactor (Burkart et al 2000; Zhou et al 2011) (Wang et al 2013). …”