Synthesis of bioengineered heparin by recombinant yeast Pichia pastoris

Abstract

Heparin, the most widely used anticoagulant drug, is mainly acquired from livestock. Variable structures and contaminations of other glycosaminoglycans of animal-sourced heparin aggravate risks in medical treatments and hamper the structure–activity relationship study. While chemically synthesized heparin products with refined structures are largely unaffordable. We present bioengineered heparin synthesized using an yeast platform. After achieving efficient expression of C5 epimerase and all the essential sulfotransferases in particular the bifunctional enzyme N-deacetylase/N-sulfotransferase, a green cell-free synthesis system for heparin was established by recruiting all the enzymes from cell lysates. The bioengineered heparin, transformed from heparosan with a ratio of 41%, exhibits comparable anticoagulant activity to commercial heparin extracted from animals. A Pichia pastoris cell factory for the de novo biosynthesis of heparin from methanol was further constructed, which enabled the production of 2.08 g L⁻¹ bioengineered heparin in fed-batch cultures. The strategies developed have potential to be scaled up to produce non-animal sourced heparin.