Metabolic Engineering OfLactococcus LactisFor The Production Of Heparosan

Abstract

AbstractHeparosan is a precursor molecule for the widely used anticoagulant heparin, which also has other uses such as certain drug delivery applications and as a scaffold for tissue engineering in biomaterials. Traditionally, pathogenic bacteria such asE.Colihave been used as a host to produce heparosan as an alternative to animal and chemoenzymatic synthesis. Using GRAS status organisms likeLactococcus Lactisas the host for production of heparosan provides a safe alternative as well as being a well-established organism for genetic manipulation and reengineering. In this study, a functional heparosan synthesis pathway was successfully expressed inLactococcus Lactisby the expression ofE.coliK5 genes KfiA and KfiC, along with the overexpression of ugd, glmu and pgma genes present natively in the host organism. The genes were activated using the tightly controlled NICE expression system. The genes were cloned into plasmid p8148 and transformed into two strains,Lactococcus LactisNZ9000 andLactococcus LactisNZ9020, totaling six different recombinant strains were created using these two hosts and various combinations of the heterologous genes. The recombinantLactococcus LactisSH6 strain, expressing the genesugd-KfiA-KfiC-pgma yielded a maximum concentration of 754 mg/l in batch bioreactor experiments and the titer was increased to 1263 mg/l in fed-batch fermentation. NMR imaging successfully determined that the structure of the product derived fromLactococcus Lactiswas indeed similar toE.coliheparosan. The molecular weight of heparosan varied from 10-20 KDa, indicating its potential use for chemoenzymatic heparin biosynthesis.