Low molecular weight heparosan is an un-sulfated polysaccharide primarily used as a precursor for heparin synthesis that has recently been used in drug delivery applications. Heparosan synthesis from recombinant bacterial systems provides a safer alternative to naturally producing pathogenic bacterial systems. In this study, we engineered a functional heparosan synthesis pathway in Bacillus megaterium by the expression of E. coli K5 kfiC and kfiA glycosyltransferase genes. Upregulation of individual UDP-sugar precursor pathway genes enhanced the heparosan production, indicating that UDP-precursor sugar concentrations were limiting the biosynthesis. The engineered B. megaterium yielded a maximum heparosan concentration of 394 mg/L in batch bioreactor. The heparosan titer was further increased to 1.32 g/L in fed-batch fermentation. Nuclear magnetic resonance analysis revealed that the chemical structure of B. megaterium derived heparosan was identical to E. coli K5 heparosan. The heparosan molecular weight varied from 31 to 60 kDa, indicating its potential as a precursor for chemoenzymatic heparin biosynthesis. This study provides an efficient process to produce heparosan from non-pathogenic B. megaterium.

glycosyltransferases, Heparosan, Bacillus megaterium

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4	bDGlcpA	102.02	?	?	?	?	174.91
2	Ac	?	21.98
	aDGlcpN	96.42	53.29	?	78.51	?	59.50


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4	bDGlcpA	4.38	3.25	3.57	3.57	3.62	-
2	Ac	-	1.92
	aDGlcpN	5.27	3.74	3.74	3.57	3.74	3.74


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4	bDGlcpA	102.02/4.38	?/3.25	?/3.57	?/3.57	?/3.62	
2	Ac		21.98/1.92
	aDGlcpN	96.42/5.27	53.29/3.74	?/3.74	78.51/3.57	?/3.74	59.50/3.74

The spectrum also has 7 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

[*]O[C@@H]1[C@@H](C(=O)O)O[C@@H](O[C@@H]2[C@@H](CO)O[C@H]([*])[C@H](NC(C)=O)[C@H]2O)[C@H](O)[C@H]1O

379.318 g/mol (C₁₄H₂₁R₂NO₁₁, R = next and previous repeats, not counted in mol weight)