title: The role of hyaluronic acid precursor concentrations in molecular weight control in Streptococcus zooepidemicus.
abstract: The biosynthetic pathway responsible for the production of hyaluronic acid (HA) has been thoroughly studied; however, many aspects remain elusive regarding the mechanisms that control molecular weight (MW). Previously, we demonstrated a positive correlation between MW and the concentration of the HA precursor sugar UDP-N acetylglucosamine (UDP-GlcNAc). To further investigate the role of UDP-GlcNAc in MW control, we increased the intracellular concentration of this monomer using both feeding strategies and genetic engineering approaches. Feeding cells glucosamine dramatically increased intracellular levels of UDP-GlcNAc, but unexpectedly, produced HA of a lower MW. This was subsequently attributed to an equally dramatic decrease in the level of the other HA precursor sugar UDP-glucuronic acid (UDP-GlcUA). Feeding cells a mixture of glucose and GlcNAc addressed this imbalance of precursor sugars, leading to an increase in both UDP-GlcNAc and UDP-GlcUA; however, no significant increase in MW was observed. Despite the increase in UDP-sugars, RNA sequencing identified no increase in the expression of the genes involved in production of HA. Returning to genetic engineering approaches to examine UDP-GlcNAc and MW, genes known to contribute to the production of UDP-GlcNAc were over-expressed, both individually and together. Using this approach, UDP-GlcNAc and MW increased. At lower levels of UDP-GlcNAc, the positive correlation between UDP-GlcNAc levels and MW was maintained, however this relationship stalled at higher concentrations of UDP-GlcNAc. Taken together, these results suggest that while optimising HA precursor levels using feeding or genetic engineering approaches can improve HA MW, there is a point at which excess availability of precursors is no longer advantageous. Once precursor concentrations are addressed, it would seem that other uncharacterised factor(s) (e.g. rate of HA synthesis) also contribute to HA MW control.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD, 4072, Australia.
authors.forename: Wendy Yiting
authors.initials: WY
authors.lastname: Chen
authors.forename: Esteban
authors.initials: E
authors.lastname: Marcellin
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Steen
authors.forename: Lars Keld
authors.initials: LK
authors.lastname: Nielsen
mesh_terms: D053898:Biosynthetic Pathways; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006820:Hyaluronic Acid / Q000378:metabolism*; D007218:Industrial Microbiology; D008970:Molecular Weight; D017423:Sequence Analysis, RNA; D018502:Streptococcus equi / Q000502:physiology*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism*
chemical_list: D014537:Uridine Diphosphate N-Acetylglucosamine; D006820:Hyaluronic Acid
references.citation: Infect Immun. 1980 Feb;27(2):444-8
references.pmid: 6991416
references.citation: Drugs. 1994 Mar;47(3):536-66
references.pmid: 7514978
references.citation: Curr Pharm Biotechnol. 2008 Aug;9(4):236-8
references.pmid: 18691081
references.citation: Biomacromolecules. 2004 Nov-Dec;5(6):2122-7
references.pmid: 15530025
references.citation: Appl Microbiol Biotechnol. 2005 Oct;68(6):705-17
references.pmid: 16088349
references.citation: J Biol Chem. 1999 Aug 27;274(35):25085-92
references.pmid: 10455188
references.citation: J Biotechnol. 2003 Jan 9;100(1):33-41
references.pmid: 12413784
references.citation: Appl Microbiol Biotechnol. 2010 Mar;86(1):273-83
references.pmid: 19862515
references.citation: Biomaterials. 2005 May;26(14):1925-33
references.pmid: 15576166
references.citation: Infect Immun. 1993 Jan;61(1):295-302
references.pmid: 8418050
references.citation: Appl Microbiol Biotechnol. 2007 Nov;77(2):339-46
references.pmid: 17805528
references.citation: Biotechnol Lett. 2007 Jan;29(1):17-25
references.pmid: 17091377
references.citation: J Biol Chem. 1997 Dec 19;272(51):32539-46
references.pmid: 9405467
references.citation: Genome Biol. 2009;10(3):R25
references.pmid: 19261174
references.citation: Metab Eng. 2008 Jan;10(1):24-32
references.pmid: 17959405
references.citation: Nat Biotechnol. 2010 May;28(5):511-5
references.pmid: 20436464
references.citation: Appl Microbiol Biotechnol. 2005 Jan;66(4):341-51
references.pmid: 15599518
references.citation: Biotechnol Bioeng. 2003 Jun 5;82(5):578-89
references.pmid: 12652481
references.citation: J Bacteriol. 1983 Apr;154(1):1-9
references.pmid: 6403500
references.citation: J Biol Chem. 1956 May;220(1):303-6
references.pmid: 13319348
references.citation: FEMS Immunol Med Microbiol. 2008 Jul;53(2):166-77
references.pmid: 18462386
references.citation: Mol Biotechnol. 2011 Oct;49(2):166-75
references.pmid: 21365475
references.citation: Appl Environ Microbiol. 2000 Aug;66(8):3519-27
references.pmid: 10919816
references.citation: Proteome Sci. 2009 Mar 28;7:13
references.pmid: 19327162
references.citation: Tissue Eng Part A. 2009 Jul;15(7):1645-53
references.pmid: 19105604
references.citation: Methods Enzymol. 1985;113:278-81
references.pmid: 3003497
references.citation: J Mol Biol. 2012 Apr 20;418(1-2):21-31
references.pmid: 22343360
references.citation: Mol Biotechnol. 2013 Jul;54(3):747-55
references.pmid: 23180221
references.citation: Eur J Pharm Biopharm. 2008 Sep;70(1):199-206
references.pmid: 18644705
references.citation: Biotechnol J. 2009 Jan;4(1):58-63
references.pmid: 19156726
references.citation: Metab Eng. 2010 Jan;12(1):62-9
references.pmid: 19782148
references.citation: J Mol Evol. 2008 Jul;67(1):13-22
references.pmid: 18551332
references.citation: Appl Environ Microbiol. 2001 Jun;67(6):2734-8
references.pmid: 11375188
references.citation: J Biol Chem. 2009 Jul 3;284(27):18007-14
references.pmid: 19451654
references.citation: Skin Pharmacol Physiol. 2004 Sep-Oct;17(5):207-13
references.pmid: 15452406
references.citation: J Biol Chem. 1999 Feb 12;274(7):4246-53
references.pmid: 9933624
references.citation: J Bacteriol. 2011 Oct;193(19):5583-4
references.pmid: 21914890
references.citation: Int J Cancer. 1999 May 5;81(3):411-6
references.pmid: 10209956