Part:BBa_K2593001
HasA gene
Biology
HasA gene, isolated from S.zooepidemicus, encodes hyaluronic acid synthase (HAS), which is 419 amino acids in length. HasA gene is part of the hyaluronic acid synthesis operon in S.zooepidemicus genome by simulataneously catalyzing gluconuronic acid and acetaminoglucose transfer reaction. In vivo, the synthesis of hyaluronic acid is achieved by the alternating aggregation of monosaccharides uridine 5 '- 2 - glucose phosphate aldehyde (uridine 5' - diphosphate - GlcUA, UDP - GlcUA) and uridine 5 '- diphosphate - acetylglucosamine (uridine 5' - diphosphate - GlcNAc, UDP - GlcNAc) into HA long polysaccharide chain. In nature HASs exist in various organisms: group A Streptococcus coccus HAS (spHAS), group C streptococcus HAS(seHAS), bacillus HAS (pmHAS), dinococcus virus HAS(cvHAS); African claw toad HAS(xlHAS1), rodent HAS(mmHAS1,2,3), human HAS(hsHAS1,2,3), etc. In the binding areas of the spHAS and seHAS structures containing myocardial phospholipids or their structural analogues, phospholipids assist HASs to form a pore structure in the bilayer of the cell membrane to transport the extended HA out of the cell membrane to realize extracellular expression of HA.
HA synthase pathway
In Streptococcus species, HA biosynthsis begins with the phosphorylation of glucose by hexokinase to produce the main precursor, glucose-6-phosphate (G6P).From here, HA synthesis pathway can then be divided into two distinct pathways that syntheses the two building blocks of HA, glucuronic acid and N-acetylglucosamine (Fig. 2). In the first set of reactions, a-phosphoglucomutase (pgcA) converts glucose-6-phosphate to glucose-1-phosphate before a phosphate group from UTP is transferred to glucose-1-phosphate by UDP-glucose pyrophosphorylase (hasC/gtaB) to produce UDP-glucose. UDP-glucose is oxidised by UDP-glucose dehydrogenase (hasB/tuaB) to yield the first HA precursor, UDP-glucuronic acid (UDP- GlcUA). In the second set of reactions, G6P is converted into fructose 6-phosphate (F6P) by glucose phosphate isomerase which is encoded by pgi gene. Fructose 6-phosphate (F6P) is then catalyzed stepwide to glucosamine 6-phosphate, glucosamine-1-Pand N-Acetyl Glucosamine-1-P by Glucosamine--fructose-6-phosphateaminotransferase (glmS), Phosphoglucosaminemutase (glmM) and N-acetyl glucosamine-1-phosphateuridyltransferase (glmU) respectively. Once the two precursors are synthesised,hyaluronan synthase (hasA) polymerises the two components in an alternate manner to produce the HA polymer.
B. Subtilis, containing two precursor synthesis pathway genes for the synthesis of the HA precursor UDP-GlcUA and UDP-GlcNAc, has been regarded as an ideal cell factory and has been widely studied for HA production.
Usage：
HasA was commercially synthesized and subconed into pAX01 integration vector at the SacII and BamHI restriction sites. pAX01 vector is able to be expressed in both E. Coli and Bacillus Subtilis chassis, along with the ability to integrate into the genome of Bacillus subtilis strains via the lacA loci. Wildtype B. Subtilis 168 strain was used in this project. Colony PCR and Enzyme digestion further confirmed that HasA gene was successfully cloned in the vector and transformed in B.subtilis.
Fig 1 the HasA gene was validated by using PCR primer HasA-F and HasA-R, the expected size is 479bp.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 247
Reference
Peng Jin, Guocheng Du, Zhen Kang. High-yield novel leech hyaluronidase to expedite the preparation of specific
hyaluronan oligomers[J].Scientific Reports, 2014 : 1-2
Jinpeng, Kangzhen, Biosynthesis of hyaluronan oligosaccharides and construction of DNA editing and assembly tools[D]Jiangnan University: Jinpeng,2016.9-10.
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