Difference between revisions of "Part:BBa K1469009"

Latest revision as of 02:11, 18 October 2018

gcaD

UDP-N-acetylglucosamine pyrophosphorylase of Bacillus megaterium. The enzyme is bifunctional and catalyses the conversion of glucosamine 1-phosphate to N-acetyl glucosamine 1-phosphate as well as the conversion of N-acetyl glucosamine 1-phosphate to UDP-N-acetyl glucosamine, a key precursor molecule for hyaluronic acid production.

Characterization by SSTi-SZGD(2018)

Usage

This is a improvement part of glmU (2014 saarland iGEM team) (also known as : GcaD in B.megaterium)

This device is used to over-express glmU gene in B. subtilis 168. glmU encodes UDP-N-acetylglucosamine pyrophosphorylase that converts GlcN-1-P to GlcNAc-1-P as well as GlcNAc-1-P to UDP-GlcNAc in the synthetic pathway of UDP-GlcNAc, one of the two HA precursors in B. subtilis. It is assumed that overexpression of glmU contributes to the elevation of UDP-GlcNAc production, which may have a positive effect on HA production.

Fig1 a. the synthesis pathway of HA . b.the construct of glmU in pP43NMK plasmid

In our project, glmU expression was regulated under the control of a constitutive promoter P43, and the recombinant pP43NMK-glmU vector was transformed into recombinant B.subtilis 168E, confirmed by colony PCR polymerization(Fig2) . It has been engineered to secrete extracellular HA. Our results showed the introduction of extra copies of glmU gene, resulted to an elevated HA accumulation in B.subtilis 168E (448mg/L, a 32% increase) (Fig3). Molecular weight analysis studies showed that HAs synthesized were high molecular weight(Fig4).

Figure 2: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of gtaB gene in pP43NMK-gtaB-tuaD using primer pair GlmU-F and GlmU-R and the expected product size is 304 bp. Right: illustration of construction of the expression vector pP43NMK-glmU. GlmU coding sequence was inserted at restriction sites HindIII and BamHI of pP43NMK plasmid.

Figure 3: CTAB analysis of HA concentration, a. Illustration of the turbidity by mixing different source of HA with CTAB solution. b: effects of overexpressing the precursor genes on HA production in recombinant B.

Figure4: molecular weights of HA produced by overexpression of precursor genes in recombinant B. subtilis 168E strains using viscometer analysis.

Sequence and Features