Part:BBa_K2593008
P43-RBS-tuaD-gtaB-T1
This part is a functional composite part, It consists of a P43 promotor, an RBS, and an operon of tuaD-gtaB which encode UDP-glucose dehydrogenase and UDP-gulcose pyrophosphoryase, respectively, to participate in the biosynthesis of HA precursor UDP- GlcUA.Promoter P43(BBa_K1628006): P43 is a constitutive promoter that common use in Bacillus subtilis168.
RBS(BBa_K2593005): A strong ribosome binding site commonly used in Bacillus species, it is part of the pP43NMK shuttle vector.
tuaD(BBa_K1469002): tuaD is one of native gene of B. subtilis, it encodes UDP-glucose 6-dehydrogenase has 461 amino acid (also know as: UDP-GlcDH in megaterium).
gtaB(BBa_K1469005): gtaB gene encodes UTP--glucose-1-phosphate uridylyltransferase in Bacillus megaterium. The enzyme catalyzes the conversion of glucose 1-phospahate to UDP-glucose 1-phosphate.
T1 terminator(BBa_B0010): it is the most used terminator in bacteria.
Usage
In our project, expression of the operon tuaD-gtaB was regulated under the control of a constitutive promoter P43, this operon is used for further increasing the production of the HA in B.subtilis, tuaD and gtaB gene, products regulate the last two steps in the synthetic pathway of UDP-GlcUA(Fig1).Cloning of operon tuaD-gtaB into B.subtilis 168E were confirmed by colony PCR polymerization .(Fig2)
Fig1 Left. the synthesis pathway of HA . Right.the construct of gtaB-tuaD in pP43NMK plasmid
Figure 2: Left: 1% agarose gel electrophoresis of colony PCR amplifying section of gtaB gene in pP43NMK-gtaB-tuaD using primer pair GtaB-TuaD-F and GtaB-TuaD-R and the expected product size is 438 bp. Right: illustration of construction of the expression vector pP43NMK-gtaB-tuaD. Operon gtaB-tuaD was inserted at the restriction sites HindIII and KpnI of pP43NMK plasmid.
In our experiment, by conducting CTAB experiments that form turbidity from a reaction between HA and CTAB solution, the results showed a remarkable increase in HA production when co-overexpressed tuaD-gtaB together (488mg/L, a 38% increase) (Figure3),In addition, Molecular weight analysis studies showed that HAs synthesized were high molecular weight(Figure4 ).
Figure 3: CTAB analysis of HA concentraton, 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.
Figure 4: molecular weights of HA produced by overexpression of precursor genes in recombinant B. subtilis 168E strains using viscometer analysis.
T Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Reference
Peng Jin, Zhen Kang, Panhong Yuan. Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168[J].Metabolic Engineering, 2016:22-25
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