IGEM:IMPERIAL/2009/Encapsulation/Phase2/Alginate Biosynthesis/Bacteria1/GeneDetails/ Details of genes

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Contents

Genes for cloning

algD

encodes GDP-mannose dehydrogenase (GMD)
catalyses essentially irreversible oxidation of GDP-mannose to GDP-mannuronic acid

the limiting step and key kinetic control point of alginate biosynthesis as GDP-mannose is common to many different pathways [1]

alg8

Alg 8 is a membrane protein and is necessary for polymerisation of GDP-mannuronic acid.

Deletion mutants did not secrete free uronic acids, showing that Alg 8 is involved in polymerisation in P.aeruginosa. [2]

The introduction of multiple copy numbers of Alg8 dramatically increased alginate production of P. aeruginosa

alg44, alg K, alg X, alg G

Alg44 are supposed to be transmembrane proteins and therefore possible subunits of the alginate polymerase.
The proteins AlgK and AlgX are periplasmic proteins, and deletion mutants showed secretion of free uronic acids presumably due to alginate lyase activity.
Together with AlgG, these proteins are supposed to be part of a scaffold surrounding the nascent alginate chain
.[2]

alg X

Proposed to be involved in scaffold formation that surrounds and protects newly formed polymers from AlgL degradation from knockout studies in P.aeruginosa

deletion mutants of genes algG, algK and algX shows secretion of free uronic acids resulting from alginate lyase (AlgL) degradation of polymannuronic acid [3]

alg 44

proposed function of Alg44 as part of the periplasmic scaffold and/or bridging Alg8 in the cytoplasmic membrane with AlgE in the outer membrane. [4]

alg L

Codes for alginate lyase, that cuts the alginate chain and determines the length of the chain.

Alg L lyase activity shown essential for alginate synthesis in P.aeruginosa. Alg L with mutations that inactivate lyase activity are still essential for alginate synthesis.[5]

Alg L is also responsible for clearing alginates in the periplasm that are not exported to the extracellular environment.

alg G

C5-epimerase that introduces single M to G changes

Cloned in Escherichia coli, and mature AlgG proteins were expressed which were localized to the periplasm .[6]

The AlgG Protein, but not its epimerase Activity, Is Needed for Alginate Polymer Formation. Therefore, it is proposed to be part of a protein scaffold. [7]

Overexpression of algG in E. coli results in the formation of a partly insoluble product [8]

alg E

Outer membrane protein involved in exporting alginate out of cell

The coding region of the algE gene was cloned and expressed in Escherichia coli, and recombinant AlgE was found mainly in the OM. Recombinant AlgE was spontaneously incorporated into planar lipid bilayers, forming ion channels which were strongly anion selective (although not definitively for alginate transport)


alg E(epimerases)

Extracellular enzymes that induce the formation of G blocks in A.vinelandii.

Expressed in e.coli, followed by harvesting of the epimerase enzymes from the cell

Transport of enzymes will be a big issue (transporter is still only guessed at in silico) [8]

We can possibly do similar to experiments in adding enzyme extract into solution.


Review of [2].
To determine the requirement of Alg8, which has been proposed as catalytic subunit of alginate polymerase, nonpolar isogenic alg8 knockout mutants of alginate-overproducing P. aeruginosa FRD1 and P. aeruginosa PDO300 were constructed, respectively. These mutants were deficient in alginate biosynthesis, and alginate production was restored by introducing only the alg8 gene.

Protein topology prediction indicated that Alg8 is a membrane protein. Fusion protein analysis provided evidence that Alg8 is located in the cytoplasmic membrane with a periplasmic C terminus. It therefore may denature in solution.

Although this paper shows that alg8 is required for production of alginate, it does not specify that it is the only necessary gene required.

The polymerization step is still not understood. The proteins Alg8, putatively encoding a glycosyltransferase, and Alg44 are supposed to be transmembrane proteins and therefore possible subunits of the alginate polymerase.
The proteins AlgK and AlgX are periplasmic proteins, and deletion mutants showed secretion of free uronic acids presumably due to alginate lyase activity.
Together with AlgG, these proteins are supposed to be part of a scaffold surrounding the nascent alginate chain.

The highest specific alginate polymerase activity was detected in the envelope fraction, suggesting that cytoplasmic and outer membrane proteins together constitute the functional alginate polymerase complex.

Useful references

Error fetching PMID 7521247:
  1. Error fetching PMID 7521247: [Alginate1]
Error fetching PMID 15489449:
  1. Error fetching PMID 15489449: [Alginate2]
Error fetching PMID 16912921:
  1. Error fetching PMID 16912921: [Alginate3]
Error fetching PMID 16321942:
  1. Error fetching PMID 16321942: [Alginate4]
Error fetching PMID 8144447:
  1. Error fetching PMID 8144447: [Alginate5]
Error fetching PMID 12775688:
  1. Error fetching PMID 12775688: [Alginate6]
Error fetching PMID 16391057:
  1. Error fetching PMID 16391057: [Alginate7]
Error fetching PMID 8830682:
  1. Error fetching PMID 8830682: [Alginate8]
Error fetching PMID 10937941:
  1. Error fetching PMID 10937941: [Alginate9]
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