IGEM:IMPERIAL/2009/Encapsulation/Phase2/Alginate Biosynthesis/Bacteria1

Summary of Useful Info:

The genes for alginate biosynthesis are all located on the bacterial chromosome, with no evidence to date of plasmid involvement.
At least 24 genes have been directly implicated in alginate biosynthesis in P. aeruginosa.

The table below shows the genes involves in alginate production for P. aeruginosa.[1]

[2]

Generally, the alginate biosynthesis pathway can be divided into four different stages:

• 1) synthesis of precursor substrate
• 2) polymerization and cytoplasmic membrane transfer
• 3) periplasmic transfer and modification
• 4) export through the outer membrane.

Gene Details

Details of genes

The Common Genes

The pathway forming alginate presented here in P. aeruginosa has some overlap with genes naturally present in E.coli. We will investigate the pathway and identify those also in E.coli. This will enable us to only insert as few genes as possible.

The initial steps in the alginate biosynthesis pathway are essentially those of general carbohydrate metabolism and the intermediates are widely utilized.
In particular, the steps up to and including GDP mannose are common to alginate and LPS biosynthesis.

Studies into the algC gene found in the biosynthesis pathway of alginate in P. aeruginosa, encodes phosphomannomutase which interconverts mannose-6-phosphate and mannose-1-phosphate. This was confirmed by successful complementation of an E.coli pgm mutation with the P. aeruginosa algC gene.

The P. aeruginosa alginate export gene (AlgE) has been functionally expressed in E.coli. [3]

• James Chappell 09:46, 23 July 2009 (EDT):I do not think it was shown to be functional, they have expressed it in E.coli and purified and did some in vitro testing but in this paper at least they never proved it was functional when expressed in E.coli cells e.g. that it would export the alginate.

True - from the paper the algE gene was successfully cloned in E.coli. It was not tested to perform the function required to transport alginate outside the cell. Further papers will be investigated.

E.coli Gene Homologues

1) Fructose 6 phosphate to mannose 6 phosphate (manA)

2) Mannose 6 phosphate to mannose 1 phosphate (manB)

3) Mannose 1 phosphate to GDP mannose (manC)

Genes We Need

By using experiments that isolate specific genes we have found that there were a number of genes we still needed to clone.

Genes we definitely need:

• algD - conversion of GDP mannose to GDP mannuronate
• alg8, alg44, algX, algK - polymerisation complex (we are yet unsure as to whether we can knock out any of these genes as they form a complex)
• algL - release of alginate (alginate lyase)
• algE - export protein
• algE1/algE2/algE6 - these genes all cause formation of G blocks which hardens the gel to form the capsule we need (not present in P.aeruginosa. Taken from A.vinelandii)
  

• James Chappell 09:48, 23 July 2009 (EDT):Try to think of genes we need in terms of the biosyn pathway. So first need to clone AlgD - this would give cytosolic GDP mannuronic acid. Then this needs to be polymerised and exported etc.

Ok so using/following the biosynthetic pathway we have reordered the genes we think we need, and added exactly what parts they are involved in. The cloning starts off with algD (conversion), then the genes for the polymerisation complex followed by the lyase gene algL, the export gene algE, and epimerases(a series of algE(s)).

Genes we may need:

• algA - found in E.coli as manA, however, manA synthesizes in the opposite direction to what we need: mannose 6 P to fructose 6 P
• algG - introduces isolated G monomers (may be in a complex with other proteins)
  
• eexD, eexE, eexF - Type I secretion machinery to transport genes for algE1/algE2/algE6 to extracellular environment

Control

The properties of pure alginates are determined by their degree of poly-merization and acetylation, and by their monomer composition and sequence.
It seems probable that it will become largely possible to control all these three parameters in bacterial alginates [4]

Issue of monomer composition

M to G transitions can be done using bacterial epimerases.

G blocks are of great biological and biotechnological significance as they are a prerequisite for the formation of strong polymer gels in the presence of divalent cations like Ca2+.

Pseudomonas species have only one mannuronan C-5- epimerase, encoded by the algG gene, and this enzyme is only able to introduce single guluronic acid residues in to the mannuronan chain.

In A. vinelandii, there are 5 AlgE genes for epimerisation. In particular, AlgE2 introduces G-blocks (gel-forming alginate). AlgE2 should probably be the gene for epimerisation that we clone

It is well-established that alginates from P. aeruginosa do not contain polyguluronate blocks, whereas those from A. vinelandii may do so

Alginates which contain polyguluronate form rigid gels in the presence of Ca2+ and are therefore important in structural roles, e.g. gel formation.

Conversely, an absence of polyguluronate, as in P. aeruginosa, produces relatively flexible gels in the presence of Ca2+ eg biofilm.

Deacetylated alginates are needed because early studies indicate that acetylation seems to protect them from epimerization of the AlgE type [4] So we probably can knock out the O-acetylation gene

• James Chappell 09:42, 23 July 2009 (EDT):What type of alignates are commercially available and used for the chemical encapsulation? From what I read you can just use polymannuronic acid...I think a good approach to look at this would be to identify how much of the biosyn pathway you need as some of the genes may not be necessary.

From the chemical method of encapsulation:
The alginate commonly used are algal alginates from brown seaweed with G-blocks (refer to Alginate properties)
The genes outlined above are all involved in making polymannuronic acid and its transport outside the cell. We have already taken out the genes for unnecessary modification (acetylation)

Production

In the lab

• James Chappell 09:50, 23 July 2009 (EDT):How much aliginate would you need to buy if you were to do this with chemical methods. Might be interesting to draw comparison, to buy 1kg of alignate for encapsulation would cost ~£108.50 from what I have seen.

For chemical encapsulation, for a method, 18 g alginate solution needs to be mixed with 1 g washed bacteria suspension.
Unsure how much this would cost biologically, but the cell production levels are not very high as seen in the graph.

Amount of alginate produced expressed as micrograms of alginate per milligram of cell dry weight [5]

Using fermenters

use A. vinelandii as producing organism, but even under optimized fermentation conditions production yields are quite low (around 4 g/l) compared to other microbial polymer fermentations. [6]

Useful Papers

1. Gacesa P. Bacterial alginate biosynthesis--recent progress and future prospects. Microbiology (Reading). 1998 May;144 ( Pt 5):1133-1143. DOI:10.1099/00221287-144-5-1133 |

2. Remminghorst U and Rehm BH. Bacterial alginates: from biosynthesis to applications. Biotechnol Lett. 2006 Nov;28(21):1701-12. DOI:10.1007/s10529-006-9156-x |

5. Hassett DJ. Anaerobic production of alginate by Pseudomonas aeruginosa: alginate restricts diffusion of oxygen. J Bacteriol. 1996 Dec;178(24):7322-5. DOI:10.1128/jb.178.24.7322-7325.1996 |

4. Rehm BH and Valla S. Bacterial alginates: biosynthesis and applications. Appl Microbiol Biotechnol. 1997 Sep;48(3):281-8. DOI:10.1007/s002530051051 |

7. Sabra W, Zeng AP, and Deckwer WD. Bacterial alginate: physiology, product quality and process aspects. Appl Microbiol Biotechnol. 2001 Aug;56(3-4):315-25. DOI:10.1007/s002530100699 |

3. Rehm BH, Boheim G, Tommassen J, and Winkler UK. Overexpression of algE in Escherichia coli: subcellular localization, purification, and ion channel properties. J Bacteriol. 1994 Sep;176(18):5639-47. DOI:10.1128/jb.176.18.5639-5647.1994 |

8. Remminghorst U and Rehm BH. In vitro alginate polymerization and the functional role of Alg8 in alginate production by Pseudomonas aeruginosa. Appl Environ Microbiol. 2006 Jan;72(1):298-305. DOI:10.1128/AEM.72.1.298-305.2006 |

6. Remminghorst U and Rehm BH. Bacterial alginates: from biosynthesis to applications. Biotechnol Lett. 2006 Nov;28(21):1701-12. DOI:10.1007/s10529-006-9156-x |