Part:BBa_K4582000
Coding sequence mucA* of P. putida MucA, resulting in alginate overproduction
Pseudomonas putida produces alginate as a polysaccharide component of its biofilm. Alginate possesses numerous properties that makes it widely useful in various fields, including biomedical and agri-food applications.
Our team focused on alginate due to its water retention capacity and aimed to overproduce it using the bacterium Pseudomonas putida.
How Pseudomonas putida Naturally Produces Alginate
:In P. putida, the natural biosynthesis of alginate is ensured by a series of enzymes encoded by genes organized in an operon.
The promoter of this operon is recognized by an alternative sigma factor, AlgU (RpoE), which is activated during membrane stress. Outside of stress conditions (e.g in "normal" conditions), the AlgU sigma factor is sequestered by an anti-sigma factor called MucA, located at the membrane. This sequestration prevents the formation of the RNA polymerase holoenzyme that recognizes the operon's promoter and, consequently, the expression of enzymes responsible for alginate production.
Our Approach to Overproduce Alginate
In order to overproduce alginate, our team constructed a "mutation tool" that allows for the replacement of the wild-type mucA gene with a mutated mucA gene encoding a truncated MucA in the C-terminal region, all through homologous recombination.
We added 50-base pair homologous sequences on both sides of the mutated gene, flanking the wild-type mucA gene on the chromosome. This procedure had previously been performed in a phylogenetically related strain, Pseudomonas aeruginosa, so we undertook the challenge of replicating it in P. putida.
Our modification involved removing 192 base pairs from the 3' end of the mucA gene, resulting in a truncated MucA with a C-terminal region corresponding to its interaction site with AlgU. Consequently, the truncated MucA would be unable to sequester AlgU, leading to alginate overproduction. We carried out cloning to insert this fragment into a non-replicative plasmid, pKNG101, and introduced the recombinant plasmid into P. putida KT2440 through conjugation. After achieving two homologous recombination events, we screened for the acquisition of mutant strains.
We successfully obtained a mutant strain, and to measure its efficiency, we compared the alginate production between the obtained mutant strain and the wild-type strain.
We successfully obtained approximatively 30 times more alginate from the mutant strain compared to the wild-type strain, demonstrating the efficiency of the mucA mutation in inducing the expression of genes under the control of the promoter recognized by AlgU and the production of alginate.
Protocols andResults can be checked on the 2023 Aix-Marseille team wiki, this part being described on their engineering page
Therefore, our work will enable future teams to easily produce alginate without the need for membrane stress from a P. putida strain.
Usage and Biology
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]
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