The ultimate goal of our project is to turn alginate into a commodity. To make it inexpensive enough that we can use it at scale in agriculture to fight drought. We’ll need a lot of engineering to achieve this goal and here is our first stone to this building.

Design

Alginate is traditionally produced from algae. Its production, extraction and purification are expensive and not environmentally friendly.

There is already research about producing alginate from bacteria. Actually, it was one of the subjects of one great iGEM project from last year: Cosmic. They tried (and unfortunately failed) to overproduce alginate in A. vinelandii by overexpressing alg8.

In our search for an alternative chassis for alginate production, we became interested in Pseudomonas sp. because:

1. certain species naturally produce this polymer
2. in the human pathogen Pseudomonas putida, genetic mutations leading to overproduction of alginate have been described in the literature
3. researchers in our host laboratory, the LISM, are specialists of Pseudomonas.

For our work, we selected Pseudomonas putida as the chassis because it is a non-pathogenic bacterium closely related to P. aeruginosa.

Alginate production is tightly regulated in the Pseudomonas genus. The genes involved in alginate biosynthesis are organized in operons with their promoter recognized by the alternative sigma factor AlgU, and the major negative regulatory element is the anti-sigma factor MucA.

Even though MucA inhibits alginate production, it is also vital for the bacteria's survival. However, researchers have identified several mucA mutations (called mucA*) resulting in a truncated MucA protein. These mutations are not lethal for the bacteria but lead to an increased production of alginate.¹. As the physiology of P. putida is similar to that of P. aeruginosa, we decided to introduce the mucA* mutations into the P. putida genome in order to obtain an alginate overproducing strain.

To do this, we constructed a mutator plasmid corresponding to the suicide plasmid pKNG101, in which we integrated the mutated mucA gene coding for a truncated MucA, along with 50 bp regions homologous to the regions flanking the mucA gene on the chromosome of P. putida KT2440. In this way, we aimed to replace the wild-type mucA gene with the mutated mucA gene through homologous recombination after plasmid integration into the strain.

Build

As our design was based on work carried out on P. aeruginosa and not on P. putida, we knew that the operation was likely to fail. The risk of failure was all the greater in that we used homologous recombination for the construction of our chassis, a technique that is itself laborious.

Four different mucA* parts were built based on the literature and introduced into the suicide plasmid pKNG101. We were tempted to introduce the mutations sequentially in the P. putida genome by homologous recombination, testing each time whether it worked, but we thought it would be much quicker to do everything in parallel.

To construct our mutator plasmid, we performed PCR using P. putida DNA as a template to obtain fragments corresponding to the truncated mutated gene with the addition of a stop codon and homologous regions. To obtain the mutated gene, we designed primers with a reverse primer hybridizing to the desired stop region of the mutated gene with the addition of a stop codon at the end. Further details are available in the results section. All fragments were integrated and assembled into the pKNG101 plasmid, digested using the NEBuilder Assembly kit, following an unsuccessful attempt with the SLIC method.

After confirming the acquisition of our mutator plasmid for the four constructed forms through sequencing, we integrated each mutator plasmid into a wild-type strain of P. putida KT2440 via conjugation. After two homologous recombination events, we aimed to screen the obtained colonies by selecting homologous recombination events that replaced the mucA gene with the mutated mucA gene by PCR on colonies. This was a challenging step since we initially wanted to differentiate the mutated mucA from the wild-type mucA on the chromosome by size, but we encountered difficulties in amplifying and distinguishing the different sizes of each mutant. Therefore, we decided to use a different approach not based on the gene size. We thought of using two different primer pairs, one pair hybridizing to the ends of the mutated gene and another pair hybridizing to the ends of the wild-type gene. This way, for a colony that replaced the wild-type mucA with the mutated one, only amplification with primers flanking the mutated gene would be detected.

Thus, we succeeded in obtaining a mutant named mucA*3₄, which replaced the wild-type mucA gene with the truncated mucA gene of 399 bp in size.

As shown in Figure 2, we succeeded in the construction of one P. putida mucA* mutant. Colony PCR indicated that the P. putida mucA3₄ clone is successfully mutated as this clone does not contain a WT copy of the mucA gene (absence of PCR amplification in the red rectangle).

The mucA* coding sequence used for this construction has been submitted as a part into the registry under reference BBa_K4582000

Test

To measure the efficiency of mucA gene mutation in alginate production, we considered performing an alginate assay with the wild-type P. putida strain under conditions of osmotic stress by adding NaCl, which is one of the best-characterized natural conditions for alginate production, under non-stress conditions (LB), and with the mutant strain obtained under non-stress conditions (LB).

The results showed a 30-fold increase in alginate production in the mutant strain compared to the wild-type strain grown in LB. This result indicates that the truncation of the MucA protein effectively prevents the sequestration of AlgU, leading to higher expression of genes encoding enzymes for alginate biosynthesis, which are naturally expressed under membrane stress conditions, resulting in improved alginate production despite optimal growth conditions. However, it does not produce more alginate than a wild-type strain under high osmotic stress conditions (0.5M NaCl).

Learn

Throughout this engineering cycle, we learned that the truncation of the MucA protein allows alginate production under non-stress conditions but not in large quantities. This can be explained by the multifactorial nature of alginate production in the wild-type strain, involving factors other than genetic regulation alone.

We observed that modifying genes in cis by homologous recombination is simpler to implement but can be more uncertain, with minimal success probabilities. In the future, other methods described as effective in P. putida could be used for genetic modification at the genome level.

We realized the importance of carrying out multiple constructions to ensure obtaining at least one successful result since, of the 4 mutator plasmids constructed, we obtained only one mutant colony. Similarly, having a backup method in a key construction step is essential, as we faced difficulties in the assembly and verification of mutant strain acquisition through colony PCR. This is a practice to keep for future constructions.

Design 2

This engineering cycle allowed us to highlight the multifactorial aspect of alginate production. To optimize its production, we have considered exploring the metabolic aspect in addition to regulation. Therefore, we aim to add the alg8 gene in trans to the mutant P. putida mucA3₄ strain under the control of an inducible promoter. Alg8 has been shown to overproduce alginate in P. aeruginosa and A. vinelandii, and it is also a path chosen by the CosMic team. alg8 is described as a key gene in alginate production because it is involved in polymerization ³. Integrating the gene into a plasmid allows us to avoid the homologous recombination step but requires the use of antibiotics to prevent plasmid loss. One solution could be to construct a plasmid based on an autotrophy system to make the plasmid essential for the strain's survival.

Sources

1. Different mutations in mucA gene of Pseudomonas aeruginosa mucoid strains in cystic fibrosis patients and their effect on algU gene expression, Giovanna Pulcrano et al. NEW MICROBIOLOGICA, 2012
2. The Cosmic 2022 iGEM project
3. Bacterial alginate production : an overview of its biosynthesis ans potential industrial production, Viviana Urtuvia et al. World J Microbiol Biotechnol, 2017