Project Description

Our project revolved around the idea of making the bacteria and yeasts in a sourdough culture produce some of the essential vitamins that many lack in their diets. Lactic acid bacteria (LAB) appear a suitable host for vitamin B12 production as many strains/species already produce the vitamin in a form that is not bioavailable to humans, so called pseudo-B12. The plan was therefore to modify the pathway pseudo-B12 to instead produce a more suitable form of B12. Previous studies, and even previous iGEM teams, have worked with introducing the required genes to make production of vitamin A viable in yeast, specifically Saccharomyces Cerevisiae. As such our plan was to introduce these genes into a strain of Saccharomyces called CEN.PK. The specific strain we were working with was developed to be dependent on externally produced B12 [1].

The targeted pathway in LAB for B12 production is originally taken from Limosilactobacillus reuteri and then assumed to apply in other LABs when direct analogs could be found to all the genes. There are several published works regarding the production of B12 in nature, and how this can be replicated in more typical lab organisms, such as E.coli [2]. Using these pathways as a template allowed us to visualize the pathway and design the lab work around: knocking out the enzyme responsible for integrating the wrong ligand, introducing/restoring the production of the desired ligand, introducing the enzyme which integrates the desired ligand and finally verifying production of desired product.

For the production of vitamin A in CEN.PK we based our approach on unpublished work by Andrea Clausen Lind and standard CRISPR integration methodology [3] utilizing both the CRISPR/Cas system as well as auxotrophic markers to select for successful integration. The previous work by Andrea focused on production of lycopene, a carotenoid closely related to the precursor of vitamin A. As such we decided to use the gene constructs provided by Andrea and design a construct for the conversion of lycopene to β-carotene. The genes CrtI, CrtE, CrtB and FAD1 bridge the terpenoid backbone synthesis in yeast and lycopene synthesis. Our alteration of exchanging CrtB for the bifunctional enzyme CrtYB, should allow for the multistep conversion of lycopene to β-carotene, which is carried out by the crtY part of the enzyme (Fig 1).

Figure 1: Biosynthetic pathways of vitamin A from glucose.
The finished design would include four constructs, two of which were to be integrated. The first one to be integrated would be the main construct carrying the genes required for β-carotene synthesis in yeast (Fig 2). The second plasmid was designed to carry the CAS9 enzyme, while the third one carried the gRNA, composed of the guiding crispr RNA (which binds to the targeted region) and the CAS9 binding tracr RNA which links it all together (Fig 3 and 4) .

Figure 2-4: Level 2 multigene construct completing pathway of vitamin A production (figure 4), Plasmid designed to carry Cas9 enzyme (figure 3), and Plasmid designed to carry gRNA (figure 4)
The fourth and final construct contains genes coding for the auxotrophic markers which the previous three plasmids used, as well as a fourth, which in theory should remove the selection pressure of maintaining the previous plasmids (Fig 5).

Figure 5: Construct containing genes coding for the auxotrophic markers.
All the designs were created using the software Snapgene. With our main objectives being a healthier world and an aid to end malnutrition, we chose to focus on the essential vitamins B12 and A for our sourdough. Besides being essential vitamins, these vitamins also have in common that they are commonly ingested through animal products. With animal husbandry representing 80% of the food sector's emissions, removing our need to eat animal based for our essential vitamins opens up the possibilities for what we eat.

The constructs were developed based on the modular cloning strategy, MoClo, which uses the Type IIS restriction enzymes BsaI and BsmBI. The MoClo system is based on a successive assembly and consists of three sets of cloning vectors Level 0, 1 or 2. Level 0 plasmids contain basic parts such as promoters, UTRs, coding sequences etc. The level 0 plasmids get then assembled into a level 1 vector, which is done using the BsaI restriction enzyme creating a simple transcriptional unit. Thereafter, several transcriptional units can be assembled into the Level 2 vector creating a multigene construct [4][5]. In this way the cloning process is simplified, as instead of having several plasmids, each containing a gene, they can be combined into one Level 2 vector. Having a big construct also simplifies integrating the genes into the yeast’s genome. The DNA fragments needed for the production of vitamin A were combined in a Level 2 plasmid presented in figure 2.

In our project, the vitamin A construct was supposed to be assembled via MoClo to be later integrated into the yeast’s genome using the already mentioned CRISPR/ Cas9 (see fig ö and ä) and homologous recombination. Constructing sgRNA and creating a plasmid coding for Cas9 could also be done using MoClo [4]. CRISPR-Cas originates from the adaptive immune system of microbes. It is based on a nuclease that, guided by noncoding RNA, is able to specifically cleave DNA by introducing double stranded breaks. This mechanism is used by bacteria and archaea to eliminate invading genetic materials such as viral DNA [6]. To achieve this precise site recognition, Cas9 needs to be complexed with crNRA and tracrRNA. Creating the double-stranded breaks in the DNA, Cas9 also requires the protospacer-associated motif, known as PAM sequence [7].

B12 and Gibson assembly

After doing some literature studies, we identified that Limosilactobacillus reuteri is known for producing pseudo vitamin B12. The chemical structure of vitamin pseudo B12 is very similar to the active form of B12, however, it cannot be utilized by the human body. In order to design the construct containing the parts identified to produce the active form of vitamin B12, we studied the metabolic pathway for the production of Cob(I)alamin in the database KEGG [8]. We tried to find which enzymes seem to be missing to complete the pathway of active form of B12 in L.Reutri. Identifying those enzymes, we tried to find if they are present in other Lactobacillus species using the KEGG database. We aimed to find the genes from Lactobacillus species that were closest to Limosilactobacillus reuteri according to the phylogenetics tree. The missing enzymes that we identified were BluB, CobT, CobC and CobS (Fig 6). According to KEGG, CobT and CobS were present in Limosilactobacillus reuteri already according to KEGG, however we decided to integrate them as the information in databases may be inadequate. As promoters, we chose Erythromycin Constitutive Promoter and Promoter CP44 and as terminator T7 and T1rrb Terminator, earlier used in L. Reuteri by iGEM Oxford 2019 [9][10][11]. For ribosome binding site (RBS), we chose Lactobacillus spp RBS, which was earlier used specifically in Limosilactobacillus reuteri [12].

Figure 6: Completed metabolic pathway for producing an active form of vitamin B12.
In order to assemble the genes for the B12 construct, a different method from MoClo was used, called GIbson assembly. In this technique, multiple DNA fragments can be efficiently assembled into one larger DNA fragment. It is based on using three key enzymes: a DNA polymerase, a 5’ exonuclease, and a DNA ligase. As the DNA fragments are designed so that they have overlapping ends, these enzymes can assemble them into a single, continuous piece of DNA [13] (Fig 7). As L.Reuteri is not as well studied as E. coli, we decided to investigate several vectors. We chose three different alternatives. The first one was shuttle vector pTRKH3-ermGFP, which was earlier used by at least two iGEM teams: Lund 2021 and Oxford 2019. The second vector was: pPBT-peRNA_GG-Puro. And the last vector was the standard MoClo vector, pYTK001. The last vector was used to amplify the construct created with Gibson assembly. The aim was to check if the bacteria could produce the active form of vitamin B12 after transformation.

Figure 7: Level 2 multigene construct completing pathway of vitamin B12 production.