Phase 3: CTP Junction Design & SPP Cleavage Verification

1. Selection of Chloroplast Transit Peptides

To improve targeting efficiency and avoid using repeated sequences, three different plant CTPs were selected:

• RbcS CTP: is derived from the Rubisco small subunit, one of the most abundant proteins in the chloroplast, and is widely used as a strong and reliable targeting signal.
• Fer2 CTP: comes from Ferredoxin-2, a chloroplast protein involved in electron transfer during photosynthesis, and is known for efficient import into the chloroplast stroma.
• RecA CTP: is derived from a chloroplast-localized RecA protein, which plays a role in DNA repair and maintenance within the chloroplast, and provides an alternative targeting signal with a different sequence composition.

These CTPs are derived from naturally chloroplast-targeted plant proteins (Arabidopsis thaliana) and are known to efficiently direct proteins into the chloroplast. Instead of using the same CTP for all seven genes, different peptides were intentionally distributed across the CODH subunits.

2. Fusion Design and Junction Engineering

Each CODH protein was fused to a CTP at its N-terminus. To make sure the protein folds correctly after cleavage, the fusion included the first 60 amino acids of each CODH protein, which were obtained using the ExPASy ProtParam tool.

A very important step was designing the junction between the CTP and the CODH protein. This region was carefully modified to include a cleavage motif recognized by the chloroplast enzyme responsible for removing the transit peptide:

(Val/Ile)-X-(Ala/Cys) ↓ Ala

To create this motif, small changes were made at the end of the CTP sequence as showed in the following sequences:

• RbcS CTP Sequence: MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVN(+AA)
• Fer2 CTP Sequence: MASTALSSAIVGTSFIRRSPAPISLRSLPSANTQSLFGLKSGTARGGRVTA(M–>A)
• RecA CTP Sequence: MDSQLVLSLKLNPSFTPLSPLFPFTPCSSFSPSLRFSSCYSRRLYSPVTVYA(+A)

This allowed a smooth transition between the CTP and the CODH protein while keeping both targeting and protein structure intact.

3. In Silico Validation of Targeting and Cleavage

All fusion sequences were analyzed using TargetP 2.0 to check two things:

• Whether the proteins are correctly targeted to the chloroplast
• Where the CTP is predicted to be cleaved

The results showed that all seven proteins are predicted to be targeted to the chloroplast, which confirms that the CTPs are working correctly:

coxD fusion: MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNAAMRHHAERDKVAERLAYAGYIPDRDLATAVWLMESLSRPLLLEGEAGVGKTEVALTLAQAN

Prediction: Chloroplast transfer peptide
CS pos: 55-56. VNA-AM. Pr: 0.5216

coxE fusion: MASTALSSAIVGTSFIRRSPAPISLRSLPSANTQSLFGLKSGTARGGRVTAAMVATAAIHESSAASAGARRKLGDFVRVLRDNGFIVGLAEAGDALTVLSRPASLTPSRLRP

Prediction: Chloroplast transfer peptide
CS pos: 51-52. VTA-AM. Pr: 0.3172

coxF fusion: MDSQLVLSLKLNPSFTPLSPLFPFTPCSSFSPSLRFSSCYSRRLYSPVTVYAAMTPTPDVLDLVNNMKARGEPFALATVVRTVSLTAAKAGAKAIILSDGTMTAGWIGGGCAR

Prediction: Chloroplast transfer peptide
CS pos: 51-52. TVY-AA. Pr: 0.4989

coxG fusion: MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNAAMDMNASQRIEASREKVYAALNDVEVLRPCIPGCESIEKISDSEMTAKVTLRIGPVKASFT

Prediction: Chloroplast transfer peptide
CS pos: 55-56. VNA-AM. Pr: 0.5923

coxL fusion: MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNAAMNIQTTVEPTSAERAEKLQGMGCKRKRVEDIRFTQGKGNYVDDVKLPGMLFGDFVRSSHA

Prediction: Chloroplast transfer peptide
CS pos: 55-56. VNA-AM. Pr: 0.4842

coxM fusion: MASTALSSAIVGTSFIRRSPAPISLRSLPSANTQSLFGLKSGTARGGRVTAAMIPGSFDYHRPKSIADAVALLTKLGEDARPLAGGHSLIPIMKTRLATPEHLVDLRDIGDL

Prediction: Chloroplast transfer peptide
CS pos: 51-52. VTA-AM. Pr: 0.7188

coxS fusion: MDSQLVLSLKLNPSFTPLSPLFPFTPCSSFSPSLRFSSCYSRRLYSPVTVYAAMAKAHIELTINGHPVEALVEPRTLLIHFIREQQNLTGAHIGCDTSHCGACTVDLDGMSVK

Prediction: Chloroplast transfer peptide
CS pos: 51-52. TVY-AA. Pr: 0.5011

Summary of the results:

Interpretation of Results

Overall, the results indicate successful design of functional targeting signals for all CODH subunits:

All constructs were confidently predicted as chloroplast-targeted proteins, confirming that the added CTPs are functional. The cleavage sites align well with the engineered junction motifs, demonstrating that the proteins are likely to be correctly processed after import.

The coxM fusion showed the highest cleavage probability (Pr = 0.7188), indicating highly efficient targeting and processing. Other subunits showed moderate probabilities (around 0.48–0.59), which are still within acceptable ranges for functional targeting. The coxE fusion presented a lower probability (Pr = 0.3172). Although this suggests potentially less efficient cleavage, the sequence still satisfies the required motif and is expected to remain functional, as variability in cleavage efficiency is common in heterologous systems.

Most constructs showed cleavage occurring exactly at the designed motif, typically between amino acid positions 51–56, depending on the transit peptide used.

However, a notable observation was made for two constructs, coxF and coxS, where the predicted cleavage site occurred slightly upstream of the engineered junction, specifically just before the designed alanine-alanine region rather than directly within it.

This slight variation in cleavage position is consistent with the known behavior of the chloroplast Stromal Processing Peptidase. Rather than recognizing a single fixed sequence, the enzyme identifies a broader structural and sequence context, which allows for some flexibility in the exact cleavage position. As a result, small shifts of one or two amino acids relative to the designed motif are commonly observed in both native and engineered proteins.

In this case, although the cleavage in coxF and coxS occurs marginally earlier than expected, it remains within a functionally acceptable region. The resulting mature proteins retain nearly identical N-terminal sequences and are not expected to lose any essential structural or functional elements. Importantly, the targeting prediction remains strong, confirming that the proteins are still efficiently directed to the chloroplast.

Therefore, this variability does not compromise the overall design. All fusion constructs are considered valid, and no redesign was required. Instead, this observation reflects the inherent flexibility of chloroplast protein processing and further validates the robustness of the engineered system.