Part:BBa_K4370002

RBS_cbbL_STR

This parts encodes the form I ribulose bisphosphate carboxylase (RuBisCO, BBa_K4370000) large subunit of Streptomyces bottropensis ATCC 25435 under the control of an RBS (BBa_B0034). The co-expression of this part with the form I RuBisCO small subunit (BBa_K4370001, BBa_K4370003) allows the expression of the complete RuBisCO of this strain.

Usage and Biology

The Calvin-Benson-Bassham cycle (CBB) is one of the six main natural CO₂ fixation cycles/pathways into biomass and potentially into products of interest in an industrial setting. This cycle is the most widespread naturally, and responsible of > 99% of the natural CO₂ fixation (approximatively 100 gigatons of carbon per year).

The ribulose-1,5-bisphosphate carboxylase/oxygenase, termed RuBisCO, is the key carboxylase of the CBB cycle, and is considered as the most abundant protein on Earth. This enzyme converts one molecule of CO₂ and one molecule of ribulose bisphosphate (RuBP) sugar into two molecules of 3-phosphoglycerate (3PG). Of note, this enzyme also catalyses an off-target reaction of oxygenation of the RuBP into 3PG and 2-phosphoglycolate (2PG) (Figure 1). This ‘parasitic’ reaction is also called photorespiration.

Figure 1: RuBisCO carboxylation

The basic functional unit of all RuBisCO is a homodimer of large subunits (L₂) that constitutes the core feature of the enzyme and contains magnesium (Mg²⁺) as a cofactor. Depending on the RuBisCO family, the enzyme may form more complex structures. For instance, the Form I RuBisCO corresponds to L₈S₈ complexes of large (L) and small (S) sub-units. The small subunit is not strictly necessary for carboxylation, but increase the efficiency of the reaction. This type of RuBisCO also generally required a chaperonine to improve folding and avoid aggregates.

Interestingly, the CBB cycle has been previously implemented in heterotrophic chassis of interest such as the bacteria Escherichia coli (Antonovsky et al., Cell, 2016) and the yeast Pichia pastoris (Gassler et al., Nature Biotechnology, 2019). This was mainly possible because only two key enzymes are required [RuBisCO and phosphoribulokinase (PRK)] to reconstitute a variant of the CBB cycle, the other enzymes being part of the ubiquitous pentose phosphate pathway.

Genes encoding RuBisCO enzymes can be identified in the genome of heterotrophic organisms. We identified very few Streptomyces (e.g. Streptomyces bottropensis ATCC 25435, Streptomyces nanshensis) that encode RuBisCO. In particular, we identified a region of 18 kb within the genome of S. bottropensis ATCC 25435 harboring genes encoding both RuBisCO, PRK and some other enzymes (Figure 2). Moreover, this DNA region is bordered by mobile sequences (encoding transposases and/or recombinases), present a lower GC than the rest of the genome (69% versus 72% GC for the whole genome) and is located in the most variable part of the genome that is enriched in specialized metabolite biosynthetic gene clusters. Altogether, these observations suggest that the RuBisCO and PRK in S. bottropensis ATCC 25435 could be part of a genomic island acquired by horizontal gene transfer. This exiting observation prompted us to characterize these enzymes in more detail.

Figure 2: Genomic island of Streptomyces bottropensis ATCC 25435 encoding notably RuBisCO and PRK genes

Finally, this genomic island constitutes a natural source of GC-rich genes coding for CBB, which is valuable in the context of our project since this type of sequence is too complex and therefore very difficult (if not impossible) to synthesize.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 391
  

References

Noam Prywes, Naiya R Phillips, Owen T Tuck, Luis E Valentin-Alvarado, David F Savage, "Rubisco function, evolution, and engineering", 2022, arXiv:2207.10773 - https://doi.org/10.48550/arXiv.2207.10773

Antonovsky N, Gleizer S, Noor E, Zohar Y, Herz E, Barenholz U, Zelcbuch L, Amram S, Wides A, Tepper N, Davidi D, Bar-On Y, Bareia T, Wernick DG, Shani I, Malitsky S, Jona G, Bar-Even A, Milo R. "Sugar Synthesis from CO2 in Escherichia coli." Cell. 2016 Jun 30;166(1):115-25. https://doi.org/10.1016/j.cell.2016.05.064

Gassler, T., Sauer, M., Gasser, B. et al. "The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2". Nat Biotechnol 38, 210–216 (2020). https://doi.org/10.1038/s41587-019-0363-0

Reference of Streptomyces bottropensis ATCC 25435 genome: GCF_000383595.1_ASM38359v1