Project Description

Figure 1: Autotrophy in plant cells. Illustration by Sokrich Ponndara (iGEM GO Paris-Saclay 2022 team member)

Synthetic autotrophy - a solution to climate change?

In many industries such as the food or pharmaceutical industry, we are still reliant on various living organisms to produce many vital resources. Most of these organisms such as the yeast or E. coli bacteria, that are widely used for insulin production (Baeshen et al., Microbial Cell Factories, 2014), are heterotrophic, which causes significant amounts of CO₂ being emitted to the atmosphere during the production process. Nonetheless, there have been successful attempts at implementing autotrophy both in Pichia pastoris industrial yeast (Gassler et al., Nature Biotechnology, 2020) as well as in E. coli (Antonovsky et al., Cell, 2016), which has been especially interesting for our project. This implementation of a variant of the Calvin cycle relies on the introduction of only two genes encoding the famous Ribulose-1,5-bisphosphate Carboxylase Oxygenase (RuBisCO) and phosphoribulokinase (PRK). Inactivation and/or punctual mutations of some genes are also required to adapt the carbon flow within the cell so that hexose, pentose and triose sugars synthesis really relies only on CO₂ fixation (Antonovsky et al., 2016), (Gassler et al., 2020), (Liang, et al., 2020). This genetic adaptation has been mainly achieved by directed evolution.

CO2CURE - antibiotics from CO₂

On the basis of these ground-breaking results, at team iGEM GO_Paris-Saclay, we have decided to implement the same strategy and introduce a variant of the Calvin cycle into Streptomyces bacteria, a new chassis at the iGEM competition! To make this new chassis known to a large iGEMer community, we have described the protocols and characteristics of several strains in a STREPTObook.

Streptomyces are soil bacteria mostly known for their complex life cycle (uni- to multi-cellular transitions, sporulation, metabolic differentiation - Figure 2) and the production of specialized metabolites. They have uncommon features for bacteria such as being multicellular (Figure 3) and having a linear genome! What a fancy chassis for the iGEM.

Streptomyces produces antibiotics along with other highly valuable molecules and specialized metabolites used in laboratories. These multicellular bacteria provide at least one third of all clinically useful, naturally obtained antibiotics (Bibb, Biochemical Society Transactions, 2013; Kieser, 2000), thus finding a solution to make them autotrophic would significantly reduce the amount of CO₂ emitted by the production of antibiotics. Beyond the environmental aspect, it will also reduce the costs of growing the bacteria, since instead of using traditional carbon sources, bacteria will be able to use the overabundant CO₂.

Figure 2: Developmental cycle of Streptomyces. Illustration by Sokrich Ponndara (iGEM GO Paris-Saclay 2022 team member)

Figure 3: Pictures of Streptomyces coelicolor. A. Colonies grown on plate several weeks. Blue and red colours are due to the production of actinorhodin and undecylprodigiosin antibiotics, respectively. Drawing of the colonies by Tiroumagale Tillay (iGEM GO_Paris-Saclay 2022 team member). B. Streptomyces filaments under the microscope. Discover a lot more of Streptomyces species in our STREPTObook!

To turn any Streptomyces into autotrophs, we needed first to introduce the genes encoding RuBisCO and PRK. At this stage, we were faced with a major challenge: these bacteria genomes are so GC-rich (approx. 70% GC!) that the synthesis of genes optimized for expression in Streptomyces is very difficult and in some cases impossible. By bioinformatic analyses, we therefore looked for an organism that naturally possesses GC-rich genes encoding RuBisCO and PRK. Amazingly, we found out that a strain of Streptomyces, namely Streptomyces bottropensis ATCC 25435, harbours a genomic region of 18 kb that may be a genomic island encoding part of the Calvin cycle that has been acquired by horizontal gene transfer! (Figure 4).

Figure 4: Genomic island of Streptomyces bottropensis ATCC 25435 encoding notably RuBisCO and PRK genes. 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. Drawn using SnapGene.

We thus cloned the native GC-rich sequences of RuBisCO and PRK genes of Streptomyces bottropensis ATCC 25435 in integrative vectors for an expression in Streptomyces. By the way, we introduce in the iGEM registry a new cloning module (BBa_K4370010) harboring promoter and RBS sequences designed for an optimal expression in Streptomyces .

Moreover, we designed sequences optimized for an expression in E. coli of RuBisCO (BBa_K4370002, BBa_K4370003) and PRK (BBa_K4370005) genes of Streptomyces bottropensis ATCC 25435 for further characterization of these enzymes.

We built a mathematical model of the implantation of RuBisCO and PRK in Streptomyces. Since our model confirmed that the depletion of the gene encoding the glycerophosphate mutase may allow a better fixation of the CO₂ by a variant of the Calvin cycle, we design single guide RNA (sgRNA) directed against this gene (BBa_K4370012). These sgRNA were introduced in a CRISPR-Cas9 plasmid and introduced by conjugation in Streptomyces.

Finally, since we noticed that antibiotic production was lower on low carbon condition, we also designed and cloned sgRNA directed against a xenogeneic silencer named Lsr2A (BBa_K4370011) to relieve the expression of specialized metabolite biosynthetic gene clusters.

Thereafter we were ready to perform all experiments to perform the proof of concept of our project!

Future of the project - beyond antibiotics

The CO2CURE project will enable a faster, more affordable, and sustainable bioproduction of antibiotics and many other useful derivatives, as we build a universal vector to transfer the Calvin cycle into various Streptomyces species. Our project also introduces fundamental progress in the field of synthetic biology.
We hope that the CO2CURE project will encourage the synthetic biology community to introduce more cycle-based processes into the industry, as they are greatly environmentally sustainable.

Project Summary

• Goal
Produce a strain of Streptomyces hemi autotrophic (semi autotrophic) and produce antibiotics.

• 2 ways
- Insert genes encoding RuBisCO and PRK into an antibiotic-producing strain
- Insert a cluster leading the biosynthesis of antibiotics into a naturally CO₂-binding strain of Streptomyces.

• Chassis
- A genus of multicellular bacteria belonging to the family Actinomycetota.
- Linear genome very rich in GC (72%).
- This genus produces more than a third of the antibiotics used in human medecine.
- These bacteria are mostly chemoorganoheterotrophic.

• Genes to add
It is possible to make E. coli CO₂ fixative (via the Calvin cycle) by inserting into their genome the genes encoding RuBisCO and PRK. The aim of CO2CURE is to render Streptomyces autotrophic in the same way.

• Purpose/Idea:
- Close the carbon cycle for the production of antibiotics.
- Produce antibiotics from Streptomyces in a less expensive way.

• Strategy
We cloned into Streptomyces vectors the GC-rich sequences encoding encoding the RuBisCO (BBa_K4370002, BBa_K4370003) and PRK (BBa_K4370005) of Streptomyces bottropensis ATCC 25435. Furthermore, to increase antibiotic production and promote the Calvin cycle, we engineered genetic tools via the CRISPR-dCas9 system to switch off the expression of Lsr2A (a xenogeneic silencer, sgRNA encoded by BBa_K4370011) and the phosphoglyceromutase (sgRNA encoded by BBa_K4370012), respectively.

Please go to the pages, engineering success, proof of concept, chassis registry results and STREPTObook to know more about our results!