Engineering

• Synthetic autotrophy

Design

We sought to introduce the Calvin Cycle into strains of Streptomyces, a genus of bacteria which produces antibiotics along with other highly valuable molecules and specialized metabolites used in laboratories. To introduce the Calvin Cycle into these bacteria, we sought to transfer the genes encoding RuBisCO (Ribulose-1,5-bisphosphate Carboxylase Oxygenase) and PRK (Phosphoribulokinase), as they are the only two missing enzymes that are needed to recreate a variant of the Calvin cycle in heterotrophic organisms (Antonovsky et al., Cell, 2016 ; Gassler et al., Nature Biotechnology, 2020).

However, to meet this challenge, we had to overcome a major problem: the Streptomyces genome has extremely high GC content (72%), which makes the chemical synthesis of genes difficult or impossible. Thus, we had to find a natural source of GC-rich genes encoding RuBisCO and PRK !

First, we thought of using type II RuBisCO (CbbM) from Rhodospirillum rubrum ATCC 11170, phosphoribulokinase (prkA) from Synechococcus elongatus PCC 7942 as it was previously done in E. coli (Antonovsky et al., Cell, 2016 ; Gassler et al., Nature Biotechnology, 2020). However, due to high GC content in Streptomyces (that makes these sequences difficult/impossible to synthesize), we decided to look for versions of those two genes that are already GC rich in nature.

Looking for such a natural source of GC rich genes, we scoured through the genomes of different representatives of the Streptomyces genus. As they were previously known to be heterotrophic, we did not expect to find any of our genes of interest. However, we made a discovery : a rather poorly described (draft genome) Streptomyces species, S. bottropensis, already possesses those two genes! The genes encoding PRK and RuBisCO are located close together and form part of a genomic island surrounded by mobile elements (transposases / recombinases) (Figure 1). This fact suggests that the region might have been acquired via a horizontal transfer from a different species.

Figure 1: Genomic island of S. bottropensis ATCC 25435 encoding notably RuBisCO and PRK genes

Thus, we thought of using it as a source of genes for our project! The region is about 69% GC rich, thus presenting a close resemblance to genes already present in Streptomyces. Furthermore, often GC rich genes cannot be chemically synthesized, so using these already existing genes seemed to us like the most optimal solution.

We also considered using Carbonactinospora thermoautotrophica (or Streptomyces thermoautotrophicus) since we also discovered that this strain is autotrophic. However, due to the fact that C. thermoautotrophica is thermophilic and that according to some sources (Volpiano et al., Systematic and Applied Microbiology, 2021), and that it is no longer considered to be a part of the genus of Streptomyces, we have decided to use S. bottropensis as the source of the genes encoding RuBisCO and PRK in our experiments.

After this brainstorming stage, we developed a roadmap for our cloning strategies described in the document entitled "IGEM_CO2_CURE_Cloning_Roadmap".

All the protocols we used to characterize the Streptomyces phenotypes are detailed in the STREPTObook.

Test

Before implementing the genes from S. bottropensis into other bacteria, we decided to do a simple test to see if the species was perhaps naturally autotrophic. If it were autotrophic, we would be sure of the functionality of RuBisCO and PRK from this species. To test it, we simply grew S.bottropensis and S.ambofaciens (a different species of Streptomyces that does not contain PRK or RuBisCO containing genes) in a minimal medium without any carbon source.

The results were quite outstanding since we have observed some growth for both S. bottropensis (containing RuBisCO and PRK genes) but also other Streptomyces including S. ambofaciens (Figure 2).

Figure 2: S. bottropensis and S. ambofaciens grown during 4 days on minimal medium devoid of any carbon source at 30°C under standard atmosphere

However, after restriking those colonies on new plates, we did not achieve the same results: the strains did not grow anymore.

Learn

This result suggests that Streptomyces can initiate a growth in absence of carbon source (maybe thanks to internal carbon stocks) but cannot maintain a prolonged autotrophic growth. Thus we discovered that S. bottropensis is in fact not autotrophic, but we decided to continue our testing.

Test

We have also compared the growth of biomass between those same two strains of Streptomyces in standard (0.0415 % CO₂) and CO₂ enriched (3 % CO₂) atmospheres. The final biomass reached by S. bottropensis in sub-minimal liquid medium under 3 % CO₂ enriched atmosphere was twice the final biomass reached in this medium in a standard atmosphere (Figure 3)!

Such a doubling in the biomass under enriched atmosphere was not observed with S. ambofaciens (our control strain) grown in the same conditions.

Figure 3: Biomass reached after 4 days of culture of Streptomyces under standard (0.0415 % CO₂) or CO₂ enriched (3 % CO₂) atmosphere. The media were inoculated with an initial biomass of approximatively 0.04 g.

Learn

Perhaps high concentration of CO₂ is a condition for induction of RuBisCO and PRK coding island expression in S. bottropensis. This result further encouraged us to use S. bottropensis, a source of our genes of interest.

Design

We have decided to work on two axes. In the first axis, we attempted to introduce the genes encoding RuBisCO and PRK from S. bottropensis into other Streptomyces species. In the second axis, we planned to introduce these same two genes optimized for E. coli into E. coli, in order to further test the functionality of the enzymes.

Build

All genes of interest were cloned under standard-GC (50 %) or high GC-version (70%) and codon-optimized for an expression in E. coli and Streptomyces, respectively

We have successfully cloned:

• cbbL-cbbS (RuBisCO) (BBa_K4370002, BBa_K4370003) optimized for an expression E. coli under the control of the tetO promoter and an RBS cloned into pGEM-T-easy plasmid.
• cbbL-cbbS-cbbX (RuBisCO), operon of S. bottropensis (BBa_K4370002, BBa_K4370003), wild type under the control of kasOp promoter (BBa_K4370006) and a strong RBS (BBa_K4370007) cloned into pSET152 integrative vector.
• prk gene of S. bottropensis (BBa_K4370005) under the control of kasOp promoter and a strong RBS cloned in pSET152 integrative vector.
• prk optimized for an expression in E. coli (BBa_K4370005) under the control of arabinose promoter cloned into pBAD33 plasmid.

Test

We performed a High Performance Liquid Chromatography (HPLC) on cell extracts of S. bottropensis as well as E. coli containing a plasmid with genes encoding the small and large subunits of S.bottropensis RuBisCO codon-optimized for E. coli and a control E. coli containing the same vector backbone with a different insert. To measure RuBisCO activity, RuBP (ribulose-1,5-bisphosphate) - a substrate of RuBisCO was added to the samples, to detect the appearance of RuBisCO’s product - PGA (3-phosphoglycerate). To do it, we measured the absorbance of the samples at 220 nm one minute after the addition of RuBP. Due to a very high baseline in E. coli samples, the results are inconclusive (Figure 4). In S.bottropensis, we detected a small spike attributed to the appearance of PGA in one of the conditions (Figure 5), correlated to the disappearance of RuBP (what is expected for a RuBisCO activity). This result is promising but needs to be confirmed by mass spectrometry analysis (to confirm the identity of the peak attributed to PGA production).

Figure 4: HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of E. coli harboring a plasmid encoding ccbL (BBa_K4370002) and cbbS (BBa_K4370003) extracts under the control of tetO promoter (BBa_R0040), or control.

Figure 5: HPLC analysis of RuBisCO assay perform with RuBP (ribulose-1-5-bisphosphate) and cell extract of S. bottropensis grown 4 days at 30°C under different conditions.

Learn

We have only performed this procedure once, due to time constraints so we suspect a problem with the execution of the experiment. The experiment would have to be repeated to gain more substantial information about the functionality of RuBisCO from S. bottropensis. However, the experiments conducted previously give us a lot of hope about the future development of the project.

Test

To test the activity of the PRK enzyme from S.bottropensis, we analyzed the impact of its overexpression in E. coli chassis. Indeed, Parikh et al. (Parikh et al., Protein Eng Des Sel, 2006) previously reported that the overexpression of the PRK from Synechococcus PCC7942 is toxic in this chassis when arabinose is used as a carbon source.

A codon-optimized version of the prk gene of S. bottropensis ATCC 25435 (BBa_K4370004, with an RBS in BBa_K4370005) was cloned under the control of an RBS (BBa_K4370005) into the L-arabinose-inducible expression vector pBAD33. This plasmid or an empty pBAD33 vector (‘pBAD33-Ø‘) were introduced in two E. coli K12 genetic backgrounds, one being able to use arabinose as a carbon source (DH5α), the other not (BW25113). Bacteria were grown on M9 minimal (Sambrook and Russell, 2001) agar plates (supplemented with 1 mM MgCl₂, 0.1 mM CaCl₂ and 0.1 % thiamine) including 0.2 % glucose and/or arabinose and 30 µg/ml chloramphenicol.

After two days of growth, we observed that the induction of the expression of BBa_K4370005 is toxic to E. coli, only when the cells use arabinose as the only carbon source. The effect is actually only visible at the lowest cell densities (maybe because at higher density, arabinose concentration is lower).

Figure 6: Growth of E. coli strains harboring pBAD33-BBa_K4370005 or a control vector on M9 supplemented minimal medium in the presence of glucose and/or arabinose. Five µl of bacteria resuspended in M9 medium devoid of carbon source were spotted on the plate. The first dilution (‘10^-1’) was adjusted to OD_{600 nm} 0.4 for all strains. The experiments were performed independently for ara⁺ (DH5) and ara^- (BW25113) strains.

Learn

This confirms that the PRK from S. bottropensis is active, and impacts E. coli metabolism when arabinose is the only source of carbon as the PRK from Synechococcus PCC7942 does.

In the course of this work, we designed a prk sequence optimized for an expression in E. coli (BBa_K4370004, with an RBS in BBa_K4370005). After constructing and testing the vectors, we learned that the expression of the biobrick is slightly toxic to these cells when arabinose is used as a source of carbon, as previously reported for another PRK. We learnt from this experiment that the genomic island present in S. bottropensis ATCC 25435 encodes a functional PRK enzyme. Thus it gives us hope that this key gene of the Calvin cycle could be introduced into other Streptomyces in order to implement carbon fixation.

Overall, we think that the experiments done to characterize both RuBisCO and PRK from S. bottropensis give a positive outlook on the feasibility of the project and that with more time and resources, our ultimate goal of producing antibiotics from CO₂, could be achieved.

Figure 7: Resume of the engineering approach of the implementation of synthetic autotrophy

• Alternative Cloning Module

Design

Streptomyces are chassis of great biotechnological interest to produce antibiotics but also antitumors, anti-cancer drugs, immunosuppressive agents, etc. Their genome is linear and highly rich in GC (circa 72 %). Their genetic modification generally relies on the introduction of plasmids by conjugation from E. coli to Streptomyces. It is therefore necessary to develop specific vectors (conjugative, GC rich, specific promoters) in order to use Streptomyces as chassis. Because of the challenging nature of working with Streptomyces, we have decided to clone the gene encoding PRK in two integrative vectors, pSET152 (mentioned earlier) and pOSV805.

In 2019, Aubry and collaborators published a set of 12 standardized and modular vectors (pOSV801 to pOSV812) designed to integrate genes of interest at specific loci in Streptomyces genomes (Table 1).

Table 1: List of the Streptomyces vectors of the pOSV collection

These vectors are composed of 5 modules: i) the replication module, ii) the antibiotic resistance cassette module (apramycin, hygromycin or kanamycin resistance), iii) the origin of transfer module, iv) the integration module (into PhiBT1, PhiC31, pSAM2 or VWB integration sites), and v) the cloning module. In this collection, this latter does not contain any Streptomyces promoter.

We designed a part containing the Streptomyces constitutive strong promoter KasOP* (BBa_K4370006) followed by a RBS from of capsid protein obtained from Streptomyces temperate phage ϕC31 (BBa_K4370007) that has been previously described as highly efficient to promote translation when combined with the kasOP* promoter (Bai et al., PNAS, 2015). The part also contains a cassette for the expression of mRFP1 (BBa_K4370008) under the control of an E. coli promoter (BBa_K1155000) and an RBS (BBa_B0034). This RFP expression module is followed by two stop codons and two terminators (BBa_B0010 and BBa_B0012). This cassette is meant to be replaced by the construction of interest and offers an easy means of visual screening the clones (pink if the E. coli cassette is still present in the plasmid i.e. if the clone contains the parental plasmid, white if not)(Figure 1).

Figure 8: Genetic organization of pOSV805 vector and of its modified version harboring BBa_K4370010 part

Since BBa_K4370010 contains two promoters (one active in Streptomyces, one active in E. coli), we had to check first that there was no promoter interference and that the mRFP1 gene could be properly expressed from this module at a level sufficient to allow a visual screening for further applications.

Build

We first introduced our biobrick (kasOP_RBS_rfp) into the pGEM-T-easy plasmid via TA cloning and cloned it in E. coli. Then, we digested this plasmid containing our biobrick, as well as the integrative plasmid - pOSV805, with the NotI restriction enzyme. This allowed introducing the biobrick into the integrative vector at the cloning module site (Figure 8).

Test

We observed the color of the colonies on the plates. We could clearly distinguish red and white colonies, harboring or not the BBa_K4370010 part, respectively (Figure 9). Thus, whereas bacteria pOV805 were blue, the bacteria transformed by pOSV805- BBa_K4370010 were now red.

Figure 9: E. coli colony phenotype after transformation with a ligation mix harboring pOSV805-BBa_K4370010 or pOSV805 devoid of cloning module

Learn

From this experiment, we learnt that pOSV805-BBa_K4370010 can now be used as a chassis vector for further cloning of gene of interest for a constitutive expression in Streptomyces, the red/white screening being a convenient means of screening the clones containing the new construction. Additionally, we learnt that the presence of the promoter active in Streptomyces does not interfere with the expression of the cassette optimized for E.coli.