Results

Chassis Registry

We conducted experiments to perform bioinformatics and phenotypic analyses (growth, antibacterial bioassays) as described in the STREPTObook on seven strains of Streptomyces. The main results are summarized in the following table.

Legend of the table: Except for [b] and [c], the color code is as follows: the bluer, the larger; the redder, the smaller. Results from [e], [f] and [g] correspond to the mean values of two independent experiments.

^aSMBGCs (specialized metabolite biosynthetic gene clusters) were predicted using AntiSMASH 6.0.

^bGeneration time was determined in exponential phase after inoculating 10⁶ spores in 10 ml of MP5 medium at 30°C under a regular atmosphere. Color code: the bluer, the shorter; the redder, the longer

^cLag phase in MP5 (as described in ^b) as considered as short if < 12h (blue), or long if > to 12 h (red).

^dThe biomass (wet weight) was measured after 5 days of growth as described in ^[b].

^eAntibacterial efficiency (ABE) (defined as the size in cm of the halo of inhibition of Micrococcus luteus growth) was determined after 3 days of growth as described in ^[b]. Antibiotic production (defined as the size in cm of the halo of inhibition of M. luteus growth) was determined after inoculating 5.10⁶ spores on MP5 solid medium, and growth during 3 days at 30°C under a regular atmosphere.

^fABE was determined after inoculating 5.10⁶ spores on sub minimal medium devoid of carbon source solid medium, and growth during 3 days at 30°C under a regular atmosphere.

^gABE was determined after inoculating 5.10⁶ spores on minimal medium containing 0.5 % glycerol (an industrial waste) devoid of carbon source solid medium, and growth during 3 days at 30°C under a regular atmosphere.

^hThe conjugation was performed for all strains in conditions optimized for Streptomyces ambofaciens engineering (page 11 of the STREPTObook).

Most of these experiments were performed only once due to time constraints. Moreover, errors could have crept into the bioinformatics analysis despite our efforts to be vigilant. These results are therefore transmitted with caution and deserve to be confirmed by the reproduction of the results and analyses by future members of the iGEMers community!

Strain Comparison

Based on all these results, our team wished to understand which of our strains is the best suited to carry the role of the main chassis in the CO₂CURE project.

• GC percent
All strains have a high proportion of GC in their genome (average 72.2 %). Therefore, it will not be a distinguishing criterion on which we will base our opinion. We will have to keep in mind that it means that to perform PCRs, the temperature of melting is high due to the high stability of GC bonds. Moreover, genes inserted in these genomes will require to be GC rich.

• Genome size
The Streptomyces genome is large: for instance, an E. coli K12’s genome is only 4.6 Mb long, almost only half the size of the one from Streptomyces rimosus ATCC 10970’s one. Streptomyces albidoflavus J1074 has the smallest genome, which can make it a chassis of choice in the context of approaches that aim to make a minimal Streptomyces genome. However there is no clear correlation between the genome size and the generation time or the number of SMBGCs. The size of the genome is therefore not an essential criterion for our project.

• SMBGCs (Specialized Metabolite Biosynthetic Gene Clusters)
At first sight, we might think that the more SMBGC the strain has, the more interesting the strain is. However, it’s not that simple. On one hand, if you prefer to maximize the production of diversified molecules, the most interesting strain is the one with the higher number of SMBGCs. This is S. rimosus ATCC 10970 with 42 SMBGCs, followed by Streptomyces venezuelae ATCC 10712 with 31. In this case, you should look for lab conditions that favor SMBGC expression (not always easy to identify…). In this context, the strains may be interesting as a source of new specialized metabolites.

On the other hand, we are often only interested in producing a type of antibiotic of interest whose pathway is either already present in the strain or can be introduced by genetic engineering. In this case, the best chassis can be the one with interesting natural capacities (e.g. production of spiramycin by S. ambofaciens, a strain used in the industry for this purpose).

Alternatively, the best chassis for heterologous expression of an SMBGC could be the one with the lowest SMBGCs. Indeed, this has at least two advantages: 1) it limits the consumption of metabolite intermediates for the production of specialized compounds other than the product of interest, 2) it avoids purifying too many contaminating specialized metabolites with the product of interest. In this case, S. albidoflavus J1074 could be considered as the best chassis.

• Genome potential regarding autotrophy
In order to add autotrophy in these organisms, we want to modify our strains by introducing the GC-rich sequences encoding RuBisCO and PRK. Fortunately, the genome of a Streptomyces, namely S. bottropensis ATCC 25435, already encodes these genes. Therefore, this strain can be both a good chassis and a source of genes for our project.

• Growth
We then decided to find out the generation rate and lag phase of each strain. The faster our strain grows, the faster we can pursue our work. On average, the generation time of all our strains is around 3 hours. The fastest strains were S. rimosus ATCC 10970 and S. bottropensis ATCC 25435 with a growth rate equal to 2.3 hours. The slowest was Streptomyces lividans TK24 with 4.4 hours. However, during our experiment, we discovered another parameter to determine the fastest strain, the duration of the lag phase. Indeed, the lag phase can be short (less than 12h) and long (more than 12h). This new criterion allowed us to separate the strains according to the duration of their lag phase. Taking into account both the generation time and the lag phase, the best chassis is S. rimosus ATCC 10970. Finally we evaluated the final biomass of the strains. S. bottropensis ATCC 25435 and S. venezuelae ATCC 10712 reach the highest biomass levels. However, more than the biomass, it is the production of antibiotics that interests us above all, which we discussed in the following section.

• Antibiotic production and impact of CO₂
To find the best chassis for antibiotic production we carried out several experiments to compare different aspects of seven Streptomyces species, the results of which are summarized in our STREPTObook. All of the strains naturally produce different antibiotics and other secondary metabolites, but we wanted to see which one would be the best candidate to introduce the Calvin Cycle into.

In one of our experiments, we grew all of the seven strains on different media, some of which contained additional CO₂ and then carried out a bioassay, in which we poured an indicator species - Micrococcus luteus around the Streptomyces colony, to observe how Streptomyces inhibits the growth of M. luteus. The results are usually in a form of a halo, the size of which indicates the efficiency of the biostatic effect of the antibiotics produced by Streptomyces (Figure 1, Figure 2).

We used three media: i) MP5 rich medium (optimized for the antibiotic production by S. ambofaciens ATCC 23877 – This medium contains 36 % glycerol, and therefore can be used to recycle industrial waste), ii) a minimal glycerol medium and iii) a subminimal medium without carbon source (as part of the objective of the project).

Figure 1: Results obtained by two Bioassays for antibacterial activity of different Streptomyces strains on sub-minimal medium (devoid of carbon source), on minimal medium + 0.5% glycerol, or on a rich medium optimized for antibiotic production by S. ambofaciens (MP5 medium containing 36 % glycerol) under regular atmosphere (0.0415 % CO₂) or under 3 % CO₂ (‘+CO₂’ in the graph). The error bars correspond to the SEM (standard error to the mean) of two independent experiments.

Figure 2: Antibacterial activity of some Streptomyces on plates with MP5 under regular atmosphere. Scale: the diameter of a plate is 8.5 cm.

From the results (Figure 1, Figure 2), we can see how the antibiotic production differs from strain to strain and medium to medium. We report for the first time the impact of CO2 on antibiotic production by Streptomyces. For most strains, the antibacterial activity tends to decrease in the presence of 3% CO₂.This decrease remains modest in most cases, so this provides proof of concept that antibiotic production in a CO₂-enriched atmosphere is possible.

Overall, from the results we can see that S. rimosus and S. venezuelae are generally more efficient in antibiotic production than other strains.

CO₂ Fixation

One of the first experiments we carried out was growing different Streptomyces species on a minimal medium containing no 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 3).

Figure 3: 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. 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. We did not get discouraged!

Beyond that, 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 4)!

Such a doubling in the biomass under enriched atmosphere was not observed with S. ambofaciens (our control strain) grown in the same conditions. A big step for the project. Perhaps this is a condition for induction of RuBisCO and PRK coding island expression in S. bottropensis.

Figure 4: 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.

Genetic engineering

We performed conjugation assays to test the ability of our strains to be modified following a standard protocol. We performed a conjugation from E. coli to Streptomyces of a test plasmid (pOSV805, Aubry et al., Appl Environ Microbiol, 2019) containing a gene for antibiotic resistance, using a protocol that was previously optimized for S. ambofaciens (Figure 5). All of the strains, except for S. rimosus, had successfully obtained the plasmid. These results indicate that all strains except S. rimosus can be easily genetically modified with an integrative plasmid and therefore are suitable for use as chassis. Despite promising results regarding antibiotic production in low carbon conditions, S. rimosus is not an optimal chassis regarding the conjugation assay. This means that this strain should either be eliminated from our list of potential candidates for the CO2CURE project, or that further experiments should be conducted to optimize the conjugation protocol for this strain. Fortunately, we were able to identify a publication in which an optimized protocol for this species had been published (Song et al., J Zhejiang Univ Sci B., 2019)). The S. rimosus chassis therefore remains potentially interesting.

Figure 5: Example of conjugation of pOSV805 integrative plasmid into S. venezuelae. The colonies correspond to exconjugants that have acquired the resistance marker (hygromycin resistance).

What makes a good chassis? survey

We then wondered what would other iGEM team pick as the best chassis among our strains. To answer such a question, we checked at the results from a form we transmitted to all iGEM teams. In this form, we asked all iGEM teams to rate from 1 to 5 some criteria to determine which characteristics from strains would be the most important to characterize to complete a chassis registry. While this book is being written, eleven iGEM teams have answered this form (Figure 6).

Figure 6: Results of the “What makes a good chassis?” survey, the teams that answered are Lund, Wageningen, Groningen, Duesseldorf, SUNY Oneonta, Pui Ching Middle School (2 times), NTHU_Taiwan, iGEM Aachen, IISc Bengaluru, iGEM IONIS.

While team teams rarely gave a rating of five, they often rated one or two the criteria, so we still can understand which ones are the most important. Among the most important ones, there is the generation time, final biomass of the production, the sensitivity of the chassis to the possible toxicity of the synthetic system and natural metabolic potential/properties of the chassis. Considering the generation time and the natural potential/properties of the chassis, S. rimosus ATCC 10970 is the best strain. Considering the final biomass, the best one would be S. bottropensis ATCC 25435/DSM40262.

Final Conclusion

Having all of this in mind, we think that S. rimosus ATCC 10970 is the best strain to conduct the CO2CURE project. It’s extremely high antibiotic production in all media accompanied by its short generation time and lag phase makes it the strain we must privilege. S. bottropensis ATCC 25435 is also quite interesting to consider since it encodes RuBisCO and PRK. This strain could be interesting as a chassis for SMBGC heterologous expression.