This year, ShanghaiTech_China developed an autotrophic sustainable multi-microbial production platform named Mini Bioproduction Cycle System (MBSC). We customized the platform for Martian conditions and launched the MBCS-Mars version. The MBCS-Mars is believed to be able to effectively use the atmospheric resources and light energy resources of Mars to continuously produce nutrients for human beings.

The core of our project is the ternary microbial symbiosis system made up of S. elongatus, A. caulinodans and E. coli. To successfully construct this symbiosis system, the circulation of nutrients, the regulation of nutrient through intercellular genetic circuit and the separate-immobilized fermentation are crucial. Therefore, the proof of concept will illustrate the above three aspects.

We hope to build effective nutrient circulations among the above three microorganisms to attribute to their symbiosis. NH₄⁺ is an ion that can be easily used by microorganisms, so there is no problem with the circulation of nitrogen sources. However, due to the preference of different microorganisms in using carbon sources, there are some problems in the circulation of carbon sources. To prove this concept, the circulation of carbon sources needs to be opened up and verified.

Overview of experimental design

For the circulation of nutrients, we firstly proved that sucrose can flow from S. elongatus to E. coli as a usable carbon source. To open up the circulation of sucrose, sucrose transporter gene cscB was integrated in the genome of S. elongatus and E. coli was transformed to express the fructofuranosidase gene SacC (See details and principles on Results page).

We detected the sucrose concentration of supernatant and OD685 value every 24 hours and lasted for 3 days. We compared the accumulation of sucrose secreted by a unit number of S. elongatus in a certain period of time by comparing the ratio of sucrose concentration in the supernatant to OD₆₈₅ value of S. elongatus (Figure 1). The results show that under conditions we tested, our engineering cyanobacteria (integrated with transporter gene cscB) have a better capacity for sucrose production than wild-type ones. In addition, the combination of IPTG (1 mM) and high salt (100mM NaCl) (Figure 1d) is most efficient in increasing secreted sucrose levels (2.5 ~ 3.2 folds).

Figure 1. The ratio of sucrose concentration in the supernatant to OD₆₈₅ value after inducing.

In our system, sucrose produced by cyanobacteria should be directly utilized by E. coli. However, some previous papers have reported that wild-type E. coli K12 strains can't utilize the sucrose to grow and produce very well^[4]. To improve the sucrose utilization efficiency, we have expressed a fructofuranosidase (SacC), an enzyme that catalyzes the hydrolysis of sucrose into fructose and glucose from Mannheimia succiniciproducens.

A series of constructs expressing SacC with different transcriptional activity was constructed and then transformed into E. coli DH5α (Figure2). Based on the activities of Anderson promoters have been well quantified, the order of expression level of these four constructs is expected as follows: J23101-SacC > J23107-SacC > J23109-SacC > Empty.

Figure 2. Four constructs expressing SacC in E. coli at different levels.The expected expression levels are shown on the right.

Then we cultured all these four strains in an M9 minimal medium which contains sucrose as the sole carbon source. Their growth is quantified by measuring optical density at 600 nm (OD₆₀₀). All of the groups expressing SacC grow faster than Empty in the first 14 hours. Furthermore, compared with J23109-SacC, the SacC high expression groups J23101-SacC and J23107-SacC showed higher growth rates, along with the dose-dependent phenomenon (Figure 3A). After culturing for 10 hours, we take out the culture medium to test the remaining sucrose. The residual sucrose concentrations of culture medium in the SacC-positive groups were significantly lower than that of Empty (Figure 3B). The higher the SacC expression level, the lower the remaining sucrose concentration. These demonstrate that the appropriate expression level of SacC can help E. coli use sucrose as the sole carbon source to grow.

Figure 3. Sucrose utilization profiles of E. coli strains in the medium containing sucrose as a sole carbon source. (A) Growth curves of four strains. (B) Remaining sucrose concentrations in culture medium after 10 hours. Blank medium: the M9 minimal medium without adding E.coli was kept at the same condition as other groups for 10 hours.

In summary, we have successfully engineered cyanobacteria S. elongatus suitable for sucrose secretion by integrating sucrose transporter gene cscB and found out the optimal condition for sucrose secretion. Also, we have successfully engineered E. coli for efficiently using of sucrose by transforming with the differently constructed fructofuranosidase gene SacC. Thus, the secreted sucrose by engineered S. elongatus would be able to support the growth of engineered E. coli.

Overview of experimental design

We established that E. coli could produce acetate, which could support the growth of A. caulinodans, a nitrogen-fixing bacteria that uses acetate as the solo carbon source. E. coli is known to produce organic acids including acetate after culture ^[3]. In mimic our symbiosis conditions, we tested whether our engineered E. coli with SacC gene could efficiently produce acetate with sucrose as the solo carbon source. We compared E. coli transformed with empty vector, J23101-SacC, J23107-SacC, and J23109-SacC with sucrose as the solo carbon source. Interestingly, we found fast-growth groups J23101-SacC and J23107-SacC had a lower PH compared to empty vector or slow-growth J23109-SacC (growth curve is showing in Figure 3), suggesting that our engineered E. coli could mimic symbiosis conditions to produce acetate.

Next, we cultured A. caulinodans in medium containing acetate as the sole carbon source (the CoBG11-Sucrose group was negative control, since when the medium contains non-metabolizing saccharides like source as the sole carbon source). The growth curve results showed that A. caulinodans can efficiently use acetate as the sole carbon source (Figure 5).

Together, these results suggest that our engineered E. coli could mimic symbiosis conditions to produce acetate, which is sufficient to support the growth of A. caulinodans.

Figure 4. pH value of medium after culturing for 48 hours. From left to right: Empty, J23101-SacC, J23107-SacC, J23109-SacC

Figure 5. The growth curve of A. caulinodans in CoBG11-Sucrose/Acetate medium.

Previous attempts at artificial microbial symbiosis mostly failed because of the mismatched rates in the consumption and production of nutrients among the subpopulations. So, it is important to induce the regulation of nutrient by building intercellular genetic circuit feedback. To prove this concept, three aspects of gene pathways were designed and verified. These genetics pathways includes the response to nutrient deficiency, the transmission of intercellular signals, and the regulation of nutrient output.

In the genetic circuit, starvation promoters in E. coli are sensors for nutrient signals. Our goal is to find a promoter with higher activity at low glucose concentrations. After three "Design-Build-Test-Learn" cycles of optimization, we finally get PcstA_Mutant1 as the best one for the nutrient response. PcstA_Mutant1 has a 5-fold activity relative to J23101 under low glucose concentration, and a fold change of 8.2. These prove that we can sense nutrient signals through a starvation promoter.

Figure 6. FI/OD₆₀₀ of PcstA variants. All the constructs are cloned into pET low-copy number backbone.

The quorum sensing luxR system was constructed and tested in E. coli as preliminary proof of concept. The results showed that the luxR system worked well in E. coli, both in the signal response and signal synthesis (Figures 7 & 8) (See details and principles on Results page).

Figure 7. Induced sfGFP expression of the receiver with 3O C6 HSL at concentrations ranging from 10^-4 to 10^-14 M.

Figure 8. Induced sfGFP expression of the receiver with the supernatant of the signal sender culture. Blank: Culture of receiver strain without treating with 3O C6 HSL

In wet lab experiments, we only successfully validated the signal communication in E. coli. The response in S. elongatus and A. caulinodans should be achieved. The luxR response gene parts have been constructed in the S. elongatus and A. caulinodans. In principle, this genetic circuit could be functional in these bacteria^{[1, 2]}. However, due to limited time and hard growth of these uncommonly used bacteria in lab, we could not functionally verify the genetic circuit in S. elongatus and A. caulinodans. To further support our principle of concept, we also used modeling to strengthen our proposals. Interestingly, through modeling, we learned that AHL molecules cannot be naturally degraded, and in order to achieve the feedback of the intercellular genetic circuit, it is necessary to introduce homoserine lactone degrading enzyme to inactivate the AHL molecule at the end of the feedback phase (See details and principles on Model page, also see below part).

For the regulation of nutrient output, we have proved the controllable output of sucrose from engineered Cyanobacteria S .elongatus. The engineering Cyanobacteria was introduced the cscB gene controlled by lac promoter, which upregulated the output of sucrose under IPTG induction (Figure 1).

Secondly, to provide nitrogen source for our symbiosis system, the controllable output of ammonium by A .caulinodans was established in literature and partially finished in our experiments. After two rounds of engineering optimization, the ΔnifA strain was successfully constructed and validated by PCR (Figure 9) and DNA sequencing (See on Results page). Due to the limitation of time and experimental conditions, but the direct experimental verification has not been done. Thankfully, the increased production of ammonium using the same strategy has been achieved the previous studies ^[1], which indirectly proved our concept.

Figure 9. Colony PCR of ΔnifA colony.

Due to the technical difficulty and time limitations, we could not verify the whole intercellular genetic circuit through experiments during the competition. Therefore, we built a series of Molecular Dynamics Models on the computer to verify the effect of the whole pathway in maintaining nutrient stability through simulation. Finally, we obtained very exciting results. In the nitrogen source regulation and carbon source regulation respectively, the nutrients in the system maintained stable oscillations with time (Figure 10 & 11) (See details and principles on Model page).

Figure 10. Simulation results of nitrogen regulation

Figure 11. Simulation results of carbon regulation

The separate-immobilized fermentation pattern we proposed should be how the three microbial symbiotic system is implemented in practice. To prove this concept, the three microorganisms should survive under some of the same medium conditions, immobilized cells in co-culture should remain viable for a period of time, and material exchange between immobilized cells needs to be verified.

It is essential to maximumly match the growth conditions for the three kinds of bacteria in the Ternary microbial symbiosis system. We made effect to find a common medium that could support the growth of all three bacteria with only minor modifications. We found that the CoBG11 media could be minimally modified to support the growth of all three bacteria. In detail, the CoBG11-Sucrose medium protocol was proposed by adding sucrose as solo carbon source to simulate the sucrose secretion of S. elongatus. It turned out that both E. coli and S. elongatus can live and grow in CoBG11-Sucrose medium (Figure 12.a&b). As mentioned above, A. caulinodans cannot live and grow when the medium contains only saccharides as solo carbon source (Figure 12.c). So the new CoBG11-Acetate medium protocol was proposed by adding acetate as solo carbon source which would ultimately come from the acetate secretion of E. coli. Later, A. caulinodans was cultured both in CoBG11-Sucrose and CoBG11-Acetate to prove that A. caulinodans can successfully live in CoBG11-Acetate medium (Figure 5) (See medium information on Experiments Page). In summary, we show that the CoBG11 media could support the growth of all three bacteria with minor modification and propose that CoBG11 media could be used as a common medium for our symbiosis system with little modifications once their metabolic flows established.

Figure 12. The growth curve of three microorganisms in CoBG11-Sucrose medium.

Three microorganisms were immobilized and cultured in the same medium condition (Figure 13). In this experiment, sucrose and acetate were added to the CoBG11 medium (called CoBG11-PLUS) to simulate the sucrose secretion of S. elongatus and the acetate secretion of E. coli (See medium information on Experiments Page).

Figure 13. Immobilized E. coli, A. caulinodans and S. elongatus in the same CoBG11-PLUS medium. In the conical flask, tagRFP-marked E. coli (pink red, see red arrow), A. caulinodans (white; see white arrow) and S. elongatus (green, see green arrow) were shown.

After a week of release of immobilized cells and OD measurement, OD survival curves did not show a significant downward trend during a week (Figure 14). The results showed that all three immobilized microorganisms can maintain high viability in the gel and in the same medium condition.

Figure 14. The survival curve of three immobilized microorganisms.

We did experimental validation of the intercellular communication in immobilized E. coli through the LuxR system as a demo of intercellular signal communication of our system as well as intuitive proof of concept that substances can circulate between immobilized cells.

After two days of co-culture and induction, the two experimental groups showed obvious fluorescence enhancement compared with the negative control groups, which indicated the success of intercellular communication in immobilized E. coli (Figure 15).

Figure 15. Intercellular communication in immobilized E. coli through LuxR system. The top dishes are the negative control group while the bottom dishes are the experimental groups. There are two parallel groups.

[1] Ryu, M.H., Zhang, J., Toth, T., Khokhani, D., Geddes, B.A., Mus, F., Garcia-Costas, A., Peters, J.W., Poole, P.S., Ané, J.M., et al. (2020). Control of nitrogen fixation in bacteria that associate with cereals. Nat Microbiol 5, 314-330.

[2] Sun, X., Li, S., Zhang, F., Sun, T., Chen, L., and Zhang, W. (2021). Development of a N-Acetylneuraminic Acid-Based Sensing and Responding Switch for Orthogonal Gene Regulation in Cyanobacterial Synechococcus Strains. ACS Synthetic Biology 10, 1920-1930.

[3] Pinhal, S., Ropers, D., Geiselmann, J., and de Jong, H. (2019). Acetate Metabolism and the Inhibition of Bacterial Growth by Acetate. J Bacteriol 201. 10.1128/JB.00147-19.

[4] Lee, J.W., Choi, S., Park, J.H., Vickers, C.E., Nielsen, L.K., and Lee, S.Y. (2010). Development of sucrose-utilizing Escherichia coli K-12 strain by cloning beta-fructofuranosidases and its application for L-threonine production. Appl Microbiol Biotechnol 88, 905-913. 10.1007/s00253-010-2825-7.