Article https://doi.org/10.1038/s41467-022-34190-z Circuit-guided population acclimation of a synthetic microbial consortium for improved biochemical production Received: 20 October 2020 Chae Won Kang1,6, Hyun Gyu Lim 1,6, Jaehyuk Won2,3,6, Sanghak Cha1, Giyoung Shin4, Jae-Seong Yang 5, Jaeyoung Sung 2,3 & Gyoo Yeol Jung 1,4 Accepted: 14 October 2022 Microbial consortia have been considered potential platforms for bioproces- Check for updates sing applications. However, the complexity in process control owing to the use 1234567890():,; 1234567890():,; of multiple strains necessitates the use of an efficient population control strategy. Herein, we report circuit-guided synthetic acclimation as a strategy to improve biochemical production by a microbial consortium. We designed a consortium comprising alginate-utilizing Vibrio sp. dhg and 3-hydroxypropionic acid (3-HP)-producing Escherichia coli strains for the direct conversion of alginate to 3-HP. We introduced a genetic circuit, named “Population guider”, in the E. coli strain, which degrades ampicillin only when 3-HP is produced. In the presence of ampicillin as a selection pressure, the consortium was successfully acclimated for increased 3-HP production by 4.3- fold compared to that by a simple co-culturing consortium during a 48-h fermentation. We believe this concept is a useful strategy for the development of robust consortium-based bioprocesses. Microorganisms grow as consortia across domains by optimizing has been performed widely, it is difficult to efficiently introduce large or community structures for their survival by acclimatizing to their complicated metabolic pathways, which are required for the utilization environments1–4. They communicate by the secretion and uptake of of non-conventional substrates (e.g., single-carbon gases, alginate) or metabolites or chemicals typically used for cellular communication5. production of complex chemicals (e.g., natural products). Microorgan- These communications facilitate diverse social interactions (e.g., isms that naturally exhibit such abilities can be considered potential commensalism, cooperation, amensalism, and competition). One of hosts; however, their use in bioprocessing is often limited owing to the important features that is observed in these community structures insufficient engineering tools or understanding16. On the contrary, the is the division of labor6–8. While community participants usually com- use of a microbial consortium does not require extensive engineering pete for limited resources, they often complement each other by and can exploit the innate abilities of microorganisms that have been performing different tasks, and also exhibit emergent properties, optimized through evolution. Furthermore, the division of labor which enables the expression of improved phenotypes (i.e., fitness) achieved by the spatial compartmentalization of metabolic pathways in compared to those expressed in independent cultures2,9–11. different cells may relieve the metabolic burden13,17 and provide addi- Based on these facts, microbial consortia have been studied as tional control for balancing various activities in the pathway18–20. Indeed, attractive bioprocessing platforms for biochemical production7,12–15. in the past years, several synthetic consortia have been developed for While single-cell engineering of genetically well-known microorganisms the conversion of various substrates (e.g., cellobiose21, cellulose22, 1 Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, Korea. 2 Creative Research Initiative Center for Chemical Dynamics in Living Cells, Chung-Ang University, 84 Heukseok-Ro, Dongjak-gu, Seoul 06974, Republic of Korea. 3Department of Chemistry, Chung-Ang University, 84 Heukseok-Ro, Dongjak-gu, Seoul 06974, Republic of Korea. 4School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, Korea. 5Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, Barcelona 08193, Spain. 6These authors contributed equally: Chae Won Kang, Hyun Gyu Lim, Jaehyuk Won. e-mail: gyjung@postech.ac.kr Nature Communications | (2022)13:6506 1 Article https://doi.org/10.1038/s41467-022-34190-z carbon monoxide23) and production of biochemicals (methyl halides24, Supplementary Data 1). Therefore, it was expected that co-culturing oxygenated taxanes19, muconic acid25, vitamin C26, hydroxytyrosol27, could be effectively used to convert alginate into 3-HP with acetate as cadaverine28). an intermediate. While the utilization of microbial consortia is promising, a critical To facilitate efficient 3-HP production from alginate, we devised a challenge is the control of process performance for the maximization genetic circuit-based population-guiding strategy (Fig. 1b). Given that of production efficiencies, which is primarily governed by the popu- only Vibrio sp. dhg serves as a beneficial strain (i.e., by providing lation composition29. Furthermore, there were also efforts in devel- acetate) for E. coli by metabolizing alginate, whereas E. coli does not oping artificial quorum sensing circuits21,30–32 for programmed growth significantly promote the growth of Vibrio sp. dhg, the two strains were control of consortium participants depending on population densities. expected to exhibit a commensal relationship, which would lead to the In addition, to achieve stability in co-cultures, symbiotic communities dominance of Vibrio sp. dhg in co-cultures. Consequently, the excess were designed mainly via creating mutualistic interactions, including growth of Vibrio sp. dhg would inevitably reduce the E. coli population, carbon cross-feeding33 and feeding a growth-inhibiting byproduct to as the former would compete for other nutrients in the medium41. To another member as a sole carbon source19,23. obtain the optimal population ratio for 3-HP production, we estab- While these efforts have successfully improved biochemical pro- lished a cooperative relationship between the two strains by introdu- duction, and also highlighted the importance of population control, it cing a population guider in E. coli. This population guider was designed remains difficult to predict and control the optimal population com- to express the bla gene for the degradation of ampicillin in the pre- position of each participant for biochemical production. In particular, sence of 3-HP in a medium. In the presence of ampicillin, which inhibits the microbial population of a consortium with different physiological the growth of the microbial strains (primarily Vibrio sp. dhg), E. coli attributes is rarely controllable simply using initial conditions, because served as a beneficial strain by improving the growth fitness of Vibrio the growth of each cell is significantly affected by the medium com- sp. dhg. We expected that strains would be acclimated to the increased position that changes dynamically throughout the cultivation period. production of 3-HP by cooperative survival under selection pressure. Therefore, the optimization process relies on pre-determined empiri- cal parameters that can only be determined through labor-intensive Cultivation of consortium without population control experimental trials. Initially, we constructed a synthetic consortium by co-culturing Vibrio In this study, we report a population-guiding strategy for sp. dhg and E. coli and examined whether 3-HP could be produced improving biochemical production by a microbial consortium based directly from alginate. To monitor the population composition, we on its synthetic acclimation. Specifically, we designed a genetic circuit, labeled Vibrio sp. dhg and ECFHP (3-HP-producing E. coli W, Supple- named “population guider”, for the degradation of specific com- mentary Data 1) strains by introducing gene expression cassettes for pounds inhibiting the growth of the consortium when a target che- the constitutive expression of mcherry and sgfp (Supplementary mical is produced. In the presence of the growth inhibitor, a Fig. 2), which led to the formation of the VDHG and ECFHPS strains, population guider tightly associates the growth of a microbial con- respectively (Supplementary Data 1). To initiate the culture, cells of the sortium with the biochemical production capability. Therefore, a VDHG and ECFHPS strains (with the cell cultures having concentra- population is expected to be autonomously acclimated for improved tions corresponding to OD600 values 0.5 and 2, respectively) were used biochemical production by its optimization even under dynamically as inoculum, considering that Vibrio sp. dhg cultured in the presence changing environments. As a model system, we applied this strategy to of alginate exhibited a considerably higher growth rate (0.83 h−1) than a consortium of Vibrio sp. dhg34,35 and an engineered E. coli strain for that of E. coli cultured in the presence of acetate (0.16 h−1) (Supple- the direct production of 3-hydroxypropionic acid (3-HP), an important mentary Fig. 3). After the culture was initiated in a minimal medium platform chemical36, from alginate, a promising non-conventional supplemented with alginate, an immediate increase in the OD600 value feedstock material obtainable from marine biomass. We chose ampi- was observed (Fig. 2a). Along with cell growth, acetate accumulation cillin as a growth-inhibiting compound and developed a population was also observed in the medium (1.67 g/L), indicating that Vibrio sp. guider to express the ampicillin degradation gene (bla) in response to dhg responded positively to the co-culture conditions (in terms of the 3-HP concentration. When Vibrio sp. dhg was co-cultivated with a 3- growth). At 12 h, 3-HP was also detected (38.45 mg/L), which confirmed HP-producing E. coli harboring the population guider in the presence that 3-HP can be produced directly from alginate using the consortium of ampicillin, notably, it was observed that 3-HP production was approach. Nevertheless, only a small quantity of 3-HP (52.95 mg/L from drastically increased (up to 4.3-fold) with the minimized accumulation 20 g/L of alginate) was produced by the consortium even when the of acetate, a key intermediate in co-cultures. A population analysis acetate level was lowered. The significant accumulation of acetate revealed that the improvement occurred due to changes in the (1.83 g/L at maximum) indicated the low efficiency of 3-HP production population compositions of co-cultures based on the concentrations (i.e., insufficient activity of the 3-HP-producing E. coli). The population of the growth-inhibiting compounds. Collectively, these results sug- composition analysis revealed that the population ratio of ECFHPS gest that the concept of synthetic acclimation can serve as an efficient reduced significantly with time and remained low (lowest: 8.25% at strategy for improving the production of diverse value-added bio- 18 h, Fig. 2b). These results indicate that the overgrowth of Vibrio sp. chemicals by microbial consortia. dhg should be avoided for efficient 3-HP production. To investigate the effect of population ratios to 3-HP production, Results we tested four different inoculum sizes of the VDHG and ECFHPS Microbial consortium for production of 3-HP from alginate strains (0.5:4, 0.5:1, 0.1:2.4, and 2.4:0.1), which were varied from the A synthetic microbial consortium was constructed for the production 0.5:2 ratio (Supplementary Fig. 4). As a result, the changes of the of 3-HP from alginate, in which an alginate-utilizing Vibrio sp. dhg population ratio affected the 3-HP production significantly; the 3-HP strain and a 3-HP producing E. coli strain were co-cultured (Fig. 1a). titers at 48 h varied from almost zero to 211.05 mg/L (a 4.0-fold Vibrio sp. dhg has a 42-kb cluster that encodes proteins that metabo- increase), implying the importance of a consortium population to 3-HP lize alginate34 and release acetate as a major byproduct. While E. coli production. In particular, when the initial amount of VDHG is high, cannot utilize alginate, its biochemical production from various car- 3-HP production greatly decreased likely due to poor growth of the bon sources, including acetate, has been performed frequently37–40. In ECFHPS strain. Despite the inoculum size control, acetate was still the current study, E. coli was engineered to produce 3-HP from acetate consistently observed at high levels, implying significant room for (Supplementary Note 1); the resulting ECFHPS strain produced further optimization. It was also found that generated 3-HP was also 272.82 mg/L of 3-HP from 10 g/L of acetate (Supplementary Fig. 1 and often decreased likely due to its degradation by either of the two Nature Communications | (2022)13:6506 2 Article https://doi.org/10.1038/s41467-022-34190-z microorganisms after alginate was depleted, indicating the necessity 5’-UTR was used (in the ECPG2 strain), the strain could grow regardless of efficient carbon utilization; the degradation of 3-HP was observed in of the presence of 3-HP, probably owing to the high basal expression of another bacterium previously42. With the confirmed importance of the bla (Supplementary Fig. 5b). Therefore, the population guider with the population ratio control, we applied the population control strategy to weak 5’-UTR for bla expression was selected for subsequent studies. optimize production by this consortium. Next, we assessed whether the growth of the consortium could be controlled by the exogenous addition of 3-HP using the developed Population guider for 3-HP-dependent growth regulation population guider (Fig. 3). We prepared an inoculum by mixing the To guide the microbial population for improved 3-HP production, we VDHG and ECPG (E. coli W harboring the pPopG plasmid, Supple- constructed a population guider and tested its controllability in terms mentary Data 1) strains at a ratio of 1:4, considering their growth rates of the growth of the consortium depending on the presence of 3-HP. (see above). We monitored the growth of the consortium in the pre- We utilized a 3-HP-responsive transcription factor (C4-lysR) and its sence of sub-lethal concentrations of ampicillin (0, 1, 2, 5, 10, and cognate promoter (PC4M) from a recently characterized 3-HP inducible 20 µg/mL, Supplementary Fig. 6). For the inducible expression of the system in Pseudomonas denitrificans43–45 (Fig. 1b). For the effective bla gene, three different concentrations of 3-HP (0, 0.5, and 2 g/L) were control of 3-HP-dependent bla expression, we assessed two 5’-UTR used. Notably, after 24 h of cultivation, successful growth control was sequences (weak and strong) with different predicted expression observed; the consortium generally exhibited better growth in the levels (Supplementary Table 1). The ECPG1 strain harboring the presence of 3-HP when the ampicillin concentrations ranged from 1 to pPopG1 plasmid with the weak 5’-UTR for bla was unable to grow in the 10 µg/mL. The differences in biomass formation depending on the absence of 3-HP (Supplementary Fig. 5a). Conversely, when the strong presence of 3-HP were generally more pronounced when the a Vibrio sp. dhg Engineered Escherichia coli DEHU KDG KDPG PYR Acetyl-CoA Acetyl-CoA Malonyl-CoA MSA Alginate Acetate 3-HP Fatty acid TCA synthesis cycle G-3-P pathway Alginate utilization pathway 3-HP production pathway b Vibrio sp. dhg grows rapidly Vibrio sp. dhg-dominant population Microbial consortium Alginate 3-HP E. coli Commensalism Acetate + Vibrio sp. dhg Competition Competition for common nutrients Nmax = 14 Population guider degrades Vibrio sp. dhg is benefited by 3-HP production-optimized population ampicillin when 3-HP is present improved growth of E. coli 3-HP Ampicillin Cooperation degradation + + + 3-HP sensor Regulator bla Competition Population guider Nmax = 14 Fig. 1 | Overall strategy of genetic circuit-guided acclimation of the microbial phosphate; MSA, malonate semialdehyde. b A comparison of the microbial consortium for improved 3-hydroxypropionic acid (3-HP) production. consortium-based strategy with genetic circuit-based population guiding. a Schematic diagram of the 3-HP biosynthesis from alginate by the microbial Without population control, the population is dominated by Vibrio sp. dhg, consortium comprising Vibrio sp. dhg and E. coli. Vibrio sp. dhg assimilates resulting in the wasteful utilization of resources and a low production of 3-HP. alginate and secretes acetate, whereas E. coli produces 3-HP by re-utilizing the The introduction of the population guider into E. coli leads to the establishment secreted acetate. Abbreviations: 3-HP, 3-hyroxypropionic acid; DEHU, 4-deoxy- of a cooperative interaction between the two microorganisms and allows the L-erythro-5-hexoseulose uronate; KDG, 2-keto-3-deoxygluconate; KDPG, 2- consortium to optimize the population for improving 3-HP production by keto-3-deoxy-6-phosphogluconate; PYR, pyruvate; G-3-P, glyceraldehyde 3- synthetic acclimation. Nature Communications | (2022)13:6506 3 Article https://doi.org/10.1038/s41467-022-34190-z 20 25 350 6 Supplementary Data 1) in the presence of ampicillin (Fig. 4a–d for the culture profiles and Fig. 4e–h for their comparisons). In the initial 300 20 5 attempt, we added 5 µg/mL of ampicillin, and the fermentation profile 15 was compared to that observed in the absence of ampicillin (Fig. 4 and 250 4 Supplementary Table 2). In the absence of ampicillin, the culture Alginate (g/L) Acetate (g/L) 3-HP (mg/L) 15 200 profiles and levels of 3-HP production (68.35 mg/L) were almost OD600 10 3 150 identical with those observed for the initial consortium, in which VDHG 10 2 and ECFHPS were co-cultured (Fig. 4a). This observation indicates that 5 100 the population guider did not affect the cultures. When 5 µg/mL of 5 ampicillin was added, the growth rate decreased marginally along with 50 1 the alginate consumption rate (Fig. 4b). Nevertheless, the increased 0 0 0 0 3-HP titer (125.40 mg/L) and low levels of accumulated acetate (1.44 g/ 0 12 24 36 48 L at most) were notable observations, indicating the improved 3-HP Time (h) production following population control. Subsequently, we investi- Total OD600 OD600 of VDHG 3-HP gated a potential change in the population of the consortium and observed that the ratio of ECFHPG increased from 18.50% to 29.15% of OD600 of ECFHPS Alginate Acetate the total population at 48 h (Fig. 4h). Such population changes resul- ted in the redirection of carbon flux toward 3-HP production, repre- Fig. 2 | 3-HP production from alginate by the microbial consortium comprising VDHG and ECFHPS strains. Fermentation profile of the co-culture of the VDHG sented by a 1.89-fold increase in the C-mole yield of 3-HP and ECFHPS strains. The left y-axis and left y-offset represent OD600 (black circles) (Supplementary Table 2). and consumed alginate (blue diamond, g/L), respectively. Estimated OD600 of the We increased the selection pressure by adding 10 µg/mL of VDHG (purple circles) and ECFHPS (green circles) strains was also provided. The ampicillin, at which point the growth of the VDHG strain was almost right y-axis and right y-offset represent 3-HP (red triangles, mg/L) and acetate (grey completely inhibited (Supplementary Fig. 6). Although the addition of inverted triangles, g/L) levels, respectively. Source data are provided as a Source excess ampicillin strongly inhibited the growth of the consortium in Data file. the absence of the population regulation module (Supplementary Fig. 7), when the population guider was used, the OD600 value of the consortium reached 13.99 (Fig. 4c). This indicates the successful p = 0.0008 p = 0.04 p = 0.04 cooperative interaction between the two microorganisms. The 0.7 p = 0.01 p = 0.05 increase in selection pressure further increased the 3-HP titer (293.55 mg/L), a 4.3-fold increase compared to that observed in the 0.6 absence ampicillin (68.35 mg/L) after a 48-h fermentation period. Acetate accumulation was significantly low (1.05 g/L at 6 h) and 0.5 undetectable after 18 h, which was accompanied by robust 3-HP pro- duction. This result indicated higher activity of the ECFHPG strain. 0.4 ΔOD600 Indeed, while the total biomass decreased, the abundance of the 0.3 ECFHPG strain increased by 2.2-fold (40.4%) compared to that in the absence of ampicillin at 48 h (Fig. 4h). It was also observed that 0.2 the C-mole yield of 3-HP increased by 4.38-fold compared to the case where ampicillin was not added (Supplementary Table 2), showing that 0.1 the population control strategy facilitated the successful acclimation of the population for improvement of 3-HP production. 0.0 We further applied a higher level of the selection pressure by 3-HP conc. (g/L) 0 0.5 2 0 0.5 2 0 0.5 2 0 0.5 2 0 0.5 2 0 0.5 2 increasing the concentration of ampicillin to 20 µg/mL to determine Ampicillin conc. (μg/mL) 0 1 2 5 10 20 whether 3-HP production could be further increased. In response to this alteration, the growth of the consortium was inhibited sig- Fig. 3 | 3-HP dependent growth control of the microbial consortium in presence nificantly; only 2.53 g/L of alginate was consumed, which resulted in of ampicillin. Comparison of biomass formation (ΔOD600, the difference in OD600 low biomass formation (final OD600 value: 5.30) (Fig. 4d). Probably due values at 0 h and 24 h) in various combinations of 3-HP (0, 0.5, and 2 g/L) and to the very small of alginate consumption and biomass formation, the ampicillin (0, 1, 2, 5, 10, and 20 µg/mL) concentrations. Error bars represent the standard deviations of triplicate biological experiments (n = 3) and their center acetate levels also remained low (below 0.23 g/L) throughout the fer- indicates a mean value. P values were calculated using Student’s t-test (two-tailed). mentation period. Nevertheless, even at such high selection pressure, White dot indicates actual data point. Source data are provided as a Source 199.40 mg/L of 3-HP was produced with the highest yield (Supple- Data file. mentary Table 2). However, 20 µg/mL of ampicillin appeared to be excessively high, given that 3-HP was produced only during the initial 12 h at a lower titer than that obtained with 10 µg/mL of ampicillin concentration of ampicillin was higher. However, when the ampicillin (293.55 mg/L). The 3-HP production was stalled probably due to concentration was excessively high (20 µg/mL), the growth benefit was unviable VDHG cells. The population analysis further confirmed the unclear, probably owing to the excessive inhibition of the growth of hypothesis that the production of 3-HP during the initial phase of both strains. Nevertheless, this result indicates the potential of the culturing resulted from the increased population ratio of ECFHPG. population guider for regulating the growth of the microbial con- sortium in response to the presence of 3-HP in the medium. Comparison with a 3-HP independent bla expression strategy We compared this 3-HP-dependent bla expression strategy (i.e., con- Acclimation of a microbial population for 3-HP production ditional cooperation) with a static bla expression strategy (i.e., simple We applied the population-guiding strategy for the robust production cooperation). For this comparison, we constructed the pC112-3HP, of 3-HP from alginate by co-culturing the VDHG strain with the ECFHPG pC100-3HP, and pC119-3HP plasmids that express bla under one of strain (an engineered ECF strain harboring the pPopG-3HP plasmid, four constitutive promoters with different strengths by more than 103- Nature Communications | (2022)13:6506 4 Article https://doi.org/10.1038/s41467-022-34190-z Fig. 4 | Improved 3-HP production upon the synthetic acclimation of the diamonds, g/L), 3-HP (red triangles, mg/L) and acetate (grey inverted triangles, g/L) microbial consortium. a–d Fermentation profiles of the co-culture of the VDHG levels, respectively. e–h Comparison of (e) 3-HP levels, (f) acetate levels, (g) con- and ECFHPG strains with the addition of (a) 0, (b) 5, (c) 10, and (d) 20 µg/mL sumed alginate, and (h) population ratios depending on ampicillin concentrations ampicillin. The left y-axis represents OD600 of cultures (black circles). Estimated (0, 5, 10, and 20 μg/mL) at 48 h. The purple and green bars indicate the population OD600 of the VDHG (purple circles) and ECFHPG (green circles) strains was also ratio of VDHG and ECFHPG strains, respectively. a–h Source data are provided as a provided. The left y-offset, right y-axis and right y-offset represent alginate (blue Source Data file. fold (PJ23112, PJ23100, and PJ23119, in the order of strengths)46. We co- with the results of the previous 3-HP-dependent bla expression cultured VDHG and ECFHPC1-3 (harboring each bla expression plas- strategy. mid instead of the population guider, Supplementary Data 1) in the We utilized mathematical modeling (see Supplementary Note 2 presence of 10 μg/mL of ampicillin (Supplementary Fig. 8). With the and Supplementary Table 3) for better understanding and comparing bla expression under PJ23119, the highest amount of final biomass was the achieved results. Due to limitations to model a dynamically chan- achieved, indicating the greatest degradation of ampicillin. Notably, ging environment, we accounted alginate consumption and remaining up to 168.00 mg/L of 3-HP was produced at 48 h, which was higher acetate for estimating cell growth of both strains and 3-HP production than that of the co-culture of the VDHG and ECFHPG strains without under each condition. As observed, the construction of the synthetic addition of ampicillin, indicating the simple cooperative interaction cooperation was expected to yield higher 3-HP production over the was also advantageous for 3-HP production. However, no significant simple co-cultivation (Supplementary Fig. 9). Moreover, a superior correlation in 3-HP production and promoter strengths was observed. production with the 3-HP dependent bla expression to that with static In addition, 1~2 g/L of acetate was consistently accumulated until 24 h, bla expression was expected. The strong expression of bla led to the suggesting the suboptimal performance of the consortia compared outgrowth of VDHG, resulting in a similar profile from the simple co- Nature Communications | (2022)13:6506 5 Article https://doi.org/10.1038/s41467-022-34190-z culture; 3-HP production was expected to be low even with the low acclimated for higher production if a gene (e.g., mcsI) for degrading expression, likely owing to insufficient growth of both microorgan- microcin S is expressed or the expression of mcsS is reduced when a isms. Collectively, we concluded that the 3-HP dependent bla expres- target chemical is produced. Furthermore, in the case that consortium sion strategy can efficiently improve the 3-HP production by the co- participants show different extent of susceptibility, multiple growth culture of Vibrio sp. dhg and 3-HP producing E. coli strains. inhibitors specifically effecting a single strain can be also utilized. (iii) To build a “population guider” for the production of other chemicals, a Discussion molecular part that can detect a target chemical must be available. In this study, we demonstrated the efficient conversion of alginate to However, with growing interest in developing synthetic circuits which 3-HP by constructing a microbial consortium of alginate-metabolizing can process diverse inputs, the capability of excavating or developing Vibrio sp. dhg and 3-HP-producing E. coli strains and introducing a protein- and RNA-biosensors has been extensively improved. For population-guiding genetic circuit to acclimatize their populations for example, parts were newly found or designed for small metabolites51,52 improving 3-HP production. Although simple co-cultivation of the and natural products53. Thus, we foresee that the “molecular catalog” strains also led to the production of 3-HP from alginate, the dominance of biosensors is more expanded and widely available. (iv) The choice of of Vibrio sp. dhg in the culture resulted in the high accumulation of the level of the selection pressure also plays a critical role and thus it acetate, which is a pathway intermediate, and the levels of 3-HP pro- needs an optimization. There was room for further optimization of the duction were also low. From the observation that the population ratio ampicillin condition between 10 and 20 μg/mL (Fig. 4). And during a significantly affected the biochemical production efficiencies, we separate repeated culture, it seemed that the level also affects the introduced the population-guiding circuit and selection pressure to degradation of 3-HP after the depletion of available carbon source establish cooperative interaction between the strains. Consequently, (Supplementary Fig. 10). Therefore, to achieve even higher produc- acclimated consortium population led to a significant improvement in tion, a detailed follow-up study about an optimal strategy to apply the 3-HP production from alginate. selection pressure needs to be carried out. The results clearly indicate that synthetic microbial consortia can One still may question “Is a consortium approach still needed serve as a powerful platform for biochemical production from non- even if genetic engineering of a non-model host becomes readily conventional substrates. Although several successful attempts have available?”. Indeed, genetic engineering of Vibrio species has been been made, the engineering of microorganisms that can utilize non- recently studied35,54,55 to leverage their rapid growth and high meta- conventional substrates is mostly difficult owing to insufficient genetic bolic capacities. Therefore, it is expected that direct conversion of information and molecular tools. However, co-culturing with micro- 3-HP from alginate using a single strain will be available soon. Although organisms with known genetic information can help overcome the there is no clear answer, as mentioned in the introduction section, the intrinsic limitations of the single-cell engineering approach. As shown consortium approach can be more efficient since it allows division of in this study, a synthetic consortium can leverage the useful properties labor, simplification of a complex metabolic pathway4. However, since of each microorganism to develop a novel pathway or function that a population control is the key to develop a successful multi-strain does not exist in a single microorganism, and resultantly, synthesize bioprocess, diverse strategies should be devised and tested. In this value-added biochemical products successfully. regard, we believe that a production-dependent synthetic cooperation To leverage the huge potential of the co-culture approach, effi- can be an effective strategy as shown with the current model con- cient population control strategy needs to be devised. However, sortium of Vibrio sp. dhg and E. coli. despite its potential, only limited strategies (e.g., programmed cell Collectively, this study shows the circuit-based population-guid- lysis depending on the level of a quorum sensing molecule) for opti- ing strategy is a promising tool for the development of efficient mizing the composition of consortia have been proposed and applied consortium-based bioprocesses. The high flexibility in the design of a for biochemical production. Although previous approaches success- microbial consortium can facilitates the development of novel pro- fully demonstrated that a microbial population is controllable to some cesses to produce diverse biochemicals from nonconventional feed- extent, since we still do not know what composition is optimal for stocks by employing multiple modularized strains. biochemical production during dynamically changing fermentation environment, the application of these strategies in bioproduction can Methods be challenging. On the contrary, a “population guider” was designed to Bacterial strains, plasmids, and reagents directly associate the “survival” of cells with biochemical production The bacterial strains and plasmids used are listed in Supplementary and to find an optimal population by autonomous acclimation of the Data 1. Methods for plasmid construction are elaborated in Supple- population toward improved production of a target. mentary Note 3. The primers were synthesized by Cosmogenetech Despite the success, there are several issues for broad applica- (Seoul, Korea) and are listed in Supplementary Data 2. Plasmid DNA tions of our approach. (i) This strategy was applied to a two-party was isolated using an ExprepTM Plasmid SV kit from GeneAll (Seoul, bacterial consortium where only one strain can utilize the substrate Korea). DNA fragments amplified by PCR were purified using an and the other strain relies on the byproduct formation. However, co- ExpinTM Gel SV kit (GeneAll). Q5 polymerase and restriction enzymes culturing two different strains are very relevant for bioprocesses, were purchased from New England Biolabs (Ipswich, MA, USA). The especially for the conversion of non-conventional feedstocks. In reagents for cell cultures were purchased from BD Bioscience (Sparks, addition, acclimation of a consortium with more than two members MD, USA). All other chemicals were obtained from Sigma (St. Louis, can be also attempted if one finds a suitable pair of a growth inhibitor MO, USA) as listed in Supplementary Data 2, unless otherwise and its degradation gene to construct a “conditional cooperation” indicated. based on target chemical production. (ii) The requirement of anti- biotics itself or potential difference in antibiotic susceptibility of each Culture conditions for Vibrio sp. dhg and E. coli strains party can act as a significant constraint47,48. We expect that ampicillin All flask-scale cell cultures were performed using 300-mL flasks con- can be replaced by any growth inhibitors which can commonly inhibit taining 50 mL of a modified minimal medium34. The medium consisted consortium participants. The use of antibiotics can be avoided by of 15 g/L NaCl, 5 g/L (NH4)2SO4, 0.5 g/L MgSO4·7 H2O, 100 mM potas- directly engineering cells to produce growth-inhibiting compounds sium phosphate buffer (pH 7), and 2 mL/L trace metal solution (ATCC (e.g., bacteriocins)49,50. For example, microcin S (encoded by mcsS) MD-TMS, pH 7.0). The trace metal solution contained 0.5 g/L ethyle- from probiotic E. coli G3/10 is known to inhibit the growth of neigh- nediaminetetraacetic acid (EDTA), 3.0 g/L MgSO4·H2O, 0.5 g/L boring microorganisms50. The population would be similarly MnSO4·H2O, 1.0 g/L NaCl, 0.1 g/L FeSO4·7H2O, 0.1 g/L Co(NO3)2·6 H2O, Nature Communications | (2022)13:6506 6 Article https://doi.org/10.1038/s41467-022-34190-z 0.1 g/L CaCl2, 0.1 g/L ZnSO4·7H2O, 0.01 g/L CuSO4·5H2O, 0.01 g/L cytometer (Brea, CA, United States). The cultured samples were AlK(SO4)2, 0.01 g/L H3BO3, 0.01 g/L Na2MoO4·2 H2O, 0.001 g/L washed using fresh minimal medium and injected into the flow cyt- Na2SeO3, 0.01 g/L Na2WO4·2 H2O, and 0.02 g/L NiCl2·6 H2O. The con- ometer after their OD600 value was adjusted to 1 (corresponding to 106 centration of NaCl in the medium was adjusted to 15 g/L for the co- cells/μL). mCherry fluorescence was detected using a 488-nm excita- culture of Vibrio sp. dhg and E. coli strains. Alginate or acetate (neu- tion laser and 610/20-nm emission channel with an energy-coupled tralized to pH 7 by the addition of NaOH) was provided as a carbon dye bandpass filter. sGFP fluorescence was detected using a 488-nm source depending on the experimental conditions. All cell culture excitation laser and a 525/40-nm emission channel with a fluorescein experiments were performed at 30 °C with continuous shaking at isothiocyanate (FITC) bandpass filter. For each sample, the fluores- 200 rpm. cence signals of at least 100,000 cells were monitored. The ratio of Seed cultures of Vibrio sp. dhg were prepared by inoculating a Vibrio sp. dhg and E. coli cells was calculated by dividing each fluor- colony into minimal medium supplemented with 10 g/L alginate. In escent cell number value by the total cell number value. A standard case of E. coli strains, minimal medium supplemented with 1 g/L yeast curve was generated using known ratios of Vibrio sp. dhg and E. coli extract and 4 g/L acetate was used. After overnight culture, the cells (Supplementary Fig. 2). were refreshed by inoculating in fresh media without yeast extract at an OD600 of 0.05. After the refreshed cells were cultured till an OD600 Statistical analysis value of 1.0 was achieved, the microorganisms were co-cultured at Statistical tests were performed using the two-tailed Student’s t-test by OD600 0.5 and 2 for Vibrio sp. dhg and E. coli, respectively. For 3-HP SigmaPlot (SigmaStat). production, isopropyl β-D-1-thiogalactopyranoside was added to a final concentration of 0.1 mM in both E. coli refreshing and co-cultures. Reporting summary Depending on the experimental conditions, various quantities of 3-HP Further information on research design is available in the Nature (0, 0.5, and 2 g/L) or ampicillin (0, 1, 2, 5, 10, and 20 μg/mL) were Research Reporting Summary linked to this article. added. For the stable maintenance of plasmids, streptomycin (50 µg/ mL) or chloramphenicol (10 µg/mL) was added to the media. Data availability A reporting summary for this article is available as a Supplementary Construction of the population guider and its testing Information file. The strains and plasmids used in this study are pro- To construct the population guider, we generated two 5’-UTR variants vided as a Supplementary Data 1 file. The primers and reagents infor- (Supplementary Table 1) with different predicted expression levels of mation used in this study are provided as a Supplementary Data 2 file. 22,884 and 115,863 using the 5’-UTR Library Designer56. The two plas- Synthetic promoter sequences were obtained from Registry of Stan- mids with each 5’-UTR of bla (pPopG1 and pPopG2 plasmids) were dard Biological Parts (http://parts.igem.org/Main_Page). The raw data introduced into the E. coli W strain, which led to the formation of the for modeling growth of Vibrio sp. dhg and E. coli strains are accessible ECPG1 and ECPG2 strains. For testing the population guider, the cul- at https://doi.org/10.5281/zenodo.7187480. Source data are provided tures were performed on a small scale (180 μL as culture volume) using with this paper. a BioscreenC MBR (Oy Growth Curves Ab, Helsinki, Finland). Seed cultures were prepared using the method used for flask-scale cultures. 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Reprints and permissions information is available at Acknowledgements http://www.nature.com/reprints This research was supported by the C1 Gas Refinery Program [NRF- 2018M3D3A1A01055754] and National Research Foundation of Korea Publisher’s note Springer Nature remains neutral with regard to grant [NRF-2019R1A2C2084631]. We are thankful to Prof. Jeong Wook jurisdictional claims in published maps and institutional affiliations. Lee for allowing the use of laboratory equipment and Dr. Myung Hyun Noh for providing insightful suggestions during discussion. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, Author contributions adaptation, distribution and reproduction in any medium or format, as C.W.K., H.G.L., and G.Y.J. designed the project. C.W.K., H.G.L., S.C., and long as you give appropriate credit to the original author(s) and the G.S. conducted the experiments. 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