Synthetic microbial consortia can leverage their expanded enzymatic reach to tackle biotechnological challenges too complex for single strains, such as biosynthesis of complex secondary metabolites or waste plant biomass degradation and valorisation. The benefit of metabolic cooperation comes with a catch-installing stable interactions between consortium members. Here, we established a mutualistic relationship in the synthetic consortium of Pseudomonas putida strains through reciprocal processing of two disaccharides-cellobiose and xylobiose-obtainable from lignocellulosic residues. Two strains were engineered to hydrolyse and metabolize these sugars: one grows on xylose and hydrolyses cellobiose to produce glucose, while the other grows on glucose and cleaves xylobiose to produce xylose. This specialization allows each strain to provide essential growth substrate to its partner, establishing a mutualistic interaction, which can be termed reciprocal substrate processing. Key enzymes from Escherichia coli (xylose isomerase pathway) and Thermobifida fusca (glycoside hydrolases) were introduced into P. putida to broaden its carbohydrate utilization capabilities and arranged in a way to instal the strain cross-dependency. A mathematical model of the consortium assisted in predicting the effects of substrate composition, strain ratios, and protein expression levels on population dynamics. Our results demonstrate that modulating extrinsic factors such as substrate concentration can help in balancing fitness disparities between the strains, but achieving this by altering intrinsic factors such as glycoside hydrolase expression levels is much more challenging. This study presents reciprocal substrate processing as a strategy for establishing an obligate dependency between strains in the engineered consortium and offers valuable insights into overcoming the challenges of fostering synthetic microbial cooperation.

Department of Experimental Biology Faculty of Science Masaryk University Kamenice 753 5 Brno 62500 Czech Republic

Systems Biology Department Centro Nacional de Biotecnología CSIC Darwin 3 Madrid 28049 Spain

Zobrazit více v PubMed

Morris  BE, Henneberger  R, Huber  H  et al.  Microbial syntrophy: interaction for the common good. FEMS Microbiol Rev  2013;37:384–406. 10.1111/1574-6976.12019 PubMed DOI

Falkowski  PG, Fenchel  T, Delong  EF. The microbial engines that drive Earth's biogeochemical cycles. Science  2008;320:1034–9. 10.1126/science.1153213 PubMed DOI

Cameron  DE, Bashor  CJ, Collins  JJ. A brief history of synthetic biology. Nat Rev Microbiol  2014;12:381–90. 10.1038/nrmicro3239 PubMed DOI

De Lorenzo  V. 15 years of microbial biotechnology: the time has come to think big—and act soon. Microb Biotechnol  2022;15:240–6. 10.1111/1751-7915.13993 PubMed DOI PMC

Brenner  K, You  L, Arnold  FH. Engineering microbial consortia: a new frontier in synthetic biology. Trends Biotechnol  2008;26:483–9. 10.1016/j.tibtech.2008.05.004 PubMed DOI

McCarty  NS, Ledesma-Amaro  R. Synthetic biology tools to engineer microbial communities for biotechnology. Trends Biotechnol  2019;37:181–97. 10.1016/j.tibtech.2018.11.002 PubMed DOI PMC

Roell  GW, Zha  J, Carr  RR  et al.  Engineering microbial consortia by division of labor. Microb Cell Factories  2019;18:35. 10.1186/s12934-019-1083-3 PubMed DOI PMC

Rapp  KM, Jenkins  JP, Betenbaugh  MJ. Partners for life: building microbial consortia for the future. Curr Opin Biotechnol  2020;66:292–300. 10.1016/j.copbio.2020.10.001 PubMed DOI

Ibrahim  M, Raajaraam  L, Raman  K. Modelling microbial communities: harnessing consortia for biotechnological applications. Comput Struct Biotechnol J  2021;19:3892–907. 10.1016/j.csbj.2021.06.048 PubMed DOI PMC

Snoeck  S, Guidi  C, De Mey  M. “Metabolic burden” explained: stress symptoms and its related responses induced by (over) expression of (heterologous) proteins in PubMed DOI PMC

Bokinsky  G, Peralta-Yahya  PP, George  A  et al.  Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered PubMed DOI PMC

Minty  JJ, Singer  ME, Scholz  SA  et al.  Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass. Proc Natl Acad Sci USA  2013;110:14592–7. 10.1073/pnas.1218447110 PubMed DOI PMC

Zhou  K, Qiao  K, Edgar  S  et al.  Distributing a metabolic pathway among a microbial consortium enhances production of natural products. Nat Biotechnol  2015;33:377–83. 10.1038/nbt.3095 PubMed DOI PMC

Shahab  RL, Brethauer  S, Davey  MP  et al.  A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose. Science  2020;369:eabb1214. 10.1126/science.abb1214 PubMed DOI

Cha  S, Lim  HG, Kwon  S  et al.  Design of mutualistic microbial consortia for stable conversion of carbon monoxide to value-added chemicals. Metab Eng  2021;64:146–53. 10.1016/j.ymben.2021.02.001 PubMed DOI

Bizukojc  M, Dietz  D, Sun  J  et al.  Metabolic modelling of syntrophic-like growth of a 1, 3-propanediol producer, PubMed DOI

Cooper  MB, Kazamia  E, Helliwell  KE  et al.  Cross-exchange of B-vitamins underpins a mutualistic interaction between PubMed DOI PMC

Wondraczek  L, Pohnert  G, Schacher  FH  et al.  Artificial microbial arenas: materials for observing and manipulating microbial consortia. Adv Mater  2019;31:e1900284. 10.1002/adma.201900284 PubMed DOI

Duncker  KE, Holmes  ZA, You  L. Engineered microbial consortia: strategies and applications. Microb Cell Factories  2021;20:211. 10.1186/s12934-021-01699-9 PubMed DOI PMC

Dinh  CV, Chen  X, Prather  KL. Development of a quorum-sensing based circuit for control of coculture population composition in a naringenin production system. ACS Synth Biol  2020;9:590–7. 10.1021/acssynbio.9b00451 PubMed DOI

Balagaddé  FK, Song  H, Ozaki  J  et al.  A synthetic PubMed DOI PMC

Scott  SR, Din  MO, Bittihn  P  et al.  A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis. Nat Microbiol  2017;2:1–9. 10.1038/nmicrobiol.2017.83 PubMed DOI PMC

Balagaddé  FK, You  L, Hansen  CL  et al.  Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science  2005;309:137–40. 10.1126/science.1109173 PubMed DOI

Rodríguez Amor  D, Dal Bello  M. Bottom-up approaches to synthetic cooperation in microbial communities. Life  2019;9:22. 10.3390/life9010022 PubMed DOI PMC

Darwin, C. (1859) On the Origin of Species by Means of Natural Selection, or Preservation of Favoured Races in the Struggle for Life PubMed DOI PMC

Castle  SD, Grierson  CS, Gorochowski  TE. Towards an engineering theory of evolution. Nat Commun  2021;12:3326. 10.1038/s41467-021-23573-3 PubMed DOI PMC

D'Souza  G, Shitut  S, Preussger  D  et al.  Ecology and evolution of metabolic cross-feeding interactions in bacteria. Nat Prod Rep  2018;35:455–88. 10.1039/C8NP00009C PubMed DOI

Losoi  PS, Santala  VP, Santala  SM. Enhanced population control in a synthetic bacterial consortium by interconnected carbon cross-feeding. ACS Synth Biol  2019;8:2642–50. 10.1021/acssynbio.9b00316 PubMed DOI

Martínez-García  E, Nikel  PI, Aparicio  T  et al.  Pseudomonas 2.0: genetic upgrading of PubMed DOI PMC

Weimer  A, Kohlstedt  M, Volke  DC  et al.  Industrial biotechnology of PubMed DOI PMC

Dvořák  P, de  Lorenzo  V. Refactoring the upper sugar metabolism of PubMed DOI

Elmore  JR, Dexter  GN, Salvachúa  D  et al.  Engineered PubMed DOI

Bujdoš  D, Popelářová  B, Volke  DC  et al.  Engineering of PubMed DOI

Ling  C, Peabody  GL, Salvachúa  D  et al.  Muconic acid production from glucose and xylose in PubMed DOI PMC

Kohlstedt  M, Weimer  A, Weiland  F  et al.  Biobased PET from lignin using an engineered cis, cis-muconate-producing PubMed DOI

Cragg  SM, Beckham  GT, Bruce  NC  et al.  Lignocellulose degradation mechanisms across the tree of life. Curr Opin Chem Biol  2015;29:108–19. 10.1016/j.cbpa.2015.10.018 PubMed DOI PMC

Ventorino  V, Aliberti  A, Faraco  V  et al.  Exploring the microbiota dynamics related to vegetable biomasses degradation and study of lignocellulose-degrading bacteria for industrial biotechnological application. Sci Rep  2015;5:1–13. 10.1038/srep08161 PubMed DOI PMC

Das  S, Rudra  S, Khatun  I  et al.  Concise review on Lignocellulolytic microbial consortia for lignocellulosic waste biomass utilization: a way forward?  Microbiology  2023;92:301–17. 10.1134/S0026261722601282 DOI

Vu  VN, Kohári-Farkas  C, Filep  R  et al.  Design and construction of artificial microbial consortia to enhance lignocellulosic biomass degradation. Biofuel Res J  2023;10:1890–900. 10.18331/BRJ2023.10.3.3 DOI

Dvořák  P, Burýšková  B, Popelářová  B  et al.  Synthetically-primed adaptation of PubMed DOI PMC

Watson  JF, García-Nafría  J. In vivo DNA assembly using common laboratory bacteria: a re-emerging tool to simplify molecular cloning. J Biol Chem  2019;294:15271–81. 10.1074/jbc.REV119.009109 PubMed DOI PMC

Wirth  NT, Kozaeva  E, Nikel  PI. Accelerated genome engineering of PubMed DOI PMC

Martínez-García  E, de  Lorenzo  V. Transposon-based and plasmid-based genetic tools for editing genomes of gram-negative bacteria. In: Weber  W, Fussenegger  M (eds.), Synthetic Gene Networks. Methods in Molecular Biology, Vol. 813, pp. 267–83. Totowa, NJ: Humana Press, 2012.   10.1007/978-1-61779-412-4_16. PubMed DOI

Volke  DC, Friis  L, Wirth  NT  et al.  Synthetic control of plasmid replication enables target-and self-curing of vectors and expedites genome engineering of PubMed DOI PMC

Görke  B, Stülke  J. Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat Rev Microbiol  2008;6:613–24. 10.1038/nrmicro1932 PubMed DOI

Rojo  F. Carbon catabolite repression in PubMed DOI

Chen  R. A paradigm shift in biomass technology from complete to partial cellulose hydrolysis: lessons learned from nature. Bioengineered  2015;6:69–72. 10.1080/21655979.2014.1004019 PubMed DOI PMC

Zobel  S, Benedetti  I, Eisenbach  L  et al.  Tn7-based device for calibrated heterologous gene expression in PubMed DOI

Schlechter  RO, Jun  H, Bernach  M  et al.  Chromatic bacteria–a broad host-range plasmid and chromosomal insertion toolbox for fluorescent protein expression in bacteria. Front Microbiol  2018;9:3052. 10.3389/fmicb.2018.03052 PubMed DOI PMC

Moraïs  S, Salama-Alber  O, Barak  Y  et al.  Functional association of catalytic and ancillary modules dictates enzymatic activity in glycoside hydrolase family 43 β-xylosidase. J Biol Chem  2012;287:9213–21. 10.1074/jbc.M111.314286 PubMed DOI PMC

Spiridonov  NA, Wilson  DB. Cloning and biochemical characterization of BglC, a β-glucosidase from the cellulolytic actinomycete PubMed DOI

Daddaoua  A, Krell  T, Ramos  JL. Regulation of glucose metabolism in PubMed DOI PMC

Van der Hoek  SA, Borodina  I. Transporter engineering in microbial cell factories: the ins, the outs, and the in-betweens. Curr Opin Biotechnol  2020;66:186–94. 10.1016/j.copbio.2020.08.002 PubMed DOI PMC

Murray  JD. Mathematical biology: I. An introduction. In: Antman SS, Marsden JE, Sirovich L, Wigging S (eds.),

Freilich  S, Zarecki  R, Eilam  O  et al.  Competitive and cooperative metabolic interactions in bacterial communities. Nat Commun  2011;2:589. 10.1038/ncomms1597 PubMed DOI

Tsoi  R, Wu  F, Zhang  C  et al.  Metabolic division of labor in microbial systems. Proc Natl Acad Sci USA  2018;115:2526–31. 10.1073/pnas.1716888115 PubMed DOI PMC

Govindaswamy  S, Vane  LM. Multi-stage continuous culture fermentation of glucose–xylose mixtures to fuel ethanol using genetically engineered PubMed DOI

Espeso  DR, Dvořák  P, Aparicio  T  et al.  An automated DIY framework for experimental evolution of PubMed DOI PMC

Bhatia  Y, Mishra  S, Bisaria  VS. Microbial β-glucosidases: cloning, properties, and applications. Crit Rev Biotechnol  2002;22:375–407. 10.1080/07388550290789568 PubMed DOI

Izert  MA, Klimecka  MM, Górna  MW. Applications of bacterial degrons and degraders—toward targeted protein degradation in bacteria. Front Mol Biosci  2021;8:669762. 10.3389/fmolb.2021.669762 PubMed DOI PMC

Keiler  KC, Waller  PR, Sauer  RT. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science  1996;271:990–3. 10.1126/science.271.5251.990 PubMed DOI

Karzai  AW, Roche  ED, Sauer  RT. The SsrA–SmpB system for protein tagging, directed degradation and ribosome rescue. Nat Struct Biol  2000;7:449–55. 10.1038/75843 PubMed DOI

Durante-Rodríguez  G, Calles  B, De Lorenzo  V  et al.  A SsrA/NIa-based strategy for post-translational regulation of protein levels in gram-negative bacteria. Bio Protoc  2020;10:e3688. 10.21769/BioProtoc.3688 PubMed DOI PMC

Lytvynenko  I, Paternoga  H, Thrun  A  et al.  Alanine tails signal proteolysis in bacterial ribosome-associated quality control. Cell  2019;178:76–90.e22. 10.1016/j.cell.2019.05.002 PubMed DOI PMC

Fritze  J, Zhang  M, Luo  Q  et al.  An overview of the bacterial SsrA system modulating intracellular protein levels and activities. Appl Microbiol Biotechnol  2020;104:5229–41. 10.1007/s00253-020-10623-x PubMed DOI

Ebrahimi  A, Schwartzman  J, Cordero  OX. Multicellular behaviour enables cooperation in microbial cell aggregates. Philos Trans R Soc Lond Ser B Biol Sci  2019;374:20190077. 10.1098/rstb.2019.0077 PubMed DOI PMC

Martínez-Gil  M, Yousef-Coronado  F, Espinosa-Urgel  M. LapF, the second largest PubMed DOI

Lahesaare  A, Moor  H, Kivisaar  M  et al. PubMed DOI PMC

Pignon  E, Holló  G, Steiner  T  et al.  Engineering microbial consortia: uptake and leakage rate differentially shape community arrangement and composition bioRxiv. 2024. 10.1101/2024.07.19.604250 PubMed DOI

Pratt  LA, Kolter  R. Genetic analysis of PubMed DOI

Salis  HM, Mirsky  EA, Voigt  CA. Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol  2009;27:946–50. 10.1038/nbt.1568 PubMed DOI PMC

Gilman  J, Walls  L, Bandiera  L  et al.  Statistical design of experiments for synthetic biology. ACS Synth Biol  2021;10:1–18. 10.1021/acssynbio.0c00385 PubMed DOI

Demko  M, Chrást  L, Dvořák  P  et al.  Computational modelling of metabolic burden and substrate toxicity in PubMed DOI PMC

Said  SB, Or  D. Synthetic microbial ecology: engineering habitats for modular consortia. Front Microbiol  2017;8:1125. 10.3389/fmicb.2017.01125 PubMed DOI PMC

Said  SB, Tecon  R, Borer  B  et al.  The engineering of spatially linked microbial consortia–potential and perspectives. Curr Opin Biotechnol  2020;62:137–45. 10.1016/j.copbio.2019.09.015 PubMed DOI PMC