Synthetic Biology Tools to Engineer Microbial Communities for Biotechnology

Trends in biotechnology, Journal Year: 2018, Volume and Issue: 37(2), P. 181 - 197

Published: Nov. 26, 2018

Microbial consortia have been used in biotechnology processes, including fermentation, waste treatment, and agriculture, for millennia. Today, synthetic biologists are increasingly engineering microbial diverse applications, the bioproduction of medicines, biofuels, biomaterials from inexpensive carbon sources. An improved understanding natural ecosystems, development new tools to construct program their behaviors, will vastly expand functions that can be performed by communities interacting microorganisms. Here, we review recent advancements biology approaches engineer consortia, discuss ongoing emerging efforts apply various biotechnological suggest future applications.

Language: Английский

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Bioremediation 3.0: Engineering pollutant-removing bacteria in the times of systemic biology

Biotechnology Advances, Journal Year: 2017, Volume and Issue: 35(7), P. 845 - 866

Published: Aug. 5, 2017

Language: Английский

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Heavy Metal Removal by Bioaccumulation Using Genetically Engineered Microorganisms

Frontiers in Bioengineering and Biotechnology, Journal Year: 2018, Volume and Issue: 6

Published: Oct. 29, 2018

Wastewater effluents from mines and metal refineries are often contaminated with heavy ions, so they pose hazards to human environmental health. Conventional technologies remove ions well-established, but the most popular methods have drawbacks: chemical precipitation generates sludge waste, activated carbon ion exchange resins made unsustainable non-renewable resources. Using microbial biomass as platform for removal is an alternative method. Specifically, bioaccumulation a natural biological phenomenon where microorganisms use proteins uptake sequester in intracellular space utilize cellular processes (e.g., enzyme catalysis, signaling, stabilizing charges on biomolecules). Recombinant expression of these import-storage systems genetically engineered allows enhanced sequestration ions. This has been studied over two decades bioremediative applications, successful translation industrial-scale virtually non-existent. Meanwhile, demands resources increasing while discovery rates supply primary grade ores not. review re-thinks how can be used proposes that it developed bioextractive applications-the recovery downstream purification refining, rather than disposal. consolidates previously tested into biochemical framework highlights efforts overcome obstacles limit industrial feasibility, thereby identifying gaps knowledge potential avenues research bioaccumulation.

Language: Английский

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Modular co-culture engineering, a new approach for metabolic engineering

Metabolic Engineering, Journal Year: 2016, Volume and Issue: 37, P. 114 - 121

Published: May 28, 2016

Language: Английский

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Metabolic division of labor in microbial systems

Proceedings of the National Academy of Sciences, Journal Year: 2018, Volume and Issue: 115(10), P. 2526 - 2531

Published: Feb. 20, 2018

Significance If contained in a single population complex metabolic pathway can impose burden on the host, decreasing system’s overall productivity. This limitation be overcome by division of labor (DOL), where distinct populations perform different steps pathway, thus reducing each population. By compartmentalizing reactions, however, DOL reduces their efficiency introducing transport barrier for metabolites and enzymes. It remains unclear how trade-off between reaction dictates potential benefit DOL. Through analysis pathways we derive general criterion establishing when outperforms Our results guide rational engineering provide insights into operation natural pathways.

Language: Английский

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Engineering living therapeutics with synthetic biology

Nature Reviews Drug Discovery, Journal Year: 2021, Volume and Issue: 20(12), P. 941 - 960

Published: Oct. 6, 2021

Language: Английский

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Engineering Robust Production Microbes for Large-Scale Cultivation

Trends in Microbiology, Journal Year: 2019, Volume and Issue: 27(6), P. 524 - 537

Published: Feb. 25, 2019

Strain engineering in the laboratory often does not consider process requirements larger-scale bioreactors. Systems and synthetic biology can be applied to design microbial strains that allow reliable robust production on a large scale. Commercial platforms should selected developed based their relevance final goals. are increasingly used examine modulate complex biological systems. As such, many issues arising during scaling-up processes addressed using these approaches. We review differences between laboratory-scale cultures provide perspective those strain characteristics especially important scaling. has been range of systems for response bioreactors fluctuations nutrients, dissolved gases, other stresses. Synthetic both assess response, engineer improve production. discuss approaches tools context use microbes applications large-scale It is well recognized produce vast compounds, from fuels commodity chemicals pharmaceuticals fine [1Zhang M.M. et al.Engineering hosts bacterial natural products.Nat. Prod. Rep. 2016; 33: 963-987Crossref PubMed Google Scholar, 2Carbonell P. al.An automated design-build-test-learn pipeline enhanced chemicals.Commun. Biol. 2018; 1: 66Crossref Scopus (114) 3Beller H.R. al.Natural products as biofuels bio-based chemicals: fatty acids isoprenoids.Nat. 2015; 32: 1508-1526Crossref Scholar]. Correspondingly, efforts broaden scope demonstrate proof-of-concept pathways new molecules gaining number [2Carbonell 4Casini A. al.A pressure test make 10 90 days: external evaluation methods biology.J. Am. Chem. Soc. 140: 4302-4316Crossref (88) 5Campodonico M.A. al.Generation an atlas chemical Escherichia coli novel pathway prediction algorithm, GEM-Path.Metab. Eng. 2014; 25: 140-158Crossref (124) However, despite development convert nearly any carbon source into desired product, rather modest cases have seen successful transition industrial-scale marketed products. Economic competitiveness with established or biosynthetic routes factor. Low titers yields setting also need overcome proceed scale-up. at level core bioconversion technology, dominant cause this dearth implementation challenge associated predicting performance The environment commercial-scale bioreactor drastically different such shake flasks. long most do perform same way two scenarios [6Humphrey Shake flask fermentor: what we learned?.Biotechnol Progress. 1998; 14: 3-7Crossref (118) Thus, industrial scale-up necessarily goes beyond [7Willrodt C. al.Guiding efficient synthesis non-natural by physicochemical properties reactants.Curr. Opin. Biotechnol. 35: 52-62Crossref (23) 8Chubukov V. al.Synthetic chemicals.NPJ Applicat. 2: 16009Crossref (152) A recent places cost large-volume $100 million $1 billion USD [9Crater J.S. Lievense J.C. Scale-up processes.FEMS Microbiol. Lett. 365 (fny138)Crossref (98) risk failure high, de-risking chassis itself aspect engineering. Process practices system fairly established, multiple reviews written topic [10Junker B.H. methodologies yeast fermentation processes.J. Biosci. Bioeng. 2004; 97: 347-364Crossref (173) 11Young T.B. Fermentation scaleup: experience total environmental approach.Ann. N. Y. Acad. Sci. 1979; 326: 165-180Crossref (9) 12Schmidt F.R. Optimization scale up processes.Appl. 2005; 68: 425-435Crossref (221) In contrast, phenotype fluctuating conditions, genotypic drift long-term cultivation, physiological factors less explored, potential impacts only recently [8Chubukov 13Rugbjerg al.Diverse genetic error modes constrain production.Nat. Commun. 9: 787Crossref best area scale-down models, where numbers generated tested under conditions representative growth production, allowing better selection will more predictably [14Neubauer Junne S. Scale-down simulators metabolic analysis bioprocesses.Curr. 2010; 21: 114-121Crossref (138) 15Baez al.Simulation CO(2) gradients system: transcriptional study recombinant coli.Biotechnol. J. 2011; 6: 959-967Crossref (20) 16Caspeta L. al.The effect heating rate metabolism, stress, temperature-induced protein: study.Biotechnol. Bioengineer. 2009; 102: 468-482Crossref (60) 17de Jonge L.P. al.Scale-down penicillin Penicillium chrysogenum.Biotechnol. 944-958Crossref (71) 18Lara A.R. al.Living heterogeneities bioreactors: understanding effects cells.Mol. 2006; 34: 355-381Crossref (281) it remains difficult develop maintain when scaled up. Advances 19Lee S.Y. Kim H.U. strategies developing strains.Nat. 1061-1072Crossref (332) Scholar] may able solutions space. Beyond improvements without plasmids inducer requirement, there some ambitious questions posed. Can create genetically phenotypically stable prevent phenotypic subculturing seed cultures? reliably increase duration phase profitability process? track subpopulations causal parameters heterogeneity within culture? dynamically regulate genes respond inhibitory byproducts stresses reduce negative selections enhance performance? There examples studies address individually which, presented collectively, reveal full approach. Most ambitiously, envision predict how behave enable us pre-emptively meet needs (Figure 1). review, focus changes ways mitigate control responses. Though yet underutilized, excellent illustrate predictive engineered strains. wide chemical, physical, negatively impact product formation bioprocess microtiter plates, flasks, bench-scale bioreactors, ultimately commercial Compounding issue fact mimicking environments encountered stages high-throughput screening initial straightforward. Moreover, challenges vary host cultivation Furthermore, measurements performed plates flasks analyses conducted 2). This exacerbates optimization resolve subsequently bioprocesses toward commercialization. One conspicuous difference operating pressure. volumes increase, height water column creates increasing hydrostatic gradient. principle, influence properties, including enzyme activity cell membrane permeability viability flux [18Lara increased bottom raises concentration gases. For instance, CO2 (dCO2) equilibrium bicarbonate carbonate ions which contribute medium osmolarity affect broth pH. dCO2 could result accumulation gas itself, osmotic changes, pH even combination all three [20Zanghi J.A. al.Bicarbonate osmolality key determinants inhibition CHO polysialylation elevated pCO(2) pH.Biotechnol. 1999; 65: 182-191Crossref Other colloidal thermodynamic bulk viscosity, emulsion stability, biomass settling, influenced pressure, among factors. Adverse persist harvest efficiency quality downstream (e.g., solid–liquid separation, chromatography, extraction, crystallization, distillation, upgrading, polymerization), resulting higher costs reduced [21Kumar D. Murthy G.S. Impact pretreatment processing technologies economics energy cellulosic ethanol production.Biotechnol. Biofuels. 4: 27Crossref (250) With volume, mixing time increases few seconds (laboratory-scale) several minutes (hundreds cubic meters) internal components, spargers, baffles, cooling coils, dead zones poor mixing, heat transfer, gas–liquid mass transfer. imperfect results microenvironments inhomogeneities, pH, temperature, oxygen (DO), dCO2, nutrients. Combined, variations lead transient permanent insults oxidative damage, nutrient limitations, stress responses viability, productivity [22Deparis Q. tolerance industrially relevant factories.FEMS Yeast Res. 2017; 17 (fox036)Crossref (108) Additional cultivations arise raw material contaminants. To costs, utilize crude materials utilized little no refinement thus introduce impurities accumulate inhibitory/toxic levels. Some real-world feedstocks include corn slurry, steep liquor, sugar cane juice, molasses, beet agricultural residues, food-processing waste, municipal solid waste Microbial contamination materials, harsh further levels deleterious titer yield (TRY) metrics 23Serate al.Controlling hydrolysis AFEX-pretreated stover switchgrass: hydrolysate composition, fermentation.Biotechnol. 8: 180Crossref (33) 24Fischer C.R. al.Selection production.Metab. Engineer. 2008; 10: 295-304Crossref (305) 25Mohamed E.T. platform ionic liquid adaptive evolution.Microb. Cell Factor. 16: 204Crossref (48) 26Mukhopadhyay Tolerance bacteria advanced chemicals.Trends 23: 498-508Abstract Full Text PDF (160) These strain-screening assays, but priority underappreciated sensitivities understudied early strain/cultivation program sterile technique very pure commonly small-scale optimization. If account risks them outset, scaling would become predictable, case platforms. Exposure heterogeneous fermenters trigger 27Enfors S.O. al.Physiological bioreactors.J. 2001; 85: 175-185Crossref (348) 28Takors R. processes: impacts, open questions.J. 2012; 160: 3-9Crossref (126) High-throughput analytics, DNA sequencing −omics (fluxomics, transcriptomics, proteomics, metabolomics), platforms, characterized simulating scaled-down experiments. Mathematical models guidance designing forecasting learnings environment. approach probability metrics, TRY, translate fully quickly manufacturing plant, thereby minimizing start-up 29Delvigne F. al.Bioprocess scale-up/down integrative enabling technology: fluid mechanics beyond.Microb. 1267-1274Crossref (40) Heterogeneity substrates nutrient, inherent population examined context. Gas O2, parameter measurable bioreactor. grown batch resulted slower by-product [30Castan al.Oxygen enriched air supply human hormone.Enzyme Microb. Technol. 2002; 30: 847-854Crossref (42) Another found needed E. green fluorescent protein (GFP), observe glutamate decarboxylase α‐ketoglutarate dehydrogenase [15Baez comparative transcript revealed fast Corynebacterium glutamicum alternating levels, appeared strongly correlated strength pCO2 stimulus [31Buchholz al.CO₂/HCO₃⁻ perturbations simulated device glutamicum.Appl. 98: 8563-8572Crossref (38) authors observed expression encoding enzymes necessary oxaloacetate hypothesized rapid availability PEP/pyruvate carboxylation. first findings were reported Oosterhuis al. [32Oosterhuis N.M. gluconic acid Gluconobacter oxydans.Biotechnol. 1985; 27: 711-720Crossref (72) studies, oxydans was cycled low high More similar intracellular 33Lara al.Fast dynamic fermentative metabolism aerobic anaerobic glucose pulses.Biotechnol. 104: 1153-1161Crossref (57) 34Limberg M.H. al.Metabolic profile 1,5-diaminopentane producing conditions: blueprint robustness inhomogeneities.Biotechnol. 114: 560-575Crossref (36) 35Käß al.Assessment against oxygen/substrate oscillations DM1933 two-compartment bioreactor.Bioprocess Biosyst. 37: 1151-1162Crossref (37) 36von Wulffen al.Rapid sampling after changing reveals dynamics.Genes (Basel). 90Crossref (13) explained alterations stoichiometry kinetics, organic acids, indicating responds rapidly [33Lara Fu [37Fu Z. al.Exometabolome hypoxia up-scaling Saccharomyces cerevisiae high-cell density fed-batch biopharmaceutical process.Microb. Fact. 13: 32Crossref carried out exometabolome high-density scale, overflow metabolism. one Liu introduced VHb (Vitreoscilla hemoglobin, facilitating O2 transport) fatty-acid-producing promote yielded 70% compared its parental [38Liu al.Enhancing Vitreoscilla hemoglobin overexpression.Biotechnol. 463-467Crossref While (see Outstanding Questions), natively resilient [35Käß bottlenecks [39Wehrs M. al.Production non-ribosomal peptide indigoidine relies respiratory state cerevisiae.Microb. 17: 193Crossref evolution [40Sandberg T.E. al.Laboratory substrate distinct strategies.Appl. Environ. 83e00410-17Crossref (52) issue. Besides leading suboptimal transfer effects, impose cultures. Recent shown adopt short- withstand regarding [41Löffler – considering ATP expenses responses.Metab. 38: 73-85Crossref 42Löffler al.Switching nitrogen limitation: unraveling dynamics coli.J. 258: 2-12Crossref (12) 43Nieß al.Repetitive short-term stimuli imposed induce adaptation experimental evidence mathematical model.Front. 1195Crossref (19) 44Simen J.D. al.Transcriptional ammonia fluctuations.Microb. 858-872Crossref (27) chrysogenum, physiology investigated imposing intermittent feeding regime culture [17de that, while remained same, significantly intermittently fed observation reduction possibly caused AMP cerevisiae, genome-wide cross-regulation limitations (C,N,P,S) chemostat identified 155 oxygen-responsive responsive macronutritional [45Boer V.M. limited carbon, nitrogen, phosphorus, sulfur.J. 2003; 278: 3265-3274Crossref (264) 46Tai S.L. al.Two-dimensional transcriptome cultures: combinatorial macronutrient limitation cerevisiae.J. 280: 437-447Abstract (120) Data obtained mirroring cellular typical derive validate adequate silico predictions performance. optimize hardware configuration conditions. reductions TRY criteria improved manufacturing. Recently, metabolically structured kinetic model chrysogenum showed sufficient accuracy simulation timescales (which minutes) [47Tang W. 9-pool describing days 1733-1743Crossref (32) Nitrogen-responsive regulation another variation, C/N balance known output. relA, regulator responsible signal molecule guanosine tetraphosphate (ppGpp) stringent activated starvation major coupled manage [48Brown D.R. al.Nitrogen coli.Nat. 5: 4115Crossref (105) Michalowski combined mechanistic knowledge maintained constant ppGpp pool independently nutritional supply, pyruvate greater towards [49Michalowski al.Escherichia HGT: throughput slowly growing resting conditions.Metab. 40: 93-103Crossref (30) Stochasticity gene considered main population, due cell-to-cell variation promoter expression, unequal transporter distribution, bet-hedging diauxic shift [2

Language: Английский

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Metabolic engineering of Pichia pastoris

Metabolic Engineering, Journal Year: 2018, Volume and Issue: 50, P. 2 - 15

Published: April 25, 2018

Besides its use for efficient production of recombinant proteins the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) has been increasingly employed as a platform to produce metabolites varying origin. We summarize here impressive methodological developments last years model and analyze metabolism P. pastoris, engineer genome metabolic pathways. Efficient methods insert, modify or delete genes via homologous recombination CRISPR/Cas9, supported by modular cloning techniques, have reported. An outstanding early example engineering in was humanization protein glycosylation. More recently cell engineered also enhance productivity heterologous proteins. The few seen an increased number pathway design mainly towards complex (secondary) metabolites. In this review, we discuss potential role engineering, strengths, major requirements future chassis strains based on synthetic biology principles.

Language: Английский

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Advances and prospects of Bacillus subtilis cellular factories: From rational design to industrial applications

Metabolic Engineering, Journal Year: 2018, Volume and Issue: 50, P. 109 - 121

Published: May 21, 2018

Language: Английский

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Complete Biosynthesis of Anthocyanins Using E. coli Polycultures

mBio, Journal Year: 2017, Volume and Issue: 8(3)

Published: June 7, 2017

ABSTRACT Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable of wide variety important products, ranging from fuels to pharmaceuticals. These have yet find industrial utilization due their inability economically compete with traditional extraction methods. Here, we engineer first time complex microbial biosynthesis an anthocyanin plant natural product, starting sugar. This was accomplished through development synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes diverse plants other microbes. synthetic consortium-based approach enables functional expression connection lengthy pathways while effectively managing accompanying metabolic burden. The de novo specific molecules, such as calistephin, has been elusive engineering target over decade. our strategy affords milligram-per-liter titers. study also lays groundwork significant advances in strain process design toward cost-competitive biochemical hosts nontraditional methodologies. IMPORTANCE To efficiently express active extensive recombinant high flux requires careful balance allocation resources ATP, reducing equivalents, malonyl coenzyme A (malonyl-CoA), well various pathway-dependent cofactors precursors. address this issue, report design, characterization, implementation polyculture. Division overexpression transcription factors 4 independent modules allowed division burden optimization module-specific metabolite needs. represents most consortia constructed date applications provides new paradigm reconstitution nonnative hosts.

Language: Английский

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