EngineeringKluyveromyces marxianusas a Robust Synthetic Biology Platform Host

ABSTRACTThroughout history, the yeastSaccharomyces cerevisiaehas played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However,S. cerevisiaehas proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeastKluyveromyces marxianusto create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates ofK. marxianuscan be made heterothallic for sexual crossing. By breeding two of these mating-type engineeredK. marxianusstrains, we combined three complex traits—thermotolerance, lipid production, and facile transformation with exogenous DNA—into a single host. The ability to crossK. marxianusstrains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering ofK. marxianusisolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establishK. marxianusas a synthetic biology platform comparable toS. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCEThe yeastKluyveromyces marxianusgrows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeastSaccharomyces cerevisiaein industrial applications. Here, we describe genetic tools for genome editing and breedingK. marxianusstrains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to usingK. marxianusas a versatile synthetic biology platform organism for industrial applications.

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Kluyveromyces marxianus as a robust synthetic biology platform host

10.1101/353680 ◽

2018 ◽

Author(s):

Paul Cernak ◽

Raissa Estrela ◽

Snigdha Poddar ◽

Jeffrey M. Skerker ◽

Ya-Fang Cheng ◽

...

Keyword(s):

Synthetic Biology ◽

Genome Editing ◽

Kluyveromyces Marxianus ◽

Complex Traits ◽

Lipid Production ◽

Carbon Sources ◽

Industrial Applications ◽

Human Society ◽

Exogenous Dna ◽

Renewable Chemicals

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Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

10.1101/166678 ◽

2017 ◽

Author(s):

Chenlu Zhang ◽

Ligia Acosta-Sampson ◽

Vivian Yaci Yu ◽

Jamie H. D. Cate

Keyword(s):

Saccharomyces Cerevisiae ◽

Directed Evolution ◽

Plant Biomass ◽

Carbon Sources ◽

Industrial Applications ◽

Yeast Saccharomyces Cerevisiae ◽

Cellulosic Biofuel ◽

Economic Production ◽

Report Analysis ◽

Selection Medium

AbstractThe economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

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Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica

Microbial Cell Factories ◽

10.1186/s12934-019-1259-x ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 5

Author(s):

Vijaydev Ganesan ◽

Michael Spagnuolo ◽

Ayushi Agrawal ◽

Spencer Smith ◽

Difeng Gao ◽

...

Keyword(s):

Metabolic Engineering ◽

Genome Editing ◽

Yarrowia Lipolytica ◽

Gene Editing ◽

Molecular Genetic ◽

Industrial Applications ◽

Cell Factory ◽

Fuel Feed ◽

Renewable Chemicals ◽

Pharmaceutical Applications

AbstractYarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR–Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

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Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.00270-20 ◽

2020 ◽

Vol 86 (12) ◽

Cited By ~ 1

Author(s):

Anna K. Wronska ◽

Meinske P. Haak ◽

Ellen Geraats ◽

Eva Bruins Slot ◽

Marcel van den Broek ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Large Scale ◽

De Novo ◽

Laboratory Strain ◽

Optimal Growth ◽

Industrial Applications ◽

Fast Growth ◽

Growth Media ◽

Free Medium ◽

Content Type

ABSTRACT Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiae BIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae. This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiae. IMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae. Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.

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The Evolutionary Rewiring of Ubiquitination Targets Has Reprogrammed the Regulation of Carbon Assimilation in the Pathogenic Yeast Candida albicans

mBio ◽

10.1128/mbio.00495-12 ◽

2012 ◽

Vol 3 (6) ◽

Cited By ~ 54

Author(s):

Doblin Sandai ◽

Zhikang Yin ◽

Laura Selway ◽

David Stead ◽

Janet Walker ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Fatty Acids ◽

Candida Albicans ◽

Isocitrate Lyase ◽

Carbon Sources ◽

Carbon Assimilation ◽

Pathogenic Yeast ◽

Content Type ◽

Catabolite Inactivation ◽

Accelerated Degradation

ABSTRACTMicrobes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested thatCandida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeastSaccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome inC. albicans. Glucose triggers the degradation of theICL1andPCK1transcripts inC. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed inC. albicans,S. cerevisiaeIcl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that likeS. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation.C. albicansIcl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed inC. albicans ubi4cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes fromS. cerevisiaebut absent from theirC. albicanshomologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure,C. albicansretains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients.IMPORTANCEPathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeastCandida albicansare strongly influenced by theSaccharomyces cerevisiaeparadigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case inC. albicansbecause there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available.

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A Minimal Set of Glycolytic Genes Reveals Strong Redundancies in Saccharomyces cerevisiae Central Metabolism

Eukaryotic Cell ◽

10.1128/ec.00064-15 ◽

2015 ◽

Vol 14 (8) ◽

pp. 804-816 ◽

Cited By ~ 16

Author(s):

Daniel Solis-Escalante ◽

Niels G. A. Kuijpers ◽

Nuria Barrajon-Simancas ◽

Marcel van den Broek ◽

Jack T. Pronk ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Systems Approach ◽

Gene Dosage ◽

Physiological Role ◽

Industrial Applications ◽

Yeast Genome ◽

Small Scale ◽

Genetic Redundancy ◽

Minimal Set ◽

Content Type

ABSTRACTAs a result of ancestral whole-genome and small-scale duplication events, the genomes ofSaccharomyces cerevisiaeand many eukaryotes still contain a substantial fraction of duplicated genes. In all investigated organisms, metabolic pathways, and more particularly glycolysis, are specifically enriched for functionally redundant paralogs. In ancestors of theSaccharomyceslineage, the duplication of glycolytic genes is purported to have played an important role leading toS. cerevisiae's current lifestyle favoring fermentative metabolism even in the presence of oxygen and characterized by a high glycolytic capacity. In modernS. cerevisiaestrains, the 12 glycolytic reactions leading to the biochemical conversion from glucose to ethanol are encoded by 27 paralogs. In order to experimentally explore the physiological role of this genetic redundancy, a yeast strain with a minimal set of 14 paralogs was constructed (the “minimal glycolysis” [MG] strain). Remarkably, a combination of a quantitative systems approach and semiquantitative analysis in a wide array of growth environments revealed the absence of a phenotypic response to the cumulative deletion of 13 glycolytic paralogs. This observation indicates that duplication of glycolytic genes is not a prerequisite for achieving the high glycolytic fluxes and fermentative capacities that are characteristic ofS. cerevisiaeand essential for many of its industrial applications and argues against gene dosage effects as a means of fixing minor glycolytic paralogs in the yeast genome. The MG strain was carefully designed and constructed to provide a robust prototrophic platform for quantitative studies and has been made available to the scientific community.

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Reconstruction and Analysis of aKluyveromyces marxianusGenome-scale Metabolic Model

10.1101/581587 ◽

2019 ◽

Cited By ~ 1

Author(s):

Simonas Marcišauskas ◽

Boyang Ji ◽

Jens Nielsen

Keyword(s):

Kluyveromyces Marxianus ◽

Kluyveromyces Lactis ◽

Carbon Sources ◽

Metabolic Model ◽

Industrial Applications ◽

Phenotypic Traits ◽

Industrial Biotechnology ◽

Thermotolerant Yeast ◽

Related Factors ◽

Insight Into

AbstractBackgroundKluyveromyces marxianusis a thermotolerant yeast with multiple biotechnological potentials for industrial applications, which can metabolize a broad range of carbon sources, including less conventional sugars like lactose, xylose, arabinose and inulin. These phenotypic traits are sustained even up to 45°C, what makes it a relevant candidate for industrial biotechnology applications, such as ethanol production. It is therefore of much interest to get more insight into the metabolism of this yeast. Recent studies suggested, that thermotolerance is achieved by reducing the number of growth-determining proteins or suppressing oxidative phosphorylation. Here we aimed to find related factors contributing to the thermotolerance ofK. marxianus.ResultsHere, we reported the first genome-scale metabolic model ofKluyveromyces marxianus, iSM996, using a publicly availableKluyveromyces lactismodel as template. The model was manually curated and refined to include missing species-specific metabolic capabilities. The iSM996 model includes 1913 reactions, associated with 996 genes and 1531 metabolites. It performed well to predict the carbon source utilization and growth rates under different growth conditions. Moreover, the model was coupled with transcriptomics data and used to perform simulations at various growth temperatures.ConclusionsK. marxianusiSM996 represents a well-annotated metabolic model of thermotolerant yeast, which provide new insight into theoretical metabolic profiles at different temperatures ofK. marxianus. This could accelerate the integrative analysis of multi-omics data, leading to model-driven strain design and improvement.

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Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides

mSphere ◽

10.1128/msphere.00099-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 17

Author(s):

Peter B. Otoupal ◽

Masakazu Ito ◽

Adam P. Arkin ◽

Jon K. Magnuson ◽

John M. Gladden ◽

...

Keyword(s):

Genome Editing ◽

Gene Disruption ◽

Genome Engineering ◽

Carbon Sources ◽

Rhodosporidium Toruloides ◽

Optimized Design ◽

Content Type ◽

Wide Range ◽

Fuels And Chemicals ◽

Microbial Biofuel

ABSTRACT Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’ engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides. Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism. IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast’s production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

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Photoheterotrophic Assimilation of Valerate and Associated Polyhydroxyalkanoate Production by Rhodospirillum rubrum

Applied and Environmental Microbiology ◽

10.1128/aem.00901-20 ◽

2020 ◽

Vol 86 (18) ◽

Author(s):

Guillaume Bayon-Vicente ◽

Sarah Zarbo ◽

Adam Deutschbauer ◽

Ruddy Wattiez ◽

Baptiste Leroy

Keyword(s):

Fatty Acids ◽

Volatile Fatty Acids ◽

Rhodospirillum Rubrum ◽

Carbon Sources ◽

Industrial Applications ◽

Purple Nonsulfur Bacteria ◽

Content Type ◽

Fermentation Processes ◽

Bicarbonate Ions ◽

Pha Production

ABSTRACT Purple nonsulfur bacteria are increasingly recognized for industrial applications in bioplastics, pigment, and biomass production. In order to optimize the yield of future biotechnological processes, the assimilation of different carbon sources by Rhodospirillum rubrum has to be understood. As they are released from several fermentation processes, volatile fatty acids (VFAs) represent a promising carbon source in the development of circular industrial applications. To obtain an exhaustive characterization of the photoheterotrophic metabolism of R. rubrum in the presence of valerate, we combined phenotypic, proteomic, and genomic approaches. We obtained evidence that valerate is cleaved into acetyl coenzyme A (acetyl-CoA) and propionyl-CoA and depends on the presence of bicarbonate ions. Genomic and enzyme inhibition data showed that a functional methylmalonyl-CoA pathway is essential. Our proteomic data showed that the photoheterotrophic assimilation of valerate induces an intracellular redox stress which is accompanied by an increased abundance of phasins (the main proteins present in polyhydroxyalkanoate [PHA] granules). Finally, we observed a significant increase in the production of the copolymer P(HB-co-HV), accounting for a very high (>80%) percentage of HV monomer. Moreover, an increase in the PHA content was obtained when bicarbonate ions were progressively added to the medium. The experimental conditions used in this study suggest that the redox imbalance is responsible for PHA production. These findings also reinforce the idea that purple nonsulfur bacteria are suitable for PHA production through a strategy other than the well-known feast-and-famine process. IMPORTANCE The use and the littering of plastics represent major issues that humanity has to face. Polyhydroxyalkanoates (PHAs) are good candidates for the replacement of oil-based plastics, as they exhibit comparable physicochemical properties but are biobased and biodegradable. However, the current industrial production of PHAs is curbed by the production costs, which are mainly linked to the carbon source. Volatile fatty acids issued from the fermentation processes constitute interesting carbon sources, since they are inexpensive and readily available. Among them, valerate is gaining interest regarding the ability of many bacteria to produce a copolymer of PHAs. Here, we describe the photoheterotrophic assimilation of valerate by Rhodospirillum rubrum, a purple nonsulfur bacterium mainly known for its metabolic versatility. Using a knowledge-based optimization process, we present a new strategy for the improvement of PHA production, paving the way for the use of R. rubrum in industrial processes.

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Defining and redefining its role in biology: Synthetic biology as an emerging field at the interface of engineering and biology

10.7287/peerj.preprints.2634 ◽

2016 ◽

Author(s):

Wenfa Ng

Keyword(s):

Synthetic Biology ◽

Directed Evolution ◽

Genome Editing ◽

Protein Design ◽

Selection Pressure ◽

Recombinant Dna ◽

De Novo ◽

Human Society ◽

Recombinant Dna Technology ◽

Dna Technology

Synthetic biology is often misunderstood as creation of artificial life or new biology using principles different from those of extant organisms around us. But, fundamentally, the field is about engineering biology in a more efficient and effective way, and endowing new functions in existing organisms using a more refined and predictable approach. Thus, synthetic biology as encapsulated by the field it helps defined, is enhanced recombinant DNA technology, an example of which is modular and orthogonal “standard swappable biological parts”. But, as the field grows and matures, various “allied” fields are subsumed into it such as metabolic engineering, protein engineering, directed evolution, origins of life research, and systems biology, which in totality represents a new perspective of how engineering principles can be utilized to expand, in scope and depth, the realms of questions that biology ask. Two parallel approaches, directed evolution and de novo protein design, are frequently used to engineer new phenotypes into organisms. Similar to evolution but with purposeful use of selection pressure to elicit progressive refinement of specific traits in an efficient manner, directed evolution is a powerful methodology that generates, at the cell level, libraries of mutants of slightly different function such as differing resistance to heavy metals, that upon exertion of continued selection pressure, led to the evolution of a strain capable of thriving under a hostile environment previously inhabitable to the organism. Taking a different approach, de novo protein design taps on advances in biomolecule structure modeling together with bioinformatic sequence search for inserting, in a structure defined manner, specific amino acids (natural or unnatural) in a protein structure to endow desired functionality, where one highly sought function is catalysis of unnatural reactions such as the Diels-Alder reaction. Long chain length DNA synthesis, on the other hand, finds utility in enabling the synthesis of a minimal genome for a bacterium, which demonstrates the huge possibilities of having a microbe with an optimized genome (free of extraneous genes) for biotechnological applications in delivering drugs and fuel at high titer with lower cost. Having assimilated other fields, synthetic biology is again redefining its role as its seeks to use, in an ethical and responsible manner, a new way of adding new functions into organisms through genome editing. For example, CRISPR/Cas9 genome editing holds enormous potential for providing life saving gene editing capability in medical treatments, while enabling fast, easy removal of undesirable genes and prophages from a production microorganism. Synthetic biologists are asking themselves deep questions on how best to regulate this powerful technology that could be as impactful on science and human society as recombinant DNA technology was in 1973.

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