scholarly journals Kluyveromyces marxianus as a robust synthetic biology platform host

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

ABSTRACTThroughout history, the yeast Saccharomyces cerevisiae has 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. cerevisiae has 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 engineer the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits– thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.

scholarly journals EngineeringKluyveromyces marxianusas a Robust Synthetic Biology Platform Host

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Paul Cernak ◽  
Raissa Estrela ◽  
Snigdha Poddar ◽  
Jeffrey M. Skerker ◽  
Ya-Fang Cheng ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Genome Editing ◽  
Kluyveromyces Marxianus ◽  
Lipid Production ◽  
Carbon Sources ◽  
Industrial Applications ◽  
Human Society ◽  
Content Type ◽  
Renewable Chemicals

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.

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

2019 ◽  
Vol 18 (1) ◽  
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.

scholarly journals Reconstruction and Analysis of aKluyveromyces marxianusGenome-scale Metabolic Model

2019 ◽  
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.

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

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.

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

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.

scholarly journals Prospection of Fungal Lignocellulolytic Enzymes Produced from Jatoba (Hymenaea courbaril) and Tamarind (Tamarindus indica) Seeds: Scaling for Bioreactor and Saccharification Profile of Sugarcane Bagasse

2021 ◽  
Vol 9 (3) ◽  
pp. 533
Author(s):  
Alex Graça Contato ◽  
Tássio Brito de Oliveira ◽  
Guilherme Mauro Aranha ◽  
Emanuelle Neiverth de Freitas ◽  
Ana Claudia Vici ◽  
...  
Keyword(s):  
Carbon Sources ◽  
Temporal Analysis ◽  
Industrial Applications ◽  
Lignocellulolytic Enzymes ◽  
Tamarindus Indica ◽  
Acetyl Xylan Esterase ◽  
Trametes Hirsuta ◽  
Hymenaea Courbaril ◽  
The Media ◽  
Main Components

The lignocellulosic biomass comprises three main components: cellulose, hemicellulose, and lignin. Degradation and conversion of these three components are attractive to biotechnology. This study aimed to prospect fungal lignocellulolytic enzymes with potential industrial applications, produced through a temporal analysis using Hymenaea courbaril and Tamarindus indica seeds as carbon sources. α-L-arabinofuranosidase, acetyl xylan esterase, endo-1,5-α-L-arabinanase, β-D-galactosidase, β-D-glucosidase, β-glucanase, β-D-xylosidase, cellobiohydrolase, endoglucanase, lichenase, mannanase, polygalacturonase, endo-1,4-β-xylanase, and xyloglucanase activities were determined. The enzymes were produced for eight filamentous fungi: Aspergillus fumigatus, Trametes hirsuta, Lasiodiplodia sp., two strains of Trichoderma longibrachiatum, Neocosmospora perseae, Fusarium sp. and Thermothelomyces thermophilus. The best producers concerning enzymatic activity were T. thermophilus and T. longibrachiatum. The optimal conditions for enzyme production were the media supplemented with tamarind seeds, under agitation, for 72 h. This analysis was essential to demonstrate that cultivation conditions, static and under agitation, exert strong influences on the production of several enzymes produced by different fungi. The kind of sugarcane, pretreatment used, microorganisms, and carbon sources proved limiting sugar profile factors.

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

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.

scholarly journals Isolation, identification and diversity of oleaginous yeasts from Kuching, Sarawak, Malaysia

2018 ◽  
Vol 19 (4) ◽  
pp. 1266-1272 ◽  
Author(s):  
MICKY VINCENT ◽  
HUANG CHAI HUNG ◽  
PATRICIA ROWENA MARK BARAN ◽  
AFIZUL SAFWAN AZAHARI ◽  
DAYANG SALWANI AWANG ADENI
Keyword(s):  
Candida Parapsilosis ◽  
Yeast Species ◽  
Lipid Production ◽  
Industrial Applications ◽  
Oleaginous Yeasts ◽  
Intracellular Lipids ◽  
Pichia Manshurica ◽  
Sudan Iv ◽  
Scientific Technical ◽  
Polymerase Chain

Vincent M, Hung MC, Baran PRM, Azahari AS, Adeni DSA. 2018. Isolation, identification and diversity of oleaginous yeastsfrom Kuching, Sarawak, Malaysia. Biodiversitas 19: 1266-1272. The present study was performed to isolate, identify and determine thediversity of oleaginous yeasts from various sources in Kuching, Sarawak (Malaysia). Microscopic observations via light and scanningelectron microscope (SEM) indicated that the yeast isolates were in sizes ranging from 2-3 μm in width and 4-8 μm in length, typical ofmost unicellular ascomycotic fungi. Polymerase Chain Reaction (PCR) and molecular identification performed on the yeast isolates,targeting the D1/D2 region of the 26S rDNA, identified 6 yeast species from the 21 isolates, namely Pichia manshurica (5/21), Candidakrusei (8/21), Candida parapsilosis (1/21), Pichia guilliermondii (2/21), Clavispora lusitaniae (1/21) and Kluyveromyces marxianus(4/21). All 21 yeast isolates accumulated intracellular lipids when grown in nitrogen-limited medium, as tested via Sudan IV staining.The present study is the first to document the production of lipids bodies in C. krusei, C. parapsilosis, and C. lusitaniae. Furtherinvestigations to assess the growth kinetics, lipid production efficiencies and lipids profiles of these oleaginous yeasts may provideinsights into the possible utilization of these isolates for a variety of scientific, technical and industrial applications.

scholarly journals Screening For Yeast Phytase Leads to the Identification of a New Cell-Bound and Secreted Activity in Cyberlindnera jadinii CJ2

2021 ◽  
Vol 9 ◽  
Author(s):  
Claudia Capusoni ◽  
Immacolata Serra ◽  
Silvia Donzella ◽  
Concetta Compagno
Keyword(s):  
High Temperature ◽  
Phytic Acid ◽  
Convenient Method ◽  
Kluyveromyces Marxianus ◽  
Food Industry ◽  
Yeast Species ◽  
Industrial Applications ◽  
Industrial Processes ◽  
Acidic Ph ◽  
Initial Screening

Phytic acid is an anti-nutritional compound able to chelate proteins and ions. For this reason, the food industry is looking for a convenient method which allows its degradation. Phytases are a class of enzymes that catalyze the degradation of phytic acid and are used as additives in feed-related industrial processes. Due to their industrial importance, our goal was to identify new activities that exhibit best performances in terms of tolerance to high temperature and acidic pH. As a result of an initial screening on 21 yeast species, we focused our attention on phytases found in Cyberlindnera jadinii, Kluyveromyces marxianus, and Torulaspora delbrueckeii. In particular, C. jadinii showed the highest secreted and cell-bound activity, with optimum of temperature and pH at 50°C and 4.5, respectively. These characteristics suggest that this enzyme could be successfully used for feed as well as for food-related industrial applications.

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