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

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.

Download Full-text

Metabolic engineering of Yarrowia lipolytica for industrial applications

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2015.08.010 ◽

2015 ◽

Vol 36 ◽

pp. 65-72 ◽

Cited By ~ 104

Author(s):

Quinn Zhu ◽

Ethel N Jackson

Keyword(s):

Metabolic Engineering ◽

Yarrowia Lipolytica ◽

Industrial Applications

Download Full-text

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

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.

Download Full-text

Bioproduction of Isoprenoids and Other Secondary Metabolites Using Methanotrophic Bacteria as an Alternative Microbial Cell Factory Option: Current Stage and Future Aspects

Catalysts ◽

10.3390/catal9110883 ◽

2019 ◽

Vol 9 (11) ◽

pp. 883 ◽

Cited By ~ 1

Author(s):

Young Chan Jeon ◽

Anh Duc Nguyen ◽

Eun Yeol Lee

Keyword(s):

Metabolic Engineering ◽

Secondary Metabolites ◽

Value Added ◽

Industrial Applications ◽

Cell Factory ◽

Methanotrophic Bacteria ◽

Microbial Cell Factory ◽

Future Outlook ◽

Metabolites Production ◽

Current Stage

Methane is a promising carbon feedstock for industrial biomanufacturing because of its low price and high abundance. Recent advances in metabolic engineering and systems biology in methanotrophs have made it possible to produce a variety of value-added compounds from methane, including secondary metabolites. Isoprenoids are one of the largest family of secondary metabolites and have many useful industrial applications. In this review, we highlight the current efforts invested to methanotrophs for the production of isoprenoids and other secondary metabolites, including riboflavin and ectoine. The future outlook for improving secondary metabolites production (especially of isoprenoids) using metabolic engineering of methanotrophs is also discussed.

Download Full-text

Comprehensive metabolic engineering for fermenting glycerol efficiently in Saccharomyces cerevisiae

10.1101/2021.02.13.430370 ◽

2021 ◽

Author(s):

Sadat M. R. Khattab ◽

Takashi Watanabe

Keyword(s):

Saccharomyces Cerevisiae ◽

Metabolic Engineering ◽

Metabolic Flux ◽

Plant Biomass ◽

Industrial Applications ◽

Cell Factory ◽

Cell Wall Polysaccharides ◽

Oxidative Pathway ◽

Biomass Decomposition ◽

Engineered Strain

ABSTRACTGlycerol is an eco-friendly solvent enhancing plant-biomass decomposition through a glycerolysis process in many pretreatment methods. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae restrains many of these scenarios. Here we outline the complete strategy for the generation of efficient glycerol fermenting yeast by rewriting the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by O2-dependent dynamic shuttle while abolishing both glycerol phosphorylation and biosynthesis pathways. By following a vigorous glycerol oxidative pathway, the engineered strain demonstrated augmentation in conversion efficiency (CE) reach up to 0.49g-ethanol/g-glycerol—98% of theoretical conversion—with production rate >1 g/L-1h-1 when supplementing glycerol as a single fed-batch on a rich-medium. Furthermore, the engineered strain showed a new capability toward ferment a mixture of glycerol and glucose with producing >86 g/L of bioethanol with 92.8% of the CE. To our knowledge, this is the highest ever reported titer in this regard. Notably, this strategy flipped our ancestral yeast from non-growth on glycerol, on the minimal medium, to a fermenting strain with productivities 0.25-0.5 g/L-1h-1 and 84-78% of CE, respectively and 90% of total conversions to the products. The findings in metabolic engineering here may release the limitations of utilizing glycerol in several eco-friendly biorefinery approaches.IMPORTANCEWith the avenues for achieving efficient lignocellulosic biorefinery scenarios, glycerol gained keen attention as an eco-friendly biomass-derived solvent for enhancing the dissociation of lignin and cell wall polysaccharides during pretreatment process. Co-fermentation of glycerol with the released sugars from biomass after the glycerolysis expands the resource for ethanol production and release from the burden of component separation. Titer productivities are one of the main obstacles for industrial applications of this process. Therefore, the generation of highly efficient glycerol fermenting yeast significantly promotes the applicability of the integrated biorefineries scenario. Besides, the glycerol is an important carbon resource for producing chemicals. Hence, the metabolic flux control of yeast from glycerol contributes to generation of cell factory producing chemicals from glycerol, promoting the association between biodiesel and bioethanol industries. Thus, this study will shed light on solving the problems of global warming and agricultural wastes, leading to establishment of the sustainable society.

Download Full-text

The generation of metabolic changes for the production of high-purity zeaxanthin mediated by CRISPR-Cas9 in Chlamydomonas reinhardtii

Microbial Cell Factories ◽

10.1186/s12934-020-01480-4 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Inhwa Song ◽

Jongrae Kim ◽

Kwangryul Baek ◽

Young Choi ◽

ByongCheol Shin ◽

...

Keyword(s):

High Purity ◽

Gene Editing ◽

Retinal Pigment ◽

Industrial Applications ◽

Age Related Macular Degeneration ◽

Parental Line ◽

Knockout Mutant ◽

Double Knockout ◽

Pharmaceutical Applications ◽

Age Related

Abstract Background Zeaxanthin, a major xanthophyll pigment, has a significant role as a retinal pigment and antioxidant. Because zeaxanthin helps to prevent age-related macular degeneration, its commercial use in personalized nutritional and pharmaceutical applications has expanded. To meet the quantitative requirements for personalized treatment and pharmaceutical applications, it is necessary to produce highly purified zeaxanthin. Results In this study, to meet the quantitative requirements for industrial applications, we generated a double knockout mutant which is gene-edited by the CRISPR-Cas9 ribonucleoprotein-mediated knock-in system. The lycopene epsilon cyclase (LCYE) was edited to the elimination of α-branch of xanthophyll biosynthesis in a knockout mutant of the zeaxanthin epoxidase gene (ZEP). The double knockout mutant (dzl) had a 60% higher zeaxanthin yield (5.24 mg L− 1) and content (7.28 mg g− 1) than that of the parental line after 3 days of cultivation. Furthermore, medium optimization improved the 3-day yield of zeaxanthin from the dzl mutant to 6.84 mg L− 1. Conclusions A Chlamydomonas strain with the elimination of lutein production by gene editing using CRISPR-Cas9 has been successfully developed. This research presents a solution to overcome the difficulties of the downstream-process for the production of high-purity zeaxanthin.

Download Full-text

Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system for Bacillus subtilis

Biotechnology for Biofuels ◽

10.1186/s13068-019-1537-1 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 15

Author(s):

Dingyu Liu ◽

Can Huang ◽

Jiaxin Guo ◽

Peiji Zhang ◽

Tao Chen ◽

...

Keyword(s):

Bacillus Subtilis ◽

Metabolic Engineering ◽

Genome Editing ◽

Gene Editing ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Gene Deletions ◽

Multiple Genes ◽

Genetic Modifications ◽

Riboflavin Operon

Abstract Background Metabolic engineering has expanded from a focus on designs requiring a small number of genetic modifications to increasingly complex designs driven by advances in multiplex genome editing technologies. However, simultaneously modulating multiple genes on the chromosome remains challenging in Bacillus subtilis. Thus, developing an efficient and convenient method for B. subtilis multiplex genome editing is imperative. Results Here, we developed a CRISPR/Cas9n-based multiplex genome editing system for iterative genome editing in B. subtilis. This system enabled us to introduce various types of genomic modifications with more satisfying efficiency than using CRISPR/Cas9, especially in multiplex gene editing. Our system achieved at least 80% efficiency for 1–8 kb gene deletions, at least 90% efficiency for 1–2 kb gene insertions, near 100% efficiency for site-directed mutagenesis, 23.6% efficiency for large DNA fragment deletion and near 50% efficiency for three simultaneous point mutations. The efficiency for multiplex gene editing was further improved by regulating the nick repair mechanism mediated by ligD gene, which finally led to roughly 65% efficiency for introducing three point mutations on the chromosome. To demonstrate its potential, we applied our system to simultaneously fine-tune three genes in the riboflavin operon and significantly improved the production of riboflavin in a single cycle. Conclusions We present not only the iterative CRISPR/Cas9n system for B. subtilis but also the highest efficiency for simultaneous modulation of multiple genes on the chromosome in B. subtilis reported to date. We anticipate this CRISPR/Cas9n mediated system to greatly enhance the optimization of diverse biological systems via metabolic engineering and synthetic biology.

Download Full-text

Multiplex genome editing by natural transformation (MuGENT) for synthetic biology inVibrio natriegens

10.1101/122655 ◽

2017 ◽

Cited By ~ 2

Author(s):

Triana N. Dalia ◽

Chelsea A. Hayes ◽

Sergey Stolyar ◽

Christopher J. Marx ◽

James B. McKinlay ◽

...

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

Genome Editing ◽

High Efficiency ◽

Natural Transformation ◽

Value Added ◽

Industrial Applications ◽

Proof Of Concept ◽

Natural Competence ◽

Low Efficiency

ABSTRACTVibrio natriegenshas recently emerged as an alternative toEscherichia colifor molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence inV. natriegensand describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented timescales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-β-hydroxybutyrate (PHB) inV. natriegensby targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ~100 times more PHB than the parent isolate and ~3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications.

Download Full-text

Establishment of a Genome Editing Tool Using CRISPR-Cas9 in Chlorella vulgaris UTEX395

International Journal of Molecular Sciences ◽

10.3390/ijms22020480 ◽

2021 ◽

Vol 22 (2) ◽

pp. 480

Author(s):

Jongrae Kim ◽

Kwang Suk Chang ◽

Sangmuk Lee ◽

EonSeon Jin

Keyword(s):

Genome Editing ◽

Chlorella Vulgaris ◽

Negative Selection ◽

Screening Strategy ◽

Feed Additive ◽

Cell Factory ◽

Dna Transformation ◽

Research Groups ◽

A Genome ◽

Food And Feed

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.

Download Full-text

Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species

Journal of Fungi ◽

10.3390/jof7070505 ◽

2021 ◽

Vol 7 (7) ◽

pp. 505

Author(s):

Ping Zhang ◽

Yu Wang ◽

Chenxi Li ◽

Xiaoyu Ma ◽

Lan Ma ◽

...

Keyword(s):

Genome Editing ◽

Fungal Pathogens ◽

Gene Editing ◽

Recombination Frequency ◽

Target Sequence ◽

Widespread Application ◽

Genetic Studies ◽

Efficient Gene ◽

Cryptococcus Species ◽

Overlap Pcr

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

Download Full-text