Application of Genome Editing Method on Kluyveromyces marxianus 17694-DH2 using CRISPR-Cas9 System for Enhanced Xylose Utilization

KSBB Journal ◽  
2019 ◽  
Vol 34 (4) ◽  
pp. 243-247
Author(s):  
Deok-Ho Kwon ◽  
Joong-Hee Park ◽  
Deok Yeol Jeong ◽  
Jae-Bum Park ◽  
Dong-Min Park ◽  
...  
mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Paul Cernak ◽  
Raissa Estrela ◽  
Snigdha Poddar ◽  
Jeffrey M. Skerker ◽  
Ya-Fang Cheng ◽  
...  

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.


2018 ◽  
Author(s):  
Paul Cernak ◽  
Raissa Estrela ◽  
Snigdha Poddar ◽  
Jeffrey M. Skerker ◽  
Ya-Fang Cheng ◽  
...  

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.


2021 ◽  
Author(s):  
Nilesh Kumar Sharma ◽  
Shuvashish Behera ◽  
Richa Arora ◽  
Sachin Kumar

Abstract Evolutionary adaptation provides stability to the strains in the challenging environment. As extension of earlier study, the evolved strains Kluyveromyces marxianus NIRE-K1.1 and K. marxianus NIRE-K3.1 were subjected for secondary adaptation on minimal salt (MS) medium with the aim to enhance xylose utilization for ethanol production together with salt tolerance. Both the strains were adapted till saturated improvement in xylose uptake i.e., 54 generations on MS medium containing xylose. Xylose utilization increased from 14.21 to 45.80% and 10.55 to 45.31%, in evolved strains KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Specific xylose reductase activity has also increased 2.04 and 3.36-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Xylitol dehydrogenase activity was also increased by 2.82 and 1.35-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively. Decrease in redox imbalance was observed in evolved strains, and hence there was a reduction in xylitol production during growth and fermentation. Xylose uptake rate increased by 2.53 and 1.5-folds in KmNIRE-K1.2 and KmNIRE-K3.2, respectively with 2.20 and 6.46-folds higher ethanol concentration, and 2.25 and 5.86-folds higher volumetric productivity, respectively. This study has demonstrated the role of evolutionary adaptation for developing robust yeast strains. KmNIRE-K1.2 and KmNIRE-K3.2 have shown enhanced ethanol production, enzyme activities and less by-product formation like xylitol during xylose metabolism.


2017 ◽  
Vol 6 (3) ◽  
pp. 162-162
Author(s):  
Liane Kaufmann ◽  
Michael von Aster
Keyword(s):  

2018 ◽  
Author(s):  
M Keller ◽  
J Dalla-Riva ◽  
A Kurbasic ◽  
M Al-Majdoub ◽  
P Spegel ◽  
...  

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