Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

Abstract

Background: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

Transcriptomic Profiles of Zymomonas mobilis 8b to Furfural Acute and Long-Term Stress in Both Glucose and Xylose Conditions

“…Other than ethanol production from pure sugars and lignocellulosic materials, Z. mobilis has been engineered for other biochemicals such as 2,3-butanediol and sorbitol, which could be engineered as an ideal microbial platform for future biomass biorefinery (Xia et al, 2019;He et al, 2014;Yang et al, 2016). In addition, the availability of genome sequences for multiple strains (Seo et al, 2005;Yang et al, 2009a;Zhao et al, 2012), metabolic modeling results (Widiastuti et al, 2011;Pentjuss et al, 2013), exogenous and native CRISPRcas genome editing tools and biological part characterization methods for strain engineering (Kerr et al, 2011;Jia et al, 2013;Zhang et al, 2013;Shen et al, 2019;Yang et al, 2019a,b;Zheng et al, 2019) also help expedite the research progress in Z. mobilis.…”

Metabolic engineering of Zymomonas mobilis for anaerobic isobutanol production

“…In addition, the genome sequence, functional re-annotation, and substantial systems biology studies and metabolic models of Z. mobilis have been reported [12,15,[17][18][19][20][21][22][23][24]. Recent development of genome editing such as exogenous and native CRISPR-cas toolkits as well as methods for biological part identification and characterization [25][26][27][28][29][30][31] will facilitate heterologous pathway engineering and unravel the underlying mechanisms for balanced production and robustness [32] in Z. mobilis.…”

CRISPR/Cas12a Mediated Genome Editing Enhances Bombyx mori Resistance to BmNPV

“…Genome editing has the potential to accurately edit the genomes of model organism ( Perez-Pinera et al, 2012 ; Burgess, 2013 ; Nakade et al, 2017 ). Cas9, Cas12a- (Cpf1), Cas12b-, Cas13-, Cas3-, and Cas14- based CRISPR systems have been explored for editing human, animals, plants and microbe genomes ( Matsoukas, 2018 ; Schindele et al, 2018 ; Moon et al, 2019 ; Morisaka et al, 2019 ; Savage, 2019 ; Shen et al, 2019 ). Cas12a is a type V CRISPR-effector protein with greater specificity for genome editing in mammals and plants ( Fagerlund et al, 2015 ; Zetsche et al, 2015 ).…”