A RecET-assisted CRISPR–Cas9 genome editing in Corynebacterium glutamicum

Abstract

BackgroundExtensive modification of genome is an efficient manner to regulate the metabolic network for producing target metabolites or non-native products using Corynebacterium glutamicum as a cell factory. Genome editing approaches by means of homologous recombination and counter-selection markers are laborious and time consuming due to multiple round manipulations and low editing efficiencies. The current two-plasmid-based CRISPR–Cas9 editing methods generate false positives due to the potential instability of Cas9 on the plasmid, and require a high transformation efficiency for co-occurrence of two plasmids transformation.ResultsHere, we developed a RecET-assisted CRISPR–Cas9 genome editing method using a chromosome-borne Cas9–RecET and a single plasmid harboring sgRNA and repair templates. The inducible expression of chromosomal RecET promoted the frequencies of homologous recombination, and increased the efficiency for gene deletion. Due to the high transformation efficiency of a single plasmid, this method enabled 10- and 20-kb region deletion, 2.5-, 5.7- and 7.5-kb expression cassette insertion and precise site-specific mutation, suggesting a versatility of this method. Deletion of argR and farR regulators as well as site-directed mutation of argB and pgi genes generated the mutant capable of accumulating l-arginine, indicating the stability of chromosome-borne Cas9 for iterative genome editing. Using this method, the model-predicted target genes were modified to redirect metabolic flux towards 1,2-propanediol biosynthetic pathway. The final engineered strain produced 6.75 ± 0.46 g/L of 1,2-propanediol that is the highest titer reported in C. glutamicum. Furthermore, this method is available for Corynebacterium pekinense 1.563, suggesting its universal applicability in other Corynebacterium species.ConclusionsThe RecET-assisted CRISPR–Cas9 genome editing method will facilitate engineering of metabolic networks for the synthesis of interested bio-based products from renewable biomass using Corynebacterium species as cell factories.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0910-2) contains supplementary material, which is available to authorized users.

Recent Advances of L-ornithine Biosynthesis in Metabolically Engineered Corynebacterium glutamicum

“…The mechanisms underlying the transportation of L-ornithine must be further investigated by new engineering techniques, such as genomics (Teusink and Smid, 2006), transcriptomics (Kim et al, 2017), and proteomics (Shen et al, 2016;Varela et al, 2018) to aid the development of high L-ornithine-producing strains. CRISPR/Cas9, a novel gene-editing technology, is a rapid and efficient method that can be applied for genetic modifications, such as gene deletion, large fragment DNA assembly, or sitedirected gene mutation (Cho et al, 2017;Jiang et al, 2017;Wang B. et al, 2018;Zhang J. et al, 2019) in C. glutamicum, which can accelerate the pathway engineering in C. glutamicum strains. Irrational breeding strategies such as induced mutation breeding, or adaptive evolution have enabled rapid progress in improving the yield of L-ornithine.…”

Efficient and Accurate Single-Base Genome Editing in Corynebacterium Glutamicum by Target-Mismatched CRISPR/Cpf1

“…The PAM sequence (5′-NYTV) of CRISPR/Cpf1 [36] does not restrict editing of the genome of industrial C. glutamicum strains with G + C contents of 53.8% [37]. Moreover, owing to the potential toxicity of CRISPR/Cas9, the CRISPR/Cpf1 system of C. glutamicum has received increasing attention [12,18,38,39].…”

“…Moreover, CRISPR/Cpf1 displayed lower toxicity in C. glutamicum cells, and was useful to engineer A + T-rich promoter regions [18]. Wang et al used DNA fragments with two homologous arms (~ 1 kb) to introduce single point mutations in the start codon of pgi with 55% e ciency, through CRISPR/Cpf1-mediated negative selection [29].…”

Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection

“…Since a non-specific binding activity associated with overexpression of Cas9 was detected at high concentrations (Cobb et al 2014;Huang et al 2015;Wang et al 2018;Cui et al 2018), we modulated expression of Cas9 by placing it under control of the arabinose inducible P BAD promoter on the kanamycin (Km) resistant pX2Cas9 plasmid (Garst et al 2016). As a control we used a defective Cas9 (dCas9) from the pdCas9 plasmid (Garst et al 2016), which binds the target DNA sequence but is unable to cleave.…”