An efficient CRISPR/Cas9‐based genome editing system for alkaliphilic Bacillus sp. N16‐5 and application in engineering xylose utilization for D‐lactic acid production

Abstract

Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16-5, an industrially interesting strain with great potential for the

Unraveling the role of intra-cellular metabolites in the lactic acid production by novel Bacillus amyloliquefaciens using sugarcane molasses as a substratum

“…The modern revolutionary genomic tool CRISPR-cas9 genome editing tool has been used to metabolically engineer the lactic acid production from glucose and cellobiose as well as xylose. 22,24 Understanding the role of metabolites in each step will lead to the addition or deletion/knock-out of the appropriate gene from the bacteria to enhance lactic acid production. 20,23…”

“…[15][16][17] Metabolomics is a powerful tool that props up the scientific community to design target-oriented upstream processes such as TILLING (Targeting Induced Local Lesions in Genomes), mutagenesis library construction, genome mining, and over-expression [18][19][20][21] or knock-out approaches through CRISPR -Cas 9 and downstream processes such as the utilization of agro-industrial waste, food, and beverages wastes would be possible. [22][23][24] Therefore, the present study is designed to unravel the role of metabolites in lactic acid production through microbial fermentation of sugarcane molasses using combined GC-MS and LC-MS analysis.…”

CRISPR Tools in Bacterial Whole-Cell Biocatalysis

“…N16-5 employing CRISPR-mediated HDR. 51 The authors replaced the endogenous L-lactic acid dehydrogenase gene (L-ldhA) with a D-lactic acid dehydrogenase gene (D-ldhA) from Lactobacillus delbrueckii. By eliminating the expression of the L-ldhA gene, the researchers aimed to obtain optically pure lactic acid from xylose.…”

“…Regulatory elements replacement, gene editing, and expression cassette integration are all examples of these modifications. For instance, enzymatic activities can be improved through structural modification or the overexpression of a key gene, while competing pathways can be downregulated, diminishing byproduct formation. ,, Furthermore, the integration of CRISPR-Cas technology with other genomic modification technologies offers an enhanced means of achieving the previously mentioned targeted modifications, greatly boosting the efficiency of the process. Using this approach, a genomic modification is incorporated via another editing technique (i.e., recombinases), and the positive clones are selected by means of CRISPR.…”

“…In another recent example of lactic acid production improvement, Huang et al reported the modification of Bacillus sp. N16-5 employing CRISPR-mediated HDR . The authors replaced the endogenous l -lactic acid dehydrogenase gene ( L-ldhA ) with a d -lactic acid dehydrogenase gene ( D-ldhA ) from Lactobacillus delbrueckii.…”