Dataset Information

Engineering of Multiple Modules to Improve Amorphadiene Production in Bacillus subtilis Using CRISPR-Cas9.

ABSTRACT: Engineering strategies to improve terpenoids' production in Bacillus subtilis mainly focus on 2C-methyl-d-erythritol-4-phosphate (MEP) pathway overexpression. To systematically engineer the chassis strain for higher amorphadiene (precursor of artemisinin) production, a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system was established in B. subtilis to facilitate precise and efficient genome editing. Then, this system was employed to engineer three more modules to improve amorphadiene production, including the terpene synthase module, the branch pathway module, and the central metabolic pathway module. Finally, our combination of all of the useful strategies within one strain significantly increased extracellular amorphadiene production from 81 to 116 mg/L after 48 h flask fermentation without medium optimization. For the first time, we attenuated the FPP-derived competing pathway to improve amorphadiene biosynthesis and investigated how the TCA cycle affects amorphadiene production in B. subtilis. Overall, this study provides a universal strategy for further increasing terpenoids' production in B. subtilis by comprehensive and systematic metabolic engineering.

SUBMITTER: Song Y

PROVIDER: S-EPMC8154554 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Engineering of Multiple Modules to Improve Amorphadiene Production in Bacillus subtilis Using CRISPR-Cas9.

Song Yafeng Y He Siqi S Abdallah Ingy I II Jopkiewicz Anita A Setroikromo Rita R van Merkerk Ronald R Tepper Pieter G PG Quax Wim J WJ

Journal of agricultural and food chemistry 20210420 16

Engineering strategies to improve terpenoids' production in Bacillus subtilis mainly focus on 2C-methyl-d-erythritol-4-phosphate (MEP) pathway overexpression. To systematically engineer the chassis strain for higher amorphadiene (precursor of artemisinin) production, a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system was established in B. subtilis to facilitate precise and efficient genome editing. Then, this system was employed to engineer ...[more]

PMID: 33877851

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Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system from Streptococcus pyogenes has been widely deployed as a tool for bacterial strain construction. Conventional CRISPR-Cas9 editing strategies require design and molecular cloning of an appropriate guide RNA (gRNA) to target genome cleavage and a repair template for introduction of the desired site-specific genome modification. Here, we present a streamlined method that leverages the existing collection of nearly 4,000 Bacillus subtilis strains (the BKE collection) with individual genes replaced by an integrated erythromycin (erm) resistance cassette. A single plasmid (pAJS23) with a gRNA targeted to erm allows cleavage of the genome at any nonessential gene and at sites nearby to many essential genes. This plasmid can be engineered to include a repair template, or the repair template can be cotransformed with the plasmid as either a PCR product or genomic DNA. We demonstrate the utility of this system for generating gene replacements, site-specific mutations, modification of intergenic regions, and introduction of gene-reporter fusions. In sum, this strategy bypasses the need for gRNA design and allows the facile transfer of mutations and genetic constructions with no requirement for intermediate cloning steps. IMPORTANCE Bacillus subtilis is a well-characterized Gram-positive model organism and a popular platform for biotechnology. Although many different CRISPR-based genome editing strategies have been developed for B. subtilis, they generally involve the design and cloning of a specific guide RNA (gRNA) and repair template for each application. By targeting the erm resistance cassette with an anti-erm gRNA, genome editing can be directed to any of nearly 4,000 gene disruptants within the existing BKE collection of strains. Repair templates can be engineered as PCR products, or specific alleles and constructions can be transformed as chromosomal DNA, thereby bypassing the need for plasmid construction. The described method is rapid and facilitates a wide range of genome manipulations.

Project description:Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool for targeted genome editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial species. A collection of vectors compatible with high-throughput (HTP) fragment exchange (FX) cloning for heterologous expression in Escherichia coli and Bacillus was previously developed. This vector catalogue was through this work supplemented with editing plasmids for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to the stable integration of the gene into the amyE locus of a seven-protease-deficient KO7 strain. The newly engineered B. subtilis strain allowed the successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering, and contribute to a cloning platform for a quick transition from HTP enzyme expression to production through the fermentation of industrially relevant B. subtilis and related strains.IMPORTANCE We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus' capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host.

Dataset Information

Engineering of Multiple Modules to Improve Amorphadiene Production in Bacillus subtilis Using CRISPR-Cas9.

Publications

Engineering of Multiple Modules to Improve Amorphadiene Production in <i>Bacillus subtilis</i> Using CRISPR-Cas9.

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