Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system

Lian, Jiazhang; HamediRad, Mohammad; Hu, Sumeng; Zhao, Huimin

doi:10.1038/s41467-017-01695-x

Cited by 260 publications

(262 citation statements)

References 56 publications

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“…Orthogonal dCas9 (SpdCas9 and SadCas9) have been used for complex and inducible gene transcription control (Y. Gao et al, ) as well as for simultaneous imaging of multiple genomic loci (Chen et al, ). More recently, a trifunctional CRISPR system that combines 3 orthogonal Cas9 variants was developed for simultaneous gene activation (using LbdCas12a), repression (using SpdCas9) and deletion (using SaCas9) in the yeast Saccharomyces cerevisiae (Lian, HamediRad, Hu, & Zhao, ). However, to the best of our knowledge, no other studies have reported the use of multiple Cas9 orthologs for metabolic engineering of E. coli .…”

Section: Discussionmentioning

confidence: 99%

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

Sung

Lin

et al. 2019

Biotech & Bioengineering

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CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9‐based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single‐guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock‐in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.

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Section: Discussionmentioning

confidence: 99%

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

Sung

Lin

et al. 2019

Biotech & Bioengineering

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“…Therefore, microbial production of pseudoionone using metabolic engineering and synthetic biology approaches (Lian et al . , ; Wang et al . ) have attracted increasing attention.…”

Section: Discussionmentioning

confidence: 99%

“…However, the supply of biologically synthesized pseudoionone is largely limited by their low concentration in nature. Therefore, microbial production of pseudoionone using metabolic engineering and synthetic biology approaches (Lian et al 2017(Lian et al , 2018Wang et al 2018) have attracted increasing attention.…”

Section: Discussionmentioning

confidence: 99%

Efficient production of Pseudoionone with multipathway engineering in Escherichia coli

Jiang

Chen²,

Lian

et al. 2019

J Appl Microbiol

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Aims This study aimed to achieve efficient production of pseudoionone in Escherichia coli. Methods and Results Firstly, a stable lycopene‐producing strain was constructed by integrating lycopene biosynthetic pathway into the chromosome. Further introduction of the Cucumis melo carotenoid cleavage dioxygenase 1 (cmCCD1) gene resulted in the production of pseudoionone. Then, the tricarboxylic acid cycle (i.e. sdhABCD and sucAB), pentose phosphate pathway (i.e. talB), and methylerythritol 4‐phosphate pathway (i.e. idi and dxs) genes were modulated to improve the production of lycopene and accordingly pseudoionone. Regulation of the expression of talB and sdhABCD for improving NADPH supplies led to a 2·81‐fold increase of pseudoionone production. Further engineering of dxs and idi increased the production of pseudoionone by 8·34‐fold. Followed by bioprocess optimization, 24 mg l−1 pseudoionone with a specific production of 8·55 mg g−1 in shake flask was achieved. Conclusions The combination of metabolic engineering and bioprocess engineering increased the production of pseudoionone by more than 185‐fold. Significance and Impact of the Study The present study promises a green and sustainable route for pseudoionone production using a microbial cell factory.

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“…As metabolic engineering often requires synergistic regulation of multiple genes, combinational strategies is highly desirable. Recently, an orthogonal trifunctional CRISPR system combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR‐AID) was developed in S. cerevisiae . Three independent CRISPR proteins with different PAM sequences and gRNA scaffolds were combined in this system.…”

Section: Crispr‐based Genome Engineeringmentioning

confidence: 99%

Whole‐Genome Regulation for Yeast Metabolic Engineering

Jiang

Dai

2019

Small Methods

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As the most thoroughly investigated eukaryotic microorganism, Saccharomyces cerevisiae is widely used as a cell factory for producing valuable compounds. Currently, strain construction and optimization still cost a vast amount of capital and time to achieve industrially relevant parameters. To efficiently excavate the genetic factors required for desired traits, many high‐throughput genome engineering tools are developed. This review focuses on the genome‐wide approaches that are applied in yeast. Through multiplex genome editing and/or transcription regulation, these tools generate cell populations with great genetic diversity, enabling identification of the critical factors and synergistic interactions in a short time.

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Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system

Cited by 260 publications

References 56 publications

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

Efficient production of Pseudoionone with multipathway engineering in Escherichia coli

Whole‐Genome Regulation for Yeast Metabolic Engineering

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