Harnessing the Crispr/dCas9 Crispri System for Rapid Assessment of Metabolic Engineering Strategies to Improve Natural Product Titers in E. coli

The field of metabolic engineering is entering a new era in which readily addressable synthetic transcription factors like RNA-guided dCas9 are opening new possibilities for dynamic regulation and for rapid assessment of multiplexed genetic interventions to alter the metabolic landscape in both workhorse and previously intractable microbes. Toward this end, our group has focused on three major applications of CRISPRi-enabled metabolic engineering.

In a recent publication, we developed a modular assembly method for traditional restriction-ligation cloning of type II-A CRISPR array libraries for multiplex, combinatorial dCas9-mediated transcriptional repression. Importantly, we demonstrated for the first time that dCas9 can be utilized for metabolic engineering in E. coli, leading to enhancement of flavonoid titers through simultaneous repression of an endogenous transcription factor and several central carbon enzymes, including partial downregulation of a synthetic lethal pair. Furthermore, we showed that this system could be utilized to generate complex phenotypes (such as dual amino acid bradytrophs), and we also demonstrated that a single plasmid can be used to attenuate virulence genes in disparate E. coli lineages by repressing capsular polysaccharide secretion in both probiotic strain Nissle 1917 and pathogenic strain K5.

Second, we have recently expanded upon this work by adapting Golden Gate Shuffling for assembly of natural type II-A CRISPR arrays, enabling randomized one-pot assembly of array libraries simultaneously repressing all combinations of a user-defined set of target genes. We test this method for improving production of glycosylated natural products—including the pharmaceutically and nutraceutically valuable polysaccharides heparin and chondroitin and the colorful class of plant natural pigments known as anthocyanins—through repression of novel knockdown targets and partial downregulation of an essential glycolytic enzyme. Critically, we show that the plasmid libraries generated with this methodology can be transformed into a variety of host strains for rapid evaluation of target combinations in distinct production chassis. Direct comparison of CRISPRi strains with analogous deletion strains (obtained through lambda-red recombineering) shows that CRISPRi can lead to production improvements comparable to gene deletions.

Finally, we constructed orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9 and covering a wide expression range. Remarkably, dCas9 orthogonality in our system is mediated by only 2-3 nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif (PAM). We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show that mismatch tolerance and orthogonality is dependent upon target copy number. Finally, a subset of these refactored promoters were incorporated into the highly branched violacein biosynthetic pathway, where they act as orthogonal, dCas9-dependent valves capable of throttling and selectively redirecting carbon flux in E. coli.