(500g) Harnessing Crispr/dCas9 and Polyculture Strategies for Enhancing Natural Product Production in Recombinant Hosts (Featured Presentation)

The field of metabolic engineering is entering a new era in which synthetic transcription factors such as RNAguided-dCas9 are providing the means for dynamic regulation and for rapid assessment of multiplexed genetic interventions to alter the cellular metabolic landscape.

In this talk we will first present a modular assembly method for traditional restriction-ligation cloning of type IIA CRISPR array libraries for multiplex, combinatorial dCas9-mediated transcriptional repression and its application in metabolic engineering of E. coli for enhancing flavonoid titers through simultaneous repression of an endogenous transcription factor and several central carbon enzymes, including partial downregulation of a synthetic lethal pair. We will also demonstrate how this method can be expanded by adapting Golden Gate Shuffling for assembly of natural type IIA CRISPR arrays, enabling randomized one-pot assembly of array libraries simultaneously repressing all combinations of a user-defined set of target genes. This approach was tested for improving production of glycosylated natural productsâ??including the pharmaceutically and nutraceutically valuable polysaccharides heparin and chondroitin and the colorful class of plant natural pigments known as anthocyaninsâ??through repression of novel knockdown targets and partial downregulation of an essential glycolytic enzyme. Direct comparison of CRISPRi strains with analogous deletion strains (obtained through λ-red recombineering) shows that CRISPRi can lead to better production improvements compared to gene deletions. Finally, we will demonstrate the construction of orthogonal variants of the classic T7lac 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 23 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 PAMproximal mismatch is insufficient to abolish dCas9-mediated repression. 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.

Finally, the presentation will also cover our work on the development and optimization of polycultures (three or more strains in co-culture) for the extension of recombinant pathways, such as the flavonoid branch pathway, in vivo. This technology has enabled, for the first time, the de novo production of flavan-3-ols and anthocyanins in E. coli. Utilizing a computationally guided optimization approach, we were able to demonstrate up to a 970-fold improvement over previously published monoculture titers.