Writing artificial logic and dynamic function into complex cellular background to achieve desired phenotypes or improved outputs calls for the development of new genetic tools as well as their innovative use. In this study, we present a sensor-regulator and RNAi-based bifunctional dynamic control network that can provide simultaneous upregulation and downregulation of cellular metabolism for engineered biosynthesis. The promoter-regulator-mediated upregulation function and its transduced downregulation function through RNAi are systematically verified and characterized. We apply this dynamic control network to regulate the phosphoenolpyruvate metabolic node in Escherichia coli and achieve autonomous distribution of carbon flux between its native metabolism and the engineered muconic acid biosynthetic pathway. This allows muconic acid biosynthesis to reach 1.8 g L⁻¹. This study also suggests the circumstances where dynamic control approaches are likely to take effects.

Co-utilization of carbon sources in microbes is an important topic in metabolic engineering research. It is not only a way to reduce microbial production costs but also an attempt for either improving the yields of target products or decreasing the formation of byproducts. However, there are barriers in co-utilization of carbon sources in microbes, such as carbon catabolite repression. To overcome the barriers, different metabolic engineering strategies have been developed, such as inactivation of the phosphotransferase system and rewiring carbon assimilation pathways. This review summarizes the most recent developments of different strategies that support microbes to utilize two or more carbon sources simultaneously. The main content focuses on the co-utilization of glucose and pentoses, major sugars in lignocellulose.

The RNA-guided CRISPR-associated Cas9 proteins have been widely applied in programmable genome recombination, base editing or gene regulation in both prokaryotes and eukaryotes. SpCas9 from Streptococcus pyogenes is the most extensively engineered Cas9 with robust and manifold functionalities. However, one inherent limitation of SpCas9 is its stringent 5′-NGG-3′ PAM requirement that significantly restricts its DNA target range. Here, to repurpose SpCas9 as a universal gene repressor, we generate and screen variants of the deactivated SpCas9 (SpdCas9) with relaxed 5′-CAT-3′ PAM compatibility that can bind to the start codon ATG of almost any gene. Stepwise structure-guided mutations of the PAM-interacting residues and auxiliary PAM-proximal residues of the SpdNG (5′-NG-3′ PAM) create a PAM-flexible variant SpdNG-LWQT that preferentially accommodates 5′-NRN-3′ PAMs. SpdNG-LWQT is demonstrated to be effective in gene repression with the advantage of customizable sgRNA design in both Escherichia coli and Saccharomyces cerevisiae. This work validates the feasibility of purposeful PAM expansion of Cas9 towards signature PAMs and establishes a universal SpdCas9-based gene repressor.