Millions of years of evolution have produced biological systems capable of efficient one-pot multi-step catalysis. The underlying mechanisms that facilitate these reaction processes are increasingly providing inspiration in synthetic chemistry. Substrate channelling, where intermediates between enzymatic steps are not in equilibrium with the bulk solution, enables increased efficiencies and yields in reaction and diffusion processes. Here, we review different mechanisms of substrate channelling found in nature and provide an overview of the analytical methods used to quantify these effects. The incorporation of substrate channelling into synthetic cascades is a rapidly developing concept, and recent examples of the fabrication of cascades with controlled diffusion and flux of intermediates are presented.

The oleaginous yeast Yarrowia lipolytica is a valuable microbial host for chemical production because it has a high capacity to synthesize, modify, and store intracellular lipids; however, rapid strain development has been hampered by the limited availability of genome engineering tools. We address this limitation by adapting the CRISPR-Cas9 system from Streptococcus pyogenes for markerless gene disruption and integration in Y. lipolytica. Single gene disruption efficiencies of 92% and higher were achieved when single guide RNAs (sgRNA) were transcribed with synthetic hybrid promoters that combine native RNA polymerase III (Pol III) promoters with tRNA. The Pol III-tRNA hybrid promoters exploit endogenous tRNA processing to produce mature sgRNA for Cas9 targeting. The highest efficiencies were achieved with a SCR1'-tRNA(Gly) promoter and Y. lipolytica codon-optimized Cas9 expressed from a UAS1B8-TEF promoter. Cotransformation of the Cas9 and sgRNA expressing plasmid with a homologous recombination donor plasmid resulted in markerless homologous recombination efficiency of over 64%. Homologous recombination was observed in 100% of transformants when nonhomologous end joining was disrupted. The end result of these studies was the development of pCRISPRyl, a modular tool for markerless gene disruption and integration in Y. lipolytica.

The yeast Yarrowia lipolytica is a promising microbial host due to its native capacity to produce lipid-based chemicals. Engineering stable production strains requires genomic integration of modified genes, avoiding episomal expression that requires specialized media to maintain selective pressures. Here, we develop a CRISPR-Cas9-based tool for targeted, markerless gene integration into the Y. lipolytica genome. A set of genomic loci was screened to identify sites that were accepting of gene integrations without impacting cell growth. Five sites were found to meet these criteria. Expression levels from a GFP expression cassette were consistent when inserted into AXP, XPR2, A08, and D17, with reduced expression from MFE1. The standardized tool is comprised of five pairs of plasmids (one homologous donor plasmid and a CRISPR-Cas9 expression plasmid), with each pair targeting gene integration into one of the characterized sites. To demonstrate the utility of the tool we rapidly engineered a semisynthetic lycopene biosynthesis pathway by integrating four different genes at different loci. The capability to integrate multiple genes without the need for marker recovery and into sites with known expression levels will enable more rapid and reliable pathway engineering in Y. lipolytica.

Proteins, which are natural heteropolymers, have evolved to exhibit a staggering array of functions and capabilities. As scientists and engineers strive to tackle important challenges in medicine, novel biomaterials continue to be devised, designed, and implemented to help to address critical needs. This review aims to cover the present advances in the use of protein engineering to create new protein and peptide domains that enable the formation of advanced functional hydrogels. Three types of domains are covered in this review: (a) the leucine zipper coiled-coil domains, (b) the EF-hand domains, and (c) the elastin-like polypeptides. In each case, the functionality of these domains is discussed as well as recent advancements in the use of these domains to create novel hydrogel-based biomaterials. As protein engineering is used to both create and improve protein domains, these advances will lead to exciting new biomaterials for use in a variety of applications.

New developments in nucleic acid nanotechnology and protein scaffold designs have enabled unparalleled control over the spatial organization of synthetic multienzyme cascade reactions. One of the goals of these new technologies is to create nanostructured enzyme cascade reactions that promote substrate channeling along the cascade and, in doing so, enhance cascade catalysis. The concept of substrate channeling has a long and rich history in biochemistry and has established methods of evaluation and quantification. In this Perspective, we review the most common of these methods and discuss them in the context of engineered multienzyme systems and natural bifunctional enzymes with known mechanisms of substrate channeling. In addition, we use experimental data and the results of simulations of coupled-enzyme reactions to develop a set of preliminary design rules for engineering multienzyme nanostructures. The design rules address the limitations on interenzyme distance and active site orientation in substrate channeling and suggest designs for promoting enhanced catalysis, specifically, that enzyme orientation should minimize interenzyme distance and that at distances greater than 1 nm between active sites, significant channeling occurs only if diffusion of the intermediate is bounded through interactions with the surface or scaffold between active sites. This field is rapidly developing and promises to create many more new and exciting technologies.