Accelerating Diverse Cell-Based Therapies Through Scalable Design.

Augmenting cells with novel, genetically encoded functions will support therapies that expand beyond natural capacity for immune surveillance and tissue regeneration. However, engineering cells at scale with transgenic cargoes remains a challenge in realizing the potential of cell-based therapies. In this review, we introduce a range of applications for engineering primary cells and stem cells for cell-based therapies. We highlight tools and advances that have launched mammalian cell engineering from bioproduction to precision editing of therapeutically relevant cells. Additionally, we examine how transgenesis methods and genetic cargo designs can be tailored for performance. Altogether, we offer a vision for accelerating the translation of innovative cell-based therapies by harnessing diverse cell types, integrating the expanding array of synthetic biology tools, and building cellular tools through advanced genome writing techniques.

Engineering of non-model eukaryotes for bioenergy and biochemical production.

The prospect of leveraging naturally occurring phenotypes to overcome bottlenecks constraining the bioeconomy has marshalled increased exploration of nonconventional organisms. This review discusses the status of non-model eukaryotic species in bioproduction, the evaluation criteria for effectively matching a candidate host to a biosynthetic process, and the genetic engineering tools needed for host domestication. We present breakthroughs in genome editing and heterologous pathway design, delving into innovative spatiotemporal modulation strategies that potentiate more refined engineering capabilities. We cover current understanding of genetic instability and its ramifications for industrial scale-up, highlighting key factors and possible remedies. Finally, we propose future opportunities to expand the current collection of available hosts and provide guidance to benefit the broader bioeconomy.

A repackaged CRISPR platform increases homology-directed repair for yeast engineering.

Inefficient homology-directed repair (HDR) constrains CRISPR-Cas9 genome editing in organisms that preferentially employ nonhomologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies used to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified the shortcomings of the conventional CRISPR platforms and developed a CRISPR platform-lowered indel nuclease system enabling accurate repair (LINEAR)-which enhanced HDR rates (to 67-100%) compared to those in previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.

Leveraging the Hermes Transposon to Accelerate the Development of Nonconventional Yeast-based Microbial Cell Factories.

We broadened the usage of DNA transposon technology by demonstrating its capacity for the rapid creation of expression libraries for long biochemical pathways, which is beyond the classical application of building genome-scale knockout libraries in yeasts. This strategy efficiently leverages the readily available fine-tuning impact provided by the diverse transcriptional environment surrounding each random integration locus. We benchmark the transposon-mediated integration against the nonhomologous end joining-mediated strategy. The latter strategy was demonstrated for achieving pathway random integration in other yeasts but is associated with a high false-positive rate in the absence of a high-throughput screening method. Our key innovation of a nonreplicable circular DNA platform increased the possibility of identifying top-producing variants to 97%. Compared to the classical DNA transposition protocol, the design of a nonreplicable circular DNA skipped the step of counter-selection for plasmid removal and thus not only reduced the time required for the step of library creation from 10 to 5 d but also efficiently removed the "transposition escapers", which undesirably represented almost 80% of the entire population as false positives. Using one endogenous product (i.e., shikimate) and one heterologous product (i.e., (S)-norcoclaurine) as examples, we presented a streamlined procedure to rapidly identify high-producing variants with titers significantly higher than the reported data in the literature. We selected Scheffersomyces stipitis, a representative nonconventional yeast, as a demo, but the strategy can be generalized to other nonconventional yeasts. This new exploration of transposon technology, therefore, adds a highly versatile tool to accelerate the development of novel species as microbial cell factories for producing value-added chemicals.

CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis.

Scheffersomyces stipitis, renowned for its native xylose-utilizing capacity, has recently demonstrated its potential in producing health-promoting shikimate pathway derivatives. However, its broader application is hampered by the low transformation efficiency and the lack of genetic engineering tools to enable sophisticated genomic manipulations. S. stipitis employs the predominant non-homologous end joining (NHEJ) mechanism for repairing DNA double-strand breaks (DSB), which is less desired due to its incompetence in achieving precise genome editing. Using CRISPR technology, here a ku70Δku80Δ deficient strain in which homologous recombination (HR)-based genome editing appeared dominant for the first time in S. stipitis is constructed. To build all essential tools for efficiently manipulating this highly promising nonconventional microbial host, the gene knockdown tool is also established, and repression efficiency is improved by incorporating a transcriptional repressor Mxi1 into the CRISPR-dCas9 platform. All these results are obtained with the improved transformation efficiency, which is 191-fold higher than that obtained with the traditional parameters used in yeast transformation. This work paves the way for advancing a new microbial chassis and provides a guideline for developing efficient CRISPR tools in other nonconventional yeasts.

Enhancing the Co-utilization of Biomass-Derived Mixed Sugars by Yeasts.

Plant biomass is a promising carbon source for producing value-added chemicals, including transportation biofuels, polymer precursors, and various additives. Most engineered microbial hosts and a select group of wild-type species can metabolize mixed sugars including oligosaccharides, hexoses, and pentoses that are hydrolyzed from plant biomass. However, most of these microorganisms consume glucose preferentially to non-glucose sugars through mechanisms generally defined as carbon catabolite repression. The current lack of simultaneous mixed-sugar utilization limits achievable titers, yields, and productivities. Therefore, the development of microbial platforms capable of fermenting mixed sugars simultaneously from biomass hydrolysates is essential for economical industry-scale production, particularly for compounds with marginal profits. This review aims to summarize recent discoveries and breakthroughs in the engineering of yeast cell factories for improved mixed-sugar co-utilization based on various metabolic engineering approaches. Emphasis is placed on enhanced non-glucose utilization, discovery of novel sugar transporters free from glucose repression, native xylose-utilizing microbes, consolidated bioprocessing (CBP), improved cellulase secretion, and creation of microbial consortia for improving mixed-sugar utilization. Perspectives on the future development of biorenewables industry are provided in the end.