Engineering of industrially important microorganisms for assimilation of cellulosic biomass: towards consolidated bioprocessing.

Conversion of cellulosic biomass (non-edible plant material) to products such as chemical feedstocks and liquid fuels is a major goal of industrial biotechnology and an essential component of plans to move from an economy based on fossil carbon to one based on renewable materials. Many microorganisms can effectively degrade cellulosic biomass, but attempts to engineer this ability into industrially useful strains have met with limited success, suggesting an incomplete understanding of the process. The recent discovery and continuing study of enzymes involved in oxidative depolymerisation, as well as more detailed study of natural cellulose degradation processes, may offer a way forward.

Microbial Upcycling of Waste PET to Adipic Acid.

Microorganisms can be genetically engineered to transform abundant waste feedstocks into value-added small molecules that would otherwise be manufactured from diminishing fossil resources. Herein, we report the first one-pot bio-upcycling of PET plastic waste into the prolific platform petrochemical and nylon precursor adipic acid in the bacterium Escherichia coli. Optimizing heterologous gene expression and enzyme activity enabled increased flux through the de novo pathway, and immobilization of whole cells in alginate hydrogels increased the stability of the rate-limiting enoate reductase BcER. The pathway enzymes were also interfaced with hydrogen gas generated by engineered E. coli DD-2 in combination with a biocompatible Pd catalyst to enable adipic acid synthesis from metabolic cis,cis-muconic acid. Together, these optimizations resulted in a one-pot conversion to adipic acid from terephthalic acid, including terephthalate samples isolated from industrial PET waste and a post-consumer plastic bottle.

Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology.

Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.

Joint Universal Modular Plasmids: A Flexible Platform for Golden Gate Assembly in Any Microbial Host.

Modular cloning standards based on Golden Gate DNA assembly allow for construction of complex DNA constructs over several rounds of assembly. Despite being reliable and automation-friendly, each standard uses a specific set of vectors, requiring researchers to generate new tool kits for novel hosts and cloning applications. JUMP vectors (Valenzuela-Ortega and French, bioRxiv 799585, 2019) combine the robustness of modular cloning standards with the Standard European Vector Architecture and a flexible design that allows researchers to easily modify the vector backbone via secondary cloning sites. This flexibility allows for JUMP vectors to be used in a wide variety of applications and hosts.

Hetero-trans-ß-Glucanase Produces Cellulose-Xyloglucan Covalent Bonds in the Cell Walls of Structural Plant Tissues and Is Stimulated by Expansin.

Current cell-wall models assume no covalent bonding between cellulose and hemicelluloses such as xyloglucan or mixed-linkage ß-d-glucan (MLG). However, Equisetum hetero-trans-ß-glucanase (HTG) grafts cellulose onto xyloglucan oligosaccharides (XGOs) - and, we now show, xyloglucan polysaccharide - in vitro, thus exhibiting CXE (cellulose:xyloglucan endotransglucosylase) activity. In addition, HTG also catalyzes MLG-to-XGO bonding (MXE activity). In this study, we explored the CXE action of HTG in native plant cell walls and tested whether expansin exposes cellulose to HTG by disrupting hydrogen bonds. To quantify and visualize CXE and MXE action, we assayed the sequential release of HTG products from cell walls pre-labeled with substrate mimics. We demonstrated covalent cellulose-xyloglucan bonding in plant cell walls and showed that CXE and MXE action was up to 15% and 60% of total transglucanase action, respectively, and peaked in aging, strengthening tissues: CXE in xylem and cells bordering intercellular canals and MXE in sclerenchyma. Recombinant bacterial expansin (EXLX1) strongly augmented CXE activity in vitro. CXE and MXE action in living Equisetum structural tissues potentially strengthens stems, while expansin might augment the HTG-catalyzed CXE reaction, thereby allowing efficient CXE action in muro. Our methods will enable surveys for comparable reactions throughout the plant kingdom. Furthermore, engineering similar hetero-polymer formation into angiosperm crop plants may improve certain agronomic traits such as lodging tolerance.