Novel biocatalytic modules for the cell-free conversion of cellodextrins to glucaric acid

Cell-free biocatalysis offers a versatile platform for the biomanufacturing of bulk or specialty chemicals due to the flexibility in assembling enzymes from different organisms in synthetic reaction pathways. Current challenges of this approach include costly enzyme preparation, low enzyme stability and efficient enzyme recycling. To overcome these challenges, we present a molecular toolbox that facilitates the simple construction of enzymes as low-cost and recyclable biocatalytic modules. The toolbox is composed of three interchangeable components: (i) inorganic matrices; (ii) matrix-specific solid-binding peptides (SBPs); and (iii) thermostable enzymes. SBPs are short amino acid sequences that can be fused genetically to proteins and direct the orientated immobilization of the resulting protein fusion onto solid matrices (1, 2). The biocatalytic module design relies on the affinity of the SBP for inorganic matrices. Single enzyme biocatalytic modules can be prepared easily consisting of one type of enzyme immobilized per matrix while a multiple enzyme biocatalytic module consists of multiple enzymes immobilized simultaneously onto the matrix. The modules can be combined rationally to generate product-specific reaction pathways and their subsequent removal from the reaction medium allows for a ‘pick, mix, and reuse’ approach, which can be optimized easily for low-cost cell-free biomanufacturing. Recently, we have shown that it is possible to assemble single and multiple enzyme biocatalytic modules using thermostable polysaccharide-degrading enzymes and that the enzymes retain their specific hydrolytic activities upon several rounds of recycling at high temperatures (2). Here, we applied the biocatalytic modules concept for the cell-free conversion of cellodextrins to glucaric acid, via a more complex seven enzyme synthetic pathway. Glucaric acid is one of the 12 top candidates for bio-based building blocks and is a precursor for polymers, including nylons and hyperbranched polyesters (3). Its bioproduction from cellodextrins, which can be derived from organic waste, provides a sustainable alternative to the fossil-derived production of polymers. Initially, single enzyme biocatalytic modules were prepared with a silica-specific SBP fused to two enzymes of the synthetic pathway allowing for their selective immobilization onto an inexpensive silica-based matrix. The SBP mediated the binding of each enzyme onto the matrix with over 85% immobilization efficiency. When comparing the enzyme activities of the biocatalytic modules against the free enzymes, 85 and 93% of their initial activities were retained upon immobilization, respectively. Furthermore, co-immobilization of these two enzymes as a multiple enzyme module resulted in similar immobilization yields. Performance of both enzymes in the multiple enzyme module in a successive reaction revealed that they retained 70% of their activity when compared to the free enzymes. Currently, the silica-specific SBP has been incorporated into other 5 enzymes of the pathway and we are proceeding with the construction of the single and multiple enzyme biocatalytic modules and pathway assembly. (1) Care A, Bergquist PL, Sunna A (2015) Trends in Biotechnology, 33: 259-268. (2) Care A, Petroll K, Gibson ESY, Bergquist PL, Sunna A (2017) Biotechnology for Biofuels 10:29. (3) Werpy T and G Petersen (2004). Results of Screening for Potential Candidates from Sugars and Synthesis Gas. National Renewable Energy Lab

Synthetic biocatalytic modules for enhanced transformation of biological waste products

Many insoluble materials can be used as carriers for the immobilisation of enzymes. Solid-binding peptides (SBPs) are short amino acid sequences that can act as molecular linkers to direct the orientated immobilisation of proteins onto solid materials without impeding their biological activity [1]. Silica-based materials like silica and zeolite have been found to be suitable matrices for enzyme immobilisation in industrial processes. They are inexpensive, offer high mechanical strength and stability, are chemically inert and can be deployed over a wide range of operating conditions. We have constructed biocatalytic modules that are based on the incorporation of a silica-binding SBP (‘linker’) sequence into several genes for thermostable enzymes to facilitate the immobilisation of the proteins onto silica-based matrices, enabling the hydrolysis of both simple and complex polysaccharides. We have shown also that the procedure is suitable for the construction of complex enzymological pathways. In proof of concept experiments, the linker (L) sequence was attached to the N- or C-terminus of three thermostable hemicellulases isolated from thermophilic bacteria using genetic engineering techniques [2]. The resulting L-enzymes remained active after fusion and displayed the same pH and temperature optima but differing thermostabilities in comparison to their corresponding enzymes without linker. The linker facilitated the rapid and simple immobilisation of each L-enzyme onto zeolite, resulting in the construction of ‘single enzyme biocatalytic modules’. All three L-enzymes co-immobilised onto the same zeolite matrix resulted in the formation of ‘multiple enzyme biocatalytic modules’, which were shown to degrade various hemicellulosic substrates effectively in a ‘one-pot’ reaction. Cell-free synthetic biology circumvents many of the limitations encountered by in vivo synthetic biology by operating without the constraints of a cell. It offers higher substrate and enzyme loading and the facile optimisation of enzyme ratios. Some of the challenges of this approach include costly enzyme preparation, biocatalyst stability, and the need for constant supplementation with co-factors. To overcome these challenges, we have developed a molecular toolbox that facilitates the construction of biocatalytic modules with predefined functions and catalytic properties. It consists of three interchangeable building blocks: (a) low-cost inorganic matrices (e.g., silica, zeolite), (b) matrix-specific SBPs and (c) thermostable enzymes. The rational combination of these building blocks allows for flexibility and a ‘pick, mix’ and re-use’ approach with multiple biocatalytic modules available for the assembly of natural and non-natural pathways. Individual immobilised enzymes can be combined rationally to assemble recyclable and product-specific reactions. We present preliminary results relating to the construction of two synthetic pathways for the conversion of organic wastes such as coffee and plant biomass. The pathway assembly process allows for rapid evaluation for proof of concept and for assessing the parameters for a synthetic pathway, which are very labour- and time-intensive by the in vivo approach. [1] Care, A, Bergquist, PL, Sunna, A. (2015) Trends Biotech. 33: 259-268 [2] Care, A, Petroll, K, Gibson, ESY, Bergquist, PL, Sunna, A. (2017) Biotech. Biofuels. 10: 2

Lsd1 ablation triggers metabolic reprogramming of brown adipose tissue

Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT- selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1- deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT

Cell-free enzymatic production of glucaric acid

Thesis by publication.Includes bibliographical references.Chapter 1. Tools and strategies for constructing cell-free enzyme pathways -- Chapter 2. Materials and methods -- Chapter 3. Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways -- Chapter 4. A novel framework for the cell-free enzymatic production of glucaric acid -- Chapter 5. Characterisation of a putative myo-inositol oxygenase from the moderately thermophilic fungus Rasamsonia emersonii -- Chapter 6. Summary and future perspectives -- Appendices.Cell-free biocatalysis with a number of enzymes is a fast growing and potentially high impact field in synthetic biology for the bio-manufacture of existing and novel fine or platform chemicals and biofuels. The assembly of multiple different enzymes to form synthetic pathways is a relatively new development in contrast to the single enzyme systems that have been used for decades. The cell-free approach employs enzymes outside of the cell, allowing for controllable reaction conditions and avoids metabolic repression. It prevents the diversion and loss of pathway intermediates or end-products into the cell's own metabolism. Multi-enzyme systems in a cell-free context are particularly attractive for the 'green' synthesis of high value compounds from inexpensive, simple and renewable substrates. These systems offer great versatility, allowing the investigator to 'pick, mix and test' without the need for genetic modification of the host organism and without interference from intracellular processes.Therefore, substrate conversion yields by cell-free biocatalysis can reach 100% of the theoretical value. However, major challenges for the industrial implementation of this approach includes costly enzyme preparation, enzyme stability and the dependency on expensive cofactors.Glucaric acid (GlucA) is one of the top 12 bio-based chemicals recognised worldwide for its potential impact and application in the synthesis of greener products. GlucA can be used in the production of (biodegradable) polymers, including (hydyroxylated) nylons and polyesters, offering a more sustainable and environmentally-friendly alternative to fossil fuel-derived products. The production of GlucA has been attempted mainly by chemical and microbial synthesis.This research describes a novel cell-free and multi-enzyme biocatalytic system developed for the synthesis of GlucA. The system is composed of a synthetic six enzyme pathway designed to facilitate the synthesis of GlucA from glucose-1-phosphate (G1P) which can be derived enzymatically from various natural polymers, such as cellulose or starch, and thus represents a promising approach to utilise crude biomass for GlucA production. An integrative framework was established to achieve an economical and efficient biocatalytic process which included; i)metabolic flux analysis for system optimisation, ii) the use of thermostable enzymes for improved stability and robustness of the system, iii) immobilisation and recycling of enzymes for reduced costs and iv) a cofactor regenerating tool to reduce cofactor requirements.To accomplish this framework, a novel analytical method was developed to engineer the GlucA production towards high titres and to monitor the pathway flux rapidly. It was based on ultrahigh performance liquid chromatography (UHPLC) and refractive index detection (RID) which enabled the simultaneous analysis of GlucA and its intermediates. All selected enzymes (mostly thermostable) were fused genetically to a synthetic peptide (referred to as the "linker") which displayed high affinity towards silica-based materials. Due to the non-invasive binding mechanism of the linker, the enzymes were co-immobilised successfully onto zeolite, a low costand commercially-available silica-based material. All the immobilised enzymes except the labile mouse myo-inositol oxygenase remained active, implying their suitability for application in a high temperature bioprocess, and allowed for their repeated use and recycling. Finally, an additional cofactor regenerating enzyme was integrated effectively into the cell-free process which maintained high cofactor levels while reducing the cofactor requirements of the pathway.In summary, this work presents the first cell-free production of GlucA and describes a powerful framework for viable cell-free biocatalysis based on an immobilised multi-enzyme synthetic pathway. Further work on the cell-free GlucA production is anticipated to extend the synthesis of GlucA from biomass such as cellulose and starch, and to allow increases in the overall efficiency of the system.Mode of access: World wide web1 online resource (241 pages) diagrams, graphs, table

Mechanotransduction of mesenchymal melanoma cell invasion into 3D collagen lattices: Filopod-mediated extension-relaxation cycles and force anisotropy

Item does not contain fulltextMesenchymal cell migration in interstitial tissue is a cyclic process of coordinated leading edge protrusion, adhesive interaction with extracellular matrix (ECM) ligands, cell contraction followed by retraction and movement of the cell rear. During migration through 3D tissue, the force fields generated by moving cells are non-isotropic and polarized between leading and trailing edge, however the integration of protrusion formation, cell-substrate adhesion, traction force generation and cell translocation in time and space remain unclear. Using high-resolution 3D confocal reflectance and fluorescence microscopy in GFP/actin expressing melanoma cells, we here employ time-resolved subcellular coregistration of cell morphology, interaction and alignment of actin-rich protrusions engaged with individual collagen fibrils. Using single fibril displacement as sensitive measure for force generated by the leading edge, we show how a dominant protrusion generates extension-retraction cycles transmitted through multiple actin-rich filopods that move along the scaffold in a hand-over-hand manner. The resulting traction force is oscillatory, occurs in parallel to cell elongation and, with maximum elongation reached, is followed by rear retraction and movement of the cell body. Combined live-cell fluorescence and reflection microscopy of the leading edge thus reveals step-wise caterpillar-like extension-retraction cycles that underlie mesenchymal migration in 3D tissue

Lsd1 Ablation Triggers Metabolic Reprogramming of Brown Adipose Tissue

Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT-selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1-deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT