Bioproducts From Euglena gracilis: Synthesis and Applications

In recent years, the versatile phototrophic protist Euglena gracilis has emerged as an interesting candidate for application-driven research and commercialisation, as it is an excellent source of dietary protein, pro(vitamins), lipids, and the β-1,3-glucan paramylon only found in euglenoids. From these, paramylon is already marketed as an immunostimulatory agent in nutraceuticals. Bioproducts from E. gracilis can be produced under various cultivation conditions discussed in this review, and their yields are relatively high when compared with those achieved in microalgal systems. Future challenges include achieving the economy of large-scale cultivation. Recent insights into the complex metabolism of E. gracilis have highlighted unique metabolic pathways, which could provide new leads for product enhancement by genetic modification of the organism. Also, development of molecular tools for strain improvement are emerging rapidly, making E. gracilis a noteworthy challenger for microalgae such as Chlorella spp. and their products currently on the market

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

Modular organisation and functional analysis of dissected modular β-mannanase CsMan26 from Caldicellulosiruptor Rt8B.4

CsMan26 from Caldicellulosiruptor strain Rt8.B4 is a modular β-mannanase consisting of two N-terminal family 27 carbohydrate-binding modules (CBMs), followed by a family 35 CBM and a family 26 glycoside hydrolase catalytic module (mannanase). A functional dissection of the full-length CsMan26 and a comprehensive characterisation of the truncated derivatives were undertaken to evaluate the role of the CBMs. Limited proteolysis was used to define biochemically the boundaries of the different structural modules in CsMan26. The full-length CsMan26 and three truncated derivatives were produced in Escherichia coli, purified and characterised. The systematic removal of the CBMs resulted in a decrease in the optimal temperature for activity and in the overall thermostability of the derivatives. Kinetic experiments indicated that the presence of the mannan-specific family 27 CBMs increased the affinity of the enzyme towards the soluble galactomannan substrate but this was accompanied by lower catalytic efficiency. The full-length CsMan26 and its truncated derivatives were unable to hydrolyse mannooligosaccharides with degree of polymerisation (DP) of three or less. The major difference in the hydrolysis pattern of larger mannooligosaccharides (DP >3) by the derivatives was determined by their abilities to further hydrolyse the intermediate sugar mannotetraose.12 page(s

A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample

Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100°C and pH 6.0, and was extremely thermostable at 90°C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.8 page(s

Immobilisation of hyperthermophilic enzymes to mineral matrices

1 page(s

Bioengineering Strategies for Protein-Based Nanoparticles

In recent years, the practical application of protein-based nanoparticles (PNPs) has expanded rapidly into areas like drug delivery, vaccine development, and biocatalysis. PNPs possess unique features that make them attractive as potential platforms for a variety of nanobiotechnological applications. They self-assemble from multiple protein subunits into hollow monodisperse structures; they are highly stable, biocompatible, and biodegradable; and their external components and encapsulation properties can be readily manipulated by chemical or genetic strategies. Moreover, their complex and perfect symmetry have motivated researchers to mimic their properties in order to create de novo protein assemblies. This review focuses on recent advances in the bioengineering and bioconjugation of PNPs and the implementation of synthetic biology concepts to exploit and enhance PNP’s intrinsic properties and to impart them with novel functionalities