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

Potential Use of Quantum Dots in Flow Cytometry

QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM) by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC) and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC

The Visually Related Posterior Pretectal Nucleus in the Non-Percomorph Teleost Osteoglossum bicirrhosum Projects to the Hypothalamus

This study was done to elucidate the ancestral (plesiomorphic) condition for visual pathways to the hypothalamus in teleost fishes. Three patterns of pretectal organization can be discerned morphologically and histochemically in teleosts. Their taxonomic distribution suggests that the intermediately complex pattern (seen in most teleost groups) is ancestral to both the elaborate pattern (seen in percomorphs) and the simple pattern (seen in cyprinids). The pretectal nuclei involved can be demonstrated with acetylcholinesterase histochemistry selectively and reliably in different species of teleosts, suggesting that the same-named nuclei are homologous in representatives of the three different patterns. Whereas there are visual pathways to the hypothalamus in both the elaborate (percomorph) and the simple (cyprinid) patterns, different pretectal and hypothalamic nuclei are involved. Thus visual hypothalamic pathways in these two patterns would not appear to be homologous. In this study, circuitry within the third, i.e., the intermediately complex, pattern is investigated. It is demonstrated that visual pathways project via the pretectum to the hypothalamus in Osteoglossum bicirrhosum and that they are very similar to the visual pathways in the elaborate pattern. This suggests that the circuitry in the intermediately complex pattern, as represented by Osteoglossum, is plesiomorphic (evolutionarily primitive) and the circuitry in both the simple pattern (seen in cyprinids) and the elaborate pattern (seen in percomorphs) is apomorphic (evolutionarily derived) for teleosts

Comparison of novel and standard diagnostic tools for the detection of Schistosoma mekongi infection in Lao People's Democratic Republic and Cambodia

Given the restricted distribution of Schistosoma mekongi in one province in Lao People's Democratic Republic (Lao PDR) and two provinces in Cambodia, together with progress of the national control programmes aimed at reducing morbidity and infection prevalence, the elimination of schistosomiasis mekongi seems feasible. However, sensitive diagnostic tools will be required to determine whether elimination has been achieved. We compared several standard and novel diagnostic tools in S. mekongi-endemic areas.; The prevalence and infection intensity of S. mekongi were evaluated in 377 study participants from four villages in the endemic areas in Lao PDR and Cambodia using Kato-Katz stool examination, antibody detection based on an enzyme-linked immunosorbent assay (ELISA) and schistosome circulating antigen detection by lateral-flow tests. Two highly sensitive test systems for the detection of cathodic and anodic circulating antigens (CCA, CAA) in urine and serum were utilized.; Stool microscopy revealed an overall prevalence of S. mekongi of 6.4% (one case in Cambodia and 23 cases in Lao PDR), while that of Opisthorchis viverrini, hookworm, Trichuris trichiura, Ascaris lumbricoides and Taenia spp. were 50.4%, 28.1%, 3.5%, 0.3% and 1.9%, respectively. In the urine samples, the tests for CCA and CAA detected S. mekongi infections in 21.0% and 38.7% of the study participants, respectively. In the serum samples, the CAA assay revealed a prevalence of 32.4%, while a combination of the CAA assay in serum and in urine revealed a prevalence of 43.2%. There was a difference between the two study locations with a higher prevalence reached in the samples from Lao PDR.; The CCA, CAA and ELISA results showed substantially higher prevalence estimates for S. mekongi compared to Kato-Katz thick smears. Active schistosomiasis mekongi in Lao PDR and Cambodia might thus have been considerably underestimated previously. Hence, sustained control efforts are still needed to break transmission of S. mekongi. The pivotal role of highly sensitive diagnostic assays in areas targeting elimination cannot be overemphasised

A placebo-controlled randomised trial of budesonide for PBC following an insufficient response to UDCA

Background & Aims: In patients with primary biliary cholangitis (PBC), the efficacy of budesonide, a synthetic corticosteroid displaying high first-pass metabolism, is unresolved. In a placebo-controlled, double-blind trial, we evaluated the added-value of budesonide in those with PBC and ongoing risk of progressive disease despite ursodeoxycholic acid (UDCA) treatment. Methods: We evaluated 62 patients with PBC who had histologically confirmed hepatic inflammatory activity, according to the Ishak score, and an alkaline phosphatase (ALP) >1.5x upper limit of normal (ULN), after at least 6 months of UDCA therapy. Participants were randomly assigned 2:1 to receive budesonide (9 mg/day) or placebo once daily, for 36 months, with UDCA treatment (12-16 mg/kg body weight/day) maintained. Primary efficacy was defined as improvement of liver histology with respect to inflammation and no progression of fibrosis. Secondary outcomes included changes in biochemical markers of liver injury. Results: Recruitment challenges resulted in a study that was underpowered for the primary efficacy analysis. Comparing patients with paired biopsies only (n = 43), the primary histologic endpoint was not met (p>0.05). The proportion of patients with ALP = 15% decrease in ALP and normal bilirubin was higher in the budesonide group than in the placebo group at 12, 24, and 36 months (p Conclusion: Budesonide add-on therapy was not associated with improved liver histology in patients with PBC and insufficient response to UDCA; however, improvements in biochemical markers of disease activity were demonstrated in secondary analyses. Lay summary: Around one-third of patients with primary biliary cholangitis (PBC) needs additional medical therapy alongside ursodeoxycholic acid (UDCA) treatment. In this clinical trial, the addition of the corticosteroid budesonide did not improve liver histology; there were however relevant improvements in liver blood tests. (C) 2020 European Association for the Study of the Liver. Published by Elsevier B.V.Peer reviewe

Boganmeldelser

Book review

Enhanced Protective Efficacy of a Chimeric Form of the Schistosomiasis Vaccine Antigen Sm-TSP-2

The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG1 and IgG3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic

norUrsodeoxycholic acid improves cholestasis in primary sclerosing cholangitis

Background & Aim: Primary sclerosing cholangitis (PSC) represents a devastating bile duct disease, currently lacking effective medical therapy. 24-norursodeoxycholic acid (norUDCA) is a side chain-shortened C-23 homologue of UDCA and has shown potent anti-cholestatic, anti-inflammatory and anti-fibrotic properties in a preclinical PSC mouse model. A randomized controlled trial, including 38 centers from 12 European countries, evaluated the safety and efficacy of three doses of oral norUDCA (500 mg/d, 1,000 mg/d or 1,500 mg/d) compared with placebo in patients with PSC. Methods: One hundred sixty-one PSC patients without concomitant UDCA therapy and with elevated serum alkaline phosphatase (ALP) levels were randomized for a 12-week treatment followed by a 4-week follow-up. The primary efficacy endpoint was the mean relative change in ALP levels between baseline and end of treatment visit. Results: norUDCA reduced ALP levels by -12.3%, -17.3%, and -26.0% in the 500, 1,000, and 1,500 mg/d groups (p = 0.029, tively, while a +1.2% increase was observed in the placebo group. Similar dose-dependent results were found for secondary end-points, such as ALT, AST, gamma-GT, or the rate of patients achieving ALP levels <1.5 x ULN. Serious adverse events occurred in seven patients in the 500 mg/d, five patients in the 1,000 mg/d, two patients in the 1500 mg/d group, and three in the placebo group. There was no difference in reported pruritus between treatment and placebo groups. Conclusions: norUDCA significantly reduced ALP values dose-dependently in all treatment arms. The safety profile of norUDCA was excellent and comparable to placebo. Consequently, these results justify a phase III trial of norUDCA in PSC patients. Lay summary: Effective medical therapy for primary sclerosing cholangitis (PSC) is urgently needed. In this phase II clinical study in PSC patients, a side chain-shortened derivative of ursodeoxycholic acid, norursodeoxycholic acid (norUDCA), significantly reduced serum alkaline phosphatase levels in a dose-dependent manner during a 12-week treatment. Importantly, norUDCA showed a favorable safety profile, which was similar to placebo. The use of norUDCA in PSC patients is promising and will be further evaluated in a phase III clinical study. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V.Peer reviewe