Increased trans-glycosylation activity of hexosaminidases for synthesis of human milk oligosaccharides

It is well known that the composition of human breast milk differs significantly from the one of ordinary bovine milk. Especially the presence of sialylated and fucosylated oligosaccharides contributes to its health and development promoting features for newborn infants. [1] Nevertheless, not all newborns and especially premature infants sometimes cannot be breast fed for different reasons. For those children it is important that they receive a proper balanced formula product containing the above mentioned human milk oligosaccharides (HMOs). With respect to this we are developing new enzymatic routes for synthesis of sialylated and fucosylated oligosaccharides, which can be used as functional ingredient for infant formula. In a previous work two candidate hexoasaminidases (both belonging to the GH20 family) were identified from a metagenomic library, which were able to synthesize the basic HMO backbone structure, Lacto-N-triose II, from chitobiose and lactose by trans-glycosylation. [2] Since the yields using these enzymes were low (2% for hex1 and 8% for hex2 based on the donor substrate chitobiose) we wanted to increase their trans-glycosylation activity to increase their applicability for a feasible process. It was decided to follow a rational design approach first to keep the screening effort low. Therefore peptide pattern recognition (PPR) [3] analysis was performed on the whole GH20 CAZy family (approx. 3000 sequences) to identify other enzymes with potential trans-glycosylation activity based on relatedness. By phylogenetic analysis of the group containing the two known enzymes (approx. 1000 sequences) and subsequent alignment of the closely related sequences a loop insertion close to the active site was identified. Homology modelling revealed that introduction of this loop structure into hex1 and hex2 would lead to a significantly narrower active site and therefore contribute to exclusion of water from the active site, which is a well-known strategy to increase trans-glycosylation activity. The proposed loop mutants were then expressed, purified and characterized towards trans-glycosylation activity. For hex2 it turned out that none of the loop mutants showed an improved trans-glycosylation activity compared to the wild-type. But for hex1 three out of four showed an up to seven-fold improved trans-glycosylation activity compared to the wild-type, which refers even to a higher trans-glycosylation activity than previously observed for the hex2 wild-type. [4] In conclusion we succeeded in engineering an enzyme towards increased trans-glycosylation activity using a custom-made rational approach utilizing available sequence analysis methods. [1] L. Bode, Glycobiology 2012, 22, 1147–1162. [2] C. Nyffenegger, R. T. Nordvang, B. Zeuner, M. Łężyk, E. Difilippo, M. J. Logtenberg, H. A. Schols, A. S. Meyer, J. D. Mikkelsen, Appl. Microbiol. Biotechnol. 2015, 99, 7997–8009. [3] P. K. Busk, L. Lange, Appl. Environ. Microbiol. 2013, 79, 3380–3391. [4] S. B. Jamek, J. Muschiol, J. Holck, P. K. Busk, L. Lange, J. D. Mikkelsen, A. S. Meyer, 2017, manuscript submitted

Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19.Fil: Sekine, Takuya. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Perez Potti, André. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Rivera Ballesteros, Olga. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Strålin, Kristoffer. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Gorin, Jean Baptiste. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Olsson, Annika. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Llewellyn Lacey, Sian. University Hospital of Wales; Reino UnidoFil: Kamal, Habiba. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Bogdanovic, Gordana. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Muschiol, Sandra. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Wullimann, David J.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Kammann, Tobias. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Emgård, Johanna. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Parrot, Tiphaine. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Folkesson, Elin. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Rooyackers, Olav. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Karolinska University Hospital; SueciaFil: Eriksson, Lars I.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Henter, Jan Inge. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Sönnerborg, Anders. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Allander, Tobias. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Albert, Jan. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Klingstrom, Jonas. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Gredmark Russ, Sara. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Björkström, Niklas K.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Sandberg, Johan K.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Price, David A.. Cardiff University School of Medicine; Reino UnidoFil: Ljunggren, Hans Gustaf. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Aleman, Soo. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Buggert, Marcus. Karolinska Huddinge Hospital. Karolinska Institutet; Sueci

Probabilistic classification of anti-SARS-CoV-2 antibody responses improves seroprevalence estimates.

OBJECTIVES: Population-level measures of seropositivity are critical for understanding the epidemiology of an emerging pathogen, yet most antibody tests apply a strict cutoff for seropositivity that is not learnt in a data-driven manner, leading to uncertainty when classifying low-titer responses. To improve upon this, we evaluated cutoff-independent methods for their ability to assign likelihood of SARS-CoV-2 seropositivity to individual samples. METHODS: Using robust ELISAs based on SARS-CoV-2 spike (S) and the receptor-binding domain (RBD), we profiled antibody responses in a group of SARS-CoV-2 PCR+ individuals (n = 138). Using these data, we trained probabilistic learners to assign likelihood of seropositivity to test samples of unknown serostatus (n = 5100), identifying a support vector machines-linear discriminant analysis learner (SVM-LDA) suited for this purpose. RESULTS: In the training data from confirmed ancestral SARS-CoV-2 infections, 99% of participants had detectable anti-S and -RBD IgG in the circulation, with titers differing > 1000-fold between persons. In data of otherwise healthy individuals, 7.2% (n = 367) of samples were of uncertain serostatus, with values in the range of 3-6SD from the mean of pre-pandemic negative controls (n = 595). In contrast, SVM-LDA classified 6.4% (n = 328) of test samples as having a high likelihood (> 99% chance) of past infection, 4.5% (n = 230) to have a 50-99% likelihood, and 4.0% (n = 203) to have a 10-49% likelihood. As different probabilistic approaches were more consistent with each other than conventional SD-based methods, such tools allow for more statistically-sound seropositivity estimates in large cohorts. CONCLUSION: Probabilistic antibody testing frameworks can improve seropositivity estimates in populations with large titer variability

The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides

Fucoidanases are endo-fucoidanases (also known as endo-fucanases) that catalyze hydrolysis of α-glycosidic linkages in fucoidans, a family of sulfated fucose-rich polysaccharides primarily found in the cell walls of brown seaweeds. Fucoidanases are promising tools for producing bioactive fucoidan oligosaccharides for a range of biomedical applications. High sulfation degree has been linked to high bioactivity of fucoidans. In this study, a novel fucoidanase, Fhf2, was identified in the genome of the aerobic, Gram-negative marine bacterium Formosa haliotis. Fhf2 was found to share sequence similarity to known endo-α(1,4)-fucoidanases (EC 3.2.1.212) from glycoside hydrolase family 107. A C-terminal deletion mutant Fhf2∆484, devoid of 484 amino acids at the C-terminus, with a molecular weight of approximately 46 kDa, was constructed and found to be more stable than the full-length Fhf2 protein. Fhf2∆484 showed endo-fucoidanase activity on fucoidans from different seaweed species including Fucus evanescens, Fucus vesiculosus, Sargassum mcclurei, and Sargassum polycystum. The highest activity was observed on fucoidan from F. evanescens. The Fhf2∆484 enzyme was active at 20–45°C and at pH 6–9 and had optimal activity at 37°C and pH 8. Additionally, Fhf2∆484 was found to be calcium-dependent. NMR analysis showed that Fhf2∆484 catalyzed hydrolysis of α(1,4) linkages between L-fucosyl moieties sulfated on C2 (similar to Fhf1 from Formosa haliotis), but Fhf2∆484 in addition released oligosaccharides containing a substantial amount of 2,4-disulfated fucose residues. The data thus suggest that the Fhf2∆484 enzyme could be a valuable candidate for producing highly sulfated oligosaccharides applicable for fucoidan bioactivity investigations

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

Engineering of enzymatic redox cascades involving oxygenases

Cascade reactions are not only of interest to chemists and biotechnologists, but also to life in general, because every metabolic reaction resembles a cascade reaction. This principle of substrate/intermediate channeling was only adapted by scientists. That way especially one-pot reactions became very attractive as for this no isolation of intermediates is necessary. Furthermore, unstable or toxic intermediates are only produced in low amounts and directly transformed in situ. In this PhD thesis two previously established cascade reactions were subject of further optimization. In the first part, a cascade reaction established in a DFG-funded project (Bo1862/6-1)in cooperation with the Vienna Technical University (Austria) for the production of chiral lactones was further optimized and extended. Therefore, on the one hand the genes encoding the needed enzymes were cloned for co-expression into a single plasmid in different arrangements to be expressed in pseudo-operon mode, with the aim to lower the metabolic burden of the cascade host cell. One out of the welve created constructs showed a reasonable activity of 15.3 ± 1.2 U · gCDW-1. On the other hand, this cascade reaction was aimed to be extended by the use of a hydroxylating enzyme to enable the use of limonene as renewable and chiral precursor for the proposed production of chiral polymers. Therefore, the feasibility of cytochrome P450-monooxygenases was studied. These turned out to be not applicable due to their bad regioselectivity for the hydroxylation of limonene or due to the difficulties of activity reconstitution. As alternative system for an initial hydroxylation step the use of a Rhodococcus equi strain, which was isolated from Cellulosimicrobium cellulans EB-8-4 and which is capable of very regioselective limonene-hydroxylation, was investigated. Therefore, the dioxygenase cluster responsible for the desired reaction was identified and especially the recombinant expression in a suitable host (Pseudomonas putida S12) was further studied. The results from these experiments revealed that the recombinant expression needs to be further optimized to enable the use of the recombinant dioxygenase in combination with the other enzymes for cascade reactions. The third part of this PhD thesis dealt with the immobilization of an established cascade reaction for the synthesis of poly-[caprolactone] precursors. Therefore, the use of a rotating bed reactor (RBR) was investigated. Preliminary studies using single enzymes involved in the desired cascade reaction demonstrated the general feasibility of this reactor concept. Especially the reusability of the catalysts was highly improved, because the catalytic particles were protected very effectively from mechanical forces within the voids of the reactor. For further work-flow optimization the immobilization was transformed into an in situ process by the application of a gas-shear device, which leads to decreased capsule size and thereby to increased mass transfer inside the particles. The developed methods were applied for encapsulation of the cells containing the enzymes needed for the reaction. After additional improvement of the reaction parameters a conversion of 93% (based on substrate depletion) was reached using catalysts produced by the established encapsulation procedure. In summary, the described cascade reactions were successfully optimized by either co-expression, extension applying a dioxygenase or immobilization. Furthermore, the general feasibility of an RBR was demonstrated.Kaskadenreaktionen sind nicht nur für Chemiker und Biotechnologen von großem Interesse, sondern auch für Lebensprozesse im allgemeinen, da jede metabolische Reaktion auch eine Reaktionskaskade ist. Dieses Prinzip der Substrat-/Intermediatweiterleitung wurde nun von Wissenschaftlern adaptiert. Auf diese Weise wurden besonders sog. ‚Eintopfreaktionen‘ sehr attraktiv, da hierfür keine Reinigung der Zwischenprodukte mehr notwendig ist. Darüberhinaus werden instabile und/oder toxische Zwischenprodukte nur in geringen Mengen gebildet und in situ direkt umgewandelt. In dieser Doktorarbeit wurden zwei bereits entwickelte Enzymkaskaden einer weiteren Optimierung unterzogen. Im ersten Teil wurde eine Redoxkaskade für die Synthese von chiralen Laktonen, die im Rahmen eines DFG-geförderten Projekts (Bo1862/6-1) in Zusammenarbeit mit der Technischen Universität Wien (Österreich) entwickelt wurde, optimiert und erweitert. Hierfür wurden einerseits die Gene, die die benötigten Enzyme kodieren, zusammen in allen möglichen Anordnungen auf ein Plasmid kloniert, um diese anschließend im Pseudo-Operon Modus zu exprimieren. Hiermit sollte der metabolische Stress für die Wirtszelle verringert werden. Eines der zwölf erstellten Konstrukte zeigte eine angemessene Aktivität von 15,3 U · gCDW-1. Andererseits sollte dieselbe Kaskade um eine initiale und enzymatische Hydroxylierung erweitert werden, um Limonen als nachwachsendes und chirales Substratmolekül zu verwenden. Dafür wurde zum einen die Anwendbarkeit von Cytochrom P450-Monooxygenasen untersucht. Diese erwiesen sich allerdings als schlecht nutzbar, da sie entweder nur eine schlechte Regioselektivität für die gewünschte Reaktion zeigten oder sich die Aktivität nicht rekonstituieren ließ. Als alternatives System für die initiale Hydroxylierung wurde dann ein Rhodococcus equi Stamm verwendet, der aus einer Cellulosimicrobium cellulans EB-8-4 Kultur isoliert wurde und in der Lage ist Limonen an der gewünschten Position höchst regioselektiv zu hydroxylieren. Der für die Reaktion verantwortliche Dioxygenase-Cluster konnte identifiziert werden und insbesondere die rekombinante Expression in einem geeigneten Wirt (Pseudomonas putida S12) wurde weiter untersucht. Die Ergebnisse aus diesen Versuchen zeigten, dass die Expression allerdings weiter optimiert werden muss, damit die rekombinante Dioxygenase in Kombination mit den anderen Enzymenm für eine Kaskadenreaktion engesetzt werden kann. Der dritte Teil dieser Arbeit befasste sich mit der Immobilisierung einer anderen bereits entwickelten Kaskadenreaktion für die Synthese von Polycaprolacton Vorstufen. Für diese Reaktion wurde insbesondere die Anwendung eines sog. rotating bed reactors (RBR) untersucht. Für diesen konnte in anfänglichen Experimenten gezeigt werden, dass enzymatische Reaktionen in diesem Reaktor prinzipiell möglich sind. Speziell wurde hierbei beobachtet, dass besonders die Wiederverwendbarkeit der Immobilisate stark erhöht wurde, da die katalytischen Partikel in den Hohlräumen des RBR vor jeglichem mechanischen Einfluss geschützt waren. Weiterhin wurde der Arbeitsablauf optimiert, indem das Einschlussverfahren durch die Anwendung einer Gas-Scher Vorrichtung in einen in situ Prozess umgewandelt wurde. Dies führte vor allem auch zu einer erhöhten Aktivität der eingeschlossenen Zellen, da die verringerte Kapselgröße den Massentransfer positiv beeinflusste. Abschließend wurden die entwickelten Verfahren für die zweite Kaskadenreaktion angewendet. Nach weiteren Optimierungen der Reaktionen konnte so ein Umsatz von 93% (bezogen auf Substratabnahme) erzielt werden. Zusammengefasst konnten die beschriebenen Kaskadenreaktionen entweder mittels Etablierung eines Co-Expressionssystems, der Erweiterung einer Kaskade durch eine Dioxygenase oder die Immobiliserung der benötigten Enzyme erfolgreich optimiert werden. Weiterhin wurde die allgemeine Anwendbarkeit von enzymatischen Reaktionen in einem neuartigen RBR demonstriert