Isolation, characterization and applications of nanocellulose produced by ancestral enzymes

Los capítulos II, III, IV, V y Vi de esta tesis están sujetos a confidencialidad por el autor. 102 p

Enzymatic upgrading of nanochitin using an ancient lytic polysaccharide monooxygenase

Numerous enzymes have the potential to upgrade biomass, converting it into high-tech materials for new applications. However, the features of natural enzymes often limit their use beyond chemical conversion of the substrate. The development of strategies for the enzymatic conversion of biomass into high-value materials may broaden the range of applications of enzymes and enzyme design techniques. A relevant case is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyzes the oxidative cleavage of glycosidic bonds. Here, we show that an ancestral LPMO can generate chitin nanocrystals. Physicochemical characterization of the chitin nanocrystals demonstrates modifications that make it superior compared to chitin obtained by chemical treatments. We show that the nanocrystals are suitable for controlled 2D and 3D cell cultures, as well as for engineering a biomatrix that combines with graphene oxide, forming a hybrid conductive bioink.This work has been supported by grants PID2019-109087RB-I00 to R.P.-J. from Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 964764 to R.P.-J. ‘Materials + Technologies’ Research Group also acknowledges UPV/EHU and the Basque Government in the frame of “Research Group” (GIU 18/216) and “Grupos Consolidados” (IT776-13), respectively. We also thank Gipuzkoako Foru Aldundia for financial Support. HEK293T cells were a kind gift from Dr. Maria Muñoz Caffarel (Biodonostia, San Sebastian, Spain)

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR–Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.This work has been supported by grant nos. PID2019-109087RB-I00 (to R.P.-J.) and RTI2018-101223-B-I00 and PID2021-127644OB-I00 (to L.M.) from the Spanish Ministry of Science and Innovation. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 964764 (to R.P.-J.). The content presented in this document represents the views of the authors, and the European Commission has no liability in respect to the content. We acknowledge financial support from the Spanish Foundation for the Promotion of Research of Amyotrophic Lateral Sclerosis. A.F. acknowledges Spanish Center for Biomedical Network Research on Rare Diseases (CIBERE) intramural funds (no. ER19P5AC756/2021). F.J.M.M. acknowledges research support by Conselleria d’Educació, Investigació, Cultura i Esport from Generalitat Valenciana, research project nos. PROMETEO/2017/129 and PROMETEO/2021/057. M.M. acknowledges funding from CIBERER (grant no. ER19P5AC728/2021). The work has received funding from the Regional Government of Madrid (grant no. B2017/BMD3721 to M.A.M.-P.) and from Instituto de Salud Carlos III, cofounded with the European Regional Development Fund ‘A way to make Europe’ within the National Plans for Scientific and Technical Research and Innovation 2017–2020 and 2021–2024 (nos. PI17/1659, PI20/0429 and IMP/00009; to M.A.M.-P. B.P.K. was supported by an MGH ECOR Howard M. Goodman Award and NIH P01 HL142494. We thank H. Stutzman for assistance with cloning plasmids, and Z. Herbert and M. Berkeley from the Molecular Biology Core Facilities at the Dana-Farber Cancer Institute for assistance with NextSeq sequencing

High performance crystalline nanocellulose using an ancestral endoglucanase

Enzymes are effective at upgrading natural materials to high-performance biomaterials. Here, an ancestral endoglucanase is used to obtain highly crystalline cellulose nanocrystals, which can act as a matrix for cell growth and be combined with graphene for conducting inks

Resurrection of efficient Precambrian endoglucanases for lignocellulosic biomass hydrolysis

Cellulases catalyze the hydrolysis of cellulose. Improving their catalytic efficiency is a long-standing goal in biotechnology given the interest in lignocellulosic biomass decomposition. Although methods based on sequence alteration exist, improving cellulases is still a challenge. Here we show that Ancestral Sequence Reconstruction can “resurrect” efficient cellulases. This technique reconstructs enzymes from extinct organisms that lived in the harsh environments of ancient Earth. We obtain ancestral bacterial endoglucanases from the late Archean eon that efficiently work in a broad range of temperatures (30–90 °C), pH values (4–10). The oldest enzyme (~2800 million years) processes different lignocellulosic substrates, showing processive activity and doubling the activity of modern enzymes in some conditions. We solve its crystal structure to 1.45 Å which, together with molecular dynamics simulations, uncovers key features underlying its activity. This ancestral endoglucanase shows good synergy in combination with other lignocellulosic enzymes as well as when integrated into a bacterial cellulosome.We thank Prof. Ed Bayer’s group for kindly providing the plasmids used in the minicellulosome constructs. Research was supported by the Basque Government grant ELKARTEK to R.P.-J, and also partly by Ministry of Economy and Competitiveness (MINECO) grant BIO2016-77390-R, BFU2015-71964 to R.P.-J., BIO2016-74875-P to J. A.G., and CTQ2015-65320-R and RYC-2016-19590 to D.D.S.; European Commission grant CIG Marie Curie Reintegration program FP7-PEOPLE-2014 to R.P.-J, and European Commission grant NMP-FP7 604530-2 (CellulosomePlus), and the ERA-IB EIB.12.022 grant (FiberFuel) funded by the MINECO (PCIN-2013-011-C02-01) to M.C.-V. We also thank Fundación Repsol and Gipuzkoako Foru Aldundia for financial support

Human mesenchymal stem cells enhance the systemic effects of radiotherapy.

Journal Article; Research Support, Non-U.S. Gov't;The outcome of radiotherapy treatment might be further improved by a better understanding of individual variations in tumor radiosensitivity and normal tissue reactions, including the bystander effect. For many tumors, however, a definitive cure cannot be achieved, despite the availablity of more and more effective cancer treatments. Therefore, any improvement in the efficacy of radiotherapy will undoubtedly benefit a significant number of patients. Many experimental studies measure a bystander component of tumor cell death after radiotherapy, which highlights the importance of confirming these observations in a preclinical situation. Mesenchymal stem cells (MSCs) have been investigated for use in the treatment of cancers as they are able to both preferentially home onto tumors and become incorporated into their stroma. This process increases after radiation therapy. In our study we show that in vitro MSCs, when activated with a low dose of radiation, are a source of anti-tumor cytokines that decrease the proliferative activity of tumor cells, producing a potent cytotoxic synergistic effect on tumor cells. In vivo administration of unirradiated mesenchymal cells together with radiation leads to an increased efficacy of radiotherapy, thus leading to an enhancement of short and long range bystander effects on primary-irradiated tumors and distant-non-irradiated tumors. Our experiments indicate an increased cell loss rate and the decrease in the tumor cell proliferation activity as the major mechanisms underlying the delayed tumor growth and are a strong indicator of the synergistic effect between RT and MSC when they are applied together for tumor treatment in this model.This work was supported by Ministerio de Economía y Competividad, MINECO: SAF2012-40011-C02-02 to JMRdA, and MINECO SAF2012-40011-CO2-01 to F.J.O and CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brasil.Ye