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      Genetic Engineering Strategies for Euglena gracilis and Its Industrial Contribution to Sustainable Development Goals: A Review

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          Abstract

          The sustainable development goals (SDGs) adopted at the 2015 United Nations Summit are globally applicable goals designed to help countries realize a sustainable future. To achieve these SDGs, it is necessary to utilize renewable biological resources. In recent years, bioeconomy has been an attractive concept for achieving the SDGs. Microalgae are one of the biological resources that show promise in realizing the “5F”s (food, fiber, feed, fertilizer, and fuel). Among the microalgae, Euglena gracilis has the potential for achieving the “5F”s strategy owing to its unique features, such as production of paramylon, that are lacking in other microalgae. E. gracilis has already been produced on an industrial scale for use as an ingredient in functional foods and cosmetics. In recent years, genetic engineering methods for breeding E. gracilis have been researched and developed to achieve higher yields. In this article, we summarize how microalgae contribute toward achieving the SDGs. We focus on the contribution of E. gracilis to the bioeconomy, including its advantages in industrial use as well as its unique characteristics. In addition, we review genetic engineering-related research trends centered on E. gracilis, including a complete nuclear genome determination project, genome editing technology using the CRISPR-Cas9 system, and the development of a screening method for selecting useful strains. In particular, genome editing in E. gracilis could be a breakthrough for molecular breeding of industrially useful strains because of its high efficiency.

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          Most cited references76

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          Methane emissions from cattle.

          Increasing atmospheric concentrations of methane have led scientists to examine its sources of origin. Ruminant livestock can produce 250 to 500 L of methane per day. This level of production results in estimates of the contribution by cattle to global warming that may occur in the next 50 to 100 yr to be a little less than 2%. Many factors influence methane emissions from cattle and include the following: level of feed intake, type of carbohydrate in the diet, feed processing, addition of lipids or ionophores to the diet, and alterations in the ruminal microflora. Manipulation of these factors can reduce methane emissions from cattle. Many techniques exist to quantify methane emissions from individual or groups of animals. Enclosure techniques are precise but require trained animals and may limit animal movement. Isotopic and nonisotopic tracer techniques may also be used effectively. Prediction equations based on fermentation balance or feed characteristics have been used to estimate methane production. These equations are useful, but the assumptions and conditions that must be met for each equation limit their ability to accurately predict methane production. Methane production from groups of animals can be measured by mass balance, micrometeorological, or tracer methods. These techniques can measure methane emissions from animals in either indoor or outdoor enclosures. Use of these techniques and knowledge of the factors that impact methane production can result in the development of mitigation strategies to reduce methane losses by cattle. Implementation of these strategies should result in enhanced animal productivity and decreased contributions by cattle to the atmospheric methane budget.
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            CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

            Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including “safe harboring” techniques shown in other organisms.
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              The Bioeconomy in Europe: An Overview

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                Author and article information

                Contributors
                Journal
                Front Bioeng Biotechnol
                Front Bioeng Biotechnol
                Front. Bioeng. Biotechnol.
                Frontiers in Bioengineering and Biotechnology
                Frontiers Media S.A.
                2296-4185
                14 July 2020
                2020
                : 8
                : 790
                Affiliations
                [1] 1RIKEN Baton Zone Program , Yokohama, Japan
                [2] 2RIKEN Center for Sustainable Resource Science , Yokohama, Japan
                [3] 3Euglena Co Ltd , Tokyo, Japan
                Author notes

                Edited by: Raul Muñoz, University of Valladolid, Spain

                Reviewed by: Pau Loke Show, University of Nottingham, United Kingdom; Changhong Yao, Sichuan University, China

                *Correspondence: Ryo Harada, ryo.harada@ 123456riken.jp

                This article was submitted to Bioprocess Engineering, a section of the journal Frontiers in Bioengineering and Biotechnology

                Article
                10.3389/fbioe.2020.00790
                7371780
                32760709
                8c5ca5d9-a487-4c9a-9a69-d63e4b267569
                Copyright © 2020 Harada, Nomura, Yamada, Mochida and Suzuki.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 28 April 2020
                : 22 June 2020
                Page count
                Figures: 2, Tables: 2, Equations: 0, References: 96, Pages: 10, Words: 0
                Categories
                Bioengineering and Biotechnology
                Review

                euglena gracilis,sdgs,bioeconomy,genetic engineering,crispr-cas9

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