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      Bioengineering of Microalgae: Recent Advances, Perspectives, and Regulatory Challenges for Industrial Application

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          Abstract

          Microalgae, due to their complex metabolic capacity, are being continuously explored for nutraceuticals, pharmaceuticals, and other industrially important bioactives. However, suboptimal yield and productivity of the bioactive of interest in local and robust wild-type strains are of perennial concerns for their industrial applications. To overcome such limitations, strain improvement through genetic engineering could play a decisive role. Though the advanced tools for genetic engineering have emerged at a greater pace, they still remain underused for microalgae as compared to other microorganisms. Pertaining to this, we reviewed the progress made so far in the development of molecular tools and techniques, and their deployment for microalgae strain improvement through genetic engineering. The recent availability of genome sequences and other omics datasets form diverse microalgae species have remarkable potential to guide strategic momentum in microalgae strain improvement program. This review focuses on the recent and significant improvements in the omics resources, mutant libraries, and high throughput screening methodologies helpful to augment research in the model and non-model microalgae. Authors have also summarized the case studies on genetically engineered microalgae and highlight the opportunities and challenges that are emerging from the current progress in the application of genome-editing to facilitate microalgal strain improvement. Toward the end, the regulatory and biosafety issues in the use of genetically engineered microalgae in commercial applications are described.

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          Most cited references333

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          Genetic engineering of algae for enhanced biofuel production.

          There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H(2) yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H(2) production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.
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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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              Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator

              Lipid production in the oleaginous microalga Nannocholoropsis gaditana is doubled by decreasing the expression of a transcriptional regulator identified through a CRISPR–Cas9 reverse-genetics approach.
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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
                03 September 2020
                2020
                : 8
                : 914
                Affiliations
                [1] 1Agricultural Biotechnology Division, National Agri-Food Biotechnology Institute (NABI) , Sahibzada Ajit Singh Nagar, India
                [2] 2Plant Cell Biotechnology Department, CSIR-Central Food Technological Research Institute (CFTRI) , Mysuru, India
                [3] 3Division of Crop Science, Indian Council of Agricultural Research , New Delhi, India
                Author notes

                Edited by: Xiaochao Xiong, Washington State University, United States

                Reviewed by: Na Pang, Michigan State University, United States; Xinqing Zhao, Shanghai Jiao Tong University, China

                *Correspondence: Gulshan Kumar gulshan.ihbt@ 123456gmail.com

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

                Article
                10.3389/fbioe.2020.00914
                7494788
                33014997
                e713f27b-1be1-4160-96a4-4d742b4de014
                Copyright © 2020 Kumar, Shekh, Jakhu, Sharma, Kapoor and Sharma.

                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
                : 08 April 2020
                : 15 July 2020
                Page count
                Figures: 2, Tables: 3, Equations: 0, References: 364, Pages: 31, Words: 25484
                Funding
                Funded by: Department of Science and Technology, Ministry of Science and Technology, India 10.13039/501100001409
                Categories
                Bioengineering and Biotechnology
                Review

                microalgae,genetic engineering,omics,genome editing,regulatory issues

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