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      Comparison of Yeasts as Hosts for Recombinant Protein Production

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          Abstract

          Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts ranging from the bacteria Escherichia coli to mammalian cells. Recombinant protein importance is represented by its market size, which reached $1654 million in 2016 and is expected to reach $2850.5 million by 2022. Among the available hosts, yeasts have been used for producing a great variety of proteins applied to chemicals, fuels, food, and pharmaceuticals, being one of the most used hosts for recombinant production nowadays. Historically, Saccharomyces cerevisiae was the dominant yeast host for heterologous protein production. Lately, other yeasts such as Komagataella sp., Kluyveromyces lactis, and Yarrowia lipolytica have emerged as advantageous hosts. In this review, a comparative analysis is done listing the advantages and disadvantages of using each host regarding the availability of genetic tools, strategies for cultivation in bioreactors, and the main techniques utilized for protein purification. Finally, examples of each host will be discussed regarding the total amount of protein recovered and its bioactivity due to correct folding and glycosylation patterns.

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          Most cited references146

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          A new efficient gene disruption cassette for repeated use in budding yeast.

          The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.
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            A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.

            Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae. Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene. We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5(+) from Schizosaccharomyces pombe and the dominant resistance marker ble(r) from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin. All five loxP--marker gene--loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency. For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or ble(r) as the yeast selection marker. The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.
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              DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways

              The assembly of large recombinant DNA encoding a whole biochemical pathway or genome represents a significant challenge. Here, we report a new method, DNA assembler, which allows the assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae. We show that DNA assembler can rapidly assemble a functional d-xylose utilization pathway (∼9 kb DNA consisting of three genes), a functional zeaxanthin biosynthesis pathway (∼11 kb DNA consisting of five genes) and a functional combined d-xylose utilization and zeaxanthin biosynthesis pathway (∼19 kb consisting of eight genes) with high efficiencies (70–100%) either on a plasmid or on a yeast chromosome. As this new method only requires simple DNA preparation and one-step yeast transformation, it represents a powerful tool in the construction of biochemical pathways for synthetic biology, metabolic engineering and functional genomics studies.
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                Author and article information

                Journal
                Microorganisms
                Microorganisms
                microorganisms
                Microorganisms
                MDPI
                2076-2607
                29 April 2018
                June 2018
                : 6
                : 2
                : 38
                Affiliations
                Grupo Engenharia de Biocatalisadores, Departamento de Biologia Celular, Instituto de Ciências Biológicas Bloco K 1º andar, Universidade de Brasília, Campus Darcy Ribeiro, CEP 70.790-900 Brasília-DF, Brazil; miltonvieirag@ 123456gmail.com (A.M.V.G.); talitacarmo@ 123456gmail.com (T.S.C.); u.lucas@ 123456gmail.com (L.S.C.), fredericombahia@ 123456gmail.com (F.M.B.)
                Author notes
                Author information
                https://orcid.org/0000-0002-0394-3819
                https://orcid.org/0000-0002-5392-4560
                Article
                microorganisms-06-00038
                10.3390/microorganisms6020038
                6027275
                29710826
                29eef491-70a0-4468-a257-b5ca85817ac0
                © 2018 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 30 March 2018
                : 24 April 2018
                Categories
                Review

                recombinant protein,yeast,saccharomyces cerevisiae,kluyveromyces lactis,yarrowia lipolytica,komagataella phaffii

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