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      The expression of glycerol facilitators from various yeast species improves growth on glycerol of Saccharomyces cerevisiae

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          Abstract

          Glycerol is an abundant by-product during biodiesel production and additionally has several assets compared to sugars when used as a carbon source for growing microorganisms in the context of biotechnological applications. However, most strains of the platform production organism Saccharomyces cerevisiae grow poorly in synthetic glycerol medium. It has been hypothesized that the uptake of glycerol could be a major bottleneck for the utilization of glycerol in S. cerevisiae. This species exclusively relies on an active transport system for glycerol uptake. This work demonstrates that the expression of predicted glycerol facilitators (Fps1 homologues) from superior glycerol-utilizing yeast species such as Pachysolen tannophilus, Komagataella pastoris, Yarrowia lipolytica and Cyberlindnera jadinii significantly improves the growth performance on glycerol of the previously selected glycerol-consuming S. cerevisiae wild-type strain (CBS 6412-13A). The maximum specific growth rate increased from 0.13 up to 0.18 h −1 and a biomass yield coefficient of 0.56 g DW/g glycerol was observed. These results pave the way for exploiting the assets of glycerol in the production of fuels, chemicals and pharmaceuticals based on baker's yeast.

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          Highlights

          • S. cerevisiae strains growing in synthetic glycerol medium with μ max up to 0.18 h −1.

          • The sole genetic modification was the expression of a predicted glycerol facilitator.

          • Facilitated diffusion can completely replace native glycerol/H + symport.

          • Biomass yield coefficients of engineered S. cerevisiae comparable to K. pastoris.

          • Exploiting the assets of glycerol as feedstock in S. cerevisiae bioprocesses visible.

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          Most cited references29

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          Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure.

          An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields > 1 x 10(6) transformants/micrograms plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 micrograms SS carrier DNA. The yield of transformants increased linearly up to 5 micrograms plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.
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            Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries.

            Metabolic engineering is the enabling science of development of efficient cell factories for the production of fuels, chemicals, pharmaceuticals, and food ingredients through microbial fermentations. The yeast Saccharomyces cerevisiae is a key cell factory already used for the production of a wide range of industrial products, and here we review ongoing work, particularly in industry, on using this organism for the production of butanol, which can be used as biofuel, and isoprenoids, which can find a wide range of applications including as pharmaceuticals and as biodiesel. We also look into how engineering of yeast can lead to improved uptake of sugars that are present in biomass hydrolyzates, and hereby allow for utilization of biomass as feedstock in the production of fuels and chemicals employing S. cerevisiae. Finally, we discuss the perspectives of how technologies from systems biology and synthetic biology can be used to advance metabolic engineering of yeast.
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              Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry.

              Although biofuels such as biodiesel and bioethanol represent a secure, renewable and environmentally safe alternative to fossil fuels, their economic viability is a major concern. The implementation of biorefineries that co-produce higher value products along with biofuels has been proposed as a solution to this problem. The biorefinery model would be especially advantageous if the conversion of byproducts or waste streams generated during biofuel production were considered. Glycerol-rich streams generated in large amounts by the biofuels industry, especially during the production of biodiesel, present an excellent opportunity to establish biorefineries. Once considered a valuable 'co-product', crude glycerol is rapidly becoming a 'waste product' with a disposal cost attributed to it. Given the highly reduced nature of carbon in glycerol and the cost advantage of anaerobic processes, fermentative metabolism of glycerol is of special interest. This review covers the anaerobic fermentation of glycerol in microbes and the harnessing of this metabolic process to convert abundant and low-priced glycerol streams into higher value products, thus creating a path to viability for the biofuels industry. Special attention is given to products whose synthesis from glycerol would be advantageous when compared with their production from common sugars.
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                Author and article information

                Contributors
                Journal
                Metab Eng Commun
                Metab Eng Commun
                Metabolic Engineering Communications
                Elsevier
                2214-0301
                29 September 2016
                December 2016
                29 September 2016
                : 3
                : 252-257
                Affiliations
                [a ]Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany
                [b ]Department of Systems Biology, Building 223, Søltofts Plads, Technical University of Denmark, Lyngby 2800 Kgs, Denmark
                Author notes
                [* ]Corresponding author. e.nevoigt@ 123456jacobs-university.de
                Article
                S2214-0301(16)30038-4
                10.1016/j.meteno.2016.09.001
                5779717
                29468128
                3d0b730b-4574-4fe0-abe5-cb9f3577a57e
                © 2016 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 3 April 2016
                : 5 August 2016
                : 28 September 2016
                Categories
                Article

                yeast,saccharomyces cerevisiae,glycerol,transport,glycerol facilitator,fps1,stl1

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