Abstract：
Background:Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts. Results:We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal. Conclusion:This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.
journal_name
Biotechnol Biofuelsjournal_title
Biotechnology for biofuelsauthors
Lee M,Rozeboom HJ,Keuning E,de Waal P,Janssen DBdoi
10.1186/s13068-019-1643-0subject
Has Abstractpub_date
2020-01-11 00:00:00pages
5issn
1754-6834pii
1643journal_volume
13pub_type
杂志文章abstract：Background:Microalgae are attracting much attention as a promising feedstock for renewable energy production, while simultaneously providing environmental benefits. So far, comparison studies for microalgae selection for this purpose were mainly based on data obtained from batch cultures, where the lipid content and th...
journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-019-1461-4
更新日期：2019-05-11 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-018-1111-2
更新日期：2018-04-23 00:00:00
abstract：Background:Insight into the function of carbohydrate-active enzymes is required to understand their biological role and industrial potential. There is a need for better use of the ample genomic data in order to enable selection of the most interesting proteins for further studies. The basis for elaborating a new approa...
journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-019-1436-5
更新日期：2019-04-30 00:00:00
abstract：:Microalgae hold great promises as sustainable cellular factories for the production of alternative fuels, feeds, and biopharmaceuticals for human health. While the biorefinery approach for fuels along with the coproduction of high-value compounds with industrial, therapeutic, or nutraceutical applications have the pot...
journal_title：Biotechnology for biofuels
pub_type： 杂志文章,评审
doi：10.1186/s13068-019-1635-0
更新日期：2019-12-23 00:00:00
abstract：BACKGROUND:Molasses is a dense and saline by-product of the sugar agroindustry. Its high organic content potentially fuels a myriad of renewable products of industrial interest. However, the biotechnological exploitation of molasses is mainly hampered by the high concentration of salts, an issue that is nowadays tackle...
journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-017-0701-8
更新日期：2017-01-31 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-016-0534-x
更新日期：2016-06-07 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-015-0370-4
更新日期：2015-11-25 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章,评审
doi：10.1186/s13068-019-1376-0
更新日期：2019-02-15 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-018-1190-0
更新日期：2018-07-09 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-020-01743-7
更新日期：2020-06-09 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章,撤回出版物
doi：10.1186/s13068-018-1086-z
更新日期：2018-04-06 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-019-1480-1
更新日期：2019-06-08 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-017-0961-3
更新日期：2017-11-30 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-016-0445-x
更新日期：2016-02-04 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-016-0589-8
更新日期：2016-08-24 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-014-0154-2
更新日期：2014-10-18 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-020-01751-7
更新日期：2020-06-24 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-014-0167-x
更新日期：2014-12-04 00:00:00
abstract：UNLABELLED: BACKGROUND:The impact of hydrothermal flowthrough (FT) pretreatment severity on pretreatment and solubilization performance metrics was evaluated for three milled feedstocks (corn stover, bagasse, and poplar) and two conversion systems (simultaneous saccharification and fermentation using yeast and fungal ...
journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/1754-6834-5-49
更新日期：2012-07-19 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/1754-6834-2-31
更新日期：2009-12-21 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/1754-6834-1-18
更新日期：2008-12-23 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/1754-6834-6-13
更新日期：2013-01-28 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/1754-6834-7-38
更新日期：2014-03-14 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章,已发布勘误
doi：10.1186/s13068-018-1185-x
更新日期：2018-07-03 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-018-1218-5
更新日期：2018-08-02 00:00:00
abstract：BACKGROUND:Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneo...
journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/1754-6834-4-58
更新日期：2011-12-10 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-017-0706-3
更新日期：2017-01-21 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-019-1411-1
更新日期：2019-04-01 00:00:00
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journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-017-0935-5
更新日期：2017-11-30 00:00:00
abstract：Background:A consolidated bioprocessing (CBP), where lignocellulose is converted into the desired product(s) in a single fermentative step without the addition of expensive degradative enzymes, represents the ideal solution of renewable routes to chemicals and fuels. Members of the genus Geobacillus are able to grow at...
journal_title：Biotechnology for biofuels
pub_type： 杂志文章
doi：10.1186/s13068-019-1540-6
更新日期：2019-08-20 00:00:00
