Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

Abstract Background Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilizati...

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Autores principales:	Jiyun Lou, Jingwen Wang, Yongfu Yang, Qing Yang, Runxia LI, Mimi Hu, Qiaoning He, Jun Du, Xia Wang, Mian Li, Shihui Yang
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Zymomonas mobilis Xylose utilization Xylose isomerase pathway Adaptive laboratory evolution (ALE) Genome resequencing RNA-Seq Fuel TP315-360 Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/1da9c48d40444d3d8bfc2b7eab8155af
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id	oai:doaj.org-article:1da9c48d40444d3d8bfc2b7eab8155af
record_format	dspace
institution	DOAJ
collection	DOAJ
language	EN
topic	Zymomonas mobilis Xylose utilization Xylose isomerase pathway Adaptive laboratory evolution (ALE) Genome resequencing RNA-Seq Fuel TP315-360 Biotechnology TP248.13-248.65
spellingShingle	Zymomonas mobilis Xylose utilization Xylose isomerase pathway Adaptive laboratory evolution (ALE) Genome resequencing RNA-Seq Fuel TP315-360 Biotechnology TP248.13-248.65 Jiyun Lou Jingwen Wang Yongfu Yang Qing Yang Runxia LI Mimi Hu Qiaoning He Jun Du Xia Wang Mian Li Shihui Yang Development and characterization of efficient xylose utilization strains of Zymomonas mobilis
description	Abstract Background Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. Results Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. Conclusions This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.
format	article
author	Jiyun Lou Jingwen Wang Yongfu Yang Qing Yang Runxia LI Mimi Hu Qiaoning He Jun Du Xia Wang Mian Li Shihui Yang
author_facet	Jiyun Lou Jingwen Wang Yongfu Yang Qing Yang Runxia LI Mimi Hu Qiaoning He Jun Du Xia Wang Mian Li Shihui Yang
author_sort	Jiyun Lou
title	Development and characterization of efficient xylose utilization strains of Zymomonas mobilis
title_short	Development and characterization of efficient xylose utilization strains of Zymomonas mobilis
title_full	Development and characterization of efficient xylose utilization strains of Zymomonas mobilis
title_fullStr	Development and characterization of efficient xylose utilization strains of Zymomonas mobilis
title_full_unstemmed	Development and characterization of efficient xylose utilization strains of Zymomonas mobilis
title_sort	development and characterization of efficient xylose utilization strains of zymomonas mobilis
publisher	BMC
publishDate	2021
url	https://doaj.org/article/1da9c48d40444d3d8bfc2b7eab8155af
work_keys_str_mv	AT jiyunlou developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT jingwenwang developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT yongfuyang developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT qingyang developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT runxiali developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT mimihu developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT qiaoninghe developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT jundu developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT xiawang developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT mianli developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis AT shihuiyang developmentandcharacterizationofefficientxyloseutilizationstrainsofzymomonasmobilis
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spelling	oai:doaj.org-article:1da9c48d40444d3d8bfc2b7eab8155af2021-12-05T12:16:45ZDevelopment and characterization of efficient xylose utilization strains of Zymomonas mobilis10.1186/s13068-021-02082-x1754-6834https://doaj.org/article/1da9c48d40444d3d8bfc2b7eab8155af2021-12-01T00:00:00Zhttps://doi.org/10.1186/s13068-021-02082-xhttps://doaj.org/toc/1754-6834Abstract Background Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. Results Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. Conclusions This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.Jiyun LouJingwen WangYongfu YangQing YangRunxia LIMimi HuQiaoning HeJun DuXia WangMian LiShihui YangBMCarticleZymomonas mobilisXylose utilizationXylose isomerase pathwayAdaptive laboratory evolution (ALE)Genome resequencingRNA-SeqFuelTP315-360BiotechnologyTP248.13-248.65ENBiotechnology for Biofuels, Vol 14, Iss 1, Pp 1-18 (2021)

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

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