Metabolic bottlenecks of Pseudomonas taiwanensis VLB120 during growth on d-xylose via the Weimberg pathway

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Philipp Nerke, Jonas Korb, Frederick Haala, Georg Hubmann, Stephan Lütz
{"title":"Metabolic bottlenecks of Pseudomonas taiwanensis VLB120 during growth on d-xylose via the Weimberg pathway","authors":"Philipp Nerke,&nbsp;Jonas Korb,&nbsp;Frederick Haala,&nbsp;Georg Hubmann,&nbsp;Stephan Lütz","doi":"10.1016/j.mec.2024.e00241","DOIUrl":null,"url":null,"abstract":"<div><p>The microbial production of value-added chemicals from renewable feedstocks is an important step towards a sustainable, bio-based economy. Therefore, microbes need to efficiently utilize lignocellulosic biomass and its dominant constituents, such as <span>d</span>-xylose. <em>Pseudomonas taiwanensis</em> VLB120 assimilates <span>d</span>-xylose via the five-step Weimberg pathway. However, the knowledge about the metabolic constraints of the Weimberg pathway<em>,</em> i.e., its regulation, dynamics, and metabolite fluxes, is limited, which hampers the optimization and implementation of this pathway for bioprocesses. We characterized the Weimberg pathway activity of <em>P. taiwanensis</em> VLB120 in terms of biomass growth and the dynamics of pathway intermediates. In batch cultivations, we found excessive accumulation of the intermediates <span>d</span>-xylonolactone and <span>d</span>-xylonate, indicating bottlenecks in <span>d</span>-xylonolactone hydrolysis and <span>d</span>-xylonate uptake. Moreover, the intermediate accumulation was highly dependent on the concentration of <span>d</span>-xylose and the extracellular pH. To encounter the apparent bottlenecks, we identified and overexpressed two genes coding for putative endogenous xylonolactonases PVLB_05820 and PVLB_12345. Compared to the control strain, the overexpression of PVLB_12345 resulted in an increased growth rate and biomass generation of up to 30 % and 100 %, respectively. Next, <span>d</span>-xylonate accumulation was decreased by overexpressing two newly identified <span>d</span>-xylonate transporter genes, PVLB_18545 and <em>gntP</em> (PVLB_13665). Finally, we combined xylonolactonase overexpression with enhanced uptake of <span>d</span>-xylonate by knocking out the <em>gntP</em> repressor gene <em>gntR</em> (PVLB_13655) and increased the growth rate and biomass yield by 50 % and 24 % in stirred-tank bioreactors, respectively. Our study contributes to the fundamental knowledge of the Weimberg pathway in pseudomonads and demonstrates how to encounter the metabolic bottlenecks of the Weimberg pathway to advance strain developments and cell factory design for bioprocesses on renewable feedstocks.</p></div>","PeriodicalId":18695,"journal":{"name":"Metabolic Engineering Communications","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2214030124000105/pdfft?md5=97d4236fe0530871599b5c6105208888&pid=1-s2.0-S2214030124000105-main.pdf","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic Engineering Communications","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2214030124000105","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

The microbial production of value-added chemicals from renewable feedstocks is an important step towards a sustainable, bio-based economy. Therefore, microbes need to efficiently utilize lignocellulosic biomass and its dominant constituents, such as d-xylose. Pseudomonas taiwanensis VLB120 assimilates d-xylose via the five-step Weimberg pathway. However, the knowledge about the metabolic constraints of the Weimberg pathway, i.e., its regulation, dynamics, and metabolite fluxes, is limited, which hampers the optimization and implementation of this pathway for bioprocesses. We characterized the Weimberg pathway activity of P. taiwanensis VLB120 in terms of biomass growth and the dynamics of pathway intermediates. In batch cultivations, we found excessive accumulation of the intermediates d-xylonolactone and d-xylonate, indicating bottlenecks in d-xylonolactone hydrolysis and d-xylonate uptake. Moreover, the intermediate accumulation was highly dependent on the concentration of d-xylose and the extracellular pH. To encounter the apparent bottlenecks, we identified and overexpressed two genes coding for putative endogenous xylonolactonases PVLB_05820 and PVLB_12345. Compared to the control strain, the overexpression of PVLB_12345 resulted in an increased growth rate and biomass generation of up to 30 % and 100 %, respectively. Next, d-xylonate accumulation was decreased by overexpressing two newly identified d-xylonate transporter genes, PVLB_18545 and gntP (PVLB_13665). Finally, we combined xylonolactonase overexpression with enhanced uptake of d-xylonate by knocking out the gntP repressor gene gntR (PVLB_13655) and increased the growth rate and biomass yield by 50 % and 24 % in stirred-tank bioreactors, respectively. Our study contributes to the fundamental knowledge of the Weimberg pathway in pseudomonads and demonstrates how to encounter the metabolic bottlenecks of the Weimberg pathway to advance strain developments and cell factory design for bioprocesses on renewable feedstocks.

台湾假单胞菌(Pseudomonas taiwanensis VLB120)通过魏姆伯格途径在二木糖生长过程中的代谢瓶颈
利用微生物从可再生原料中生产增值化学品是实现可持续生物经济的重要一步。因此,微生物需要有效利用木质纤维素生物质及其主要成分,如木糖。台湾假单胞菌(Pseudomonas taiwanensis VLB120)通过五步魏姆伯格途径同化二木糖。然而,人们对魏姆伯格途径的代谢限制(即其调节、动态和代谢通量)了解有限,这阻碍了该途径在生物过程中的优化和实施。我们从生物量增长和途径中间产物的动态两方面描述了台湾褐藻 VLB120 的魏姆伯格途径活性。在批量培养过程中,我们发现中间产物 d-xylonolactone 和 d-xylonate 积累过多,这表明 d-xylonolactone 的水解和 d-xylonate 的吸收存在瓶颈。此外,中间产物的积累与二木糖的浓度和细胞外 pH 值密切相关。为了解决这些明显的瓶颈问题,我们发现并过表达了两个编码假定内源性木糖醇内酯酶 PVLB_05820 和 PVLB_12345 的基因。与对照菌株相比,过表达 PVLB_12345 可使生长率和生物量产生率分别提高 30% 和 100%。接下来,通过过表达两个新发现的尼龙酸盐转运体基因 PVLB_18545 和 gntP(PVLB_13665),减少了尼龙酸盐的积累。最后,我们通过敲除gntP抑制基因gntR(PVLB_13655),将木尼醇内酯酶的过表达与增强对d-木尼醇酸盐的吸收结合起来,使搅拌罐生物反应器中的生长速率和生物量产量分别提高了50%和24%。我们的研究丰富了假单胞菌魏姆伯格途径的基础知识,并展示了如何突破魏姆伯格途径的代谢瓶颈,从而推动菌株开发和细胞工厂设计,促进可再生原料的生物加工。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信