通过实验室自动适应性进化(ALE)提高台湾假单胞菌 VLB120 对 5-(羟甲基)糠醛(HMF)的耐受性

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Metabolic Engineering Communications Pub Date : 2024-05-10 DOI:10.1016/j.mec.2024.e00235
Thorsten Lechtenberg, Benedikt Wynands, Moritz-Fabian Müller, Tino Polen, Stephan Noack, Nick Wierckx
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引用次数: 0

摘要

5-(hydroxymethyl)furfural (HMF)醛对循环生物经济具有重要意义。它是一种可再生的平台化学品,可转化为一系列有用的化合物,以替代石油产品,如绿色塑料单体 2,5-呋喃二甲酸(FDCA)。然而,它也表现出微生物毒性,例如阻碍了木质纤维素水解物的高效生物技术价值化。因此,迫切需要耐受性更强、适用于全细胞生物催化的生物。在这里,我们通过机器人辅助的适应性实验室进化(ALE)技术,设计出了一种氧化缺陷衍生物,它是一种天然强健的新兴生物技术主力 P. taiwanensis VLB120。通过删除 HMF 氧化酶,首次实现了在醛的持续选择压力下的进化,产生了在有毒物质存在下生长特性持续改善的菌株。对进化克隆的基因组测序发现,编码 mexT 的 LysR 型转录调节器发生了功能缺失突变,从而阻止了相关外排泵 mexEF-oprN 的表达。有了这些知识,即使在 HMF 氧化机制活跃或过度表达的背景下,也能逆向工程培育出耐醛性更强的菌株,这证明了两种不同耐醛机制的协同效应。
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Improving 5-(hydroxymethyl)furfural (HMF) tolerance of Pseudomonas taiwanensis VLB120 by automated adaptive laboratory evolution (ALE)

The aldehyde 5-(hydroxymethyl)furfural (HMF) is of great importance for a circular bioeconomy. It is a renewable platform chemical that can be converted into a range of useful compounds to replace petroleum-based products such as the green plastic monomer 2,5-furandicarboxylic acid (FDCA). However, it also exhibits microbial toxicity for example hindering the efficient biotechnological valorization of lignocellulosic hydrolysates. Thus, there is an urgent need for tolerance-improved organisms applicable to whole-cell biocatalysis. Here, we engineer an oxidation-deficient derivative of the naturally robust and emerging biotechnological workhorse P. taiwanensis VLB120 by robotics-assisted adaptive laboratory evolution (ALE). The deletion of HMF-oxidizing enzymes enabled for the first time evolution under constant selection pressure by the aldehyde, yielding strains with consistently improved growth characteristics in presence of the toxicant. Genome sequencing of evolved clones revealed loss-of function mutations in the LysR-type transcriptional regulator-encoding mexT preventing expression of the associated efflux pump mexEF-oprN. This knowledge allowed reverse engineering of strains with enhanced aldehyde tolerance, even in a background of active or overexpressed HMF oxidation machinery, demonstrating a synergistic effect of two distinct tolerance mechanisms.

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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.
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