UNSW Water Research Centre, School of Civil and Environmental Engineering, University of New South Wales, Sydney, NSW, Australia.
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.
mSystems. 2023 Apr 27;8(2):e0011923. doi: 10.1128/msystems.00119-23. Epub 2023 Mar 21.
Recent discoveries of isoprene-metabolizing microorganisms suggest they might play an important role in the global isoprene budget. Under anoxic conditions, isoprene can be used as an electron acceptor and is reduced to methylbutene. This study describes the proteogenomic profiling of an isoprene-reducing bacterial culture to identify organisms and genes responsible for the isoprene hydrogenation reaction. A metagenome-assembled genome (MAG) of the most abundant (89% relative abundance) lineage in the enrichment, Acetobacterium wieringae, was obtained. Comparative proteogenomics and reverse transcription-PCR (RT-PCR) identified a putative five-gene operon from the A. wieringae MAG upregulated during isoprene reduction. The operon encodes a putative oxidoreductase, three pleiotropic nickel chaperones (2 × HypA, HypB), and one 4Fe-4S ferredoxin. The oxidoreductase is proposed as the putative isoprene reductase with a binding site for NADH, flavin adenine dinucleotide (FAD), two pairs of canonical [4Fe-4S] clusters, and a putative iron-sulfur cluster site in a Cys-bonding environment. Well-studied strains, such as A. woodii DSM 1030, A. wieringae DSM 1911, or A. malicum DSM 4132, do not encode the isoprene-regulated operon but encode, like many other bacteria, a homolog of the putative isoprene reductase (~47 to 49% amino acid sequence identity). Uncharacterized homologs of the putative isoprene reductase are observed across the , , , , , , and , suggesting the ability of biohydrogenation of unfunctionalized conjugated doubled bonds in other unsaturated hydrocarbons. Isoprene was recently shown to act as an electron acceptor for a homoacetogenic bacterium. The focus of this study is the molecular basis for isoprene reduction. By comparing a genome from our isoprene-reducing enrichment culture, dominated by Acetobacterium wieringae, with genomes of other lineages that do not reduce isoprene, we shortlisted candidate genes for isoprene reduction. Using comparative proteogenomics and reverse transcription-PCR we have identified a putative five-gene operon encoding an oxidoreductase referred to as putative isoprene reductase.
最近发现的异戊二烯代谢微生物表明，它们可能在全球异戊二烯预算中发挥重要作用。在缺氧条件下，异戊二烯可用作电子受体，并被还原为甲基丁烯。本研究描述了异戊二烯还原细菌培养物的蛋白质基因组分析，以鉴定负责异戊二烯氢化反应的生物和基因。从富集物中最丰富的（相对丰度 89%）谱系的宏基因组组装基因组（MAG）中获得了醋酸杆菌 wieringae 的基因组。比较蛋白质基因组学和反转录-PCR（RT-PCR）鉴定了来自 A. wieringae MAG 的一个假定的五基因操纵子，该操纵子在异戊二烯还原过程中上调。该操纵子编码一种假定的氧化还原酶、三种多效性镍伴侣（2×HypA、HypB）和一种 4Fe-4S 铁氧还蛋白。该氧化还原酶被提议为假定的异戊二烯还原酶，具有与 NADH、黄素腺嘌呤二核苷酸（FAD）、两对典型的[4Fe-4S]簇以及假定的铁-硫簇结合位点的结合位点在一个 Cys 结合环境中。像 A. woodii DSM 1030、A. wieringae DSM 1911 或 A. malicum DSM 4132 这样的研究充分的菌株不编码异戊二烯调节操纵子，但像许多其他细菌一样，编码假定的异戊二烯还原酶的同源物（~47 到 49%的氨基酸序列同一性）。在 、 、 、 、 、 和 中观察到假定的异戊二烯还原酶的未表征同源物，这表明在其他不饱和烃中未官能化的共轭双键的生物氢化能力。最近表明，异戊二烯可以作为同型乙酰生成菌的电子受体。本研究的重点是异戊二烯还原的分子基础。通过将我们的异戊二烯还原富集培养物中的基因组与不还原异戊二烯的其他 谱系的基因组进行比较，我们列出了异戊二烯还原的候选基因。使用比较蛋白质基因组学和反转录-PCR，我们已经鉴定出一个假定的五基因操纵子，该操纵子编码一种氧化还原酶，称为假定的异戊二烯还原酶。
