Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
Institut für Mikrobiologie, Ernst-Moritz-Arndt Universität, Greifswald, Germany.
Appl Microbiol Biotechnol. 2017 Nov;101(21):7945-7960. doi: 10.1007/s00253-017-8508-x. Epub 2017 Sep 27.
The actinomycete Gordonia polyisoprenivorans strain VH2 is well-known for its ability to efficiently degrade and catabolize natural rubber [poly(cis-1,4-isoprene)]. Recently, a pathway for the catabolism of rubber by strain VH2 was postulated based on genomic data and the analysis of mutants (Hiessl et al. in Appl Environ Microbiol 78:2874-2887, 2012). To further elucidate the degradation pathway of poly(cis-1,4-isoprene), 2-dimensional-polyacrylamide gel electrophoresis was performed. The analysis of the identified protein spots by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry confirmed the postulated intracellular pathway suggesting a degradation of rubber via β-oxidation. In addition, other valuable information on rubber catabolism of G. polyisoprenivorans strain VH2 (e.g. oxidative stress response) was provided. Identified proteins, which were more abundant in cells grown with rubber than in cells grown with propionate, implied a putative long-chain acyl-CoA-dehydrogenase, a 3-ketoacyl-CoA-thiolase, and an aldehyde dehydrogenase. The amino acid sequence of the latter showed a high similarity towards geranial dehydrogenases. The expression of the corresponding gene was upregulated > 10-fold under poly(cis-1,4-isoprene)-degrading conditions. The putative geranial dehydrogenase and a homolog were purified and used for enzyme assays. Deletion mutants for five aldehyde dehydrogenases were generated, and growth with poly(cis-1,4-isoprene) was investigated. While none of the mutants had an altered phenotype regarding growth with poly(cis-1,4-isoprene) as sole carbon and energy source, purified aldehyde dehydrogenases were able to catalyze the oxidation of oligoisoprene aldehydes indicating an involvement in rubber degradation.
放线菌 Gordonia polyisoprenivorans 菌株 VH2 以有效降解和分解天然橡胶（聚（顺-1,4-异戊二烯））而闻名。最近，根据基因组数据和突变体分析，提出了菌株 VH2 分解橡胶的途径（Hiessl 等人，应用环境微生物学 78:2874-2887, 2012）。为了进一步阐明聚（顺-1,4-异戊二烯）的降解途径，进行了二维聚丙烯酰胺凝胶电泳。通过基质辅助激光解吸/电离飞行时间串联质谱分析鉴定的蛋白质斑点，证实了推测的细胞内途径，表明橡胶通过β-氧化降解。此外，还提供了关于 Gordonia polyisoprenivorans 菌株 VH2 橡胶分解代谢的其他有价值的信息（例如氧化应激反应）。在以橡胶为唯一碳源和能源的条件下，与以丙酸为唯一碳源和能源的条件相比，鉴定出的蛋白质丰度更高，这暗示了一种可能的长链酰基辅酶 A 脱氢酶、3-酮酰基辅酶 A 硫解酶和醛脱氢酶。后者的氨基酸序列与牻牛儿醛脱氢酶具有高度相似性。在聚（顺-1,4-异戊二烯）降解条件下，相应基因的表达上调了>10 倍。纯化了推测的牻牛儿醛脱氢酶及其同源物，并用于酶测定。生成了五个醛脱氢酶的缺失突变体，并研究了它们在聚（顺-1,4-异戊二烯）上的生长情况。虽然没有一个突变体在聚（顺-1,4-异戊二烯）作为唯一碳源和能源的生长方面表现出改变的表型，但纯化的醛脱氢酶能够催化低聚异戊二烯醛的氧化，表明其参与了橡胶的降解。
