Badur Ahmet H, Plutz Matthew J, Yalamanchili Geethika, Jagtap Sujit Sadashiv, Schweder Thomas, Unfried Frank, Markert Stephanie, Polz Martin F, Hehemann Jan-Hendrik, Rao Christopher V
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
Pharmaceutical Biotechnology, Institute of Pharmacy, Ernst Moritz Arndt University of Greifswald, D-17489 Greifswald, Germany.
J Biol Chem. 2017 Aug 4;292(31):13056-13067. doi: 10.1074/jbc.M117.787192. Epub 2017 Jun 7.
Polysaccharide degradation by marine microbes represents one of the largest and most rapid heterotrophic transformations of organic matter in the environment. Microbes employ systems of complementary carbohydrate-specific enzymes to deconstruct algal or plant polysaccharides (glycans) into monosaccharides. Because of the high diversity of glycan substrates, the functions of these enzymes are often difficult to establish. One solution to this problem may lie within naturally occurring microdiversity; varying numbers of enzymes, due to gene loss, duplication, or transfer, among closely related environmental microbes create metabolic differences akin to those generated by knock-out strains engineered in the laboratory used to establish the functions of unknown genes. Inspired by this natural fine-scale microbial diversity, we show here that it can be used to develop hypotheses guiding biochemical experiments for establishing the role of these enzymes in nature. In this work, we investigated alginate degradation among closely related strains of the marine bacterium One strain, 13B01, exhibited high extracellular alginate lyase activity compared with other strains. To identify the enzymes responsible for this high extracellular activity, we compared 13B01 with the previously characterized 12B01, which has low extracellular activity and lacks two alginate lyase genes present in 13B01. Using a combination of genomics, proteomics, biochemical, and functional screening, we identified a polysaccharide lyase family 7 enzyme that is unique to 13B01, secreted, and responsible for the rapid digestion of extracellular alginate. These results demonstrate the value of querying the enzymatic repertoires of closely related microbes to rapidly pinpoint key proteins with beneficial functions.
海洋微生物对多糖的降解是环境中最大且最快速的有机物质异养转化过程之一。微生物利用互补的碳水化合物特异性酶系统将藻类或植物多糖（聚糖）解构为单糖。由于聚糖底物的高度多样性，这些酶的功能往往难以确定。解决这个问题的一种方法可能在于自然存在的微观多样性；在密切相关的环境微生物中，由于基因丢失、复制或转移，酶的数量不同，从而产生了类似于实验室中用于确定未知基因功能的基因敲除菌株所产生的代谢差异。受这种自然的微观尺度微生物多样性的启发，我们在此表明，它可用于提出假设，指导生化实验以确定这些酶在自然界中的作用。在这项工作中，我们研究了海洋细菌密切相关菌株之间的藻酸盐降解情况。其中一个菌株13B01与其他菌株相比表现出较高的细胞外藻酸盐裂解酶活性。为了鉴定导致这种高细胞外活性的酶，我们将13B01与先前已表征的12B01进行了比较，12B01的细胞外活性较低，并且缺少13B01中存在的两个藻酸盐裂解酶基因。通过基因组学、蛋白质组学、生化和功能筛选相结合的方法，我们鉴定出一种多糖裂解酶家族7的酶，它是13B01特有的，可分泌，并负责细胞外藻酸盐的快速消化。这些结果证明了查询密切相关微生物的酶库以快速确定具有有益功能的关键蛋白质的价值。
