Doi Hidetaka, Tokura Yuriko, Mori Yukiko, Mori Kenichi, Asakura Yoko, Usuda Yoshihiro, Fukuda Hiroo, Chinen Akito
Process Development Laboratories, Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-8681, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Appl Microbiol Biotechnol. 2017 Feb;101(4):1581-1592. doi: 10.1007/s00253-016-8021-7. Epub 2016 Dec 3.
Alginate is a marine non-food-competing polysaccharide that has potential applications in biorefinery. Owing to its large size (molecular weight >300,000 Da), alginate cannot pass through the bacterial cell membrane. Therefore, bacteria that utilize alginate are presumed to have an enzyme that degrades extracellular alginate. Recently, Vibrio algivorus sp. SA2 was identified as a novel alginate-decomposing and alginate-utilizing species. However, little is known about the mechanism of alginate degradation and metabolism in this species. To address this issue, we screened the V. algivorus genomic DNA library for genes encoding polysaccharide-decomposing enzymes using a novel double-layer plate screening method and identified alyB as a candidate. Most identified alginate-decomposing enzymes (i.e., alginate lyases) must be concentrated and purified before extracellular alginate depolymerization. AlyB of V. algivorus heterologously expressed in Escherichia coli depolymerized extracellular alginate without requiring concentration or purification. We found seven homologues in the V. algivorus genome (alyB, alyD, oalA, oalB, oalC, dehR, and toaA) that are thought to encode enzymes responsible for alginate transport and metabolism. Introducing these genes into E. coli enabled the cells to assimilate soluble alginate depolymerized by V. algivorus AlyB as the sole carbon source. The alginate was bioconverted into L-lysine (43.3 mg/l) in E. coli strain AJIK01. These findings demonstrate a simple and novel screening method for identifying polysaccharide-degrading enzymes in bacteria and provide a simple alginate biocatalyst and fermentation system with potential applications in industrial biorefinery.
藻酸盐是一种海洋非食用竞争型多糖，在生物炼制中具有潜在应用价值。由于其分子量大（分子量>300,000道尔顿），藻酸盐无法穿过细菌细胞膜。因此，利用藻酸盐的细菌被推测具有降解细胞外藻酸盐的酶。最近，食藻弧菌SA2菌株被鉴定为一种新型的藻酸盐分解和利用藻酸盐的物种。然而，对于该物种中藻酸盐降解和代谢的机制知之甚少。为了解决这个问题，我们使用一种新型的双层平板筛选方法，从食藻弧菌基因组DNA文库中筛选编码多糖分解酶的基因，并确定alyB为候选基因。大多数已鉴定的藻酸盐分解酶（即藻酸裂解酶）在细胞外藻酸盐解聚之前必须进行浓缩和纯化。在大肠杆菌中异源表达的食藻弧菌AlyB可使细胞外藻酸盐解聚，而无需浓缩或纯化。我们在食藻弧菌基因组中发现了七个同源物（alyB、alyD、oalA、oalB、oalC、dehR和toaA），它们被认为编码负责藻酸盐运输和代谢的酶。将这些基因导入大肠杆菌，使细胞能够将食藻弧菌AlyB解聚的可溶性藻酸盐作为唯一碳源进行同化。在大肠杆菌AJIK01菌株中，藻酸盐被生物转化为L-赖氨酸（43.3毫克/升）。这些发现证明了一种简单而新颖的筛选细菌中多糖降解酶的方法，并提供了一种具有潜在工业生物炼制应用价值的简单藻酸盐生物催化剂和发酵系统。
