Characterization of genes for chitin catabolism in Haloferax mediterranei
Characterization of genes for chitin catabolism in Haloferax mediterranei
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Haloferax mediterranei 几丁质分解代谢基因的特征
DOI：
10.1007/s00253-013-4969-8
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发表时间：
2014-02-01
影响因子：
5
通讯作者：
Xiang, Hua
中科院分区：
文献类型：
--
作者：
Hou, Jing;Han, Jing;Xiang, Hua
甲壳素是仅次于纤维素的第二丰富的天然多糖。但几丁质的降解在盐生古生菌中从未见过报道。在本研究中，我们揭示了一种代谢多功能性的卤虫，可以利用胶体或粉状甲壳素进行生长和聚(3-羟基丁酸酯-3-羟基戊酸酯)的积累，并通过实验鉴定了甲壳素分解代谢途径的基因簇(HFX_5025-5039)。首先，逆转录聚合酶链式反应结果表明，四种可能的几丁质酶基因(ChiAHme、ChiBHme、ChiCHme和ChiDHme，HFX_5036-5039)、LmbE样去乙酰基酶(DacHme，HFX_5027)和糖苷酶(GlyAHme，HFX_5029)的表达被胶体或几丁质粉末诱导，chia HME、Chib HME和chic HME共转录。当以几丁质为唯一碳源时，chiABC HME或chid HME基因敲除对细胞生长和PHBV产量有显著影响，而chiABCD HME基因敲除突变体丧失了利用甲壳素的能力。DAC HME或GALA HME的敲除也减少了PHBV在甲壳素上的积聚。这些结果表明ChiABCDHme、DacHme和GlyAHme确实参与了甲壳素的降解。此外，几丁质酶活性测定表明，各几丁质酶对胶体或粉状几丁质均有水解性，且ChiABCDHme对胶体几丁质的主要水解物为二乙酰基壳二糖，可能被DacHme、GlyAHme等相关酶进一步降解为单糖，促进细胞生长和PHBVDNA的合成。综上所述，本研究首次揭示了海洋古生菌中参与甲壳素分解代谢的基因和酶，并揭示了甲壳素在甲壳素生物转化中作为全细胞生物催化剂的潜力。
Chitin is the second most abundant natural polysaccharide after cellulose. But degradation of chitin has never been reported in haloarchaea. In this study, we revealed that Haloferax mediterranei, a metabolically versatile haloarchaeon, could utilize colloidal or powdered chitin for growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) accumulation, and the gene cluster (HFX_5025-5039) for the chitin catabolism pathway was experimentally identified. First, reverse transcription polymerase chain reaction results showed that the expression of the genes encoding the four putative chitinases (ChiAHme, ChiBHme, ChiCHme, and ChiDHme, HFX_5036-5039), the LmbE-like deacetylase (DacHme, HFX_5027), and the glycosidase (GlyAHme, HFX_5029) was induced by colloidal or powdered chitin, and chiA Hme, chiB Hme, and chiC Hme were cotranscribed. Knockout of chiABC Hme or chiD Hme had a significant effect on cell growth and PHBV production when chitin was used as the sole carbon source, and the chiABCD Hme knockout mutant lost the capability to utilize chitin. Knockout of dac Hme or glyA Hme also decreased PHBV accumulation on chitin. These results suggested that ChiABCDHme, DacHme, and GlyAHme were indeed involved in chitin degradation in H. mediterranei. Additionally, the chitinase assay showed that each chitinase possessed hydrolytic activity toward colloidal or powdered chitin, and the major product of colloidal chitin hydrolysis by ChiABCDHme was diacetylchitobiose, which was likely further degraded to monosaccharides by DacHme, GlyAHme, and other related enzymes for both cell growth and PHBV biosynthesis. Taken together, this study revealed the genes and enzymes involved in chitin catabolism in haloarchaea for the first time and indicated the potential of H. mediterranei as a whole-cell biocatalyst in chitin bioconversion.