卫亚红等《Biotechnology for Biofuels and Bioproducts》2024年

论文题目：Lignin bioconversion based on genome mining for ligninolytic genes in  Erwinia billingiae  QL-Z3

论文作者：Shuting Zhao, Dongtao Deng, Tianzheng Wan, Jie Feng, Lei Deng, Qianyi Tian, Jiayu Wang, Umm E. Aiman, Balym Mukhaddi, Xiaofeng Hu, Shaolin Chen, Ling Qiu, Lili Huang*, Yahong Wei*

论文摘要：Bioconversion of plant biomass into biofuels and bio-products produces large amounts of lignin. The aromatic biopolymers need to be degraded before being converted into value-added bio-products. Microbes can be environment-friendly and efficiently degrade lignin. Compared to fungi, bacteria have some advantages in lignin degradation, including broad tolerance to pH, temperature, and oxygen and the toolkit for genetic manipulation. Our previous study isolated a novel ligninolytic bacterial strain  Erwinia billingiae  QL-Z3. Under optimized conditions, its rate of lignin degradation was 25.24% at 1.5 g/L lignin as the sole carbon source. Whole genome sequencing revealed 4556 genes in the genome of QL-Z3. Among 4428 protein-coding genes are 139 CAZyme genes, including 54 glycoside hydrolase (GH) and 16 auxiliary activity (AA) genes. In addition, 74 genes encoding extracellular enzymes are potentially involved in lignin degradation. Real-time PCR quantification demonstrated that the expression of potential ligninolytic genes were significantly induced by lignin. 8 knock-out mutants and complementary strains were constructed. Disruption of the gene for  ELAC_205  (laccase) as well as  EDYP_48  (Dyp-type peroxidase),  ESOD_1236  (superoxide dismutase),  EDIO_858  (dioxygenase),  EMON_3330  (monooxygenase), or  EMCAT_3587  (manganese catalase) significantly reduced the lignin-degrading activity of QL-Z3 by 47%-69%. Heterologously expressed and purified enzymes further confirmed their role in lignin degradation. Fourier transform infrared spectroscopy (FTIR) results indicated that the lignin structure was damaged, the benzene ring structure and groups of macromolecules were opened, and the chemical bond was broken under the action of six enzymes encoded by genes. The abundant enzymatic metabolic products by EDYP_48, ELAC_205 and ESOD_1236 were systematically analyzed via liquid chromatography-mass spectrometry (LC-MS) analysis, and then provide a speculative pathway for lignin biodegradation. Finally, The activities of ligninolytic enzymes from fermentation supernatant, namely LiP、MnP and Lac were 367.50 U/L, 839.50 U/L, and 219.00 U/L by orthogonal optimization. Our findings provide that QL-Z3 and its enzymes have the potential for industrial application and hold great promise for the bioconversion of lignin into bioproducts in lignin valorization. 

将植物生物质生物转化为生物燃料和生物产品时会产生大量的木质素。芳香族生物聚合物在转化为增值生物产品之前需要进行降解。微生物对环境更友好且能有效降解木质素。与真菌相比，细菌在木质素降解方面具有对pH、温度和氧气具有广泛耐受性，且便于遗传操作等优势。本研究分离到一株新型木质素降解菌 Erwinia billingiae  QL-Z3。在优化条件下，以1.5 g/L木质素为唯一碳源时，其木质素降解率为25.24%。全基因组测序结果显示，QL-Z3基因组共包含4556个基因。4428个蛋白编码基因中，CAZyme基因有139个，其中糖苷水解酶（GH）基因54个，辅助活性（AA）基因16个，74个编码胞外酶的基因可能参与木质素降解。实时荧光定量PCR结果表明，木质素可显著诱导木质素降解基因的表达。构建了8个敲除突变体和互补菌株，发现ELAC_205（漆酶）以及EDYP_48 （dyp型过氧化物酶）、ESOD_1236（超氧化物歧化酶）、EDIO_858（双加氧酶）、EMON_3330（单加氧酶）或EMCAT_3587（锰过氧化氢酶）基因的破坏显著降低了QL-Z3的木质素降解活性，降低幅度为47%-69%。异源表达并纯化上述酶进一步证实了它们在木质素降解中的作用。傅里叶变换红外光谱（FTIR）结果表明，在基因编码的6种酶的作用下，木质素结构被破坏，大分子苯环结构和基团被打开，化学键断裂。通过液相色谱-质谱（LC-MS）分析，系统分析了EDYP_48、ELAC_205和ESOD_1236丰富的酶代谢产物，推测了其木质素的降解途径。通过正交优化，发酵上清液中木质素降解酶LiP、MnP和Lac的活性分别为367.50 U/L、839.50 U/L和219.00 U/L。研究结果表明，QL-Z3及其酶具有工业应用潜力，在木质素增值中将木质素生物转化为生物产品方面具有广阔的前景。

文章链接：https://doi.org/10.1186/s13068-024-02470-z