【作者】 李志伟；

【导师】 罗晓春；

【作者基本信息】 华南理工大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 全球每年约产生数百万吨的羽毛废弃物,大多数没有被合理回收利用,不仅产生环境污染,同时对羽毛这一富含高蛋白的生物资源造成极大的浪费。因此,废弃羽毛的回收利用越来越受到人们的关注。本文从微生物的角度出发,探索利用生物法降解废弃羽毛,将其转化为高附加值的饲料蛋白资源,同时探索相关菌株高效降解羽毛的具体机制。本文首先进行了角蛋白酶的异源表达,成功获得三株产角蛋白酶的枯草芽孢杆菌工程菌株,通过发酵对比其中B.subtilis WB600-p MA5q-ker K的角蛋白酶活最高,达到982.26U/m L,但随后进行的实验表明单一的角蛋白酶并不能降解天然羽毛,需要还原剂和角蛋白酶共同作用。在此基础上,课题组通过大量的分离筛选从家禽羽毛废弃物长期堆积的土壤中获得一株新的羽毛降解微生物,该菌能够在36小时内完全降解鸡羽毛（包括羽毛轴）。对该菌株从形态学,生理生化水平以及分子水平对该菌株进行了表征和鉴定,最终将筛选的高效降解羽毛降解菌株命名为Streptomyces sp.SCUT-3。随后对SCUT-3降解羽毛产物在动物、植物和微生物中进行了初步应用试验,羽毛降解产物能够部分替代鱼粉作为动物饲料蛋白源,能够促进植物的生长并能提高微生物工程菌株的产酶量。为了对该菌株高效降解羽毛的机制进行研究,课题组挑选了三株与供试菌株亲缘关系较近的链霉菌进行降解羽毛机制的初步研究,进一步证实了链霉菌降解羽毛需要还原力和蛋白酶的共同作用。同时扫描电子显微镜及琼脂穿透实验证实了链霉菌降解羽毛的过程中菌丝机械作用力起着重要作用。还原力实验表明只有在完整菌体存在的情况下才能够还原二硫键,这可能与细胞膜表面氧化还原系统有关。链霉菌降解羽毛的过程中发现了游离巯基的存在,表明天然羽毛的二硫键被打开,经检测发现该菌株发酵上清中存在还原剂亚硫酸盐。为了从分子水平进一步探究链霉菌高效降解羽毛的相关机制,对该菌株进行了比较转录组测序。转录组学分析显示显著性差异表达的基因有1,459个,其中871个基因显著上调,588个基因显著下调。对显著性差异的基因进行GO功能注释,其中细胞过程,代谢过程,单一活性过程,催化活性,细胞膜相关以及转运活性功能方面差异的基因数量最多。其中跨膜转运活性,ABC转运活性以及肽和氨基酸跨膜转运活性相关的基因富集较为显著。KEGG代谢通路分析表明群体感应,氨基酸代谢,ABC转运系统,氧化磷酸化和脂肪酸代谢都有着非常显著的上调表达。通过转录组分析和实验测定表明亚硫酸盐和巯基化合物提供了SCUT-3降解羽毛的还原力。同时发现氧和Fe²⁺也能够明显影响SCUT-3对羽毛的降解。从显著性差异的基因中选取10个与羽毛降解相关的基因,包括亚硫酸盐产生及转运途径相关基因,巯基化合物（MSH）产生及转运途径相关基因和蛋白酶基因进行q RT-PCR验证,其结果与RNA-Seq结果相一致。本文首次对Streptomyces sp.SCUT-3的进行了全基因组测序和转录组测序并基于此提出了其降解羽毛的复合机制,Streptomyces sp.SCUT-3高效降解羽毛不是单一因素的作用,包括菌丝机械压力,亚硫酸盐和含巯基化合物的还原力和蛋白酶水解共同作用的结果。我们的研究结果将有助于后续深入了解链霉菌对角蛋白的降解机制,并且为羽毛的水解工艺研究提供理论支持。更多还原

【Abstract】 Millions of tons of feather waste are generated worldwide each year,and most of them are not properly recycled,which not only produces environmental pollution,but also causes great waste to feather,a high-protein biological resource.Therefore,the recycling of waste feathers is receiving more and more attention.From the perspective of microorganisms,this paper explores the use of biological methods to degrade waste feathers and convert them into high-value-added feed protein resources.At the same time,it explores the specific mechanism of efficient degradation of feathers by related strains.In this paper,we first performed heterologous expression of keratinase,and successfully obtained three keratinase-producing Bacillus subtilis engineering strains.B.subtilis WB600-p MA5q-ker K showed the highest keratinase activity by fermentation,reaching 982.26 U/m L,but Subsequent experiments have shown that a single keratinase cannot degrade natural feathers and requires a reducing agent and keratinase to work together.Based on this,the research team obtained a new feather-degrading microorganism from a long-term accumulation of soil from poultry feather waste through a large number of separations and screenings,which can completely degrade chicken feathers（including feather shafts）within 36 hours.The strain was characterized and identified from the morphological,physiological,biochemical and molecular levels,and the screened high-efficiency feather-degrading strain was named Streptomyces sp.SCUT-3.Subsequently,the preliminary application test of SCUT-3 degraded feather products in animals,plants and microorganisms was carried out.Feather degradation products can partially replace fish meal as an animal feed protein source,which can promote the growth of plants and increase the enzyme production of microbial engineering strains.In order to study the mechanism of high-efficiency degradation of feathers by this strain,the research team selected three strains of Streptomyces that are closely related to the test strain to conduct preliminary research on the mechanism of feather degradation.Scanning electron microscopy and agar penetration experiments at the same time confirmed that the mechanical force of hypha played an important role in the degradation of feathers by Streptomyces.Reducing power experiments show that disulfide bonds can be reduced only in the presence of intact bacteria,which may be related to the redox system on the cell membrane surface.The presence of free sulfhydryl groups was found during the degradation of feathers by Streptomyces,indicating that the disulfide bonds of natural feathers were opened.It was found through experiments that the reducing agent of sulfite was present in the fermentation supernatant of this strain.On this basis,in order to further explore the related mechanism of efficient degradation of feathers by Streptomyces at the molecular level,a comparative transcriptome sequencing of this strain was performed.Transcriptomics analysis showed that 1,459 genes were significantly differentially expressed,of which 871 genes were significantly up-regulated and588 genes were significantly down-regulated.Significantly different genes were annotated for GO function,among which the cellular process,metabolic process,single activity process,catalytic activity,cell membrane-related,and transport activity-related functions had the largest number of genes.Among them,transmembrane transport activity,ABC transport activity,and gene enrichment related to peptide and amino acid transmembrane transport activity are more significant.Analysis of the KEGG metabolic pathway showed that quorum sensing,amino acid metabolism,ABC transport system,oxidative phosphorylation,and fatty acid metabolism all have very significant up-regulated expression.Transcriptome analysis and experimental measurements indicate that sulfite and thiol compounds provides the reducing power of SCUT-3 to degrade feathers.Transcriptome analysis also found that oxygen and Fe²⁺can also significantly affect SCUT-3 degradation of feathers.Ten genes that may be related to feather degradation were selected from the significantly different genes,including genes related to sulfite production and transport pathways,genes related to MSH production and transport pathways,and protease genes for q RT-PCR verification.The results are consistent with the RNA-Seq results.In this paper,the whole genome sequencing and transcriptome sequencing of Streptomyces sp.SCUT-3 are performed for the first time.Based on this,a composite mechanism for degrading chicken feathers is proposed.The efficient degradation of feathers by Streptomyces sp.SCUT-3 is not a single factor,including mycelium machinery.The combined effect of stress,sulfite and thiol compounds reducing power and proteolytic hydrolysis.The results of our research will help further understand the degradation mechanism of keratin by Streptomyces,and provide theoretical support for the research on the hydrolysis process of feathers.更多还原