组学工具研究拟威克酵母变种的槐糖脂合成酶系及调控机制

Surfactants are known as "industrial monosodium glutamate", have attracted more attention for their increasing application in a variety of industrial area. Biosurfactants are produced by microorganisms and take great advantages over chemically-synthesized surfactants and the replacement of chemical surfactants by biosurfactants can mitigate resources and environmental crisis. Sophorolipids produced by a selected species of yeasts were one of the most important biosurfactants. The synthetic enzymes and the regulation and control network involved in sophorolipid production were very complicated, genetic information was almost unavailable. The sophorolipid-producing patent strain, Wickerhamiella domercqiae var. sophorolipid, was mutated to obtain several mutant strains of different traits by low energy nitrogen ion implantation, the whole genome of wild strain was being sequenced. On the basis of the previous studies, the work aims to investigate the main genes and enzymes involved in the synthetic pathway of sophorolipids. Cloning, gene knockout and overexpression of the main enzyme genes related with sophorolipid prodcution will be used to study the function of these genes in sophorolipid synthesis; In addition, by exploring those genes that caused the changes of phenotypes including high production of total SLs or the increase of percentage of lactonic SLs or acidic SLs in total SLs , the regulation and control mechanism of sophorolipid synthesis and the genetic basis of high sophorolipid production of mutant strains will be investigated. In addition, the responses of the yeast strain to nitrogen sources were also studied under different nitrogen source and at different period of cultivation of the yeast strain by transciptomic technique, aims to understand the regulation mechanism of sophorolipid synthesis on the transcriptive level. The results will be of great significance for genetic manipulation of SL-producing strains to improve the production and lowering the production cost of sophorolipid. The results will also provide important information for the regulation mechanism of the production of other biosurfactants that are structrally similar to sophorolipid (such as rhamnolipid). All the studies are of theoretic importance.

以微生物发酵得到的生物表面活性剂替代石化来源的化学合成的表面活性剂，是克服资源、环境危机的战略举措。槐糖脂的合成酶系及其合成调控网络非常复杂，对于相关酶系的分子生物学研究仍然较少。我国具有自主知识产权的产槐糖脂的拟威克酵母菌株变种经低能氮离子注入技术获得了多种不同特性的突变株,其原始菌株正在开始进行全基因组测序，拟在此基础上，应用基因组学技术研究槐糖脂合成调控网络中的主要基因的调控，对主要酶基因进行克隆,进行基因敲除/过表达,通过转录组学研究在不同的氮源条件下此酵母菌株槐糖脂合成相关基因对氮源的响应情况，获得槐糖脂合成相关基因的表达谱，深入阐明槐糖脂在转录水平上的合成调控机制。研究结果将为菌株的遗传改造提供合理的靶点,有助于提高槐糖脂的产量和降低成本，具有重要的实践意义，同时其研究成果可为阐明其他具有相似结构的表面活性剂（如鼠李糖脂）的合成调控机制提供重要参考，具有重要的理论意义。

以微生物发酵得到的生物表面活性剂替代石化来源的化学表面活性剂，是克服资源、环境危机的战略举措。槐糖脂的合成酶系及其合成调控网络非常复杂，对于相关酶系的分子生物学研究仍然较少。产槐糖脂的拟威克酵母变种专利菌株的全基因组测序已经完成。.主要研究内容、重要结果和关键数据：.1 对基因组中与槐糖脂合成相关的基因进行基因组注释，应用组学工具对槐糖脂合成调控网络中的主要基因进行了基因克隆、敲除、过表达，研究各种基因工程菌株中的槐糖脂合成的变化情况。.这些基因主要包括7种脂肪酸羟基化酶基因CYP52-M1, CYP52-N1, CYP52-N3, CYP52-E1, CYP52-E2, CYP51, CYP-X，4种酸性蛋白酶基因axpA, axp B, axpC, axpD，槐糖脂内酯化相关的基因MoA和MoB，以及槐糖脂合成调控相关基因palA等。.（2）对槐糖脂合成中的关键基因，包括脂肪酸羟基化的CYP52-M1基因，两种UDP葡萄糖基转移酶基因ugTI和ugTⅡ基因，糖异生途径的关键基因G6P以及4种酸性蛋白酶基因axpA, axpB, axpC, axpD分别进行了异源表达，并纯化得到了纯酶，并对其催化作用在体外进行了研究。.2 长链脂肪酸转运蛋白-ALCS的发现及研究.（1）通过蛋白的同源模建，找到了一个长链脂肪酸转运蛋白-ALCS。发现Alcs基因受长链脂肪酸的诱导表达，构建了alcs的敲除株及过表达菌株，对于工程菌株利用脂肪酸的能力分别进行了研究，并研究了Alcs基因的缺失对槐糖脂的组分的影响。.（2）异源表达了有活性的ALCS蛋白（71kDa），并进行了酶催化实验。对催化产物进行了质谱鉴定。.3 单加氧酶MoA基因和MoB基因的发现及研究.（1）对MoA和MoB基因进行了基因敲除和基因过表达，同时对敲除株和过表达株的槐糖脂组分进行了分析。.（2）将MoA酶进行异源表达并纯化，随后将纯酶用于催化两种纯的槐糖脂分子C18:2 DASL和C18:1 DASL，结果发现C18:1 DASL没有发生变化，而C18:2 DASL消失，表明MoA酶和C18:2DASL这种特异性分子的内酯化相关。.此研究结果将有利于菌株的遗传改造，从而提高槐糖脂的产量和降低成本，具有重要的实践意义，同时对于理性设计包括槐糖脂在内的生物表活剂的合成调控机制提供重要参考，具有重要的理论意义。