在丝状真菌中开发连续基因打靶的新工具

Filamentous fungi are important in industry and medicine as they secret a diverse range of enzymes (e.g., cellulase, proteases, lipases), as well as primary (e.g., organic acids ) and secondary metabolites (e.g., the antibiotics penicillin and cephalosporin). To overcome the limited availability of antibiotic resistance markers and low homologous recombination (HR) frequency in filamentous fungi, we designed two new tools for marker rescue and sequential gene targeting.. In eukaryotes, two main recombination pathways have been identified, differing as to whether double-strand break (DSB) repair depends on DNA sequence homology. The first, HR, involves interaction between homologous sequences, whereas the second, nonhomologous end-joining (NHEJ), involves direct ligation of the strand ends independent of DNA homology. HR is, in principle, the most efficient method of disrupting, modifying, or replacing a target gene. Although homologous integration occurs readily in Saccharomyces cerevisiae, the NHEJ pathway seems to be the dominant mode of DNA integration in other fungi. The DNA binding KU70/80 heterodimer is an essential component of the NHEJ machinery.. Recent studies show that the homologous integration frequency can be improved in mycelial fungi by deletion of KU70. However, the fact that such strains lack an important DNA repair pathway needs to be considered as it may impact fitness. Therefore, when gene targeting has been completed in an NHEJ deficient strain it is advisable to restore the NHEJ activity before further characterization of the mutant strain. This can be done by time-consuming sexual back-crossing or, if this is not possible, by an additional round of gene targeting. To facilitate gene targeting with NHEJ deficient strains, we are developing a system based on resiliently disrupted KU70 using Hypocrea jecorina as a model organism. In our system, KU70 function can be rapidly regenerated by a simple selection scheme after the desired genetic manipulations have been completed.. Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid. And the Cre recombinase is integrated in genome. Although expressed under the control of the inducible promoter, leakage expression is detected. Prolonged, low levels of Cre activity permit undesired recombination. This urges for a careful method in conditional gene modification. We designed a method wherein Cre could be transient expressed. After marker deletion the Cre gene should be lost. It will be a powerful tool in fungi.

丝状真菌被广泛应用于生物工程来生产化学药品（有机酸）、药物（青霉素、头孢霉素等）及酶（纤维素酶、蛋白酶、脂酶等）。为了扩大其生产能力，合成生物学研究是必要的。但筛选标记缺乏和同源重组率低限制了丝状真菌底盘的开发。为了克服上述问题，本申请书以里氏木霉为模式菌株设计基因操作新工具——“瞬时表达获救系统”和“NHEJ弹性系统”，可实现标记获救和连续高效基因打靶。. 充分分析前人研究的不足，提出的“瞬时表达获救系统”是采用“不恒定Cre的整体式结构”的设计，即在标记获救的过程中，Cre等外源基因同时缺失。其次，基于对KU70蛋白表达的精确调控，设计出“NHEJ弹性系统”，相比前人“先敲除，再回补”的操作，节约了工作量和时间。这两个工具，可以提高同源重组效率，实现筛选标记获救；并且无Cre基因残留，NHEJ机制完整，保证宿主基因组的稳定性；希望能够成为真核基因操作中有力的工具。

丝状真菌被广泛应用于生物工程来生产化学药品（有机酸）、药物（青霉素、头孢霉素等）及酶（纤维素酶、蛋白酶、脂酶等）。为了扩大其生产能力，合成生物学是研究方向。但筛选标记缺乏和同源重组率低限制了丝状真菌底盘的开发。为了克服上述问题，本申请书以里氏木霉为模式菌株设计了基因操作新工具——“瞬时表达获救系统”和“NHEJ弹性系统”，可实现标记获救和连续高效基因打靶。. 本项目基于Cre/loxP系统构建稳定存在的单质粒筛选标记系统LML2.0、LML2.1；通过木糖诱导Cre重组酶表达，一步法完成筛选标记和Cre基因的去除，实现筛选标记的重复利用，避免了cre重组酶基因残留在基因组中导致的染色体不稳定等风险。本研究创新性的使用了融合的cre基因，将含有内含子的内源基因片段与cre基因的编码序列进行融合。而这样的融合结构完全阻碍了cre基因在原核生物中的渗漏表达，使得单质粒系统LML2.0、LML2.1的构建成为可能。其次，本项目利用人工转录因子构建光诱导型筛选标记自切除系统LML3.0。用光作为诱导剂避免了LML系统对里氏木霉宿主的依赖。在本项目中，LML3.0已经成功应用到其他真菌中，如，Neurospora crassa, Aspergillus niger 和Metarhizium anisopliae等。而且由于光诱导的机制是全人工构建的，和宿主代谢无关，因此本项目构建的LML3.0系统，可以实现跨种属的应用，可以成为整个真菌系统的精确控制基因表达的新工具。. 最后，本项目对丝状真菌里氏木霉的NHEJ途径构建了人工可控的开关系统——OFN1.0，实现“NHEJ弹性系统”。本研究在NHEJ 途径的关键基因tku70的编码序列两端连入一对反向的loxP位点，成功构建了可控的tku70编码序列翻转的表达盒。通过诱导Cre的表达，完成tku70基因的转录开关切换的精确调控。通过转录水平分析，得出OFN1.0D关闭状态的严谨程度最好。OFN1.0D可以弹性地重复实现NHEJ途径的开启和关闭，简化了真菌的常规基因操作过程。并且，本项目设计的开关系统可以实现对基因转录精确的控制，在丝状真菌的表达调控上有着广泛的应用前景。