组蛋白甲基化与去乙酰化对蛇足石杉内生真菌盘长孢状刺盘孢合成石杉碱甲的影响

以蛇足石杉Huperzia serrata内生真菌盘长孢状刺盘孢Cg01菌株为研究对象,利用PEG介导的同源重组转化体系,对Cg01组蛋白甲基化酶基因（histone methyltransferases,HMT）CgClr4和组蛋白去乙酰化酶基因（histone deacetylase,HDAC）CgClr3、CgSir2进行基因敲除与回补,并通过实时荧光定量PCR（RT-qPCR）检测了回补株中对应基因表达量以及高效液相色谱HPLC检测突变体菌株中石杉碱甲huperzine A（HupA）产量。结果显示3个基因敲除突变体菌株ΔCgClr4、ΔCgClr3、ΔCgSir2的HupA产量分别为255μg/L、270μg/L、244μg/L,与野生型菌株相比分别下降了21.3%、16.6%、24.7%。在基因回补突变体菌株ΔCgClr4/CgClr4、ΔCgClr3/CgClr3、ΔCgSir2/CgSir2中,相应回补基因表达均与野生型无显著性差异,其HupA产量分别为351.9μg/L、334.7μg/L、331μg/L,回补菌株的HupA产量回复到野生型水平。结果表明这3个基因均具有调控内生真菌盘长孢状刺盘孢Cg01合成HupA的作用,为研究蛇足石杉内生真菌中石杉碱甲的合成调控机制提供了理论基础和新的思路。     

一种高效无选择标记的黑曲霉基因组编辑方法

黑曲霉(Aspergillus niger)是一种重要的工业生产菌株,被广泛地应用于生产酶制剂和有机酸,但仍需要进行基因组改造提高它的应用潜力。CRISPR/Cas9技术是一种被广泛采用的黑曲霉基因组编辑技术,但由于需要在基因组中整合选择标记或基因编辑效率还有待提高,影响了其在工业菌株改造中的应用。本研究建立了一种基于CRISPR/Cas9技术的高效无选择标记的基因编辑方法。首先,利用5S rRNA启动子启动sgRNA的表达,构建了一个含有AMA1(autonomously maintained in Aspergillus)复制起始片段的sgRNA和Cas9共表达质粒;同时通过敲除kusA基因构建非同源末端连接(non-homologous end joining pathway,NHEJ)修复缺陷的高效同源重组菌株;最后利用含有AMA1片段质粒的不稳定性,通过无抗平板传代丢失含有sgRNA和Cas9共表达质粒。利用该方法,在采用同源臂长度仅为20bp的无选择标记供体DNA进行基因编辑时,基因编辑效率可达到100%。该方法为黑曲霉基因功能的研究和细胞工厂的构建奠定了基础。     

UTH1基因破坏提高重组酿酒酵母分泌β-葡萄糖苷酶

异源蛋白质分泌效率低限制了重组酿酒酵母的多种药用蛋白和工业酶生产。挖掘促进蛋白质生物合成和分泌的关键基因,是提高异源蛋白质生产效率的重要手段。酿酒酵母细胞壁完整性影响异源蛋白质分泌,本研究利用基于CRISPR/Cas9的基因组编辑技术,破坏了重组酿酒酵母Y294-BGL1中参与细胞壁合成的未知功能基因UTH1,发现所获得的突变体胞外β-葡萄糖苷酶酶活比出发菌株提高112.9%,而细胞壁完整性下降。对促进产酶的分子机理进行探索,发现突变体产酶条件下与细胞壁完整性相关的关键基因和与蛋白质分泌途径相关的基因转录出现明显差异,提示UTH1基因破坏不仅影响细胞壁完整性关键基因的表达,也影响蛋白质分泌途径。本文的研究结果有助于深入理解UTH1的基因功能,并为构建异源蛋白质高分泌酵母菌株提供了借鉴。     

曲霉高效基因编辑技术研究进展

曲霉Aspergillus spp.是自然界中分布极为广泛的一大类真菌,在工业上如黑曲霉等已经被人们广泛利用于食品添加剂等生产。因此,曲霉在工业、农业、医药领域均发挥着重要的作用。然而,有些曲霉如黄曲霉能够分泌致癌物黄曲霉毒素从而污染农产品;在临床上还有一类以烟曲霉为主的引起侵袭性真菌感染的条件致病曲霉菌。因此,曲霉就像一把双刃剑影响着人们的生活。曲霉菌丝具有发达的隔膜形成多细胞菌丝体并能在分生孢子梗上产生大量的分生孢子进行无性繁殖。研究曲霉的基因编辑技术对于控制医学和农业上有害曲霉的增殖和促进工业上有益曲霉的生长和代谢都具有非常重要的意义。长期以来,同源重组和随机整合一直是被用于研究曲霉基因功能的传统基因编辑方法,然而其操作费时费力且效率低、难以达到预期的要求。随着第三代基因编辑技术CRISPR/Cas9即成簇的规律间隔的短回文重复序列及其相关系统CRISPR/Cas9（Clustered regulatory interspaced short palindromic repeats-associated protein 9）建立以来,CRISPR/Cas9以编辑高效、操作简便等优势被广泛应用到不同物种中。目前,世界上多个研究团队已经建立了有效地用于不同曲霉物种的CRISPR-Cas9 基因编辑体系。本文概述了有关曲霉基因编辑的历史和进展,以期为尚未建立完整遗传编辑体系的其他曲霉物种或者丝状真菌引入高效CRISPR-Cas9体系提供帮助。     

苜蓿中华根瘤菌氮代谢调控相关非编码RNA的研究

为了探究非编码RNA（non-coding RNA,ncRNA）在氮代谢调控过程的作用,以苜蓿中华根瘤菌（Sinorhizobium meliloti）为出发菌株,鉴定并获得了与ntrC基因表达相关的ncRNA,利用lncPRO软件分析苜蓿中华根瘤菌中ncRNA与调控蛋白NtrC相互作用的可能性,最终确定了评分较高的4个基因,即SM2011_c06191、SM2011_c06248、SM2011_c07102和SM2011_c07132,并对其中得分最高的SM2011_c06248基因进行研究。利用Northern blot验证ncRNA的表达,发现富氮处理后其表达水平呈先升高后降低的趋势。实验构建了SM2011_c06248基因敲除菌株,通过qRT-PCR发现SM2011_c06248在自然生长条件下表达无明显规律,富氮处理后,野生型菌株ncRNA表达水平呈先降低后升高再降低的趋势,与自然生长相比,ncRNA在20 min后表达水平明显上调;野生型菌株ntrC、nar基因表达水平呈先降低后升高再降低的趋势;与野生型相比,SM2011_c06248基因敲除菌株SM∷248中ntrC、nar基因表达水平明显下调。实验中构建SM2011_c06248、NtrC双突变菌株SM∷248-NtrC和SM2011_c06248过表达菌株SM:dld-248,qRT-PCR分析表明,与野生型相比,富氮处理后,SM∷248-NtrC菌株nar基因表达量无明显变化,SM:dld-248菌株ntrC、nar基因表达水平显著上调。ncRNA SM2011_c06248可响应环境中氮元素信号变化,对ntrC、nar基因的表达有正调控作用,且ntrC对nar有上位基因效应。     

一种拟南芥基因高效编辑体系研究

CRISPR/Cas9基因编辑系统操作简单易行,无需引入外源基因,生物安全性高。但怎样快速筛选获得不含外源转化元件的基因编辑后代是一个关键技术问题。本研究创造性的将拟南芥种皮特异性启动子At2S3与荧光筛选标记基因mCherry组装进植物基因组定点编辑CRISPR载体pHDE中,以拟南芥as1为靶基因,构建一种通过荧光标记筛选、实现转化后代中Cas9 Free的基因高效编辑体系。结果表明,通过同源重组方法构建的带有筛选标记的CRISPR载体与设计相符,外源插入片段正确。挑选转化后种皮上带有红色荧光标记的阳性种子培育得到T₁代植株,经PCR验证,成功获得as1定点敲除的纯合突变植株,纯合子比率达到40%;挑选T₁代纯合突变上不带荧光的种子,培育得到的T₂代植株中,PCR检测不到Cas9片段,实现了编辑后代的Cas9 Free。本研究构建的一种带有可视化筛选标记的基因高效编辑体系,成功实现编辑后代中无外源插入的Cas9等转化元件,生物安全性高,为基因组定点编辑技术在植物遗传资源改良中的高效利用提供了借鉴与参考。     

yhcZ基因在苏云金芽胞杆菌生长中的功能研究

在枯草芽胞杆菌和蜡样芽胞杆菌中,yhcZ基因和yhcY基因组成双组分系统调控细菌生长,但yhcZ基因在苏云金芽胞杆菌中发挥的生物学功能尚未明确。本研究通过基因功能注释、上下游基因排列分析和氨基酸序列比对,证实苏云金芽胞杆菌库斯塔克亚种HD73中HD73_5824基因为yhcZ基因,推测其与HD73_5825基因(yhcY基因)共同组成双组份系统调控细菌生长。利用同源重组技术敲除HD73菌株中的yhcZ基因获得缺失突变体HD (ΔyhcZ),其在LB和SSM培养基中生长均慢于野生型HD73,而互补菌株HD(ΔyhcZ::yhcZ)菌株则能够部分恢复生长,表明yhcZ基因的缺失影响了该菌株细胞的生长。在以0.4%葡萄糖为唯一碳源的M9培养基中,HD (ΔyhcZ)生长速度快于HD73,表明yhcZ基因在该菌株吸收利用葡萄糖的过程中发挥重要作用。Biolog实验显示HD (ΔyhcZ)的单孔颜色变化率低于HD73,且对D/L-丝氨酸、甲酸、D-葡糖酸、L-组胺,D-乳酸甲酯以及柠檬酸等的吸收利用能力低于HD73,表明yhcZ基因能显著影响HD73菌株对碳源的利用。同时,HD(ΔyhcZ)对8% NaCl的耐受能力弱于HD73,表明该基因可能参与细菌细胞应力响应相关基因的表达与调控。以上结果表明yhcZ基因在HD73菌株生长过程中对葡萄糖及其他碳源的利用具有重要的促进作用。本研究结果为解析yhcZ基因调控葡萄糖及碳源利用的分子机制奠定基础,且为进一步研究细菌生长及发酵提供参考。     

耐热黑曲霉3.316菌株全基因组测序及分析

黑曲霉3.316是一株耐热型丝状真菌,最高生长温度达47℃,在工业发酵中有着巨大的应用潜力.为了更加充分地在工业发酵中利用其耐热特性,需要对菌株信息进行全面了解.通过PacBio Sequel测序平台的CLR测序方式对黑曲霉3.316菌株进行全基因组测序.结果表明,基因组最后得到15个contigs,总长度为34 95...     

Directed Evolution of the Epoxide Hydrolase from Aspergillus niger

An efficient and genetically stable expression system for the directed evolution of epoxide hydrolase from Aspergillus niger (ANEH) has been constructed. Error prone polymerase chain reaction (PCR) with defined mutation rates was used to create biodiversity in two libraries of mutants. Screening for activity allowed the isolation of clones with improved properties. One of these clones shows an expression level 3.4 higher than the original wild type clone in E. coli SG13009 and a 3.3 fold increased catalytic efficiency on 4-(p-nitrophenoxy)-1,2-epoxybutane. In addition, a screening assay for determining the enantioselectivity in the kinetic resolution of styrene oxide has been established using mass spectrometry.     

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9(CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs(sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies.Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.     

Biotransformation of monoterpenoids, (−)- and menthols, terpinolene and carvotanacetone by Aspergillus species

Four monoterpenoids, (−)- and (+)-menthols, terpinolene and carvotanacetone were biotransformed by Aspergillus niger and several related species. Aspergillus niger converted (−)-menthol to 1-, 2-, 6-, 7-, and 9-hydroxymenthols and the mosquito repellent-active 8-hydroxymenthol. On the other hand, (+)-menthol was smoothly biotransformed by A. niger to give 7-hydroxymenthol. Aspergillus cellulosae biotransformed (−)-menthol specifically to 4-hydroxymenthol. Terpinolene and (−)-carvotanacetone were converted by A. niger to two , β-unsaturated ketones, a fenchane-type compound and diastereoisomeric p-menthane-2,9-diols and 8-hydroxycarvomenthol, respectively.     

Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris

The industrial yeast Pichia pastoris has been harnessed extensively for production of proteins, and it is attracting attention as a chassis cell factory for production of chemicals. However, the lack of synthetic biology tools makes it challenging in rewiring P. pastoris metabolism. We here extensively engineered the recombination machinery by establishing a CRISPR-Cas9 based genome editing platform, which improved the homologous recombination (HR) efficiency by more than 54 times, in particular, enhanced the simultaneously assembly of multiple fragments by 13.5 times. We also found that the key HR-relating gene RAD52 of P. pastoris was largely repressed in compared to that of Saccharomyces cerevisiae. This gene editing system enabled efficient seamless gene disruption, genome integration and multiple gene assembly with positive rates of 68–90%. With this efficient genome editing platform, we characterized 46 potential genome integration sites and 18 promoters at different growth conditions. This library of neutral sites and promoters enabled two-factorial regulation of gene expression and metabolic pathways and resulted in a 30-fold range of fatty alcohol production (12.6–380 mg/l). The expanding genetic toolbox will facilitate extensive rewiring of P. pastoris for chemical production, and also shed light on engineering of other non-conventional yeasts.     

CRISPR-Cas9研究进展及在基因治疗上的应用

CRISPR-Cas9[Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated （Cas）9]是近年兴起的一种高特异性和高效的基因编辑新技术,由向导RNA（single guide RNA,sgRNA）和cas9（CRISPR-associated 9）蛋白组成,引起DNA位点特异性双链断裂（double-strand breaks,DSBs）,引发同源重组修复（homology-directed repair,HDR）或非同源末端连接修复（non-homologous end joining,NHEJ）,达到靶基因修饰的作用。CRISPR-Cas9技术自发现以来,因其便于操作、花费较低、高特异性、可同时打靶任意数量基因等优点而被应用。近年研究显示,对于一些遗传性疾病,可通过CRISPR-Cas9精确的基因编辑破坏致病的内源基因、改正引起疾病的突变体或插入新的保护性基因进行治疗,该技术为基因治疗开启了一个新方向。主要从CRISPR-Cas9结构、作用机制及在疾病基因治疗上的应用等方面进行了综述。     

黑曲霉中生物合成赭曲霉毒素A的非核糖体肽合成酶基因的鉴定

赭曲霉毒素A(ochratoxin A,OTA)是国际癌症研究机构认定的"2B"类致癌物。黑曲霉Aspergillus niger是美国食品药品监督局认可的食品安全菌。然而近年来陆续发现某些黑曲霉菌株能够产生OTA,这会对人类健康构成潜在威胁。阐明黑曲霉生物合成OTA的关键基因有助于理解OTA生物合成机制,这对OTA污染的防控具有重要意义。本研究克隆了产OTA黑曲霉中非核糖体肽合成酶(NRPS)编码基因(An15g07910),并对其进行了生物信息学分析,在此基础上采用同源重组的方法敲除了该基因,获得了一株性能稳定的敲除突变株Δnrps。与野生株相比,Δnrps突变株的表型在CYA培养基中并无明显改变,但在7d培养期间完全失去了合成赭曲霉毒素α(ochratoxinα,OTα)和OTA的能力,而赭曲霉毒素β(ochratoxinβ,OTβ)的合成不受影响。在野生株培养过程中,该nrps基因前4d表达量逐渐增大,并在第4天达到最高,随后基因表达量逐渐下降并趋于稳定,这与OTA的含量变化基本一致。结果表明该nrps基因(An15g07910)参与OTA的生物合成,其编码的NRPS可能负责催化苯丙氨酸部分和二氢异香豆素部分的交联。     

Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System

An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the Escherichia coli genome by using the Streptococcus pyogenes type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in Tatumella citrea, another species of the Enterobacteriaceae, with highest efficiency of 100%.     

Biochemical characterisation and kinetic properties of a purified lipase from Aspergillus niger in bulk phase and monomolecular films

An isolate of Aspergillus niger was used as source of lipase which was purified to a specific activity of 729 U/mg. It has an acidic pH optimum and has a half-life of 42 h at pH 4.4, which can be increased to 138 h in the presence of 10 mM calcium ions. For the first time a lipase from Aspergillus niger was characterised using the monomolecular film technique. The lipase was classified to have a sn-1 selectivity using diacylglycerols and R-isomer hydrolytic preference with pseudolipids representing triacylglycerols in which two of the ester bonds were replaced with ether and amide linkages.