基因组重排技术选育乳链菌肽高产菌株

目的采用基因组重排(Genome shuffling)技术选育乳链菌肽高产菌株。方法以乳酸乳球菌(Lactococcus lactis)G7分别经超声波、硫酸二乙酯(Diethyl suilate,DES)及超声波和DES复合诱变后获得的3株突变株S-1、D-1、F-1为出发菌株,制备原生质体,并进行再生及灭活,采用基因组重排技术选育乳链菌肽高产菌株。结果原生质体的制备率和再生率分别为98.5%±1.2%和13.6%±0.9%;原生质体的灭活条件为紫外线灭活120s及60℃热处理20min,原生质体的致死率可达100%;通过2轮基因组重排,成功选育出1株高产乳链菌肽菌株R2-6,其比原始菌株G7的乳链菌肽的产量提高了69.8%。结论已成功选育出了1株产量大幅提高、遗传稳定的乳链菌肽菌株。     

ARTP-DES复合诱变结合前体耐受性选育达托霉素高产菌株

为提高玫瑰孢链霉菌（Streptomyces roseosporus）AN-126的达托霉素（Daptomycin）产量，对菌株AN-126进行ARTP-DES复合诱变，并结合癸酸钠耐受性筛选，最终获得一株达托霉素高产菌株RR-447，摇瓶产量为101.41 mg/L，是出发菌株的2.44倍。传代实验表明，该菌株高产性能稳定遗传。对突变前后菌株的形态特征以及基因指纹图谱进行分析，结果显示突变菌株与出发菌株在形态学特征有明显差异，且高产菌株RR-447 基因组DNA在1.5 kb~2.0 kb之间存在一条特异性条带。通过在5L发酵罐中流加前体物质癸酸钠，菌株RR-447达托霉素产量达到521.39 mg/L。研究结果表明，ARTP-DES复合诱变结合癸酸钠耐受性是选育达托霉素高产菌株的一种有效育种手段，为达托霉素工业微生物育种以及其他菌株改良提供了技术参考。     

基因组再造与重排构建细胞工厂

细胞工厂能利用微生物细胞制备人类所需能源、药物和化学品。底盘细胞和外源代谢路径的适配是构建高效细胞工厂的核心难题。基因组再造指利用化学合成的核苷酸分子“自下而上”构建生物基因组,基因组诱导重排指通过在全基因组尺度进行DNA序列与结构的人为调控。基因组再造和诱导重排实现了对生命体的创造,增强了模式底盘细胞的遗传稳定性和操作柔性。基因组的适度精简和密码子简化改善细胞对底物、能量的利用效率,提高细胞生理性能的预测性和可控性。基因组重排可诱导染色体发生随机删除、复制、移位和倒置等结构变异,可产生大量性状优良的模式底盘细胞,进而加速代谢路径优化,提高路径和底盘细胞的适配性,为人工细胞工厂快速构建和优化提供了新策略。     

基因组再造与重排构建细胞工厂

细胞工厂能利用微生物细胞制备人类所需能源、药物和化学品。底盘细胞和外源代谢路径的适配是构建高效细胞工厂的核心难题。基因组再造指利用化学合成的核苷酸分子"自下而上"构建生物基因组,基因组诱导重排指通过在全基因组尺度进行DNA序列与结构的人为调控。基因组再造和诱导重排实现了对生命体的创造,增强了模式底盘细胞的遗传稳定性和操作柔性。基因组的适度精简和密码子简化改善细胞对底物、能量的利用效率,提高细胞生理性能的预测性和可控性。基因组重排可诱导染色体发生随机删除、复制、移位和倒置等结构变异,可产生大量性状优良的模式底盘细胞,进而加速代谢路径优化,提高路径和底盘细胞的适配性,为人工细胞工厂快速构建和优化提供了新策略。     

紫外-ARTP复合诱变选育达巴万星前体高产菌株

采用紫外-常压室温等离子体(ARTP)技术对达巴万星前体产生菌野野村放线菌属(Nonomuriaspp.)菌株DW-3-19进行复合诱变,并进行链霉素抗性突变选育,获得了具有稳定遗传性的高产菌株,该菌株发酵单位比出发菌株提高了68.7%。表明,ARTP技术可有效应用于放线菌的诱变选育,紫外-ARTP复合诱变可提高达巴万星前体发酵单位,降低生产成本,为短时间内获得稳定遗传的高产菌株提供了可行的方法。     

选育纤维素乙醇生产的高效产酶工程菌技术的研究进展

综述了近年来选育高效产纤维素酶工程菌取得的最新理论研究及工艺进展,并就复合理化诱变获取高效产酶菌,以及利用原生质体融合、原生质体诱变、基因工程等技术对各种产纤维素酶、半纤维素酶菌株进行遗传改造,对纤维素酶高效产酶工程菌的选育进展进行了总结,进一步展望了选育高产纤维素酶菌株的研究方向。     

基于基因组重排技术的多杀菌素高产菌株选育

利用课题组前期通过不同物理化学诱变和分子生物学方法获得的8株多杀菌素产生菌为出发菌株,采用96孔板发酵培养结合生物检测的高通量筛选方法,探索了原生质体制备、再生、双亲灭活和原生质体融合等条件,并通过多轮基因组重排获得了多杀菌素高产菌株。结果表明:当菌龄为65 h、溶菌酶浓度为4 mg·ml-1,39℃处理时间20 min时,原生质体的制备率及再生率分别为92.30%和7.66%;60℃恒温水浴90 min以上和紫外照射200s以上为双亲灭活条件;PEG浓度为50%,在32℃下处理15 min时,原生质体融合率约为1.18%。最终获得1株产量较出发菌株提高了36.07%且遗传稳定的融合株。     

原生质体诱变提高亚麻刺盘孢ST对底物DHEA的耐受性和转化率

采用原生质体诱变技术选育出一株耐高浓度底物去氢表雄酮（DHEA）的高产亚麻刺盘孢菌株（Colletotrichum lini） ST-0317,并研究了其转化特性。以C. lini ST为出发菌株,考察了其原生质体制备与再生的最适条件,随后对其原生质体进行等离子诱变,最终选育得到优势突变株ST-0317。该菌株在底物耐受性提高的同时也具有良好的遗传稳定性,在DHEA投料浓度高达10g/L的条件下,产物7α,15α-diOH-DHEA摩尔得率可达36.9%,比出发菌株提高了50%。     

农抗120生产菌高能电子流诱变育种与发酵性能研究

通过诱变育种提高农抗120生产菌株的发酵效价。采用不同剂量高能电子流辐射孢子芽管悬液、琼脂移块 法初筛、摇瓶管碟法复筛得到高产菌株,然后在发酵罐中放大培养。选育得到一株高产突变株D3226,其摇瓶效价达 9260 μg·mL-1,比出发菌株O11提高了100．4％,并且传代稳定;在100 L和10 t发酵罐中的分批培养实验表明:pH值 下降到最低点5．7时为抗生素累积高峰,pH值可以作为一种新的有效的放罐指标。得到的高产菌株及发酵规律可以应 用于工业生产。     

基于基因组重排技术的多杀菌素高产菌株选育

利用课题组前期通过不同物理化学诱变和分子生物学方法获得的8株多杀菌素产生菌为出发菌株,采用96孔板发酵培养结合生物检测的高通量筛选方法,探索了原生质体制备、再生、双亲灭活和原生质体融合等条件,并通过多轮基因组重排获得了多杀菌素高产菌株。结果表明：当菌龄为65 h、溶菌酶浓度为4 mg·ml^-1,39℃处理时间20 min时,原生质体的制备率及再生率分别为92.30%和7.66%;60℃恒温水浴90 min以上和紫外照射200 s以上为双亲灭活条件;PEG浓度为50%,在32℃下处理15 min时,原生质体融合率约为1.18%。最终获得1株产量较出发菌株提高了36.07%且遗传稳定的融合株。     

DNA及基因组改组在代谢工程中的应用

代谢工程是应用分子生物学与反应工程的结合,已发展成为菌种改进的平台技术.后基因组学时代的基因组学、转录组学、蛋白质组学及代谢物组学等为代谢工程的发展提供了极好的机遇.DNA改组及基因组改组可以构建新的代谢途径、对已知或未知的代谢途径进行快速优化,极大地促进了代谢工程的进一步发展和应用.DNA改组及基因组改组技术可以加深对代谢网络及其分子调节机制的理解,是对合理代谢设计策略的完善和补充.本文首先分别介绍了DNA改组、基因组改组在代谢工程中的应用,进而展望了基于基因组改组的代谢工程方法步骤及发展方向.     

基因组改组及代谢通量分析在产核黄素Bacillus subtilis性能改进中的应用

综合应用基因组改组和DNA重组技术改进了核黄素生产菌B.subtilis 24/pMX45的性状,在B.subtilis染色体上整合和扩增了多个B.subtilis 24的核黄素操纵子拷贝,并经过两轮的基因组改组后筛得菌株B.subtilis RH33/pMX45,在以12%葡萄糖为碳源的分批发酵中,其核黄素产量约为B.subtilis 24/pMX45的2倍,并具有快速同化利用葡萄糖、生长迅速、可形成感受态和进行基因整合、扩增等优良性状.随后比较分析了三株产核黄素B.subtilis在间歇培养条件下的代谢通量分布,通量分析和实验结果表明,B.subtilis RH33/pMX45中核黄素合成的主要通量限制因素存在于从5-磷酸核酮糖到GTP的一系列反应中.     

提高微生物抗逆性技术的研究进展

微生物体内存在多种抗逆基因或机制,这些机制的发现为定向提高微生物的抗逆性奠定了基础。提高微生物抗逆性的技术主要有过表达抗逆基因,长期适应性进化, genome shuffling（基因组改组）和异源表达抗逆基因等。利用这几种技术增强微生物的抗逆性,在以微生物为主的工业生产和环境污染物降解方面有着广阔的应用前景。     

Enhancing Animal Disease Resistance,Production Efficiency,and Welfare through Precise Genome Editing

The major goal of animal breeding is the genetic enhancement of economic traits. The CRISPR/Cas system, which includes nuclease-mediated and base editor mediated genome editing tools, provides an unprecedented approach to modify the mammalian genome. Thus, farm animal genetic engineering and genetic manipulation have been fundamentally revolutionized. Agricultural animals with traits of interest can be obtained in just one generation (and without long time selection). Here, we reviewed the advancements of the CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) genome editing tools and their applications in animal breeding, especially in improving disease resistance, production performance, and animal welfare. Additionally, we covered the regulations on genome-edited animals (GEAs) and ways to accelerate their use. Recommendations for how to produce GEAs were also discussed. Despite the current challenges, we believe that genome editing breeding and GEAs will be available in the near future.     

Differential Proteomics Analysis of Bacillus amyloliquefaciens and Its Genome-Shuffled Mutant for Improving Surfactin Production

Genome shuffling technology was used as a novel whole-genome engineering approach to rapidly improve the antimicrobial lipopeptide yield of Bacillus amyloliquefaciens. Comparative proteomic analysis of the parental ES-2-4 and genome-shuffled FMB38 strains was conducted to examine the differentially expressed proteins. The proteome was separated by 2-DE (two dimensional electrophoresis) and analyzed by MS (mass spectrum). In the shuffled strain FMB38, 51 differentially expressed protein spots with higher than two-fold spot density were detected by gel image comparison. Forty-six protein spots were detectable by silver staining and further MS analysis. The results demonstrated that among the 46 protein spots expressed particularly induced in the genome-shuffled mutant, 15 were related to metabolism, five to DNA replication, recombination and repair, six to translation and post-translational modifications, one to cell secretion and signal transduction mechanisms, three to surfactin synthesis, two to energy production and conversion, and 14 to others. All these indicated that the metabolic capability of the mutant was improved by the genome shuffling. The study will enable future detailed investigation of gene expression and function linked with surfactin synthesis. The results of proteome analysis may provide information for metabolic engineering of Bacillus amyloliquefaciens for overproduction of surfactin.     

Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling

Evolutionary protein engineering is now proceeding to a newstage in which novel technologies, besides the conventionalpoint mutations, to generate a library of proteins, are required.In this context, a novel method for shuffling and rearrangingDNA blocks (leading to protein libraries) is reported. A cycleof processes for producing combinatorial diversity was devisedand designated Y-ligation-based block shuffling (YLBS). Methodologicalrefinement was made by applying it to the shuffling of module-sizedand amino acid-sized blocks. Running three cycles of YLBS withmodule-sized GFP blocks resulted in a high diversity of an eight-blockshuffled library. Partial shuffling of the central four blocksof GFP was performed to obtain in-effect shuffled protein, resultingin an intact arrangement. Shuffling of amino acid monomer-sizedblocks by YLBS was also performed and a diversity of more than10¹⁰ shuffled molecules was attained. The deletion problemsencountered during these experiments were shown to be solvedby additional measures which tame type IIS restriction enzymes.The frequency of appearance of each block was skewed but waswithin a permissible range. Therefore, YLBS is the first generalmethod for generating a huge diversity of shuffled proteins,recombining domains, exons and modules with ease.     

利用DNA改组筛选高活性抗凝血酶Ⅲ

目的　利用DNA改组技术大幅度提高抗凝血酶Ⅲ (AT -Ⅲ )的生物活性。方法　采用RQ1DNaseⅠ不完全酶解ST -Ⅲ基因 ,产生长度为 5 0～ 10 0bp的随机片段 ,用低熔点胶回收。无引物PCR从小片段组装成大片段 ,通过有引物PCR得到正确大小的片段。线性化的质粒通过电转转入毕赤酵母GS115细胞。结果　获得了 5株高活性的克隆。结论　探索了在毕赤酶母进行DNA改组和筛选的方法和路线 ,成功进行了AT Ⅲ的改组 ,并为其它分子的改组提供借鉴。     

Engineering Properties of Sweet Potato Starch for Industrial Applications by Biotechnological Techniques including Genome Editing

Sweet potato (Ipomoea batatas) is one of the largest food crops in the world. Due to its abundance of starch, sweet potato is a valuable ingredient in food derivatives, dietary supplements, and industrial raw materials. In addition, due to its ability to adapt to a wide range of harsh climate and soil conditions, sweet potato is a crop that copes well with the environmental stresses caused by climate change. However, due to the complexity of the sweet potato genome and the long breeding cycle, our ability to modify sweet potato starch is limited. In this review, we cover the recent development in sweet potato breeding, understanding of starch properties, and the progress in sweet potato genomics. We describe the applicational values of sweet potato starch in food, industrial products, and biofuel, in addition to the effects of starch properties in different industrial applications. We also explore the possibility of manipulating starch properties through biotechnological means, such as the CRISPR/Cas-based genome editing. The ability to target the genome with precision provides new opportunities for reducing breeding time, increasing yield, and optimizing the starch properties of sweet potatoes.