应用基因组改组选育耐糖L-乳酸高产菌株

本研究应用基因组改组提高鼠李糖乳杆菌的耐糖能力,进而提高L-乳酸的产量。通过紫外线和亚硝基胍诱变方法获得8株改良出发菌株。考察了氨基酸前处理和变溶菌素对原生质体形成的影响。利用双亲灭活的原生质体进行递进式融合,经过两轮基因组改组和碳酸钙高糖平板及瓶筛选,选育出了耐糖改组菌株F2-2。在初糖浓度为150g/L发酵罐中,F2-2的乳酸产量为140g/L,是野生型乳杆菌的1.8倍。结果表明基因组改组技术能够快速提高乳酸菌耐糖和产酸能力两个表型。     

微生物菌种选育中基因组重排技术应用的研究进展

基因组重排(genome shuffling)技术是通过传统诱变育种结合原生质体融合技术开发出的一种新型微生物菌种选育和改良技术,具有易操作、效率高和适用广等特点。本文对基因组重排技术的原理、操作过程及其在增加次生代谢产物产量、增强菌株耐受性和提高底物利用能力等方面的应用进行了综述。     

基因组改组技术对L-乳酸产生菌耐热性的影响

采用紫外线与亚硝基胍(NTG)两种诱变方法获得用于基因组重组的出发菌株,应用灭活的多亲原生质体再融合获得活性融合子的方法,有效地提高了基因组改组后的筛选效率.采用低pH值平板和碳酸钙平板连续筛选的方法,筛选得到干酪乳杆菌改组菌株,具有较强耐酸性能,够在pH3.6 MRS平板上生长繁殖,产酸量为原始菌株的3.4倍,发酵温度提高到40℃,摇瓶发酵结果表明,改良菌株的代谢途径未发生变化.     

基因组改组技术改善植物乳杆菌C88的益生作用

采用基因组改组技术得到融合子菌株,并筛选益生性最优的融合子。采用基因组改组方法,结合紫外和亚硝基胍诱变,对突变菌株进行原生质体融合,经体外实验对融合子的耐酸性、耐高胆盐浓度、黏附相关性和抗氧化能力进行分析,筛选并获得益生性提高的融合子。筛选获得的两株融合子为F1-9和F1-17,均能够在低p H值和高胆盐浓度环境中生长;与原始菌株相比,融合子在表面疏水性、自聚性和与致病菌共聚性方面均有显著提高;在抗氧化方面较原始菌株也有所提高。融合子突变株连续传代培养,遗传稳定性良好。基因组改组技术能够使菌株的益生性得到明显提高,并且能够稳定遗传。     

改良Genomeshuffling技术选育埃博霉素B高产菌株

目的：用改genomeshuffling技术选育埃博霉素高产菌株。方法：本研究对genomeshu御ing技术进行了改良,具体是在递归原生质体融合过程中对原生质体进行了紫外诱变;通过UV和DES复合诱变方法获得T4株出发菌株,并用改良genomeshuffling技术选育埃博霉素B高产菌株。结果：通过三轮基因组重组成功选育到了2株遗传稳定的高产埃博霉素B重组菌株,其中一株重组菌株S0073-45的埃博霉素B产量达到T42．5mg／L。结论：该研究证明改良后的基因组重组技术不仅能有效提高埃博霉素B的产量,而且比传统的基因组重组技术更为高效。     

基因重组技术在工业微生物菌种选育中应用的研究进展

全基因组改组(Crenome shuffling)技术作为一种新型的菌种选育方法,与常见的育种方法相比,具有快速有效、简单易行和实用性强等特点,近年来不断被应用于各种工业微生物菌种的改良研究中.文中介绍了Genome shuffling技术在一系列菌种改良实例中应用现状,为该技术进一步的推广应用莫定基础.     

基因组改组选育氨肽酶和自溶度高的植物乳杆菌

利用基因组改组方法筛选出促进干酪成熟的非发酵剂乳酸菌。采用紫外线和亚硝基胍两种传统的诱变方法对植物乳杆菌进行诱变,通过检测自溶度、氨肽酶、产酸能力指标获得6株氨肽酶活性和自溶度均有所提高的突变菌株。以获得的突变菌株为出发菌株,应用灭火双亲原生质体融合后致死损伤得到的互补获得活性融合子的方法,对其进行基因组改组,经自溶度、氨肽酶、产酸能力筛选,获得1株遗传性能稳定的菌株,其氨肽酶活力为34.01个酶活单位,比出发菌株提高了3.09倍,自溶度为56.45%,比出发菌株提高了3.16倍。     

产γ-氨基丁酸贝莱斯芽孢杆菌CLYB1的鉴定及基因组改组选育研究

为了提高γ-氨基丁酸产量，本研究采用紫外诱变和基因组改组技术处理筛选鉴定的产γ-氨基丁酸菌株CLYB1，并对改组后的菌株进行溶血试验和抗生素敏感性试验。结果表明：产γ-氨基丁酸菌株CLYB1为贝莱斯芽孢杆菌Bacillus velezensis，产量为3.95 g/L。对菌株CLYB1进行紫外诱变，得到菌株CLYB1-Y,γ-氨基丁酸产量为10.26 g/L，比出发菌株CLYB1的γ-氨基丁酸产量提高160%。通过基因组改组得到菌株CLYB1-YC,γ-氨基丁酸产量为20.19 g/L，比出发菌株CLYB1提高411%。改组菌株进行菌株溶血试验和抗生素敏感性试验，CLYB1-YC没有溶血环出现，无溶血性，对青霉素、氨苄西林、头孢曲松、庆大霉素、四环素、红霉素、环丙沙星、林可霉素、氯霉素、复方新诺明10种常见抗生素均敏感，菌株安全性良好。贝莱斯芽孢杆菌CLYB1通过基因组改组可以提高γ-氨基丁酸产量，菌株具有更好的应用开发价值。     

基因组改组快速提高谷氨酸棒杆菌L-鸟氨酸产量

以谷氨酸棒杆菌ATCC13032为出发菌,应用基因组改组技术快速提高L-鸟氨酸产量。经过紫外线、亚硝基胍和甲基磺酸乙酯分别诱变处理,获得6株产量有所提高的突变株,以此构建用于基因组改组的候选菌库。考察培养基成分对原生质体再生率的影响。经过两轮的灭活原生质体递推式融合,以磺胺胍和氟化钠为双抗性筛选标记,共筛选出2株遗传性能稳定的改组菌株。其中改组菌株F2-6摇瓶发酵72h,积累L-鸟氨酸产量为2.99g/L,是出发菌株的13.6倍。结果表明基因组改组技术能够在短期内使谷氨酸棒杆菌的L-鸟氨酸产量得以提高。     

干酪乳杆菌基因组改组菌株耐酸性表征

本实验对干酪乳杆菌基因组改组菌株的耐酸性进行了研究,结果表明,冷冻( － 80℃)和临界pH 值(pH3.2)对干酪乳杆菌基因组改组菌株影响不显著,HCl 和游离乳酸对该菌株生长有一定影响,游离乳酸影响更大。在pH5.0 条件下发酵60h,干酪乳杆菌改组菌株获得的菌体干重为6.34g/L,乳酸产量为87.6g/L,比原始菌株耐酸性有显著提高。     

微生物降解植物甾醇侧链生产雄甾烯二酮的研究进展

甾体激素类药物是我国医药领域的重要门类,雄甾烯二酮是甾体激素类药物不可替代的中间体。微生物选择性降解植物甾醇侧链生成雄甾烯二酮,能替代复杂的多步化学合成法,并减轻目前由于薯蓣皂素为原料造成的资源紧缺,对合理利用我国的甾体植物资源,推动制药行业的发展有着重要的意义。本文结合相关的研究进展,综述了微生物选择性降解植物甾醇侧链菌种的选育与改良,水相体系中转化条件的优化及几种非水相转化系统的研究状况,并针对传统诱变育种耗时、费力、正突变率低、突变性状比较分散等缺陷,提出通过基因组改组技术实现突变性状优化重组,为构造甾醇微生物转化高产菌种提供广阔的发展空间。     

基因组改组技术及其在工业微生物改良中的应用

基因组改组技术是一种以原生质体融合为手段,实现多基因组重组的育种策略。文中从基因组改组基本原理出发,以相关研究成果为基础,系统介绍了基因组改组的策略与方法,并对该技术未来发展方向予以展望。     

Genome shuffling to improve fermentation properties of top-fermenting yeast by the improvement of stress tolerance

Fermentation properties of top-fermenting yeast are under the control of multiple genes difficult to manipulate directly by classical breeding, metabolic engineering, or other genetic methods with specific genes or pathways as target. Here, genome shuffling is introduced to improve fermentation performance (such as the viability of the yeast, flavor of beer, and the fermentation time) by improving wort and ethanol tolerance of top-fermenting yeast. The strategy was performed not based on polyethylene glycol (PEG)-mediated protoplast fusion but using yeast sexual and asexual reproduction by itself. The best performing strain W3-8 was selected on the selective plates after 3 rounds of genome shuffling. The fermentation time of W3-8 was not only markedly shortened, but also, most flavor compounds were distinctly improved. In particular, ethanol yield was increased by up to 67% after the 3^rd pitching compared with the control. Furthermore, W3-8 promoted desired amounts of esters and higher alcohols, in accordance with specific consumer preferences. Significant improvement in the fermentation traits of the top-fermenting yeast was achieved using genome shuffling.     

基因改组选育高产杆菌肽地衣芽孢杆菌及改组菌株差异分析

为了提高地衣芽孢杆菌肽产量,采用全基因组改组技术进行菌株选育。以地衣芽孢杆菌TJ12为原始菌株,通过紫外诱变、亚硝基胍诱变、常压室温等离子体诱变构建了TJ12菌的突变体库。在优化其原生质体制备和再生条件的基础上,以其中4 株诱变菌株（U11、U23、H1、L71）作为亲本,采用聚乙二醇介导的方法进行3 轮多亲本的全基因组改组,同时,结合双亲灭活的筛选方法,最终选出1 株杆菌肽产量提高并能稳定遗传的优良菌株F3,对其摇瓶发酵36 h,杆菌肽A产量达760 mg/L,为野生菌株TJ12的1.7 倍。与野生菌株TJ12相比,改组菌株提前进入生长稳定期,两者发酵过程pH值几乎无差异;最终改组菌生长量虽低于野生菌,但菌株单位细胞还原糖产量及杆菌肽产量都高于野生菌株;其合成基因和调控基因表达量相对野生菌株都上调,其中合成基因上调幅度较大。推测改组菌株自身有了更强大的杆菌肽耐受机制,且合成基因相关部位可能发生了改变。     

战略与机遇:迈进烟草基因组时代

中国烟草基因组计划是中国烟草行业实施的科技重大专项之一,是烟草行业对国家创新发展战略的具体落实,是顺应当前生物技术和生命科学发展趋势、加强基础前沿研究、抢占科技制高点、增强行业核心竞争力的重要举措。本文介绍了中国烟草基因组计划的提出背景、研究进展和主要成就,展望了未来烟草基因组研究的方向。中国烟草基因组计划旨在从源头上攻克烟叶生产关键技术的瓶颈制约,推动中国烟草基础与应用研究步入基因时代,实现从传统育种向精准育种的战略跨越,全面引领烟草科技发展未来。      

Reduced acetaldehyde production by genome shuffling of an industrial brewing yeast strain

High levels of acetaldehyde produced by yeast during fermentation can be of concern to product quality. A novel approach, based on genome shuffling, was applied to reduce the production of acetaldehyde by industrial brewing strain YS86. Four isolates with different impacts of acetaldehyde concentration were obtained from populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis. These yeast strains were then subjected to recursive pool‐wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a recombinant YSF2–9 strain produced less acetaldehyde than wild‐type strain YS86, by 64.5 and 66.2% in laboratory and pilot plant fermentations, respectively. The shuffled yeast strain YSF2–9 was genetically stable and may have a potential application in brewing industry for managing acetaldehyde in beer. Copyright © 2017 The Institute of Brewing & Distilling     

Genome shuffling of Zygosaccharomyces rouxii to accelerate and enhance the flavour formation of soy sauce

BACKGROUND: The purpose of this study was to achieve rapid improvement of the flavour of soy sauce by increasing the salt stress resistance of Zygosaccharomyces rouxii. Here, we describe genome shuffling to improve the salt tolerance of Z. rouxii while simultaneously enhancing and accelerating flavour formation of soy sauce. RESULTS: A mutant, S3‐2, with a stronger resistance to salt, was selected after three rounds of genome shuffling. S3‐2 not only grew well in peptone/yeast extract/dextrose medium containing a high salt content with wide range of pH, but also exhibited stronger stress resistance to potassium chloride and lithium chloride. In high‐salt liquid fermentation, S3‐2 obviously accelerated flavour formation of soy sauce, thus decreasing the total time required for development of the aroma. In addition, S3‐2 gave high amino acid nitrogen and good flavour. In particular, the ethyl acetate content was 2.38 times that in the control. S3‐2 distinctly improved the formation of 4‐hydroxy‐2 (or 5) ‐ethyl‐5 (or 2) ‐methyl‐3 (2H) ‐furanone by up to 75%. Another important flavour component, 4‐ethylguaiacol, was also detected. CONCLUSIONS: Genome shuffling was successfully used to achieve significant improvements in flavour formation. The selected strain improved the main flavour components and amino acid nitrogen, thereby enhancing the quality of soy sauce. Copyright © 2009 Society of Chemical Industry     

植物乳杆菌细菌素高产菌株的诱变选育及其对肉丸的防腐保鲜作用

为了提高细菌素产量并研究细菌素对肉制品的保鲜效果,本研究以植物乳杆菌JL-A65为出发菌株,对其进行常温等离子(ARTP)诱变、甲基硝基亚硝基胍(MNNG)诱变与基因组改组,并将细菌素与双乙酸钠复配后添加到肉丸中。结果表明,ARTP诱变最佳处理时间为40 s,经筛选得到两株突变株A7-10和A8-110,细菌素产量提高率分别为45.1%和48.9%。MNNG诱变的最佳处理浓度为1.5 mg/mL,经筛选得到两株突变株M2-58和M7-111,细菌素产量提高率分别为46.6%和31.3%。对植物乳杆菌进行基因组改组最终得到一株融合菌株F4-23,细菌素产量为413 mg/L,较原始菌株提高了103.48%。细菌素与双乙酸钠之间存在协同作用,可以使肉制品保质期较对照延长5 d。结论:利用理化诱变结合基因组改组可以获得细菌素高产菌株,细菌素与双乙酸钠复配后可以使肉丸保质期较对照延长5 d。细菌素对肉丸具有较好的防腐保鲜效果。