阿特拉津降解菌Arthrobacter sp. AG1降解基因研究

菌株Arthrobacter sp. AG1能以4000 mg/L的阿特拉津（AT）为唯一碳源、氮源和能源生长。通过设计特异引物从AG1中扩增出阿特拉津氯水解酶基因trzN的全序列,该基因与已报道的trzN基因序列相似性为99%。AG1菌株中含有两个大于100kb的质粒,Southern杂交结果显示trzN和atzB基因均位于其中较大的一个质粒pAG1上。将AG1菌株在LB液体培养基中转接三代后,发现34%的细菌细胞丢失了降解活性,但却未发现丢失质粒,PCR扩增结果表明突变子丢失了trzN基因,但atzB和atzC基因未丢失,说明降解活性的缺失是trzN基因片段从质粒上丢失的结果,表明trzN基因在环境中存在水平转移现象,暗示菌株AG1中的阿特拉津降解基因是基因的水平转移重组的结果。     

阿特拉津降解菌Arthrobacter sp. AG1降解基因研究

菌株Arthrobacter sp. AG1能以4000 mg/L的阿特拉津（AT）为唯一碳源、氮源和能源生长。通过设计特异引物从AG1中扩增出阿特拉津氯水解酶基因trzN的全序列,该基因与已报道的trzN基因序列相似性为99%。AG1菌株中含有两个大于100kb的质粒,Southern杂交结果显示trzN和atzB基因均位于其中较大的一个质粒pAG1上。将AG1菌株在LB液体培养基中转接三代后,发现34%的细菌细胞丢失了降解活性,但却未发现丢失质粒,PCR扩增结果表明突变子丢失了trzN基因,但atzB和atzC基因未丢失,说明降解活性的缺失是trzN基因片段从质粒上丢失的结果,表明trzN基因在环境中存在水平转移现象,暗示菌株AG1中的阿特拉津降解基因是基因的水平转移重组的结果。     

mhbR和mhbT基因移码突变对3-羟基苯甲酸代谢的影响*

Klebsiellapneumoniae M5a1菌株能以 3 羟基苯甲酸作为唯一碳源和能源生长 ,编码分解代谢 3 羟基苯甲酸的基因被克隆至一 8kb的DNA片段上 (pBSI质粒 ) ,含有该质粒的大肠杆菌获得了以 3 羟基苯甲酸作为唯一碳源和能源生长的能力。经测序并与GenBank中的已知基因产物进行氨基酸同源性比较后 ,发现其中的mhbR与转录调节基因和mhbT与底物转运基因具有较高同源性 ,推断可能具有相应的功能。含有mhbR的移码突变子或     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

一株有色金属矿山1-亚硝基-2-萘酚降解菌的筛选、鉴定和降解特性

1-亚硝基-2-萘酚是一类有色选矿药剂代表性的新型浮选药剂,在采选冶行业为了提高低品位矿物资源的利用率,被大量投入使用,该药剂的高稳定性进一步增大了矿山环境中重金属与有机选冶药剂复合污染的治理难度。微生物修复作为稠环芳烃(polycyclic aromatic hydrocarbons,PAHs)类污染物重要技术手段之一,具有安全、经济、高效和无二次污染等特点。【目的】本研究从我国广西河池市周边的典型有色金属尾矿环境中分离出1株高效降解1-亚硝基-2-萘酚的菌株,并分析其降解特性及其潜在的代谢途径,从而探究矿山复合污染生态系统中稠环芳烃类污染物的微生物修复技术的应用前景。【方法】从有色金属尾矿样本中筛选到以1-亚硝基-2-萘酚为唯一碳源的菌株,经16S rRNA基因序列鉴定,结合气相色谱-质谱联合(gas chromatography-mass spectrometer,GC-MS)检测分析菌株对1-亚硝基-2-萘酚的降解特性及中间代谢产物,并推测可能的代谢途径。【结果】筛选获得1株高效降解1-亚硝基-2-萘酚的Pseudomonas putida CUGB-JL11菌株,经鉴定为革兰氏阴性杆的恶臭假单胞菌。该菌株最适温度为30 ℃左右,最适pH值范围为6-8条件下,菌株5 d内对40 mg/L 1-亚硝基-2-萘酚的降解率高达81%。降解中间代谢产物主要为苯环类物质：苯甲羟肟酸甲酯和苯丙胺,但其母体及大部分中间产物都降解为小分子物质或者被完全降解。【结论】恶臭假单胞菌P. putida CUGB-JL11具有良好的1-亚硝基-2-萘酚降解能力和较强的环境适应性,有进一步被开发为微生物菌剂以用于稠环芳烃类污染修复的巨大潜力,为有色金属矿山生态系统中重金属和有机选冶药剂复合污染的微生物修复研究提供了理论依据和可利用的微生物资源。     

一株多菌灵降解细菌的分离、鉴定及系统发育分析

从被多菌灵污染的湖南红壤土中分离到一株能以多菌灵为唯一碳源和能源生长的菌株1_1,该菌在含酵母膏多菌灵(500mg/L)的无机盐培养基中与无酵母膏的多菌灵(500mg/L)无机盐培养基中,24d对多菌灵的降解率分别为95.56％和19.6％。根据表型特征、G+C含量及16S rDNA序列分析将菌株1-1鉴定为β-Proteobacteria中的Ralstonia sp.（罗尔斯通氏菌）。     

两株真菌降解菜籽饼中植酸的研究

利用选择性培养基从土壤中分离到两株能降解植酸的丝状真菌。这些菌株能利用肌醇作为唯一的碳源和能源而生长。在液态发酵中植酸的降解率分别为74．4％和95．0％;在固态发酵中植酸的降解率为40％左右。某些金属离子对菌株的降解率的提高具有一定的促进作用。对温度、pH和水分等影响因子也进行了初步的探讨。经初步鉴定,这两株菌株中有一株为拟青霉（Paecilomycessp）,另一株为青霉（Penicilliumsp．）,它们均不产黄曲霉素素。     

高效芘降解菌N12的分离鉴定与降解特性

以芘为目标降解物,利用选择性富集培养方法,从沈抚灌区污染土壤中分离到一株高效芘降解菌N12,经生理生化试验和16S rDNA测序分析,该菌被鉴定为分枝杆菌属（Mycobacterium sp.）.菌株N12能以菲、苊、芴和芘为唯一碳源和能源生长,不能以蒽、萘和苯并芘为唯一碳源和能源生长.在菲和芘共同存在的情况下菌株N12可降解苯并芘,9 d内对苯并芘降解率可达79.0%.摇瓶降解试验表明,菌株N12可在7 d内将100 mg·L^-1的芘降解94.4%,14 d内将其完全降解;可将600 mg·L^-1的芘在7 d内降解56.1%,14 d内降解95.5%.添加葡萄糖可促进N12对芘的降解.菌株N12是一株优良的多环芳烃降解菌,可作为多环芳烃污染土壤生物修复的菌种资源.     

混合菌株降解褐藻胶的筛选、鉴定及产酶条件优化

为获得仿刺参（Apostichopus japonicus）肠道菌群中可有效降解褐藻胶的混合菌株,以海藻酸钠为唯一碳源配制培养基,以透明圈法进行初筛,DNS法和紫外法复筛,从已驯化仿刺参肠道中筛选得到4株高酶活力褐藻胶降解菌株S1、S2、S10和S11,经16S rDNA序列分析、电镜观察,确定菌株S2与S11分别为微杆菌属（Microbacterium sp.）和微小杆菌属（Exiguobacterium sp.）。对该4株菌株分别进行混合培养,获得菌株S2与S11最佳配比组合,并通过单因素试验及响应面优化试验对影响混合菌株产酶条件的发酵初始pH值、NaCl质量浓度、装液量和发酵温度4个因素进行优化。得到混合菌株最佳产酶条件为pH 8,NaCl质量浓度为40 g/L,装液量80 mL,温度28 ℃。在最佳发酵条件下,混合菌株酶活力可达94.78 U/mL,相比于优化前提高了43.9%,优化后混合菌株的酶活力显著提高。     

一株霍氏肠杆菌对四环素的降解作用及其降解产物的毒性评估

【目的】抗生素作为新兴污染物,已经引起社会的极大关注。针对四环素有效降解菌株缺乏这一现状,本研究旨在筛选和鉴定具有降解四环素功能的菌株,分析其降解特性和降解作用类型、初步探讨其降解活性物质定位并评估其降解产物的生理毒性。【方法】以四环素为唯一碳源,从受四环素污染的猪场污泥中筛选四环素降解菌株;结合菌落形态学特征、生理生化特征、扫描电镜观察和16S rRNA基因测序鉴定菌株,通过不同外源碳、pH及去除动力学阐明菌株对四环素的降解特性,提取菌株不同成分探讨其去除四环素的作用类型,并进一步从细胞内液和细胞外液开展生物降解的活性物质定位,最后评估降解产物的生理毒性。【结果】筛选鉴定得到一株霍氏肠杆菌(Enterobacter hormaechei) MEH2305,pH为7.0和添加10 g/L外源碳胰蛋白胨是其发挥降解作用的最适条件。MEH2305通过非生物降解和生物降解的共同作用,在培养第7天对四环素总去除率达到68% (对土霉素和盐酸强力霉素去除率分别为53%和56%),其分泌的细胞内液和细胞外液对四环素的去除率分别为40.77%和31.18%。同时,与未经MEH2305处理的四环素对照组比较,MEH2305降解四环素的产物对革兰氏阴性大肠杆菌(Escherichia coli) K88和革兰氏阳性枯草芽孢杆菌(Bacillus subtilis) 168的生理毒性作用显著降低。【结论】MEH2305可以作为一株潜在的有效且安全的四环素降解菌株,应用于抗生素的环境治理领域。     

Effect of light on carotenoid and lipid production in the oleaginous yeast Rhodosporidium toruloides

ABSTRACT

The oleaginous yeast Rhodosporodium toruloides is receiving widespread attention as an alternative energy source for biofuels due to its unicellular nature, high growth rate and because it can be fermented on a large-scale. In this study, R. toruloides was cultured under both light and dark conditions in order to understand the light response involved in lipid and carotenoid biosynthesis. Our results from phenotype and gene expression analysis showed that R. toruloides responded to light by producing darker pigmentation with an associated increase in carotenoid production. Whilst there was no observable difference in lipid production, slight changes in the fatty acid composition were recorded. Furthermore, a two-step response was found in three genes (GGPSI, CAR1, and CAR2) under light conditions and the expression of the gene encoding the photoreceptor CRY1 was similarly affected.     

Co‐utilization of acidified glycerol pretreated‐sugarcane bagasse for microbial oil production by a novel Rhodosporidium strain

Acidified glycerol pretreatment is very effective to deconstruct lignocellulosics for producing glucose. Co‐utilization of pretreated biomass and residual glycerol to bioproducts could reduce the costs associated with biomass wash and solvent recovery. In this study, a novel strain Rhodosporidium toruloides RP 15, isolated from sugarcane bagasse, was selected and tested for coconversion of pretreated biomass and residual glycerol to microbial oils. In the screening trails, Rh. toruloides RP 15 demonstrated the highest oil production capacity on glucose, xylose, and glycerol among the 10 strains. At the optimal C:N molar ratio of 140:1, this strain accumulated 56.7, 38.3, and 54.7% microbial oils based on dry cell biomass with 30 g/L glucose, xylose, and glycerol, respectively. Furthermore, sugarcane bagasse medium containing 32.6 g/L glucose from glycerol‐pretreated bagasse and 23.4 g/L glycerol from pretreatment hydrolysate were used to produce microbial oils by Rh. toruloides RP 15. Under the preliminary conditions without pH control, this strain produced 7.7 g/L oil with an oil content of 59.8%, which was comparable or better than those achieved with a synthetic medium. In addition, this strain also produced 3.5 mg/L carotenoid as a by‐product. It is expected that microbial oil production can be significantly improved through process optimization.     

Behavior of R388rep(Ts)::Tn5, a thermosensitive Tn5 vector,and its derivatives inErwinia carotovora and some species of rhizobiaceae

R388rep(Ts)::Tn5 a thermosensitive, Tn5 vector (pCHR81) developed by Sasakawa and Yoshikawa [12], was found to be compatible with two strains ofErwinia carotovora and a strain ofRhizobium meliloti. pCHR81 was introduced into these organisms at lower temperatures and rendered suicidal at higher temperatures, giving rise to Tn5 transposed. To the transconjugants ofE. carotovora, which were cured of the R388 moiety and carrying Tn5 transposed, another Tn5 vector R388rep(Ts)::Tn5-Tcl (pCHR82) was re-introduced; this is a derivative of R388rep(Ts)::Tn5 with a tetracycline resistance marked instead of the original antibiotic resistances of Tn5. Gua⁺ gene ofE. carotovora was transferred by the cultures carrying only R388rep(Ts)::Tn5 or by those carrying R388rep(Ts)::Tn5-Tc and transposed Tn5. Though one strain of each ofAgrobacterium tumefaciens andA. radiobacter showed restriction to R388rep(Ts)::Tn5 plasmid maintenance, derivatives devoid of R388 and carrying Tn5 transposed were obtained. Streptomycin resistance gene on Tn5 was expressed in the cultures of all four species.     

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.     

<Emphasis Type="Italic">Erwinia carotovora</Emphasis> elicitors and <Emphasis Type="Italic">Botrytis cinerea</Emphasis> activate defense responses in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Background  

Vascular plants respond to pathogens by activating a diverse array of defense mechanisms. Studies with these plants have provided a wealth of information on pathogen recognition, signal transduction and the activation of defense responses. However, very little is known about the infection and defense responses of the bryophyte, Physcomitrella patens, to well-studied phytopathogens. The purpose of this study was to determine: i) whether two representative broad host range pathogens, Erwinia carotovora ssp. carotovora (E.c. carotovora) and Botrytis cinerea (B. cinerea), could infect Physcomitrella, and ii) whether B. cinerea, elicitors of a harpin (HrpN) producing E.c. carotovora strain (SCC1) or a HrpN-negative strain (SCC3193), could cause disease symptoms and induce defense responses in Physcomitrella.     

Genome-scale model of Rhodotorula toruloides metabolism

The basidiomycete red yeast Rhodotorula toruloides is a promising platform organism for production of biooils. We present rhto-GEM, the first genome-scale model (GEM) of R. toruloides metabolism, that was largely reconstructed using RAVEN toolbox. The model includes 852 genes, 2,731 reactions, and 2,277 metabolites, while lipid metabolism is described using the SLIMEr formalism allowing direct integration of lipid class and acyl chain experimental distribution data. The simulation results confirmed that the R. toruloides model provides valid growth predictions on glucose, xylose, and glycerol, while prediction of genetic engineering targets to increase production of linolenic acid, triacylglycerols, and carotenoids identified genes—some of which have previously been engineered to successfully increase production. This renders rtho-GEM valuable for future studies to improve the production of other oleochemicals of industrial relevance including value-added fatty acids and carotenoids, in addition to facilitate system-wide omics-data analysis in R. toruloides. Expanding the portfolio of GEMs for lipid-accumulating fungi contributes to both understanding of metabolic mechanisms of the oleaginous phenotype but also uncover particularities of the lipid production machinery in R. toruloides.     

Compositional profiles of Rhodosporidium toruloides cells under nutrient limitation

Lipid production by the red yeast Rhodosporidium toruloides was explored under nutrient limitation. To determine the compositional profiles of R. toruloides cells, samples were prepared using a continuous cultivation process under nutrient limitation and analyzed via several methods, including Fourier transform infrared spectroscopy and elemental analysis. Under nitrogen limitation, as the dilution rate increased, the cellular lipid content decreased but the carbohydrate and protein contents increased. Under carbon limitation, the cellular lipid, protein, and carbohydrate contents remained relatively constant at the different dilution rates. Moreover, the cellular elemental composition was essentially identical under nitrogen and carbon limitation at a high dilution rate of 0.20 h⁻¹. We also analyzed the consumed carbon to nitrogen (C/N) under different nutrition conditions. The results indicated that the consumed C/N had a major influence on cell metabolism and product formation, which contributed to our understanding of the physiological characteristics of R. toruloides.

     

Combined mutagenesis of <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> for improved production of carotenoids and lipids

Objectives

To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains.

Results

Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW.

Conclusions

A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.