碳源对manA基因突变大肠杆菌代谢的影响

ManA基因编码的甘露糖-6-磷酸异构酶在大肠杆菌中催化D-甘露糖和D-果糖的异构化,促进大肠杆菌对碳源的代谢吸收。本文通过研究manA基因突变大肠杆菌对碳源的利用和编码糖代谢基因情况,探讨甘露糖-6-磷酸异构酶对大肠杆菌糖代谢的影响。采用Ⅱ型内含子逆转录突变方法构建manA基因突变大肠杆菌,分析manA基因突变大肠杆菌对不同碳源的利用情况和manA基因突变对大肠杆菌糖代谢相关基因表达的影响,结果显示,大肠杆菌BL21(DE3)ΔmanA以甘露糖、果糖为碳源时,菌株生长受到显著抑制;以淀粉为碳源时,BL21(DE3)ΔmanA菌株的生长显著优于野生型大肠杆菌;以葡萄糖为碳源时,manA基因突变对大肠杆菌的生长无显著影响。通过基因表达分析,发现大肠杆菌BL21(DE3)ΔmanA中甘露糖代谢相关基因的表达显著性降低;果糖代谢途径中6-磷酸果糖激酶Ⅰ亚基的编码基因(pfkA)显著下调表达;水解淀粉的α-淀粉酶编码基因(malS)显著性上调表达。ManA基因突变影响大肠杆菌甘露糖、果糖和淀粉代谢途径中相关基因的表达,从而影响大肠杆菌对碳源的利用。     

Isolation and characterization of an alkaliphilic bacterium utilizing pyrene as a carbon source

Alkaliphilic Mycobacterium sp. strain MHP-1, which can grow on pyrene as a sole carbon and energy source, was isolated from a soil sample. At the optimum pH for growth (pH 9), about 50% of pyrene (final concentration at 0.1% [w/v]) was degraded during 7 d of incubation, and 4,5-phenanthrenedioic acid, 4-phenanthroic acid and phthalic acid were identified as metabolic intermediates. Strain MHP-1 was found to possess aromatic-ring dioxygenase genes, which are highly homologous to the known nidAB genes from pyrene-degrading mycobacteria.     

苍耳内生真菌的分离鉴定及抗菌活性研究

从植物苍耳中分离内生真菌,选出8株进行抑菌活性测定,有6株对番茄早疫病菌抑制率大于50%。菌株SC008对5种植物病原菌的抑制率均大于50%。对内生真菌SC008进行形态学观察及ITS序列鉴定,初步判定菌株SC008归属于黑孢霉属(Nigrospora Zim.)。碳氮源实验表明,SC008在以乳糖和蔗糖为碳源及以酵母提取物和牛肉膏为氮源的培养基上生长良好。     

乳酸克鲁维酵母常温常压等离子体诱变选育及其突变株整细胞催化特性

采用常温常压等离子体（ARTP）诱变方法,以耐20 g/L的苯乙酮毒性和酮基还原酶酶活作为筛选标记,对乳酸克鲁维酵母（Kluyveromyces lactis）进行选育,得到酮基还原酶酶活2.72 U/mL的突变菌株KL5,并研究了该菌株在不同碳源、氮源中的生长特性及对底物苯乙酮转化能力。结果表明,蔗糖、酵母浸粉分别是突变株的最佳碳源和氮源;在一次添加苯乙酮20 g/L底物,蔗糖22.0 g/L,酵母浸粉10.0 g/L,磷酸二氢钾3.0 g/L,七水合硫酸镁1 g/L的催化条件下,28 ℃、200 r/min转化48 h,诱变菌株KL5对苯乙酮转化率可达到91.8%,比出发菌株提高2.5倍。该菌株具有广阔的生物催化应用前景。     

生物合成天然2-苯乙醇细菌的筛选及合成途径分析

从上海国家森林公园土壤样本中筛选到1?株菌株,经过生理生化鉴定和16S rDNA序列分析,该菌属肠杆菌属并命名为Enterobacter sp. MF024。Enterobacter sp. MF024全基因组测序结果表明该菌株包含从头合成途径和艾氏途径合成2-苯乙醇所有关键酶的编码基因。分别以葡萄糖、L-苯丙氨酸为底物进行生物转化实验,并以苯乙醛、苯丙酮酸等合成途径中间产物为底物进行验证,气相色谱-质谱法、红外光谱分析结果进一步说明Enterobacter sp. MF024具备两种2-苯乙醇合成途径,且该菌利用从头合成途径和艾氏途径产2-苯乙醇产量分别达到0.56、1.15 g/L。该菌株以葡萄糖为碳源生物合成2-苯乙醇极具应用前景。     

西索米星生产菌株的诱变选育及工艺优化

利用原生质体常压室温等离子体（ARTP）诱变方法选育西索米星高产菌株,经诱变筛选获得一株高产菌株I4-10,其西索米星的生物效价达到1 389 U/mL,较原始菌株提高了35.4%。通过对高产菌株I4-10进行碳源、氮源优化,确定可溶性淀粉和牛肉粉是突变菌株I4-10的最适碳源和氮源。进一步采用响应面实验设计方法优化发酵培养基,结果表明黄豆饼粉和DL-蛋氨酸的添加量为显著影响因素,经优化其最适添加质量浓度分别为32.78 g/L和1.75 g/L。在此最适工艺下,西索米星的生物效价提高至1 849 U/mL,较原始菌株提高了80%。最后对原始菌及高产突变株中西索米星代谢途径及菌株生长相关的关键酶进行转录水平比较分析,初步解析了突变菌株I4-10高产西索米星的机制。     

The putative monocarboxylate permeases of the yeast Saccharomyces cerevisiae do not transport monocarboxylic acids across the plasma membrane

We have characterized the monocarboxylate permease family of Saccharomyces cerevisiae comprising five proteins. We could not find any evidence that the monocarboxylate transporter-homologous (Mch) proteins of S. cerevisiae are involved in the uptake or secretion of monocarboxylates such as lactate, pyruvate or acetate across the plasma membrane. A yeast mutant strain deleted for all five MCH genes exhibited no growth defects on monocarboxylic acids as the sole carbon and energy sources. Moreover, the uptake and secretion rates of monocarboxylic acids were indistinguishable from the wild-type strain. Additional deletion of the JEN1 lactate transporter gene completely blocked uptake of lactate and pyruvate. However, uptake of acetate was not even affected after the additional deletion of the gene YHL008c, which had been proposed to code for an acetate transporter. The mch1-5 mutant strain showed strongly reduced biomass yields in aerobic glucose-limited chemostat cultures, pointing to the involvement of Mch transporters in mitochondrial metabolism. Indeed, intracellular localization studies indicated that at least some of the Mch proteins reside in intracellular membranes. However, pyruvate uptake into isolated mitochondria was not affected in the mch1-5 mutant strain. It is concluded that the yeast monocarboxylate transporter-homologous proteins perform other functions than do their mammalian counterparts.     

New insights into galactose metabolism by Schizosaccharomyces pombe: isolation and characterization of a galactose-assimilating mutant

The fission yeast Schizosaccharomyces pombe cannot use galactose as a carbon or energy source, and little is known about galactose metabolism in this species. Here we report isolation of a galactose-assimilating mutant that grows on a medium containing galactose as a sole carbon source through use of a proofreading-deficient DNA polymerase δ variant encoded by cdc6-1. Based on comparative analysis of gene expression profiles in the wild-type and the mutant (FG2-8), we found that SPBPB2B2.10c (gal7+), SPBPB2B2.12c (gal10+) and SPBPB2B2.13 (gal1+), homologous to Saccharomyces cerevisiae GAL7, GAL10 and GAL1, respectively, and SPBPB2B2.08, SPBPB2B2.09c, and SPBPB2B2.11 that localize close to the gal genes, were highly expressed and dramatically induced by addition of galactose. The gal7Δ strain, carrying an integrated ura4+ marker at the gal7+ locus, grew on 5-fluoroorotic acid (5-FOA)-containing medium. In contrast, the FG2-8 gal7Δ strain could not grow on 5-FOA medium. In addition, expression of gal7+, SPBPB2B2.13, gal10+ and gal1+ genes increased in the wild-type strain when carried on a vector, and these transformants grew on galactose medium. We suggest that gal7+, gal10+, and gal1+ are localized close to a chromosomal terminal repressed by gene silencing in S. pombe. In contrast, gene silencing was defective in the FG2-8 strain making galactose assimilation possible.     

富含核糖核酸酿酒酵母的选育及其高密度发酵工艺

该研究首先以酿酒酵母（Saccharomyces cerevisiae）J-5为出发菌株进行诱变选育,筛选出一株核糖核酸（RNA）含量优于菌株J-5的诱变菌株J-5-9,其RNA含量在摇瓶中达到了13.12%,比出发菌株提高了10.62%。选取糖蜜为碳源,应用正交试验优化菌株J-5-9发酵培养基组成为：糖蜜3%,酵母浸粉2%,磷酸二氢钾0.01%,谷氨酸钠0.2%,硫酸亚铁0.1%。最后利用优化发酵培养基和碳源、氮源和磷源的流加补料工艺,在10 L发酵罐中培养诱变菌株J-5-9,RNA含量达到8.11%,比优化前提高了18.22%,同时细胞生物量达到188 g/L（湿质量）,实现了酿酒酵母高产RNA的高密度发酵。这说明诱变菌株J-5-9是一株很有潜力的工业化生产菌株。     

基于代谢组学分析Slr0643蛋白在调控集胞藻PCC6803葡萄糖混养适应中的重要性

本研究文采用集胞藻PCC6803野生型藻株和Slr0643敲除突变体(Δslr0643)藻株,通过生理实验和基于气相色谱-质谱联用(GC-MS)技术的植物代谢组学分析,对Slr0643蛋白调控集胞藻PCC6803葡萄糖混养适应的机制进行探讨.在2.5 mM葡萄糖混养条件下,野生型藻株的生长速率较光自养条件明显加快,而Δ...     

重离子诱变对木聚糖酶产生菌产酶的影响

以1株由青藏高原牦牛粪中分离出的链霉菌为出发菌株,该菌株在发酵培养基中能产生胞外木聚糖酶(3 227.346 U/mL)。以此菌株为出发菌株,对其进行重离子辐照诱变处理,从大量突变株中筛选出木聚糖酶高产菌株SZ10-7,其酶活力达到5 338.42 U/mL,与出发菌株相比较,突变株SZ10-7的酶活力提高了1.65倍。对突变株SZ10-7的发酵条件进行了优化研究,结果表明,该菌株的木聚糖酶活力得到进一步提高,达到5 850.20U/mL,其最适发酵条件为:培养基(g/100 mL)为玉米芯∶麸皮(体积比1∶1)5,酵母膏0.8,K2 HPO4.3H2 O 0.1,MgSO4.7H20 0.5,NaCl 0.3,pH 7.0,培养温度25℃振荡培养时间96 h,实验结果表明,重离子辐照诱变技术是一种有效的微生物诱变育种新技术。     

碳、氮源对红曲黄色素色价和色调值的影响

本文对红曲黄色素发酵条件进行了探究,红曲菌sjs-6发酵所得红曲黄色素,仅在420 nm处有单吸收峰。不同碳源种类对色素产量有较大影响,对产色素种类影响不大;氮源对黄色素种类和产量均有较大影响,有机氮源可促使橙、红色素的产生,而无机氮源更有利于单产黄色素。发酵进程曲线显示,72¹20 h,菌体量处于稳定水平,是红曲菌sjs-6产黄色素的关键时期,120 h之后随着碳源耗尽,菌体自溶,产色素能力下降,色价保持稳定。通过正交实验可得,当以6%（w/v）玉米淀粉为碳源,0.4%（w/v）硫酸铵为氮源时,培养基初始p H为5.0,发酵温度为30℃时,发酵144 h,黄色素色价可达378 U/m L。产量是国内现有报道水平（110 U/m L）的3倍多。由于菌株非突变株,具有较好的稳定性。由此,为实现红曲黄色素的工业化生产奠定了基础。      

Biodegradation of long-chain n-paraffins from waste oil of car engine by Acinetobacter sp

Microorganisms that degrade long-chain n-paraffins from used car engine oil were isolated from soil. For the screening, a fraction of n-paraffin prepared from car engine oil was applied as the sole carbon source. The strain was identified as Acinetobacter sp. The ability of the strain to assimilate long-chain n-paraffins was assessed and characterized. The strain mineralized long-chain n-paraffins (0.1% w/v) in the minimal medium after cultivation for 96 h and also reduced the weight of the waste oil added (1% w/v) by 20% after 72 h without an extracellular biosurfactant. When n-hexadecane was fed as substrate, 1-hexadecanol and 1-hexadecanoic acid were detected as the intermediates by gas chromatography/mass spectrometry. This indicates that the long-chain n-paraffins were metabolized via the terminal oxidation pathway of n-alkane.     

产聚半乳糖醛酸酶的黑曲霉诱变育种

利用透明圈法对黑曲霉SH312菌株进行初筛,得到黑曲霉SH312-26菌株。对其进行紫外线诱变,筛选出SH312-26-19菌株,该菌株聚半乳糖醛酸酶酶活力为113.68kU/mL,较出发菌株SH-312-26提高了1.44倍;果胶酯酶酶活力为14.34kU/mL,较出发菌株提高了0.14倍,其幅度远小于聚半乳糖醛酸酶酶活的提高幅度。突变株SH312-26-19经斜面传代培养了5代,产酶遗传特性稳定。     

Isolation of a mutant of Kluyveromyces sp. Y-85 resistant to catabolite repression

The production of inulinase in Kluyveromyces sp. Y-85 is repressed by the carbon catabolites. To screen for derepression mutants, the strain of Kluyveromyces sp. Y-85 was treated with the mutagenic agent, ethyl methane sulfonate (EMS). Initially, mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated. One such mutant, Kluyveromyces sp. Y-85 K6, was found to exhibit stable, 2-fold higher expression of inulinase than the wild-type in glucose-containing medium, indicating that it is a promising strain for industrial production.     

传统锦州虾酱中产蛋白酶嗜盐菌的分离与鉴定

目的:从传统锦州虾酱中分离产蛋白酶嗜盐菌,并对其进行鉴定。方法:应用1.0%脱脂乳Gibbons培养基分离产蛋白酶嗜盐菌,经形态学、生理生化和分子生物学对其鉴定。采用SDS-PAGE分析虾酱中可溶性蛋白的变化。结果:获得16株产蛋白酶嗜盐菌,菌株CW0-1、CW0-2、CW0-3和CW0-4是弧菌属,菌株CW1-1、CW2-1和CW2-2是尼泊尔葡萄球菌,菌株CW2-3、CW5-1、CW5-2、CW5-3是马胃葡萄球菌,菌株CW3-1、CW3-2、CW4-1、CW4-2是枝芽孢杆菌,菌株CW4-3是独岛枝芽孢杆菌。弧菌属主要存在于原料虾中,嗜盐性葡萄球菌出现在发酵2个月和5个月,枝芽孢杆菌出现在发酵3⁴个月。葡萄球菌和枝芽孢杆菌呈现交替演变,是虾酱发酵前期的优势产蛋白酶嗜盐菌。虾酱中的总可溶性蛋白随着发酵时间的延长逐渐下降。结论:葡萄球菌和枝芽孢杆菌是发酵前期虾酱中的优势产蛋白酶嗜盐菌,也是降解蛋白的主体微生物类群。      

高产Monacolin K红曲霉菌种的诱变选育及液态发酵工艺优化

从29株红曲霉菌（Monascus sp.）中筛选得到的M2为出发菌株,进行UV照射、LiCl诱变以及硫酸二乙酯处理与LiCl复合诱变三次诱变,筛选获得Monacolin K的产量显著提高的菌株MS-12。对液态发酵培养基、发酵温度、接种量以及摇床转速等因素进行优化。在最优条件下MS-12液态发酵Monacolin K的产量显著提高,达到264.7μg/mL,桔霉素含量仅为0.026ng/mL。      

利用木糖高产谷胱甘肽酵母菌株的诱变选育及培养条件优化

以CT1463(热带假丝酵母)为出发菌株,经紫外诱变结合镉盐平板筛选,获得-株高产谷胱甘肽的突变株CV26,其谷胱甘肽产量和舍量分别为45.4mg/L和7.05mg/g,较出发菌株提高了41%和52%.对CV26发酵条件进行了优化,在优化发酵条件下谷胱甘肽产量和含量分别为128.70 mg/L和14.31 mg/g,分...     

Formaldehyde-limited cultivation of a newly isolated methylotrophic bacterium, Methylobacterium sp. MF1: enzymatic analysis related to C1 metabolism

Formaldehyde is a highly toxic compound to most living organisms. We have isolated a bacterial strain that is able to efficiently degrade formaldehyde and use it as a sole carbon source. The isolated strain was identified as Methylobacterium sp. MF1, which could grow on formaldehyde and methanol. Methylobacterium sp. MF1 was grown in batch culture using 1.2 g/l formaldehyde as a sole carbon source, which was all consumed within 200 h. In order to decompose formaldehyde more efficiently, formaldehyde-limited chemostat cultivation of Methylobacterium sp. MF1 was investigated. Formaldehyde was consumed at 1.7 g/l/d when the dilution rate was 0.012 h(-1). Under these conditions, the cell turbidity (OD610) reached 2.0. Furthermore, when the initial turbidity was adjusted to 3.0 using methanol-grown cells, continuous cultivation could be started at an initial dilution rate of 0.008 h(-1). Using these conditions, consumption of formaldehyde could be continued for at least 600 h. The enzyme activities of cells growing as a chemostat culture, using methanol or formaldehyde as a sole carbon source, were compared to that of C1 metabolism. No difference was detected in the enzyme activities for the oxidation and assimilation of C1 compounds between the two cell-free extracts. Furthermore, methanol dehydrogenase activity was detected at the same level when formaldehyde was used as a sole carbon source. These results suggest that the resistance to the toxic effects of formaldehyde exhibited by Methylobacterium sp. MF1 is related to factors other than C1 metabolism.