维生素B12的光解研究及发酵液中维生素B12检测新方法的建立

采用HPLC检测法研究5,脱氧腺苷钴胺素和甲基基钴胺素的水溶液在不同光源及不同照度下的光解情况,结果表明随着光能量的增加光解速度加快。利用脱氧腺苷钴胺素的光解性质,探索了一种检测发酵液中维生素B12含量的新方法。将含有钴胺素的细胞破碎液完全光解后,测定光解后的产物羟基钴胺素,以此来确定维生素B12产量。此方法简便快速、重复性好,可应用于发酵生产维生素B12的各个环节。     

增大罐压提高脱氮假单胞菌发酵生产维生素B12的产量

【目的】提高发酵罐的罐压,增加维生素B12的产率。【方法】利用常规代谢通量分析(MFA)方法,对脱氮假单胞菌生产维生素B12的发酵过程进行研究。【结果】发现随着VB12合成速率的加快,磷酸烯醇式丙酮酸(PEP)羧化生成草酰乙酸(OAA)的通量明显加大,以满足维生素B12合成对前体的需求。根据该分析结果,对发酵工艺进行了改进,即在脱氮假单胞菌进入合成维生素B12阶段时,提高发酵罐的罐压,增加发酵液中二氧化碳的溶解度,从而强化了羧化回补途径。维生素B12的产率明显增加,发酵160 h的产物浓度为176 mg/L,比对照批次终浓度147 mg/L高出了19.7%。【结论】通过增大罐压提高了脱氮假单胞菌进入合成维生素B12的产量。     

脱氮假单胞菌生产维生素B12过程中粪卟啉Ⅲ的测定及发酵调控

脱氮假单胞菌发酵生产维生素B₁₂过程中,副产物粪卟啉Ⅲ的积累对产物的代谢合成和分离提取有很大的影响。建立了发酵液中粪卟啉Ⅲ的HPLC快速测定方法,发酵液处理后直接进样测定,检测线性范围为12~275 μg/ml,重复性好。研究了不同供氧水平、二氧化碳浓度和pH值对发酵过程中维生素B₁₂和粪卟啉Ⅲ代谢合成的影响,并在120吨发酵罐中进行了发酵过程优化控制。结果表明:在发酵过程产物的合成期控制适当的供氧水平、控制二氧化碳浓度在8.6±0.8%、维持pH值在7.0±0.12能明显抑制卟啉Ⅲ的生物合成,同时使维生素B₁₂产量提高15%。     

谷氨酸棒杆菌SYPS-062合成L-丝氨酸的代谢通量分析

以一株由自然界筛选获得的能够利用糖质原料直接产L-丝氨酸的谷氨酸棒杆菌Corynebacterium glutamicum SYPS-062为研究对象,考察了一碳单元循环中的辅因子—叶酸和维生素B12对菌株生长、蔗糖消耗及L-丝氨酸生成的影响,同时对处于对数生长期的菌株进行了代谢流量分析。结果发现,添加扰动因子叶酸和维生素B12对磷酸戊糖途径(HMP)碳流影响较大,碳源主要用于细胞生长及合成能量,而流向目的产物L-丝氨酸的碳流减少。同时在添加维生素B12时,增大了G3P节点的L-丝氨酸合成途径的分流比,但造成三羧酸循环(TCA)的流量不足,需要大量回补,从而限制了产物合成速率的进一步提高。     

透明颤菌vgb基因在脱氮假单胞菌中的表达及对维生素B12合成的碳中心代谢流分析

在为维生素B₁₂生产菌株脱氮假单胞菌确立合适的接合转移操作条件的基础上,通过单交换的方式,将vgb基因整合到脱氮假单胞菌染色体上,获得了vgb重组菌株Pvgb-16,并通过¹³C同位素标记实验,探索VHb蛋白对脱氮假单胞菌碳中心代谢流变化和维生素B₁₂合成的影响。研究结果表明,在相同的供氧条件下,vgb重组菌株Pvgb-16拥有更高的比生长速率和比产物合成速率,与出发菌株相比分别提升了22%和52%。碳代谢通量分布分析表明,vgb重组菌株Pvgb-16的PP途径改善,提升了NADPH合成通量;甘氨酸由甜菜碱合成的通量上升,促进了前体物质氨基乙酰丙酸的合成,进一步加速维生素B₁₂的合成。总体来看,含vgb基因的重组菌株与出发菌株相比在促进菌体的生长、维生素B₁₂的合成速率及得率上都有显著效果,对进一步的发酵生产应用研究具有重要意义。     

添加物对枯草芽胞杆菌γ-聚谷氨酸合成代谢的影响

在枯草芽孢杆菌HCUL-B115代谢网络和发酵特性研究的基础上,通过添加适量的氨基酸、有机酸和维生素对聚γ谷氨酸(γPGA)发酵进行合成代谢进行研究。结果发现,大部分添加物对聚γ谷氨酸的积累都有一定的影响,特别是L谷氨酸、L苯丙氨酸、L精氨酸、L天冬氨酸、L缬氨酸、延胡索酸、草酸、丙二酸、烟酸、维生素B6和抗坏血酸等添加物对菌株HCUL-B115合成聚γ谷氨酸有明显促进作用,添加后产率比不添加任何物质提高20%左右。从代谢层面上分析,这些添加物除了促进菌体自身生长之外,同时防止了菌体对各添加物的过量合成,强化了菌株HCUL-B115合成聚γ谷氨酸的代谢途径。     

植物3-羟基-3-甲基戊二酰辅酶A还原酶基因研究进展

细胞分裂素、赤霉素、脱落酸、叶绿素、萜类等类异戊二烯物质,是植物中广泛存在的一类代谢产物,在植物生长发育过程中起着非常重要的作用。一些萜类化合物作为药物的合成前体或有效的药用成分在工农业及医药生产上具有重要的经济价值。类异戊二烯物质主要通过甲羟戊酸代谢途径中的一系列酶催化合成,其中,3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)是该代谢途径中的第一个关键限速酶,能够将3-羟基-3-甲基戊二酰辅酶A转化成中间代谢产物甲羟戊酸。对植物HMGR基因的克隆、酶结构和功能分析、基因组织表达及调控等方面进行了综述,旨在为其在重要农作物的遗传改良、代谢产物工程植物创制以及植物亲缘关系分析中的应用等研究提供理论依据。     

大肠杆菌产L-苯丙氨酸发酵调控及代谢通量分析

考察了外源添加中间代谢产物、维生素B1和硫酸镁对大肠杆菌发酵产L-苯丙氨酸的影响,结果表明,添加1g/L柠檬酸三钠、1g/Lα-酮戊二酸、150mg/L维生素B1及3g/L硫酸镁均对L-苯丙氨酸的合成有利。根据构建的大肠杆菌合成L-苯丙氨酸的生化反应网络,利用代谢通量分析其原因。结果表明,这些物质的添加均可以调节G6P和PEP节点处的代谢通量分布,为L-苯丙氨酸的合成提供更多的前体物质赤藓糖四磷酸(E4P)、磷酸烯醇式丙酮酸(PEP)和还原力NADPH。通过补料分批发酵实验得出,优化后菌体对总葡萄糖的消耗提高了24.49%,菌体终浓度提高了23.50%,L-苯丙氨酸的终浓度提高了62.87%,L-苯丙氨酸的收率提高了30.88%,乙酸的合成降低了56.51%。     

真菌诱导子在发酵工业中的应用现状及展望

真菌诱导子是一类能诱导植物和微生物产生次级代谢产物的活性物质,它一经识别,将通过信号转导途径,引起相关基因表达发生变化,从而调节次级代谢产物合成途径中相关酶的活性,诱导特定次级代谢产物的积累。近年来国内外在真菌诱导子诱导途径及机制方面进行了深入研究,同时在生物工业领域,尤其在发酵工业中的应用也引起了广泛关注。以下结合本实验室的研究工作,重点介绍了真菌诱导子在植物和微生物细胞次级代谢产物合成方面的应用现状、诱导机制和存在的问题及展望。     

温度调节基因开关调控大肠杆菌发酵合成L-丙氨

【目的】L-丙氨酸的存在导致Escherichia coli的生长速率显著降低,最终会降低发酵过程中L-丙氨酸的体积合成速率。用温度调节基因开关(λpR-pL)高效、动态调控重组E. coli菌株菌体生长与L-丙氨酸合成过程,使两者相协调。【方法】以野生型E. coli B0016为出发菌株,敲除乙酸、甲酸、乙醇、琥珀酸、乳酸代谢产物合成途径以及丙氨酸消旋酶编码基因(ackA-pta、pflB、adhE、frdA、ldhA、dadX),获得菌株B0016-060B。将嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus)来源的L-丙氨酸脱氢酶基因(alaD)克隆于pL启动子下游,并在B0016-060B菌株中表达,获得菌株B0016-060B/pPL-alaD,进行摇瓶和发酵罐发酵考察菌体生长和L-丙氨酸发酵性能。【结果】竞争代谢途径的敲除显著降低了副产物合成量,仅形成极少量的乙酸、琥珀酸和乙醇。28 °C下菌株B0016-060B/pPL-alaD几乎不合成L-丙氨酸,可保证菌体快速生长;而在42 °C下可高效合成L-丙氨酸。经发酵罐发酵,可合成67.2 g/L L-丙氨酸,体积生产强度达到2.06 g/(L·h)。【结论】通过发酵培养温度的简单切换,分阶段实现了细胞的快速增量和L-丙氨酸的高强度合成。     

Influence of 5,6-dimethylbenzimidazole (DMB) on vitamin B12 biosynthesis by strains of Propionibacterium

Work on vitamin B₁₂ biosynthesis in whey permeate using 5,6-dimethylbenzimidazole (DMB) as a precursor has often been carried out, but with no reference to the stage of fermentation at which it is to be added to the fermentation medium. In the present paper we report 168 h incubation as the optimum time for vitamin B₁₂ biosynthesis and the effect of DMB addition at four phases of fermentation, on vitamin B₁₂ productivity, growth and substrate utilization by three strains of Propionibacterium. The infusion of DMB at the end of 144 h of incubation (24 h before the end of fermentation) has been found optimum for maximum metabolic efficiency of the cultures.     

Bacillus megaterium Has Both a Functional BluB Protein Required for DMB Synthesis and a Related Flavoprotein That Forms a Stable Radical Species

Despite the extensive study of the biosynthesis of the complex molecule B₁₂ (cobalamin), the mechanism by which the lower ligand 5,6-dimethylbenzimidazole (DMB) is formed has remained something of a mystery. However, recent work has identified and characterized a DMB-synthase (BluB) responsible for the oxygen-dependent, single enzyme conversion of FMN to DMB. In this work, we have identified BluB homologs from the aerobic purple, nonsulfur, photosynthetic bacterium Rhodobacter capsulatus and the aerobic soil bacterium Bacillus megaterium and have demonstrated DMB synthesis by the use of a novel complementation assay in which a B₁₂ deficient strain, substituted with the precursor cobinamide is recovered either by the addition of DMB or by the recombinant expression of a bluB gene. The DMB-synthetic activity of the purified recombinant BluB enzymes was further confirmed in vitro by providing the enzyme with FMNH₂ and oxygen and observing the formation of DMB by HPLC. The formation of a 4a-peroxyflavin intermediate, the first step in the oxygen dependent mechanism of DMB biosynthesis, is reported here and is the first intermediate in the enzyme catalysed reaction to be demonstrated experimentally to date. The identification and characterization of an FMN-binding protein found on the cobI operon of B. megaterium, CbiY, is also detailed, revealing an FMN-containing enzyme which is able to stabilize a blue flavin semiquinone upon reduction with a 1-electron donor.     

Dichloromethane as the sole carbon source for an acetogenic mixed culture and isolation of a fermentative, dichloromethane-degrading bacterium.

Dichloromethane (DCM) is utilized by the strictly anaerobic, acetogenic mixed culture DM as a sole source of carbon and energy for growth. Growth with DCM was linear, and cell suspensions of the culture degraded DCM with a specific activity of 0.47 mkat/kg of protein. A mass balance of 2 mol of chloride and 0.42 mol of acetate per mol of DCM was observed. The dehalogenation reaction showed similar specific activities under both anaerobic and aerobic conditions. Radioactivity from [14C]DCM in cell suspensions was recovered largely as 14CO2 (58%), [14C]acetate (23%), and [14C]formate (11%), which subsequently disappeared. This suggested that formate is a major intermediate in the pathway from DCM to acetate. Efforts to isolate from culture DM a pure culture capable of anaerobic growth with DCM were unsuccessful, although overall acetogenesis and the partial reactions are thermodynamically favorable. We then isolated bacterial strains DMA, a strictly anaerobic, gram-positive, endospore-forming rod, and DMB, a strictly anaerobic, gram-negative, endospore-forming homoacetogen, from culture DM. Both strain DMB and Methanospirillum hungatei utilized formate as a source of carbon and energy. Coculture of strain DMA with either M. hungatei or strain DMB in solid medium with DCM as the sole added source of carbon and energy was observed. These data support a tentative scheme for the acetogenic fermentation of DCM involving interspecies formate transfer from strain DMA to the acetogenic bacterium DMB or to the methanogen M. hungatei.     

Statistical optimization of a thermostable and neutral glucoamylase production by a thermophilic mold <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> in solid-state fermentation

Glucoamylase production by a thermophilic mold Thermomucor indicae-seudaticae was optimized in solid-state fermentation (SSF) by conventional one variable at a time approach and further statistically using response surface methodology (RSM). Glucoamylase secretion was strongly affected by three variables (moisture ratio, inoculum level and incubation time), and therefore, these three factors were further optimized using response surface methodology. The glucoamylase production in flasks containing wheat bran, under the conditions optimized by RSM, was 455 ± 23 U/g of dry moldy bran (DMB), while the predicted value by a polynomial model was 433.30 U/g DMB. The enzyme titre (455 ± 23 U/g DMB) attained in the validation experiment of this investigation is higher than those reported in the literature. When the large-scale production was attempted in enamel trays, a marginally lower enzyme titres were attained. An overall 1.8-fold increase in glucoamylase production was achieved in SSF due to statistical optimization in comparison with that of one variable at a time approach (250 ± 13 U/g DMB). A 10-fold enhancement in glucoamylase production was recorded in SSF as compared to that in submerged fermentation.     

RNAi沉默lovF基因实现土曲霉一步法发酵生产辛伐他汀

辛伐他汀是重要的降胆固醇处方药物。莫那可林J是辛伐他汀合成过程的关键中间体,也是洛伐他汀生物合成的中间产物。为获得莫那可林J并通过一步法发酵生成辛伐他汀,构建了洛伐他汀生物合成关键基因lov F基因的RNAi载体p MHJ137,通过农杆菌介导的方法转化土曲霉F001,筛选整合到染色体的阳性菌,并在阳性菌株MJ1-24的发酵过程中加入了DMB-S-MMP验证其直接合成辛伐他汀的效率。结果显示MJ1-24不再产生洛伐他汀,产物中仅有莫那可林J累积。如果在发酵过程中加入前体物质,可得到产物辛伐他汀。综上,RNAi技术能够有效实现土曲霉基因沉默。此项技术推进了一步法发酵生产辛伐他汀的工艺开发。     

Identification of 5,6-dimethylbenzimidazole as the Co alpha ligand of the cobamide synthesized by Salmonella typhimurium. Nutritional characterization of mutants defective in biosynthesis of the imidazole ring.

The Co beta-cyano derivative of the cobamide isolated from Salmonella typhimurium was identified as Co alpha-(alpha-5,6-dimethylbenzimidazolyl)-Co beta-cyanocobamide, indicating that this bacterium synthesizes 5,6-dimethylbenzimidazole (DMB) de novo. We found that mutants deficient in the synthesis of DMB can incorporate benzimidazole without modification to form Co alpha-(alpha-benzimidazolyl)cobamide, a cobamide that is physiologically active. The analysis of the nutritional requirements of mutants deficient in DMB synthesis identified 4,5-dimethylphenylenediamine as a putative intermediate in the synthesis of the imidazole ring of DMB. Our results suggest that the CobII region of the cob operon of S. typhimurium only encodes functions involved in the synthesis of the imidazole ring of DMB.     

Potential of Aspergillus oryzae CFTRI 1480 for producing proteinase in high titres by solid state fermentation

Aspergillus oryzae CFTRI 1480, an isolate from a spoiled moist sample of casein, produced 59,105 units of an extracellular proteinase/g dry mouldy bran (DMB) at 72 h in an arbitrarily formulated wheat bran medium in a solid state fermentation system. The enzyme production was significantly affected by mineral salt content and pH of the liquid used for moistening the wheat bran. Enzyme titres were enhanced 1.34-fold with the addition of 0.4% corn starch. Optimization of key parameters, i.e., initial moisture content, age and size of inoculum, increased the enzyme production to 191,869 units/g DMB and reduced the fermentation time to 48 h. Such high titres in a simple medium, surpassing most of the literature reports, indicate the industrial importance of the culture. The properties of acetone-precipitated enzyme, viz, the optimum pH of 10.0, more than 95% activity between pH 7.0 and 10.0, temperature optimum at 55° C and more than 90% activity between 10 and 27°C, are similar to those of commercially available fungal proteinases employed in animal feed, leather and other industries. Correspondence to: B. K. Lonsane     

Formation of a square-planar Co(I) B12 intermediate. Implications for enzyme catalysis.

X-ray edge and extended x-ray absorption fine structure (EXAFS) techniques provide powerful tools for analysis of local molecular structure of complexes in solution. We present EXAFS results for Co(I) B12 that demonstrate a four-coordinate (distorted) square-planar configuration. Comparison of EXAFS solutions for Co(I) and Co(II) B12 (collected previously; Sagi et al. 1990. J. Am. Chem. Soc. 112:8639-8644) suggest that modulation of the Co-N bond to the axial 5,6-dimethylbenzimidazole (DMB), in the absence of changes in Co-N (equatorial) bond distances, may be a key mechanism in promoting homolytic versus heterolytic cleavage. As Co-C bond homolysis occurs, the Co-N (DMB) bond becomes stronger. However, for heterolytic cleavage to occur, earlier electrochemical studies (D. Lexa and J. M. Saveant. 1976. J. Am. Chem. Soc. 98:2652-2658) and recent studies of methylcobalamin-dependent Clostridium thermoaceticum (Ragsdale et al. 1987. J. Biol. Chem. 262:14289-14297) suggest that removal of the DMB ligand (before Co-C bond cleavage) favors formation of the four-coordinate square-planar Co(I) species while inhibiting formation of the five-coordinate Co(II) B12 complex. This paper presents the first direct evidence that formation of the Co(I) B12 intermediate must involve breaking of the Co-N (DMB) bond.     

Demethyleneberberine induces cell cycle arrest and cellular senescence of NSCLC cells via c-Myc/HIF-1α pathway

BackgroundDemethyleneberberine (DMB) is a natural active component of medicinal plant Cortex phellodendri chinensis with favorable bioactivity. However, the role of DMB in suppressing non-small cell lung cancer (NSCLC) remains unknown.PurposeIn this study, we aimed to examine the effect and underlying mechanism of DMB in suppressing NSCLC.MethodsCCK8 assay and colony formation assay were utilized to assess the efficiency of DMB on the viability and colony formation capacity of NSCLC cells. Flow cytometry and β-Galactosidase Staining Kit were utilized to determine the efficiency of DMB on the cell cycle and cellular senescence of NSCLC cells. RT-qPCR and Western blot were used to detect the effect of DMB on cell cycle and cellular senescence related gene and protein expression of NSCLC cells. In vivo tumor model was established to evaluate the anti NSCLC effect of DMB. In addition, RNA-seq analysis was performed to detect the differential gene expression after DMB treatments.ResultsIn this study, we revealed that DMB exhibits efficient inhibitory effect on NSCLC cell proliferation and tumor xenografts growth in vivo. We also demonstrated that DMB could inhibit cell migration by suppressing epithelial-mesenchymal transition (EMT) and trigger cell cycle arrest by down-regulating the expression of cell cycle related genes in NSCLC cells. In addition, DMB treatment efficiently induces cellular senescence of NSCLC cells. From the RNA-seq analysis, we found that DMB accelerates senescence through suppressing HIF-1α expression, which was further elucidated by overexpressing HIF-1α in NSCLC to reduce the inhibitory effect of DMB. Furthermore, we also revealed that DMB decreases the expression of c-Myc, an up-stream protein of HIF-1α.ConclusionsTaken together, we first report that DMB inhibits NSCLC progress through inducing cell cycle arrest and triggering cellular senescence by downregulating c-Myc/HIF-1α pathway.