灵芝液态深层发酵三萜类化合物研究进展

三萜类化合物是灵芝中主要的活性化学成分之一,由于其具有多种重要的生理活性,现已成为国内外学者研究灵芝的热点。本文总结了灵芝三萜发酵工艺的优化及其生物合成中的信号转导等方面的进展,并在此基础上提出了灵芝发酵研究中存在的问题,以期为灵芝三萜液态深层发酵的调控研究及发酵生产工艺的开发提供参考和启示。     

灵芝三萜的生物合成和发酵调控

灵芝作为一种名贵的药用真菌,在我国已有2 000多年的利用历史。灵芝三萜是灵芝的关键药效成分之一。从灵芝三萜的结构和构效关系、灵芝三萜的合成途径和相关关键酶,以及灵芝三萜的深层发酵调控策略和技术等方面,综述了灵芝三萜生物合成和发酵调控方面的新进展,进一步提出了当前存在的主要问题,并对今后的主要研究策略和研究方向进行了分析与展望。     

油酸促进灵芝三萜液态深层发酵的工艺研究及规模化验证

灵芝三萜是灵芝中主要的活性成分之一,前期研究发现油酸可以促进灵芝三萜液态深层发酵下的发酵合成。本研究主要对油酸促进灵芝三萜液态深层发酵的工艺进行优化,并进行3L发酵罐规模的验证。通过单因素实验考察油酸的添加方式、添加时间和添加浓度对灵芝三萜的影响,结合响应面实验,获得最优工艺条件并进行验证：在发酵第32h添加1.21%高温灭菌油酸,最高灵芝三萜含量为42.69mg/g;在发酵第7h添加1.35%过滤除菌油酸,最高三萜含量为43.38mg/g,分别比对照提高2.04倍和2.08倍。在1 000mL摇瓶中添加高温灭菌油酸和过滤除菌油酸,灵芝三萜含量分别为32.18和32.48mg/g,为对照的1.96倍和1.95倍;在3L发酵罐规模下灵芝三萜含量分别为28.66和25.13mg/g,为对照的1.62倍和1.42倍。本研究系统优化了油酸促进灵芝三萜液态深层发酵的工艺条件,并在与工业生产相对应的3L发酵罐上进行验证。该研究可为灵芝三萜的规模化发酵提供重要参考和借鉴。     

灵芝液态发酵胞内外多糖的研究进展

灵芝具有悠久的药用历史,其活性物质灵芝多糖具有广泛的药理活性,利用液态发酵技术获取灵芝多糖展现出独特优势。近年来,关于灵芝多糖液态发酵的研究报道越来越多,引起了研究者的广泛关注。本文从灵芝多糖的生物合成代谢及调控、液态发酵的培养基及工艺优化等方面对近期的研究结果进行总结,以期为灵芝液态发酵制备胞内外多糖及其产业化应用提供参考和依据。     

灵芝三萜合成调控与抗肿瘤药理活性的研究进展

三萜类化合物是灵芝的重要药效成分之一,是一类活性天然产物,不仅在保肝、护肾、降血糖、抗缺氧等方面有着重要的应用价值,而且还具有很好的抗肿瘤活性。现就灵芝三萜的发酵培养优化和抗肿瘤药理活性的研究进展进行综述,以期为酸性和中性灵芝三萜类化合物的研究及进一步开发相应抗癌药物提供科学基础。     

灵芝菌丝体三萜及其药理活性的研究进展

菌丝体作为灵芝生长发育过程中的一个特定生理阶段,会产生不同于子实体和孢子的特定结构的灵芝酸类三萜化合物。本文总结了灵芝菌丝体中已发现和鉴定的三萜化合物结构、生物活性及其构效关系的研究进展,以期为灵芝菌丝体三萜在生物合成、代谢调控等的基础研究及相关产品的开发应用提供科学参考。     

灵芝β-葡萄糖苷酶基因的克隆与功能分析

灵芝是我国著名的药用真菌,具有抗癌、抗肿瘤等功效。灵芝酸属于三萜类化合物,是灵芝的主要活性成分,并已成为评价灵芝品质的重要指标之一。β-葡萄糖苷酶(β-glucosidase,BG)是次生代谢产物合成途径中的关键限速酶,能够调节次生代谢产物的生物合成。本研究通过同源序列比对,注释获得了灵芝β-葡萄糖苷酶基因(GlBG),并通过RNAi技术对灵芝β-葡萄糖苷酶进行功能分析。序列分析结果显示GlBG基因的DNA全长为2 759 bp,包含7个外显子和6个内含子,编码793个氨基酸,其编码的蛋白序列中含有β-糖苷水解酶的2个保守结构域。灵芝β-葡萄糖苷酶基因的沉默转化子中灵芝酸含量比野生型菌株的灵芝酸含量平均降低了38%,并且灵芝酸生物合成途径中的关键酶基因(hmgs、hmgr、fps、sqs和osc)的表达量也显著下降,实验结果表明灵芝β-葡萄糖苷酶在灵芝酸生物合成过程中具有重要作用,并为灵芝次生代谢途径及其调控机制提供了参考。     

茉莉酸甲酯对灵芝三萜生物合成的影响及其灵芝应答基因的差异表达

灵芝三萜类化合物是灵芝的主要药用成分之一,灵芝三萜含量的多少是衡量灵芝质量高低的重要指标。经研究发现茉莉酸甲酯能显著提高灵芝三萜含量并上调灵芝三萜生物合成途径中的羟甲基戊二酰辅酶A还原酶、法尼酯焦磷酸合成酶和羊毛甾醇合成酶等基因的表达。通过在灵芝中建立的cDNA-AFLP体系,采用64对引物组合对灵芝的转录组进行分析。64对引物组合共产生3 910个TDFs。茉莉酸甲酯不同诱导条件下约有919个TDFs的表达谱发生了变化,占表达谱的23.5%,其中上调表达的有703个,下调表达的有216个。选择458个TDFs进行回收、TA克隆、测序后,获得390个高质量的TDFs片段。通过蛋白数据库同源性检索,并对蛋白功能进行GO分类,结果表明:390个高质量的TDFs中,BlastX比对后没有显著匹配的序列占77.1%。在有匹配蛋白的序列中,与碳代谢及能量相关的基因为最大一组,占35.2%,转录调控类及信号传导类基因分别占14.3%和11.0%。通过荧光定量PCR技术考察了茉莉酸甲酯对20个基因转录水平的影响,与cDNA-AFLP分析的结果相一致。并通过灵芝基因过量表达技术和基因沉默技术对筛选到的相关基因进行沉默和过量表达,研究筛选到的基因在灵芝三萜生物合成与调控中的作用,获得大量参与灵芝三萜生物合成与调控的候选基因,进一步阐明了茉莉酸甲酯诱导下灵芝基因表达的变化图。     

复合有机氮源对灵芝三萜液态深层发酵的影响

以灵芝为材料,在前期研究基础上,以不同酵母粉作为氮源,研究复合有机氮源对灵芝三萜液态深层发酵的影响。首先,由单因素实验考察3种不同的酵母粉对灵芝菌丝体合成灵芝三萜的影响,确定酵母粉的最适宜浓度范围。在此基础上,根据中心组合实验设计,对3种酵母粉分别采用2种复合和3种复合的方式,优化复合有机氮源的最佳组合配比。结果表明：当基础培养基中添加6.6g/L的酵母粉N-1与6.6g/L的酵母粉N-2时,灵芝三萜产量可达0.478g/L（理论产量为0.485g/L）,比添加单一酵母粉N-1、N-2、N-3分别提高了21%、139%、103%,其氮源用量为两种组合时最低。当基础培养基中酵母粉N-1、N-2与N-3添加量分别为5.07g/L、3.78g/L、7.63g/L时,灵芝三萜产量达0.514g/L（理论产量为0.510g/L）,比单因素对照组分别提高了30%、157%和74%。本研究优化的复合氮源添加方式可明显提高灵芝菌丝体液态深层发酵生产灵芝三萜的产量,为其规模化液态深层发酵的生产提供科学数据。     

HPLC法测定灵芝孢子粉中三萜含量

为准确测定灵芝孢子粉中三萜的含量,运用高效液相建立适合孢子粉的分析测定方法。通过对前处理条件的优化,确定40%乙醇为孢子粉中等极性三萜酸类的最佳提取溶剂,浓缩倍数是子实体提取条件的50倍。通过色谱柱和洗脱条件的优化,建立了包括灵芝酸I、灵芝烯酸C、灵芝酸C2等13种标准品测定方法,方法学考察显示该分析方法精密度、重复性、稳定性的RSD值均小于5%,可以用于灵芝孢子粉中三萜类成分的定量检测。通过5组样品的分析发现,灵芝酸C6、灵芝酸G、灵芝酸A、灵芝酸D、灵芝酸F是灵芝孢子粉中的主要三萜类成分,其中灵芝酸A含量最高,平均占样品三萜总量的比例达19.71%;三萜类成分的溶出量与是否破壁没有相关性。三萜类成分在灵芝孢子粉和灵芝孢子油产品中的含量非常低,孢子粉的三萜含量为14.24-99.70μg/g,仅为子实体的1/100,灵芝孢子油中三萜含量也均低于50μg/g,因此三萜类成分不适合作为灵芝孢子粉及其相关产品的定量检测指标。     

A novel Ganoderma lucidum G0119 fermentation strategy for enhanced triterpenes production by statistical process optimization and addition of oleic acid

A novel enhanced triterpenes fermentation production process by Ganoderma lucidum G0119 with the addition of oleic acid in the medium has been developed and optimized. All of the six exogenous additives tested were found to exhibit stimulatory effect on mycelial growth and triterpenes biosynthesis by G. lucidum. The results show that oleic acid addition had significant role in promoting triterpenes production. The optimal concentration and time of oleic acid addition were determined to be 30 mL/L and 0 h, respectively. Furthermore, three significant factors influencing triterpenes production were identified as glucose, magnesium sulfate and temperature using the Plackett–Burman design. The optimized conditions by central composite design were 27.83 g/L glucose, 1.32 g/L magnesium sulfate, 26.2°C temperature. The triterpenes fermentation yield with the optimized medium based on actual confirmatory experimental data in 6 L fermentor was 1.076 g/L versus the statistical model predicted value of 1.080 g/L. Our innovatively developed triterpenes fermentation production technology and process has been proven to produce high triterpenes productivity and yield conceivably useful for industrial production.     

An unstructured kinetic model for the improvement of triterpenes production by Ganoderma lucidum G0119 based on nitrogen source effect

The kinetics of cell growth and triterpenes production for liquid submerged fermentation of the medicinal mushroom Ganoderma lucidum were investigated. A kinetic model was developed based on the Logistic and Luedeking-Piret equations for cell growth, substrate consumption and triterpene formation. The kinetic parameters of the model were optimized by specifically designed Runge-Kutta genetic algorithms. The mathematical model simulated the experimental data well and was capable of explaining the behavior of triterpenes production. The predictions of the kinetics from this model are very good both for normal fermentation kinetics under nitrogen limitation as well as for predictions of transitions to sluggish fermentations. The resulting model is very useful for scaling up liquid submerged fermentation of the mushroom G. lucidum and its application to the industrial production of triterpene.     

The role of oxidative stress on carotene production by Blakeslea trispora in submerged fermentation

In aerobic metabolism, reactive oxygen species (ROS) are formed during the fermentation that can cause oxidative stress in microorganisms. Microbial cells possess both enzymatic and non-enzymatic defensive systems that may protect cells from oxidative damage. The antioxidant enzymes superoxide dismutase and catalase are the two key defensive enzymes to oxidative stress. The factors that induce oxidative stress in microorganisms include butylated hydroxytoluene (BHT), hydrogen peroxide, metal ions, dissolved oxygen tension, elevated temperature, menadione, junglone, paraquat, liquid paraffin, introduction to bioreactors of shake flask inocula and synthetic medium sterilized at initial pH 11.0. Carotenes are highly unsaturated isoprene derivatives. They are used as antioxidants and as coloring agents for food products. In fungi, carotenes are derived via the mevalonate biosynthesis pathway. The key genes in carotene biosynthesis are hmgR, ipi, isoA, carG, carRA and carB. Among microorganisms, Βlakeslea trispora is the main microorganism used for the production of carotenes on the industrial scale. Currently, the synthetic medium is considered the superior substrate for the production of carotenes in a pilot plant scale. The fermentation systems used for the production of carotenes include shake flasks, stirred tank fermentor, bubble column reactor and flat panel photobioreactor. This review summarizes the oxidative stresses in microorganisms and it is focused on the current status of carotene production by B. trispora including oxidative stress induced by BHT, enhanced dissolved oxygen levels, iron ions, liquid paraffin and synthetic medium sterilized at an initial pH 11.0. The oxidative stress induced by the above factors increases significantly the production of carotenes. However, to further reduce the cost of carotene production, new biotechnological methods with higher productivity still need to be explored.     

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase

Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA), an antitumor triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, is considered as a key precursor for biosynthesizing other ganoderic acids (GAs) with superior antitumor activities. Our previous study identified CYP5150L8 from G. lucidum as a lanosterol oxidase, and achieved heterologous biosynthesis of GA-HLDOA in Saccharomyces cerevisiae. However, low production of GA-HLDOA in either G. lucidum or heterologous host hindered its further investigation and application. In this study, we constructed a dual tunable system for balancing the expression of CYP5150L8 and a Ganoderma P450 reductase iGLCPR, and performed a comprehensive optimization of CYP5150L8 expression, iGLCPR expression, and glycerol usage. Then, we investigated the fermentation behavior of the best strain in optimized condition in flask and achieved 154.45 mg/L GA-HLDOA production, which was 10.7-fold higher compared with previous report. This study may facilitate the wide-spread application of GA-HLDOA and the discovery of unknown cytochrome P450s in downstream GAs biosynthesis.     

免疫抑制剂他克莫司生物合成的研究进展

他克莫司(FK506)是一种具有免疫抑制活性的23元大环内酯类化合物,临床上广泛用于防止器官移植术后的免疫排斥反应。生物合成法是他克莫司制备方法的研究热点,但他克莫司生物合成的研究还存在一定生产技术上的瓶颈。基于此,本文主要从他克莫司代谢途径改造和发酵过程控制等方面对他克莫司生物合成进展进行综述,以期为今后突破他克莫司生物合成的技术瓶颈提供参考,进而利用代谢工程、发酵工程等技术提升他克莫司的生物合成水平。     

基于代谢组学分析的O-琥珀酰-L-高丝氨酸发酵调控

【目的】O-琥珀酰-L-高丝氨酸(O-succinyL-L-homoserine, OSH)是合成L-蛋氨酸、L-草铵膦等重要前体，在医药、农药、食品等领域具有重要的应用前景，其绿色高效制造受到广泛关注。本研究通过解析OSH发酵过程代谢途径和代谢产物变化规律，建立OSH发酵调控策略，提升其产量和糖酸转化率。【方法】运用代谢组学技术，系统考察OSH生产菌在发酵不同时间段的代谢物变化情况，探究与OSH合成显著关联的代谢途径，通过在不同时间外源添加关键代谢物，平衡关键代谢物及其前体通量，减少旁路途径对前体的竞争性利用。【结果】在5 L发酵罐中产量达70.1 g/L，糖酸转化率达0.52 g/g (葡萄糖)。【结论】研究结果表明，基于代谢组学分析技术的OSH发酵体系优化和发酵过程调控显著提升了目标产物生产效率，奠定了OSH的产业化基础。     

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

Summary A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.     

Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production

The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas–liquid volumetric mass transfer coefficient k _L a positively. The k _L a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k _L a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r _DHA (9.75 mg l⁻¹ h⁻¹) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.     

Performance analyses of a pH-shift and DOT-shift integrated fed-batch fermentation process for the production of ganoderic acid and Ganoderma polysaccharides by medicinal mushroom Ganoderma lucidum

Investigations on Ganoderma lucidum fermentation suggested that the responses of the cell growth and metabolites biosynthesis to pH and dissolved oxygen tension (DOT) were different. The ganoderic acid (GA) production of 321.6 mg/L was obtained in the pH-shift culture by combining a 4-day culture at pH 3.0 with the following 6-day culture at pH 4.5, which was higher by 45% and 300% compared with the culture at pH 3.0 and 4.5, respectively. The GA production of 487.1 mg/L was achieved in the DOT-shift culture by combining a 6-day culture at 25% of DOT with a following 6-day culture at 10% of DOT, which was higher by 43% and 230% compared with the culture at 25% and 10% of DOT, respectively. A fed-batch fermentation process by combining the above-mentioned pH-shift and DOT-shift strategies resulted in a significant synergistic enhancement of GA accumulation up to 754.6 mg/L, which is the highest reported in the submerged fermentation of G. lucidum in stirred-tank bioreactor.