溶氧对L-苏氨酸发酵的影响

探索溶氧对L-苏氨酸发酵过程的影响及其控制方法。通过摇瓶装液量试验、不同溶氧控制方式考察发酵过程中溶氧对L-苏氨酸合成的影响。采用补料分批发酵工艺发酵L-苏氨酸,利用氨基酸分析仪测定发酵液中L-苏氨酸的产量,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。可以得出溶氧对L-苏氨酸生物合成有重要影响,并建立了最佳溶氧控制条件。     

L-缬氨酸的发酵工艺研究

目的:以黄色短杆菌XV0505为生产菌株,探讨发酵培养基和发酵控制条件对L-缬氨酸的产量和糖酸转化率的影响。方法:应用单因素实验确定发酵的工艺条件;利用纸层析-色斑洗脱比色法测定发酵液中L-缬氨酸的含量。结果:在最优发酵条件下,通过10L罐流加发酵72h,产酸量可达53.4g/L,糖酸转化率为26.7%,分别比补料分批发酵提高11.9%和3.5%。结论:环境因子和发酵控制工艺对发酵生产L-缬氨酸具有重要影响。     

丙氨酸转氨酶修饰对大肠杆菌L-色氨酸合成的影响

探究大肠杆菌细胞内负责L-丙氨酸合成的转氨酶对菌株代谢及L-色氨酸合成的影响。运用Red重组技术分别对编码L-丙氨酸转氨酶的基因alaA、alaC和avtA进行敲除。通过摇瓶和50 L罐中探究其对L-色氨酸积累、L-丙氨酸代谢及菌体生长变化情况。结果显示,除3种L-丙氨酸转氨酶全部缺失的工程菌L-丙氨酸合成受阻、菌体生长受到较强抑制外,其它各任意一种或两种丙氨酸转氨酶缺失菌株的生长并未有较大差异,但色氨酸的合成变化显著。其中alaA和alaC双基因缺失的E.coli FS-T4工程菌,摇瓶发酵L-色氨酸产量达6.08 g/L,L-丙氨酸含量仅0.16 g/L,较出发菌株分别提高了26.7%和降低了91.0%。在50 L罐中E.coli FS-T4工程菌L-色氨酸产量最高可达41.9 g/L,糖酸转化率达20.5%,分别较出发菌株提高了13.8%和5.1%。转氨酶AlaA和AlaC的同时缺失,既可以满足细胞整体氨基酸池的需要,而且有利于减少杂酸的积累,使得更多的碳源流向L-色氨酸的合成。     

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。     

ppc基因敲除对大肠杆菌L-色氨酸发酵的影响

目的：减少大肠杆菌L-色氧酸前体物质磷酸烯醇式丙酮酸向草酰乙酸的代谢流,提高其L-色氨酸的产量。方法：以大肠杆菌TRTH0709为出发菌株,利用Red重组敲除技术敲除磷酸烯醇式丙酮酸羧化酶（Ppc）编码基因PPc,并经测序和酶活性检测确证;对出发菌株和基因敲除菌株进行L-色氖酸发酵,对比分析发酵结果。结果：测序和酶活性检测结果表明ppc基因被成功敲除。发酵结果表明,与出发菌株相比,基因敲除菌株TRTH0709△ppc生长速度减慢,最终生物量减少32％,L-色氯酸产量降低27％,但糖酸转化率提高6％;向发酵培养基中添加1％琥珀酸后,TRTH0709△ppc的生长速率和产酸量有所提高,但仍与出发菌株有一定差距。结论：虽然ppc基因敲除对菌体生长和产酸量影响较大,但能有效提高其糖酸转化率;选育Ppc弱化的突变株以达到减弱代谢流且不影响菌体生长,以及增加,L-色氨酸积累的目的,将是本研究今后的主要方向。     

重组大肠杆菌利用废纸水解液发酵产L-乳酸

前期通过基因工程手段,构建了一株大肠杆菌工程菌E.coli WL204,该菌株可以有效利用木糖为底物发酵产L-乳酸。以废纸为发酵原料,研究该菌株利用木质纤维素发酵产乳酸的特性。原料以稀硫酸预处理后,经纤维素酶酶解,得到的水解液用Ca(OH)2脱毒后,接种E.coli WL204,在7L发酵罐中发酵72h,每100g废纸可以产生31g乳酸,糖酸转化率为79%。结果表明,E.coli WL204可以木质纤维素原料为底物发酵生产L-乳酸,具有一定的工业化开发潜力。     

氮源对L-苏氨酸发酵的影响

以L-苏氨酸生产菌TRFC为供试菌株,研究了氮源对L-苏氨酸发酵的产量和糖酸转化率的影响。首先通过摇瓶实验确定发酵的最佳无机氮源和有机氮源分别为硫酸铵和酵母粉,进一步利用10L罐补料分批发酵确定硫酸铵和酵母粉的最佳用量,继续优化培养条件,采用发酵中后期流加硫酸铵和糖氨混合补料等措施,L-苏氨酸产量得到进一步的提高。在最优发酵条件下,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。     

玉米芯稀酸水解及L-乳酸发酵研究

对玉米芯稀硫酸水解条件及糖化液发酵L-乳酸进行了初步研究。结果表明,玉米芯木聚糖最适水解条件为2%H2SO_4、120℃、30 min、固液比1:10,糖化液还原糖含量可达40.8 g/L,主要成分为木塘。细菌A-19可以利用水解液中的葡萄糖和木糖产酸,最适发酵条件为45℃、pH 6.5,从45℃～51℃、pH 5.5～pH 6.5产量均较高。用未浓缩的水解液发酵24 h,L-乳酸产量为30.6g/L,残糖为1.6 g/L,糖酸转化率为82.6%;用浓缩1倍的水解液发酵48 h,L-乳酸产量为41.4 g/L,残糖4.1g/L,糖酸转化率为68.2%,在发酵48 h后继续补料发酵至72 h(补料液为浓缩3倍的水解液),L-乳酸产量为50.9 g/L,残糖6.3 g/L,糖酸转化率为71.8%。该研究为利用木质纤维素生产L-乳酸奠定了一定基础。     

重组大肠杆菌转化富马酸生产L-丙氨酸

通过克隆来源于睾丸酮丛毛单胞菌的L-天冬氨酸-β-脱羧酶基因,在一株具有L-天冬氨酸酶生产能力的大肠埃希氏菌CICC 11022S中异源表达,构建转化富马酸生产L-丙氨酸的重组工程菌。结果发现:重组工程菌9 h转化富马酸产生112.7 g/L的L-丙氨酸,生产速率12.5 g/(L·h),转化率93.8%。富马酸价格较低,有效降低L-丙氨酸生产的原料成本。通过构建重组工程菌,以富马酸为底物,高效生产L-丙氨酸,结果表明该方法具有较好的工业应用潜力。     

L—天门冬氨酰胺一水合物的合成

<正> L-天门冬氨酰胺是构成蛋白质的一种氨基酸成分。因此是一种重要的生化试剂。在医药上,它是氨基酸输液(20种氨基酸)的一个组分。目前,国内只报导了用抽提法生产L-天门冬氨酰胺的工艺,由于原料来源有限,生产量满足不了市场上的需要。而在国外则主要以化学合成法生产L-天门冬氨酰胺。如日本就是采用化学合成法,由L-天     

Intracellular metabolite profiling of Fusarium oxysporum converting glucose to ethanol

The filamentous fungus Fusarium oxysporum is known for its ability to produce ethanol by simultaneous saccharification and fermentation (SSF) of cellulose. However, the conversion rate is low and significant amounts of acetic acid are produced as a by-product. In this study, the growth characteristics of F. oxysporum were evaluated in a minimal medium using glucose as the sole carbon source in aerobic, anaerobic and oxygen-limited batch cultivations. Under aerobic conditions the maximum specific growth rate was found to be 0.043 h(-1), and the highest ethanol yield (1.66 mol/mol) was found under anaerobic conditions. During the different phases of the cultivations, the intracellular profiles were determined under aerobic and anaerobic conditions. The profiles of the phosphorylated intermediates indicated that there was a high glycolytic flux at anaerobic growth conditions, characterized by high efflux of glyceraldehyde-3-phosphate (G3P) and fructose-6-phosphate (F6P) from the pentose phosphate pathway (PPP) to the Embden-Meyerhof-Parnas (EMP) pathway, resulting in the highest ethanol production under these conditions. The amino acid profile clearly suggests that the TCA cycle was primarily active under aerobic cultivation. On the other hand, the presence of high levels of gamma-amino-n-butyric acid (GABA) under anaerobic conditions suggests a functional GABA bypass and a possible block in the TCA cycle at these conditions.     

餐厨垃圾协同剩余污泥发酵产酸的生物过程与影响因素研究进展

资源化利用是应对餐厨垃圾(Kitchen waste,KW)和剩余污泥(Excess sludge,ES)快速增加的有效方法,而厌氧发酵获得挥发性脂肪酸(Volatile fatty acids,VFAs)是其中的重要方式之一,但单一底物限制了VFAs的高效生产。近年来,不同底物厌氧共发酵产生VFAs被广泛研究与应用,文中分析了KW和ES单独和协同发酵产酸过程的特点,总结了厌氧发酵产酸过程及其生物代谢机制,阐述了环境因子及微生物群落结构对厌氧发酵产物类型及系统产物回收效率的影响。并进一步提出了针对区域饮食习惯、接种外源微生物构建稳定高效的定向产酸发酵体系以及KW和ES与原位污水间的耦联作用的研究方向。以期减少垃圾回收站及污水处理厂的运行成本,为实现城市有机固体垃圾处理与污水处理共赢提供参考。     

A novel and efficient enzymatic method for the production of peptides from unprotected starting materials

We report herein the development of a novel and efficient enzymatic method for the production of oligopeptides. This newly discovered method is a simple, cost-effective process, using unprotected amino acids as substrates in an aqueous solution and producing peptides in high yield. The target of our initial screen was l-alanyl-L-glutamine, a dipeptide of significant industrial interest by virtue of its widespread use in infusion therapy. By means of the screening of microorganisms that can catalyze the peptide-forming reaction producing l-alanyl-L-glutamine from L-alanine methylester (acyl donor) and L-glutamine (nucleophile), we discovered that Empedobacter brevis ATCC 14234 produced l-alanyl-L-glutamine most efficiently. The newly found enzyme purified from E. brevis ATCC 14234 facilitates significantly high production yields of l-alanyl-L-glutamine from L-alanine methylester and L-glutamine in an aqueous solution--more than 80% yield based on L-alanine methylester. In addition, this enzyme has wide substrate specificity--both for acyl donors and nucleophiles--and can catalyze peptide-forming reactions not only to produce various dipeptides from the corresponding amino acid esters and amino acids but also to produce various oligopeptides from the corresponding amino acid esters and peptides.     

Formation of fermentation products and extracellular protease during anaerobic growth of Bacillus licheniformis in chemostat and batch-culture

For a relaxed (rel-), protease producing (A-type) and a stringent (rel+), not-protease producing (B-type) variant of Bacillus licheniformis we determined fermentation patterns and products, growth parameters and alkaline protease-production (if any) in anaerobic, glucose-grown chemostats and batch-cultures. Glucose is dissimilated via glycolysis and oxidative pentose phosphate pathway simultaneously; the relative share of these two routes depends on growth phase (in batch) and specific growth rate (in chemostat). Predominant products are lactate, glycerol and acetaldehyde for A-type batches and acetaldehyde, ethanol, acetate and lactate for B-type batches. Both types show a considerable acetaldehyde production. In chemostat cultures, the fermentation products resemble those in batch-culture. From the anaerobic batches and chemostats, we conclude that the A-type (with low ATP-yield) will have a YATPmax of probably 12.9 g/mol and the B-type (with high ATP-yield) a YATPmax of about 10.1 g/mol. For batch-cultures, both types have about the same, high Yglucose (12 g/mol). So, the slow-growing A-type has a relatively high efficiency of anaerobic growth (i.e. an efficient use of ATP) and the fast-growing B-type a relatively low efficiency of anaerobic growth. In aerobic batch-cultures, we found 48, respectively 41% glucose-carbon conversion into mainly glycerol and pyruvate, respectively acetate as overflow metabolites in the A- and B-type. In both aerobic and anaerobic batch-cultures of the A-type, protease is produced predominantly in the logarithmic and early stationary phase, while a low but steady production is maintained in the stationary phase. Protease production occurs via de novo synthesis; up to 10% of the total protease in a culture is present in a cell-associated form. Although anaerobic protease production (expressed as protease per amount of biomass) is much higher than for aerobic conditions, specific rates of production are in the same range as for aerobic conditions while, most important, the substrate costs of anaerobic production are very much higher than for aerobic conditions.     

Exploitation of olive oil mill wastewater for combined biohydrogen and biopolymers production

The present study aimed to the investigation of the feasibility of the combined biohydrogen and biopolymers production from OMW (Olive oil Mill Wastewater), using a two stage system. H₂ and volatile fatty acids (VFAs) were produced via anaerobic fermentation and subsequently the acidified wastewater was used as substrate for aerobic biodegradable polymer production. Two different bioreactors, one of CSTR type and a SBR were used for the anaerobic and the aerobic process respectively. The anaerobic reactor was operated at different hydraulic retention times (HRTs) with OMW, diluted 1:4 (v/v) with tap water, as feed. The main VFAs produced were acetate, butyrate and propionate, in different ratios depending on the HRT. Valerate, isovalerate and isobutyrate were also detected in small quantities. Selective effluents of the acidogenic/hydrogen producing reactor were subsequently used as feed for the aerobic reactor. The aerobic reactor was inoculated with an enriched PHAs producing bacteria culture, and was operated in sequential cycles of nitrogen offer (growth phase) and nitrogen limitation (PHAs accumulation phase). The operational program of the SBR was determined according to the results from batch test, and its performance was evaluated for a period of 100 days. During the accumulation phase butyrate was consumed preferably, indicating that the dominant PHA produced is polyhydroxybutyrate. The higher yield of PHAs observed was 8.94% (w/w) of dry biomass weight.     

基于转录和翻译调控的氨基酸高产菌株筛选及构建策略

氨基酸的工业化生产和应用有近50年的历史。除了调味料和其他食品用途外,氨基酸还被用于动物饲料、药物和化妆品等许多领域。如今大多数氨基酸通过微生物发酵生产,极低的价格对它们在工业中的应用至关重要。通常采用挑选营养缺陷型或具有类似物抗性的菌株来筛选氨基酸生产菌。然而,因部分氨基酸生产菌的生产效率低下,限制了其工业化发展。主要梳理了营养缺陷型、氨基酸类似物和富含稀有密码子标记基因3种不同筛选方法及其实际应用,旨为高产菌株的筛选与构建提供候选方案。     

Effect of oxygen and shear stress on molecular weight of hyaluronic acid

Dissolved oxygen (DO) and shear stress have pronounced effects on hyaluronic acid (HA) production, yet various views persist about their effect on the molecular weight of HA. Accordingly, this study investigated the effects of DO and shear stress during HA fermentation. The results showed that both cell growth and HA synthesis were suppressed under anaerobic conditions, and the HA molecular mass was only (1.22+/-0.02) x 106 Da. Under aerobic conditions, although the DO level produced no change in the biomass or HA yield, a high DO level favored the HA molecular mass, which reached a maximum value of (2.19+/- 0.05) x 106 Da at 50% DO. Furthermore, a high shear stress delayed the rate of HA synthesis and decreased the HA molecular weight, yet had no clear effect on the HA yield. Therefore, a high DO concentration and mild shear environment would appear to be essential to enhance the HA molecular weight.     

生物强化污泥厌氧发酵产酸研究进展*

污泥厌氧发酵生产挥发性脂肪酸相较产甲烷,是更具应用价值的污泥稳定途径及资源化利用方式,得到国内外学者的普遍重视。考虑到产酸量低和产酸过程的不稳定性是限制污泥发酵产酸的主要问题,采用生物强化方法实现挥发性脂肪酸的大量积累,与物理和化学方法相比,具有成本低、无二次污染等优点。根据生物强化制剂的类型,归纳了微生物纯培养物、微生物混合培养物及生物酶强化对污泥厌氧发酵产酸的影响,并在此基础上对生物强化技术控制污泥定向产酸、调控奇偶数碳比率等方面的应用进行讨论。此外,分析了影响挥发性脂肪酸产量和组分的因素,如pH、温度、底物、水力停留时间和污泥龄等。最后对生物强化技术的发展方向进行了展望,以期为深入探究污泥资源化利用提供借鉴。     

Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate

Most reported efforts to enhance production of the industrially valuable specialty chemical succinate have been done under anaerobic conditions, where E. coli undergoes mixed-acid fermentation. These efforts have often been hampered by the limitations of NADH availability, poor cell growth, and slow production. An aerobic succinate production system was strategically designed that allows E. coli to produce and accumulate succinate efficiently and substantially as a product under absolute aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhAB, icd, iclR) and acetate pathways (poxB, ackA-pta) of E. coli were created to construct the glyoxylate cycle for aerobic succinate production. Experiments in flask studies showed that 14.28 mM of succinate could be produced aerobically with a yield of 0.344 mole/mole using 55 mM glucose. In aerobic batch reactor studies, succinate production rate was faster, reaching 0.5 mole/mole in 24 h with a concentration of 22.12 mM; further cultivation showed that succinate production reached 43 mM with a yield of 0.7. There was also substantial pyruvate and TCA cycle C(6) intermediate accumulation in the mutant. The results suggest that more metabolic engineering improvements can be made to this system to make aerobic succinate production more efficient. Nevertheless, this aerobic succinate production system provides the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network.