Higher intracellular levels of uridinemonophosphate under nitrogen-limited conditions enhance metabolic flux of curdlan synthesis in Agrobacterium species

Changes of intracellular nucleotide levels and their stimulatory effects on curdlan synthesis in Agrobacterium species were investigated under different culture conditions. Under nitrogen-limited conditions where curdlan synthesis was stimulated, intracellular levels of UMP were as high as 87 and those of AMP were 78 nmol/mg of cellular protein, while those under nitrogen-sufficient conditions were lower than 45 nmol/mg-protein. The levels of other nucleotides such as UDP, UTP, UDP-glucose, ADP, ATP, and ADP-glucose were lower than 30 nmol/mg-protein under both nitrogen-limited and sufficient conditions. The time profiles of curdlan synthesis and cellular nucleotide levels showed that curdlan synthesis had a positive relationship with intracellular levels of UMP and AMP. After the ammonium concentration in the medium fell below 0.1 g/L, intracellular levels of UMP and AMP increased, followed by curdlan synthesis. However, no significant changes in the specific activities of UMP kinase, UDP kinase, and UDP-glucose pyrophosphorylase were observed during cultivation. In vitro enzyme reactions for the synthesis of UDP-glucose, which serve as a precursor for curdlan synthesis, demonstrated that the synthesis of UDP-glucose increased with the increase of UMP concentration. In contrast, AMP had no effect on UDP-glucose synthesis at all. Addition of UMP in the medium increased the curdlan synthesis, whereas curdlan synthesis was inhibited in the presence of AMP. From these results, we concluded that only the higher intracellular UMP levels caused by nitrogen limitation in the medium enhance the metabolic flux of curdlan synthesis by promoting cellular UDP-glucose synthesis.     

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45–60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD⁺, NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20–50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.     

Metabolic engineering of Agrobacterium sp. for UDP-galactose regeneration and oligosaccharide synthesis

Curdlan-producing Agrobacterium sp. is unique in possessing a highly efficient UDP-glucose regeneration system. A broad-host-range expression strategy was successfully developed to exploit the unique metabolic capability for UDP-galactose regeneration during oligosaccharide synthesis. The engineered Agrobacterium cells functioned as a UDP-galactose regeneration system, allowing galactose-containing disaccharides to be synthesized from glucose or other simple sugars. Unexpectedly, a lag period of 24h preceded the active synthesis, which could be eliminated with rifampicin. An intracellular nucleotide profiling revealed that the UMP level was elevated by 3.8 fold in the presence of rifampicin, suggesting that rifampicin simulated a nitrogen-limitation condition that triggered the metabolic change. Product selectivity was improved nearly 40-fold by using high acceptor concentration and restricting glucose supply. N-acetyllactosamine concentration near 20 mM (7.5 g/l) was obtained, demonstrating the effectiveness of the engineered strain in UDP-galactose regeneration. This organism could be engineered to regenerate other UDP-sugar nucleotides using the same strategy as illustrated here.     

葡萄糖和麦芽糖碳源底物对粪产碱杆菌合成凝胶多糖的胞内代谢流影响*

麦芽糖和葡萄糖对粪产碱杆菌发酵合成凝胶多糖有着显著的影响,为了详细分析两种底物对凝胶多糖合成的影响机制,利用恒化培养实验及稳态碳平衡代谢分析,研究发现在稀释速率为0.1h^-1时,利用麦芽糖和葡萄糖为碳源底物的条件下粪产碱杆菌的微观代谢途径通量有较大的差异。以麦芽糖为底物时凝胶多糖的摩尔得率为53.8%,比葡萄糖为碳源时的摩尔得率(36.9%)高出了45.8%以上。同时以麦芽糖为碳源时HMP途径的绝对代谢通量比葡萄糖时的通量提升了40%以上。这条途径通量的增加,提升了NADPH还原力供给速率,促进了依赖于还原力NADPH的凝胶多糖合成途径通量,提升了碳源底物向产物的摩尔转化速率。而且代谢流分析结果显示ED途径通量和能量提供也是影响粪产碱杆菌凝胶多糖合成效率的关键因素。麦芽糖作为碳源底物过程中维持的较低的残留葡萄糖浓度解除了高葡萄糖浓度条件下对凝胶多糖合成的抑制,能够实现更高通量的ATP能量提供效率,更加促进了凝胶多糖合成通量。     

Optimal pH control of batch processes for production of curdlan by Agrobacterium species

We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species. The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin. The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters. The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion. By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L⁻¹) compared to that of constant pH operation (36 g L⁻¹). Received 24 November 1998/ Accepted in revised form 17 June 1999     

High-yield anthocyanin biosynthesis in engineered Escherichia coli

Anthocyanins are red, purple, or blue plant water-soluble pigments. In the past two decades, anthocyanins have received extensive studies for their anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, and cardioprotective properties. In the present study, anthocyanin biosynthetic enzymes from different plant species were characterized and employed for pathway construction leading from inexpensive precursors such as flavanones and flavan-3-ols to anthocyanins in Escherichia coli. The recombinant E. coli cells successfully achieved milligram level production of two anthocyanins, pelargonidin 3-O-glucoside (0.98 mg/L) and cyanidin 3-O-gluside (2.07 mg/L) from their respective flavanone precursors naringenin and eriodictyol. Cyanidin 3-O-glucoside was produced at even higher yields (16.1 mg/L) from its flavan-3-ol, (+)-catechin precursor. Further studies demonstrated that availability of the glucosyl donor, UDP-glucose, was the key metabolic limitation, while product instability at normal pH was also identified as a barrier for production improvement. Therefore, various optimization strategies were employed for enhancing the homogenous synthesis of UDP-glucose in the host cells while at the same time stabilizing the final anthocyanin product. Such optimizations included culture medium pH adjustment, the creation of fusion proteins and the rational manipulation of E. coli metabolic network for improving the intracellular UDP-glucose metabolic pool. As a result, production of pelargonidin 3-O-glucoside at 78.9 mg/L and cyanidin 3-O-glucoside at 70.7 mg/L was achieved from their precursor flavan-3-ols without supplementation with extracellular UDP-glucose. These results demonstrate the efficient production of the core anthocyanins for the first time and open the possibility for their commercialization for pharmaceutical and nutraceutical applications.     

ntrB基因对Agrobacterium sp.ATCC31749合成胞外多糖的调控研究

利用三亲本接合方法将含有ntrB基因两端同源序列的自杀载体pJQ-ntrB-cat导入土壤杆菌(Agrobacterium sp.ATCC 31749)中,获得了ntrB基因突变株.结果表明,ntrB突变株对NH4Cl和KNO3的利用能力有所下降.当分别以谷氨酸和谷氨酰胺为氮源时,ntrB突变株生长状况与野生菌相同.n...     

pH控制对热凝胶发酵的影响

热凝胶 (Ｃｕｒｄｌａｎ)是一种直链结构的 β 1,3 葡聚糖 ,由Ａｌｃａｌｉｇｅｎｅｓｆａｅｃａｌｉｓｖａｒ.ｍｙｘｏｇｅｎｅｓ发酵生产而来 ,是一种新型的微生物胞外多糖[1 ] ,其分子量在 5 0万左右。热凝胶在中性条件下不溶于水 ,但能溶于碱溶液中。加热含有热凝胶的水浊液可形成两种类型的凝胶 ,一种是弹性较低的类似琼脂的可逆胶 ;另外一种是凝胶强度大、弹性好的热不可逆胶。由于热凝胶具有独特的热成胶性能 ,在食品工业 ,特别是高温制作的食品领域具有广阔的应用前景。热凝胶的胶体可以包容和控制药物的扩散 ,所以可以用来作为药物…     

Genome sequence of the curdlan-producing Agrobacterium sp. strain ATCC 31749

Agrobacterium sp. ATCC 31749 is an industrial strain for the commercial production of curdlan, an important exopolysaccharide with food and medical applications. Here we report the genome sequence of the curdlan-producing strain ATCC 31749. Genome sequencing is the first step toward the understanding of regulation of curdlan biosynthesis.     

Endo-(1 → 3)β-D-glucanase activity secreted by Bacillus sp. no. 215

The production of an extracellular endo-(1 → 3)-β-D-glucanase by Bacillus sp. no. 215 was induced during growth with (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU as carbon and energy source. Maximum levels of activity (0.26 U ml^-1 resp. 1.40 U mg^-1) were detected in cell-free culture supernatant fluid after 25 h of aerobic growth at 55°C. The cells secreted an endo-(1 → 3)-β-D-glucanase with low electrophoretic mobility that used curdlan from C. flavigena strain KU and from Agrobacterium sp. (formerly Alcaligenes faecalis var. myxogenes ) as substrates. The enzyme activity was highest at pH 7.0 and 55°C. It exhibited a remarkably low thermal stability with a half-life of 14 min at 55°C in the presence of substrate. Divalent metal cations were required for enzyme activity.     

Improvement of welan gum production and redistribution of metabolic flux under pH control process in Alcaligenes sp. CGMCC2428

Batch fermentations of welan gum from Alcaligenes sp. CGMCC2428 at pH values of 5.5 ～ 7.0 were studied. Based on the kinetic analysis, a pH control process for improving welan production was developed. By maintaining the culture pH at 7.0, the process significantly improved the maximal welan concentration and productivity to reach 25.1 ± 0.65 g/L and 0.42 ± 0.003 g/L/h, respectively, compared with those in native pH processes where pH value would decrease from 7.0 to 5.1 (18.5 ± 0.72 g/L and 0.28 ± 0.002 g/L/h). This improvement may be due to the increased metabolic flux of glucose-1-phosphate to welan induced by pH control process. Furthermore, scale-up fermentation under controlled pH was implemented using 300-L fermentor. The highest welan concentration of 27.5 ± 0.97 g/L was obtained. This simplified process proved effective in industry fermentation for high welan production.     

氮源NH4Cl浓度对粪产碱杆菌发酵生产热凝胶的影响

研究了利用粪产碱杆菌(Alcaligenes faecalis)发酵生产热凝胶的发酵条件,氮源是菌体生长的限制性底物,单纯地提高初始底物(氮源)浓度并不一定能促进细菌的生长和产物的合成.在分批发酵过程中,底物消耗导致培养环境pH的改变也是影响细菌进一步生长和产物合成的重要因素.通过增加培养基中初始氯化铵的浓度并同时控制发酵过程的pH条件,得到了较高的菌体浓度,热凝胶的合成水平也得到了显提高.当培养基中NH4Cl浓度提高到3.6g/L时,菌体浓度达到7.2g/L,热凝胶合成的产量可达30.5g/L,比原来NH4Cl浓度为1.1g/L时提高了51.7%.提高菌体浓度意味着需要提高溶氧水平来满足细菌的生长和代谢.初始氮源NH4Cl浓度的增加虽然能使菌体浓度得到提高,但发酵过程对溶氧的需求也相应增加,需要提高搅拌转速和通风以增加供氧水平.但高搅拌速率产生的高剪切力对热凝胶的凝胶性能将产生破坏作用,因此在发酵过程中需要综合考虑细菌培养密度对合成热凝胶产量和质量的影响.     

Optimization of uracil addition for curdlan (β-1→3-glucan) production by Agrobacterium sp.

Uracil, acting as a precursor of UDP-glucose, served as an activator on the production of curdlan with Agrobacterium sp. (ATCC 31750). The time of adding uracil was important to improve curdlan production. When uracil was added after ammonium depletion (at 26 h), it was used as a nitrogen source for cell growth. Although the cell concentration increased, the curdlan production was decreased. If uracil was added at 46 h, then uracil was degraded slowly but still activated curdlan production. With the addition of both sucrose (200 g) and uracil (1.5 g), the curdlan production was increased up to 93 g l^–1 after 160 h fermentation.     

Optimal production of curdlan by Agrobacterium sp. with feedback inferential control of optimal pH profile

The pH control was important for curdlan production with Agrobacterium sp. ATCC31750. Specific cell growth rate was the highest at pH 7 and the specific curdlan production rate was at pH 5.5. The pH profiles maximizing curdlan production was changed from pH 7 optimal for cell growth to pH 5.5 optimal for curdlan production after ammonium consumption. The feedback inferential control methods, with easily measurable variables such as NaOH addition for pH control and dissolved oxygen (DO), were also applied. The pH was successfully controlled to follow optimal profiles and the maximal production of curdlan (60 g l^–1 in 120 h) was achieved with feedback optimal control.     

氨水流加用于粪产碱杆菌热凝胶发酵

热凝胶是粪产碱杆菌(Alcaligenes faecalis)在氮源限制条件下生成的水不溶性胞外多糖,分泌到胞外后就附着在菌体外壁,因此在细胞生长期提高生物量对促进热凝胶合成有重要意义。热凝胶分批发酵时, 起始NH4Cl浓度提高到3.6 g/L时能促进菌体生长和热凝胶合成,但是过量NH4Cl会抑制热凝胶合成,且生物量提高不是很明显。为了进一步提高菌体浓度, 在菌体生长期, 氨水取代NaOH溶液进行流加控制pH为7.0, 随后又用2 mol/L NaOH控制pH 5.6。实验表明, 氨水流加使菌体浓度大大提高,流加24 h使菌体浓度达到18.8 g/L。但是菌体浓度过高也会抑制热凝胶的合成,在氨水流加14 h时,菌体浓度在11.9 g/L左右, 热凝胶产量最高（72 g/L）。     

Enzymatic Studies on the Conversion of Erythritol Fermentation to d-Mannitol Fermentation in n-Alkane-grown Yeast Candida zeylanoides

In the polyol fermentation by Candida zeylanoides KY6166, which occurred preferentially by keeping the pH of medium at acidic side (below 4.0), phosphate ion played a precise role in the conversion of erythritol fermentation to d-mannitol fermentation. Enzymatic studies on the conversion mechanism provided the following evidences.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphate ion.

The enzymes involved in pentosephosphate cycle were considerably depressed in polyol production phase in which intracellular pH ranged from 5.5 to 5.7. Particularly transaldolase responsible for the synthesis of erythrose 4-phosphate and fructose 6-phosphate from glyceraldehyde 3-phosphate plus d-sedoheptulose 7-phosphate was significantly depressed at pH 5.5. Besides, transketolase which participated directly in the formation of erythrose 4-phosphate from fructose 6-phosphate was significantly inhibited by phosphate ion. Glucose 6-phosphate dehydrogenase was slightly inhibited by phosphateion. From these results, the alteration from erythritol fermentation to mannitol fermentation by phosphate ion was explained as the result of the change in the level of erythrose 4-phosphate and fructose 6-phosphate which was caused by the inhibition of transketolase.