从代谢流量分析角度探讨培养条件改变下对放射型根瘤茵WSH2601合成辅酶Q10的影响

分析了放射型根瘤菌(R．radiobacter)WSH2601生物合成辅酶Q10的代谢途径网络,并在溶氧条件改变和培养基中添加玉米浆条件下对辅酶Q10发酵细胞内代谢途径流量变化作定量的分析,结果表明：提高溶氧浓度(20％)5-磷酸核酮糖(RuSP)物流(r7)增加26．6,即糖酵解途径(EMP)途径向磷酸戊糖途径(HMP)转移;添加1％玉米浆r7增加17．2,EMP与HMP途径物流比值与三羧酸循环(TCA)途径物流都下降,而癸异戊烯基焦磷酸(DPP)生成物流通量(绝对值)变化都较小,即辅酶Q10的生物合成更大程度地取决于辅酶Q10生物合成途径中催化DPP的合成和4-羟基苯甲酸(PHB)与DPP的缩合反应的两种关键酶活性。6-磷酸葡萄糖(G6P)节点是辅酶Q10生物合成代谢途径的柔性节点,而丙酮酸节点是半柔性节点。细胞生物量的提高与HMP途径物流增加有关。     

营养条件和流加发酵对放射型根瘤菌(Rhizobium radiobacter)产辅酶Q10的影响

利用放射型根瘤菌WSH2 6 0 1(RhizobiumradiobacterWSH2 6 0 1)重点考察了葡萄糖、蔗糖、玉米浆和蛋白胨、添加物以及流加发酵对细胞生长和产辅酶Q1 0 的影响 ,结果表明 ,葡萄糖和蔗糖适合于生产辅酶Q1 0 的最佳浓度分别为 30g L和 40g L ;辅酶Q1 0 发酵时玉米浆和蛋白胨的最适浓度分别为 11g L和 16g L ;添加蕃茄汁、玉米浆能提高发酵液的生物量 ,玉米浆、异戊醇、L 甲硫氨基酸等能促进辅酶Q1 0 的积累 ;与分批发酵相比 ,在 7L罐上流加蔗糖其细胞生物量 (DCW)和辅酶Q1 0 积累量增加 ,若在流加蔗糖的同时流加适当浓度的玉米浆能显著提高辅酶Q1 0 的产量 ,最大产量达到 5 2 .4mg L ;最大生物量 (DCW)和胞内辅酶Q1 0 含量 (C B值 )分别达到 2 6 .4g L和 2 .38mg g DCW ,比不流加的分批发酵分别提高 5 3 %和 33% ,比只流加蔗糖分别提高 2 4%和 2 6 %。     

光合细菌产辅酶Q10发酵条件的研究

利用均匀设计原理进行实验设计 ,对光合细菌R .capsulatusMT1131产辅酶Q10培养基配方及培养条件进行优化 ,结果当培养基中酵母膏质量浓度为 3 .13g·L- 1 ,硫酸铵 0 .8g·L- 1 ,Mg2 + 0 .6 4g·L- 1 ,Fe2 + 45 .2mg·L- 1 ,Mn2 + 18mg·L- 1 ,Co2 + 16mg·L- 1 ,培养基初始pH值为 7.0时 ,于 30℃ ,光照强度为 2 0 0 0Lx条件下培养 4天后 ,菌体中辅酶Q10质量浓度由 15 .2 13mg·L- 1提高至 2 0 .36 5mg·L- 1 ,产量提高约 33.87%。     

辅酶Q10生物合成与生产研究进展

微生物发酵法是生产辅酶Q10的最佳工艺.辅酶Q10的生物合成途径包括异戊二烯焦磷酸合成、聚十异戊二烯焦磷酸合成、苯环修饰等过程.1-脱氧-D-木酮糖-5-磷酸合成酶、聚十异戊二烯焦磷酸合成酶、对羟基笨甲酸聚十异戊二烯焦磷酸转移酶等是Q10合成的关键酶.生产辅酶Q10的菌种可通过诱变、基因重组和支路敲除等方法获得.氧化还原电位控制、pH控制补料分批发酵、发酵萃取耦合技术等新工艺逐浙应用于辅酶Q10生产.     

产辅酶Q10酵母的发酵条件研究

研究了豆油、豆粉、胡萝卜汁、西红柿汁、烟叶、β-胡萝卜素、桔子皮汁等自然物的添加对酵母发酵生产CoQ10的影响,结果表明它们均能大幅度提高酵母菌中CoQ10的含量。其中豆油、豆粉、西红柿汁、桔子皮汁是富含CoQ10。和胡萝卜素合成途经中的前体物质因而提高了CoQ10的产量;烟叶和β-胡萝卜素阻断了合成β-胡萝卜素的途经从而起到提高CoQ10合成的作用;胡萝卜汁的作用可能两兼而有之。因此可以得出以下结论,微生物中Co10的合成与β-胡萝卜素的合成密切相关。     

促进剂对发酵生产辅酶Q10的影响

研究了几种促进剂对放射型根瘤菌细胞生长及其辅酶Q10发酵的影响。结果表明:在培养基中适量添加VB1、乙酸钠、花生油均可明显提高辅酶Q10的产量,使用正交试验优化后的促进剂组合可使辅酶Q10产量达到50.7 mg/L,比对照组提高33%。     

提高微生物发酵辅酶Q10产量的研究进展

辅酶Q10具有很高的保健和临床应用价值,开发潜力巨大。主要从菌种筛选、发酵条件优化以及提取方法改进三方面介绍了如何提高微生物发酵辅酶Q10产量的一些研究进展。     

紫罗酮和麦角固醇对酵母生长及产辅酶Q10的影响

研究了β-紫罗酮和麦角固醇对酵母生长及产辅酶Q10的影响。研究发现,β-紫罗酮能促进菌体积累辅酶Q10,当培养基中β-紫罗酮的添加量为0·208×10-3mol/L时,菌体中CoQ10的含量提高了28·3%;少量麦角固醇能促进菌体产辅酶Q10,当麦角固醇的添加量为0·15×10-4mol/L时,菌体中CoQ10的含量提高了31·8%,而增加麦角固醇的添加量为0·60×10-4mol/L时则会抑制菌体产辅酶Q10;同时添加β-紫罗酮和麦角固醇时,菌体中CoQ10的含量提高了36·1%。研究结果表明,β-紫罗酮和麦角固醇能有效地促进菌体产辅酶Q10,这为发酵法生产辅酶Q10提供了一条新的研究思路。     

辅酶Q10产生菌的抗性筛选及发酵条件优化

以根癌土壤杆菌(Agrobacterium tumefaciens)WSHAT12为出发菌株,通过硫酸二乙酯诱变,获得遗传稳定性好的抗L-乙硫氨酸(Eth)突变株WSH-E01,通过进一步的诱变处理,获得L-乙硫氨酸和维生素K3(VK3)双抗性突变株WSH-V01,以双抗性突变株WSH-V01为出发菌株,再进行诱变处理,获得一株X-gal利用能力提高的突变株WSH-X01,与出发菌株WSHAT12相比,突变株WSH-X01的辅酶Q10产量提高幅度达50.6%,同时,对突变株WSH-E01的发酵条件进行优化。出发菌株WSHAT12、突变株WSH-E01、WSH-V01和WSH-X01在优化后的发酵条件下辅酶Q10产量分别达到23.1mg/L、26.8mg/L、29.5mg/L和34.8mg/L。     

Association of colony morphology with coenzyme Q(10) production and its enhancement from Rhizobium radiobacter T6102W by addition of isopentenyl alcohol as a precursor

Rhizobium radiobacter T6102 was morphologically purified by the aniline blue agar plates to give two distinct colonies; white smooth mucoid colony (T6102W) and blue rough colony (T6102B). The coenzyme Q(10) (CoQ(10)) was produced just by T6102W, showing 2.0 mg/g of CoQ(10) content, whereas the T6102B did not produce the CoQ(10). All of the used CoQ(10) biosynthetic precursors enhanced the CoQ(10) production by T6102W. Specifically, the supplementation of 0.75 mM isopentenyl alcohol improved the CoQ(10) concentration (19.9 mg/l) and content (2.4 mg/g) by 42% and 40%, respectively.     

Effects of dissolved oxygen concentration and DO-stat feeding strategy on CoQ10 production with Rhizobium radiobacter

The cell growth and CoQ₁₀ (coenzyme Q₁₀) formation of Rhizobium radiobacter WSH2601 were investigated in a 7-1 bioreactor under different dissolved oxygen (DO) concentrations. A maximal CoQ₁₀ content (C/B) of 1.91 mg/g dry cell weight (DCW) and CoQ₁₀ concentration of 32.1 mg/l were obtained at the appropriate DO concentration of 40% (of air saturation). High DO concentration was favourable to the cell growth of Rhizobium radiobacter WSH2601. In order to achieve the maximal yield of CoQ₁₀ production, a new DO-stat feeding strategy was proposed, which significantly improved cell growth and CoQ₁₀ formation. With this strategy, the maximal CoQ₁₀ concentration and DCW reached 51.1 mg/l and 23.9 g/l, respectively, which were 67 and 44.8% higher than those obtained in the batch culture with DO concentration controlled.     

Lactate increases coenzyme Q10 production by Agrobacterium tumefaciens

By the optimization of nitrogen source for coenzyme Q₁₀ (ubiquinone, CoQ₁₀) production in Agrobacterium tumefaciens KCCM 10413 culture, the highest CoQ₁₀ production was achieved in medium containing corn steep powder (CSP). Components for a stimulatory effect on the production of CoQ₁₀ in CSP were screened, and lactate was found to increase dry cell weight (DCW) and the specific CoQ₁₀ content. In a fed-batch culture of A. tumefaciens, supplementation with 1.5 g of lactate l⁻¹ further improved DCW, the specific CoQ₁₀ content, and CoQ₁₀ production by 16.0, 5.8, and 22.8%, respectively. It has been reported that lactate stimulates cell growth and acts as an accelerator driving the tricarboxylic acid (TCA) cycle (Roberto et al. 2002, Biotechnol Let 24:427–431; Matsuoka et al. 1996, Biosci Biotechnol Biochem 60:575–579). In this study, lactate supplementation increased DCW and the specific CoQ₁₀ content in A. tumefaciens culture, probably by accelerating TCA cycle and energy production as reported previously, leading to the increase of CoQ₁₀ production.     

Biotechnological production and applications of coenzyme Q10

An efficient whole cell biotransformation process using Lactobacillus kefir was developed for the asymmetric synthesis of tert-butyl (3R, 5S) 6-chloro-dihydroxyhexanoate, a chiral building block for the HMG-CoA reductase inhibitor. The effects of buffer concentration, temperature, pH and oxygen on the asymmetric reduction were investigated in batch reactions. Improvements in final product concentration and yields of 153% (120 mM) and 79% (0.85 mol/mol) with respect to the batch-process were achieved in an optimised fed-batch process. The pure substrate tert-butyl-6-chloro-3,5-dioxohexanoate was dispersed as microdroplets into the reaction system. This resulted in a space-time yield of 4.7 mmol l⁻¹ h⁻¹. A diastereomeric excess of >99% was measured for (3R, 5S) and (3S, 5S) tert-butyl 6-chloro-dihydroxyhexanoate.     

Effect of culture conditions on producing and uptake hydrogen flux of biohydrogen fermentation by metabolic flux analysis method

In this work, metabolic flux analysis (MFA) method was used to estimate the effects of the culture conditions on both the producing and uptake hydrogen flux inside the cell of Klebsiella pneumoniae ECU-15. The results indicated that higher temperature could reduce the amount of the uptake hydrogen and enhance the hydrogen production from the NADH pathway. Moreover, both the producing hydrogen flux from formate and the uptake hydrogen flux were attained to the maximum at pH 7.0-7.5. The producing hydrogen flux was higher at 5 g/L initial glucose than that of the other concentrations, and the uptake hydrogen flux showed the minimum value under the same condition. The apparent hydrogen generation was caused by the combined action of producing hydrogenase, uptake hydrogenase and bidirectional hydrogenase. These results were helpful to deeply understand the mechanism of the biohydrogen evolving process and establish the suitable molecular strategies for improving hydrogen production.     

Factors affecting broth viscosity and coenzyme Q10 production by Agrobacterium species

Summary In the production of coenzyme Q₁₀ (CoQ₁₀) by Agrobacterium sp. the culture broth becomes highly viscous. In an attempt to improve the production process, the effects of chemical and physical factors on broth viscosity and CoQ₁₀ production were studied, using Agrobacterium sp. KY-8593. A particular concentration ratio of sugar to ammonium-nitrogen (NH₄–N) in the medium could effectively enhance CoQ₁₀ production without increasing broth viscosity. An increase in culture temperature to between 32°C and 34°C lowered broth viscosity without reducing CoQ₁₀ production. NH₄–N concentration and temperature had a correlative effect on broth viscosity. At a temperature of about 33°C, there was a wide range of NH₄–N concentration which was optimal for both broth viscosity and CoQ₁₀ production. In optimal conditions with 8% sugar the apparent broth viscosity was reduced to less than 10 pseudo-cP and CoQ₁₀ production was increased to more than 80 mg/l.     

Plasma coenzyme Q10 response to oral ingestion of coenzyme Q10 formulations

Plasma coenzyme Q10 (CoQ10) response to oral ingestion of various CoQ10 formulations was examined. Both total plasma CoQ10 and net increase over baseline CoQ10 concentrations show a gradual increase with increasing doses of CoQ10. Plasma CoQ10 concentrations plateau at a dose of 2400 mg using one specific chewable tablet formulation. The efficiency of absorption decreases as the dose increases. About 95% of circulating CoQ10 occurs as ubiquinol, with no appreciable change in the ratio following CoQ10 ingestion. Higher plasma CoQ10 concentrations are necessary to facilitate uptake by peripheral tissues and also the brain. Solubilized formulations of CoQ10 (both ubiquinone and ubiquinol) have superior bioavailability as evidenced by their enhanced plasma CoQ10 responses.     

Current state of coenzyme Q10 production and its applications

Coenzyme Q₁₀ (CoQ₁₀), an obligatory cofactor in the aerobic respiratory electron transfer for energy generation, is formed from the conjugation of a benzoquinone ring with a hydrophobic isoprenoid chain. CoQ₁₀ is now used as a nutritional supplement because of its antioxidant properties and is beneficial in the treatment of several human diseases when administered orally. Bioprocesses have been developed for the commercial production of CoQ₁₀ because of its increased demand, and these bioprocesses depend on microbes that produce high levels of CoQ₁₀ naturally. However, as knowledge of the biosynthetic enzymes and the regulatory mechanisms modulating CoQ₁₀ production increases, approaches arise for the genetic engineering of CoQ₁₀ production in Escherichia coli and Agrobacterium tumefaciens. This review focused on approaches for CoQ₁₀ production, strategies used to engineer CoQ₁₀ production in microbes, and potential applications of CoQ₁₀.