酸奶生产菌株的诱变选育

通过诱变选育高产酸力菌株;首先采用紫外线和亚硝基胍对乳酸菌进行单因素诱变,确定紫外线照射剂量为150s,并得到一株产酸力为57.85°T的菌株,比原菌株产酸力提高了3.05%;NTG的诱变剂量是0.3mg/mL,处理时间为60min,得到产酸力为58.02°T的菌株,较出发菌株提高了6.52%。接着以紫外线和NTG对高产酸菌株进行了2轮复合诱变,最终得到产酸力达83.66°T的诱变株,较出发菌株产酸力提高了30.64%。     

乳酸菌低温菌株的复合诱变选育

【目的】采用复合诱导的方法,选育出能在低温条件下性状稳定、生长良好且具有工业生产价值的低温乳酸菌菌株。【方法】使用紫外照射和亚硝基胍处理的方法对分离的Q1菌株进行诱变处理,将选育得到的低温菌株Q1-4-6与出发菌株Q1在15°C、20°C、37°C下,比较其生长速率、产酸量和糖降解量。【结果】从玛曲牧民自制酸奶中分离得到一株乳酸菌Q1,根据其形态学特征、生理生化特性,同时结合16SrDNA序列分析结果,将其鉴定为肠球菌属棉籽糖肠球菌(Enterococcus raffinosus)。经过多轮诱变选育出一株能在低温下良好生长、遗传性状稳定的菌株Q1-4-6,将出发菌株与诱变菌株在15°C、20°C比较其生长、产酸和糖降解量发现,诱变菌株均好于出发菌株。在37°C的自然条件下,诱变菌株和出发菌株的生长速率差异不大,但均略高于出发菌株。在15°C培养发现,诱变菌株生长速率、产酸量均高于购买菌株干酪乳杆菌(CL04)和嗜热链球菌(CL05)。【结论】通过UV+NTG复合诱变,最终选育出一株在低温下生长良好、遗传性状稳定的菌株Q1-4-6。     

紫外线与亚硝酸钠复合诱变选育L-组氨酸产生菌

以1株谷氨酸棒杆菌(Corynebacterium glutamicum)S_6作为出发菌株,利用亚硝酸钠(NaNO_2)、紫外线(UV)进行诱变,通过实验证明亚硝酸钠诱变时间在180 s后致死率达到80%,紫外线在照射30 s后致死率达到80%。诱变后的突变菌株经6-巯基嘌呤结构类似物的抗性平板筛选,最终筛得3株菌,Y_1产L-组氨酸量达到331 mg/L,比出发菌株S_6高了5.08%,Z_2产L-组氨酸量达到325 mg/L,比出发菌株S_6高了1.9%,F_6产L-组氨酸量达到330 mg/L,比出发菌株S_6高了7.14%。结果显示,经紫外线与亚硝酸钠复合诱变后的菌株F_6产L-组氨酸的产量最高,比亚硝酸钠诱变后的菌株产L-组氨酸量提高2.06%,比紫外线诱变后的菌株产L-组氨酸量提高5.24%。     

L-谷氨酸温度敏感突变株的选育

采用黄色短杆菌TJ1为出发菌株,根据代谢控制发酵原理,利用紫外线、硫酸二乙酯进行诱变,定向选育出具有寡霉素抗性、谷氨酸氧肟酸盐抗性的温度敏感突变株TMGO106。然后,以温度敏感突变株TMGO106和产酸率高（10.5%以上）的天津短杆菌TG961为新株,通过原生质体融合技术,成功地选育出了产酸率高的融合子CN1021（13．6g/dl,糖酸转化率达60％）,在6m^3发酵罐上中试其L－谷氨酸产量达14．6％,糖酸转化率达62．8％,并且该菌株系温度敏感型菌株,可用于谷氨酸强度发酵。     

L-精氨酸高产菌的选育及基于代谢流量分布的育种机制

从分析钝齿棒杆菌(Corynebacterium crenatum)的精氨酸合成途径入手,提出了一种通过选育脯氨酸结构类似物抗性突变株以提高其精氨酸合成能力的育种思路。采用亚硝基胍(NTG)诱变处理出发菌株YD8(His-,SGr1.2 mg/mL,D-Argr15 mg/mL),经含15 mg/mL的脯氨酸结构类似物S-甲基半胱氨酸(S-MC)的抗性筛选获得精氨酸高产突变株YDM403(His-,SGr1.2 mg/mL,D-Argr15 mg/mL,S-MCr15 mg/mL),产酸水平可达29.4 g/L,较出发菌株YD8的产酸高出55.0%。通过代谢流量分布分析了菌株YD8和YDM403代谢网络的变化,结果表明,菌株YD8可能存在顺序反馈抑制作用,出发株YD8解除了Arg对Glu到Arg的反馈抑制,而YDM403又解除了Pro对Glu到Pro的反馈抑制,从而使中间物Glu累积量下降而对-αKG到Glu不再有反馈抑制,其通量提高,与此同时从Glu向Arg的代谢通量也相应增加。     

离子注入结合紫外线及~(60)Co-γ射线诱变选育碱性果胶酸裂解酶高产菌株的研究

以碱性果胶酸裂解酶产生菌芽孢杆菌WZ008为出发菌株,经形态鉴定和16S鉴定为类芽孢杆菌,命名为Paenibacillus sp.WZ008,通过N~+注入诱变、紫外线诱变、~(60)Co-γ射线诱变等多次反复诱变,选育得到一株产碱性果胶酸裂解酶性能稳定且酶活明显提高的突变株,其酶活为97.8U/mL,比出发菌株产碱性果胶酸裂解酶能力提高了1.04倍。     

离子注入L-乳酸产生菌诱变选育研究

采用离子注入技术对一株产L -乳酸的干酪乳杆菌L110Z进行诱变处理。结果表明 ,在离子注入剂量为 1× 10 14 N+ /cm2 时 ,诱变效果较好 ,从正变菌株中反复筛选 ,得到了一株产酸量高的菌株L110Z5 ,产酸量达到了 92g/L ,比出发菌株产酸提高了 18.0 % ,经过连续传代试验 ,其遗传性状稳定 ,表明L110Z5是一株极具工业化前景的高产菌     

L-异亮氨酸高产菌选育及其培养基优化

旨在诱变选育L-异亮氨酸高产菌,并探索突变株最佳发酵条件。利用传统化学诱变结合常压室温等离子体生物诱变体系对实验室保藏的Brevibacterium flavum I-12进行逐级诱变,选育2-噻唑丙氨酸(2-TA)和磺胺胍(SG)高抗性和在琥珀酸平板上能快速生长的突变菌株。随后,在单因素实验的基础上,利用响应面设计优化出目的突变株摇瓶发酵培养基组分的最佳参数水平。结果显示,经过一系列诱变和筛选,成功选育出一株在40 g/L的2-TA和5 g/L的SG,且以琥珀酸为唯一碳源的培养基上快速生长突变株,命名为B. flavum TA-6,该菌株产酸达26.2±0.5 g/L,比出发菌株提高了44.75%,而副产物L-缬氨酸和L-亮氨酸积累量明显降低。经响应面法优化发酵条件后,突变株产酸可达27.8±0.5 g/L,比优化前提高了6.1%。通过传统化学诱变结合ARTP生物诱变体系,成功选育出一株杂酸降低的L-异亮氨酸高产菌TA-6,该菌株具有潜在生产应用价值。     

复合诱变选育高产GSH的菌株

目的:选育出GSH的高产菌株.方法:以产GSH的产朊假丝酵母(Candida utilis)为出发菌株,利用紫外-超声波复合诱变.结果:筛选得到ZnCl2抗性突变株UU3,GSH菌体含量为53.50mg·g-1,比出发菌株提高137.03%.结论:突变株UU3遗传性能稳定,可做进一步研究.     

诱变选育具有磺胺胍抗性的L-色氨酸产生菌的研究

采用紫外线、甲基磺酸乙酯(EMS)及半导体激光诱变的方法,处理产色氨酸谷氨酸棒状杆菌GA22(Phe-+Tyr-+5MTr)选育磺胺胍抗性菌株以提高L-色氨酸的产量。用紫外照射20S、EMS处理40 min及半导体激光辐照16 min,筛选得到抗性突变株GA507(Phe-+Tyr-+5MTr+SGr),产色氨酸的量达到5.35 g/L,较出发菌株提高50.3%,并具有良好的遗传稳定性。     

定向选育衣康酸高产菌株的研究

以衣康酸生产菌土曲霉Ａ９００２为出发菌株,经紫外线及亚硝基胍复合诱变后再行定向选育,即在以衣康酸为唯一碳源的培养基中富集不能同化衣康酸的菌种,将将它们涂布在含乌头酸酶抑制剂（单氟醋酸）的高糖、高衣康酸平板培养基上,最后从中而筛选出一支衣康酸氧化酶弱,乌头酸酶活强,并耐自身代谢产物的高产突变株Ａ９００３。此菌株在摇瓶培养７２ｈ后,产酸为９．２％,转化率为５８．１％,在１００ｍ＾３发酵罐生产性试验中,     

衣康酸生产菌种的定向选育和产酸条件的研究

通过紫外线—高温复合诱变处理衣康酸生产菌株土曲霉构Aspergillus terreus As3.2811,用以琥珀酸为唯一碳源的选择性乎板定向筛选高产菌株,获得产酸率较其亲株提高了5倍以上的突变株。用正交试验的方法对突变株的适宜产酸条件进行了研究,通过分批补糖发酵可提高其产酸率高达39．92％。     

Enhanced production of itaconic acid from corn starch and market refuse fruits by genetically manipulated Aspergillus terreus SKR10

A potent itaconic acid producing strain, Aspergillus terreus SKR10, was isolated from horticulture waste. Market refuse, apple and banana, were explored as novel substrates for itaconic acid production with yields of 20+/-2.0 and 20.0+/-1.0 g l(-1), respectively. Itaconic acid yields of 28.5+/-2.2 and 31.0+/-1.7 g l(-1) were obtained with acid and alpha-amylase hydrolyzed corn starch. The efficiency of itaconic acid production by this wild type strain was improved by ultraviolet, chemical and mixed mutagenic treatments. Two high itaconic acid yielding mutants, N45 and UNCS1 were obtained by gradient plating. These two mutants were capable of producing twice the yield of itaconic acid as the parent strain.     

Efficient Itaconic acid production via protein–protein scaffold introduction between GltA,AcnA, and CadA in recombinant Escherichia coli

Itaconic acid, which is a promising organic acid in synthetic polymers and some base-material production, has been produced by Aspergillus terreus fermentation at a high cost. The recombinant Escherichia coli that contained the cadA gene from A. terreus can produce itaconic acid but with low yield. By introducing the protein–protein scaffold between citrate synthesis, aconitase, and cis-aconitase decarboxylase, 5.7 g/L of itaconic acid was produced, which is 3.8-fold higher than that obtained with the strain without scaffold. The optimum pH and temperature for itaconic acid production were 8.5 and 30°C, respectively. When the competing metabolic network was inactivated by knock-out mutation, the itaconic acid concentration further increased, to 6.57 g/L.     

Reduced by-product formation and modified oxygen availability improve itaconic acid production in Aspergillus niger

Aspergillus niger has an extraordinary potential to produce organic acids as proven by its application in industrial citric acid production. Previously, it was shown that expression of the cis-aconitate decarboxylase gene (cadA) from Aspergillus terreus converted A. niger into an itaconic acid producer (Li et al., Fungal Genet Bio 48: 602–611, 2011). After some initial steps in production optimization in the previous research (Li et al., BMC biotechnol 12: 57, 2012), this research aims at modifying host strains and fermentation conditions to further improve itaconic acid production. Expression of two previously identified A. terreus genes encoding putative organic acid transporters (mttA, mfsA) increased itaconic acid production in an A. niger cis-aconitate decarboxylase expressing strain. Surprisingly, the production did not increase further when both transporters were expressed together. Meanwhile, oxalic acid was accumulated as a by-product in the culture of mfsA transformants. In order to further increase itaconic acid production and eliminate by-product formation, the non-acidifying strain D15#26 and the oxaloacetate acetylhydrolase (oahA) deletion strain AB 1.13 ?oahA #76 have been analyzed for itaconic acid production. Whereas cadA expression in AB 1.13 ?oahA #76 resulted in higher itaconic acid production than strain CAD 10.1, this was not the case in strain D15#26. As expected, oxalic acid production was eliminated in both strains. In a further attempt to increase itaconic acid levels, an improved basal citric acid-producing strain, N201, was used for cadA expression. A selected transformant (N201CAD) produced more itaconic acid than strain CAD 10.1, derived from A. niger strain AB1.13. Subsequently, we have focused on the influence of dissolved oxygen (D.O.) on itaconic acid production. Interestingly, reduced D.O. levels (10–25 %) increased itaconic acid production using strain N201 CAD. Similar results were obtained in strain AB 1.13 CAD + HBD2.5 (HBD 2.5) which overexpressed a fungal hemoglobin domain. Our results showed that overexpression of the hemoglobin domain increased itaconic acid production in A. niger at lower D.O. levels. Evidently, the lower levels of D.O. have a positive influence on itaconic acid production in A. niger strains.     

Production of (S)-(+)-citramalic acid from itaconic acid by resting cells of Alcaligenes denitrificans strain MCI2775

(S)-(+)-Citramalic-acid-producing activity in microorganisms was studied with resting cells in a reaction mixture containing itaconic acid. Itaconic-acid-utilizing bacteria were found to produce (S)-(+)-citramalic acid from itaconic acid. The strain, which showed the best productivity among those studied, was identified taxonomically as Alcaligenes denitrificans strain MCI2775. (S)-(+)-Citramalic acid produced by this strain was present in a 99.9% enantiometric excess. The culture and reaction conditions for the production were optimized for this strain. Addition of Mn²⁺, d-pantothenic acid and l-leucine to the culture medium enhanced the (S)-(+)-citramalic acid-producing activity. Under optimal conditions, 27 g (S)-(+)-citramalic acid/l was produced in 30 h. The yield to itaconic acid added was 69.0 mol%. Correspondence to: Y. Asano     

Itaconic acid production by immobilizedAspergillus terreus on sucrose medium

Summary The itaconic acid production by immobilizedAspergillus terreus TTK 200-5-3 mycelium was optimized in shake flask fermentations using statistical experimental design and empirical modelling. The maximum itaconic acid concentration was calculated to be 13.3 g/l in the investigated experimental area when initial sucrose concentration was 10%, ammonium nitrate concentration 0.275% and initial pH 3. The itaconic acid product concentration using immobilized mycelium was about double of that obtained with the free mycelium.     

Itaconic acid production by immobilized Aspergillus terreus with varied metal additions

Summary The effect of trace and alkaline metals on itaconic acid production by polyurethane-foam-immobilized Aspergillus terreus was examined in repeated shake-flask cultures according to a statistical experimental design. An increase in the glucose or copper concentration increased the need for earth alkaline metals. The experimentally obtained highest itaconic acid concentration of 51 g/l from 15% glucose with a total productivity of 3.67 g/l per day was reached during the first 14-day batch fermentation. In the fourth batch the calculated highest itaconic acid concentration of 19 g/l was reached with 25% glucose, 5 g/l of magnesium sulphate, 13 mg/l of copper sulphate and 10 g/l of calcium chloride. The immobilization of the mycelium increased the itaconic acid concentration obtained by as much as eightfold.Offprint requests to: H. Kautola     

Preparation of Radioactive Gibberellins A20, A5 and A8

Many yeasts were isolated from natural sources in the tropics and subtropics by enrichment culture technique, using medium which contained a surfactant. The medium was acidified with citric acid. A strain S–10 belonging to the genus Candida was found to produce itaconic acid. Under suitable conditions in shake culture, a mutant derived from this strain produced the acid at about 35 % yield on the basis of glucose supplied.     

Catabolism of itaconic acid by preformed mats of aspergillus terreus

Summary The breakdown of itaconic acid by preformed mats of a local strain of Aspergillus terreus was studied using the zone-strip technique. Low p_H values restrained the uptake of itaconic acid and the accumulation of various other acids while higher p_H values exerted an opposite effect. The results obtained when using various enzyme inhibitors were those anticipated on basis of the direct transformation of itaconic to aconitic acid through the fixation of CO₂ and of the existence of the T.A.C. (tricarboxylic acid cycle) as a main metabolic channel operating in this organism.