应用基因组改组选育
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摘要
对本实验室自主筛选的琥珀酸产生菌Actinobacillus succinogenes CGMCC 1593进行了钠离子耐受性研究,将菌株分别进行X-射线诱变、紫外线诱变和EMS化学诱变,筛选得到3株发酵性能有改良的菌株(X-8、UV-17及SE-6),将其与实验室保存的经NTG诱变的高产菌株(SF-9)进行基因组改组方法选育高产耐钠菌株,获得4株稳定性较好的耐高浓度钠离子高产突变株,并对其中一株(F3-10)进行了初步发酵性能研究。
通过菌种特性研究,确定采用菌龄5 h的菌体进行诱变处理,并确定了筛选平板中NaCl临界浓度为0.8 mol/L;通过致死曲线的测定,确定了X-射线诱变照射时间40 min,紫外诱变照射时间20 s,EMS诱变处理浓度为2.0%,处理时间为20 min。出发菌株分别经X-射线诱变、紫外线诱变和EMS诱变,采用高浓度钠离子平板进行初筛,然后进行厌氧瓶发酵HPLC复筛获得3株钠离子耐受性较好、琥珀酸产量较高的菌株X-8、UV-17及SE-6。
对菌种原生质体制备及再生的影响因素进行了考察,确定采用菌龄为5 h的菌体,酶解时溶菌酶浓度为0.1 mg/mL,37℃酶解30 min,渗透压稳定剂为0.3 mol/L的蔗糖,再生培养方式采用双层平板再生。对原生质体灭活标记条件进行了研究,确定紫外灭活5 min,55℃热灭活25 min后原生质体灭活率达100%。对原生质体融合条件进行了研究,确定融合条件为采用浓度为40%的PEG6000为助融剂,融合温度为37℃,融合时间2 min,此时融合率达2.78×10-5。
采用多母本原生质体递近融合技术,经过三轮改组后筛选得到4株稳定性较好的高产突变株F3-2、F3-10、F3-13、F3-15,其中一株F3-10在厌氧瓶发酵培养基中发酵48 h,琥珀酸积累39.7 g/L,较原始菌株SW0580提高了36.0%,而在含0.2 mol/LNaCl培养基中发酵时琥珀酸产量为30.3 g/L,较原始菌株SW0580的17.6 g/L提高了72.1%;以甘蔗糖蜜为原料在5 L发酵罐中采用Na2CO3控制发酵pH厌氧发酵48 h,琥珀酸积累40.6 g/L,较出发菌SW0580(26.8 g/L)提高56.2%。F3-10菌株的耐钠和发酵产酸性能较原菌株有明显改善。
初步考察了突变株的发酵性能,在5 L发酵罐中分别以甘蔗糖蜜、麦芽糖糖浆及葡萄糖为碳源进行琥珀酸发酵,发现利用甘蔗糖蜜作碳源时发酵产琥珀酸效果最好;分别考察了酵母膏、玉米浆浓度对发酵罐中发酵产琥珀酸的影响,发现培养基中不添加酵母膏时,琥珀酸产量明显降低,增大培养基中玉米浆浓度时,琥珀酸产量相应提高。以麦芽糖浆为原料在25 L发酵罐中对琥珀酸发酵进行了放大试验,发酵48 h,琥珀酸积累33.2 g/L,基本能维持5 L发酵罐中以麦芽糖浆为原料时的产酸水平。
In this thesis, Na+-tolerance characteristic of Actinobacillus succinogenes CGMCC 1593 isolated by our laboratory was studied. Three Na+-tolerance mutant strains (X-8, UV-17, SE-6), were obtained by X-ray, ultraviolet irradiation and EMS mutagenesis respectively. These mutant strains, as well as strain SF-9 which was obtained by NTG mutagenesis in previous work were used as a parent library in recursive fusion. Through there rounds of genome shuffling, four mutant strains with high yield and good Na+-tolerance were obtained, and the fermentation performance of one strain F3-10 was studied.
After the characteristic study of strain SW0580, screening plate containing 0.8 mol/L NaCl and 5 h pre-cultured cells were used in the mutation study. The mutation conditions were as follows: X-ray treatment for 40 min, ultraviolet irradiation for 20 s, 2.0% EMS for 20 min. The mutant strains with high yield, namely X-8, UV-17 and SE-6, were obtained by X-ray, ultraviolet irradiation and EMS mutagenesis, respectively.
The optimized conditions for protoplast preparation and regeneration were as follows: the cells being cultured for 5 h; lysozyme concentration of 0.1 mg/mL; incubation at 37℃for 30 min to allow cell wall lysis; 0.3 mol/L of sucrose as osmotic stabilizer; and double-lay plate for regeneration. The protoplast inactivation methods were determined to be ultraviolet irradiation for 5 min and heat treatments for 25 min. Under the optimized conditions for protoplast fusion, 40% PEG 6000, incubation at 37℃for 2 min, the fusion rate was 2.78×10-5.
Four mutant strains namely F3-2, F3-10, F3-13 and F3-15 with high yield were obtained by three rounds of genome shuffling. Using strain F3-10, succinic acid concentration of 39.7 g/L and 30.3 g/L were obtained after 48 h of fermentation in medium containing 0 and 0.2 mol/L NaCl in anaerobic bottles respectively, this is an increase of 36.0% and 72.1% respectively. When batch fermentation was conducted using Na2CO3 for pH control in 5-L bioreactor using cane molasses, the production of succinic acid in 48 h(40.6 g/L)was increased by 56.2% compared with that of wild strain (26.8 g/L). The fermentation performance and Na+-tolerance of the mutant strain F3-10 were significantly improved, and F3-10 showed genetic stability.
The primary study on fermentation performance of the strain F3-10 was carried out. Batch fermentation in 5-L bioreactor using cane molasses, maltose syrup and glucose as carbon sources was conducted, highest succinic acid yield was observed using cane molasses as carbon source. The effect of yeast extract and corn steep liquor on succinic acid fermentation was also investigated. The results showed that succinic acid yield decreased evidently without yeast extract, and the yield of succinic acid increased with the addition of corn steep liquor. Fermentation using maltose syrup in a 25 L bioreactor was also carried out, and the final succinic acid concentration of 33.2 g/L was achieved at 48 h, which was comparable with that in 5-L bioreactor.
通过菌种特性研究,确定采用菌龄5 h的菌体进行诱变处理,并确定了筛选平板中NaCl临界浓度为0.8 mol/L;通过致死曲线的测定,确定了X-射线诱变照射时间40 min,紫外诱变照射时间20 s,EMS诱变处理浓度为2.0%,处理时间为20 min。出发菌株分别经X-射线诱变、紫外线诱变和EMS诱变,采用高浓度钠离子平板进行初筛,然后进行厌氧瓶发酵HPLC复筛获得3株钠离子耐受性较好、琥珀酸产量较高的菌株X-8、UV-17及SE-6。
对菌种原生质体制备及再生的影响因素进行了考察,确定采用菌龄为5 h的菌体,酶解时溶菌酶浓度为0.1 mg/mL,37℃酶解30 min,渗透压稳定剂为0.3 mol/L的蔗糖,再生培养方式采用双层平板再生。对原生质体灭活标记条件进行了研究,确定紫外灭活5 min,55℃热灭活25 min后原生质体灭活率达100%。对原生质体融合条件进行了研究,确定融合条件为采用浓度为40%的PEG6000为助融剂,融合温度为37℃,融合时间2 min,此时融合率达2.78×10-5。
采用多母本原生质体递近融合技术,经过三轮改组后筛选得到4株稳定性较好的高产突变株F3-2、F3-10、F3-13、F3-15,其中一株F3-10在厌氧瓶发酵培养基中发酵48 h,琥珀酸积累39.7 g/L,较原始菌株SW0580提高了36.0%,而在含0.2 mol/LNaCl培养基中发酵时琥珀酸产量为30.3 g/L,较原始菌株SW0580的17.6 g/L提高了72.1%;以甘蔗糖蜜为原料在5 L发酵罐中采用Na2CO3控制发酵pH厌氧发酵48 h,琥珀酸积累40.6 g/L,较出发菌SW0580(26.8 g/L)提高56.2%。F3-10菌株的耐钠和发酵产酸性能较原菌株有明显改善。
初步考察了突变株的发酵性能,在5 L发酵罐中分别以甘蔗糖蜜、麦芽糖糖浆及葡萄糖为碳源进行琥珀酸发酵,发现利用甘蔗糖蜜作碳源时发酵产琥珀酸效果最好;分别考察了酵母膏、玉米浆浓度对发酵罐中发酵产琥珀酸的影响,发现培养基中不添加酵母膏时,琥珀酸产量明显降低,增大培养基中玉米浆浓度时,琥珀酸产量相应提高。以麦芽糖浆为原料在25 L发酵罐中对琥珀酸发酵进行了放大试验,发酵48 h,琥珀酸积累33.2 g/L,基本能维持5 L发酵罐中以麦芽糖浆为原料时的产酸水平。
In this thesis, Na+-tolerance characteristic of Actinobacillus succinogenes CGMCC 1593 isolated by our laboratory was studied. Three Na+-tolerance mutant strains (X-8, UV-17, SE-6), were obtained by X-ray, ultraviolet irradiation and EMS mutagenesis respectively. These mutant strains, as well as strain SF-9 which was obtained by NTG mutagenesis in previous work were used as a parent library in recursive fusion. Through there rounds of genome shuffling, four mutant strains with high yield and good Na+-tolerance were obtained, and the fermentation performance of one strain F3-10 was studied.
After the characteristic study of strain SW0580, screening plate containing 0.8 mol/L NaCl and 5 h pre-cultured cells were used in the mutation study. The mutation conditions were as follows: X-ray treatment for 40 min, ultraviolet irradiation for 20 s, 2.0% EMS for 20 min. The mutant strains with high yield, namely X-8, UV-17 and SE-6, were obtained by X-ray, ultraviolet irradiation and EMS mutagenesis, respectively.
The optimized conditions for protoplast preparation and regeneration were as follows: the cells being cultured for 5 h; lysozyme concentration of 0.1 mg/mL; incubation at 37℃for 30 min to allow cell wall lysis; 0.3 mol/L of sucrose as osmotic stabilizer; and double-lay plate for regeneration. The protoplast inactivation methods were determined to be ultraviolet irradiation for 5 min and heat treatments for 25 min. Under the optimized conditions for protoplast fusion, 40% PEG 6000, incubation at 37℃for 2 min, the fusion rate was 2.78×10-5.
Four mutant strains namely F3-2, F3-10, F3-13 and F3-15 with high yield were obtained by three rounds of genome shuffling. Using strain F3-10, succinic acid concentration of 39.7 g/L and 30.3 g/L were obtained after 48 h of fermentation in medium containing 0 and 0.2 mol/L NaCl in anaerobic bottles respectively, this is an increase of 36.0% and 72.1% respectively. When batch fermentation was conducted using Na2CO3 for pH control in 5-L bioreactor using cane molasses, the production of succinic acid in 48 h(40.6 g/L)was increased by 56.2% compared with that of wild strain (26.8 g/L). The fermentation performance and Na+-tolerance of the mutant strain F3-10 were significantly improved, and F3-10 showed genetic stability.
The primary study on fermentation performance of the strain F3-10 was carried out. Batch fermentation in 5-L bioreactor using cane molasses, maltose syrup and glucose as carbon sources was conducted, highest succinic acid yield was observed using cane molasses as carbon source. The effect of yeast extract and corn steep liquor on succinic acid fermentation was also investigated. The results showed that succinic acid yield decreased evidently without yeast extract, and the yield of succinic acid increased with the addition of corn steep liquor. Fermentation using maltose syrup in a 25 L bioreactor was also carried out, and the final succinic acid concentration of 33.2 g/L was achieved at 48 h, which was comparable with that in 5-L bioreactor.
引文
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