应用基因组改组技术选育真菌α-淀粉酶高产菌株
摘要
以实验室保藏的米曲霉FS-16为原始出发菌株,通过传统诱变方法构建正突变库。通过致死曲线的测定,确定紫外诱变照射剂量为160s,微波诱变辐射剂量为25s。出发菌株分别经紫外诱变、微波诱变,采用淀粉平板透明圈初筛、液体发酵复筛,最终从2000多个突变株中筛选到α-淀粉酶产量较高稳定性较好的突变株FU-8、FU-26、FW-111及FW-130。
通过单因素实验获得米曲霉原生质体制备和再生的最佳条件：菌龄15h,酶解温度28℃、裂解时间2.5h、0.6mol/L NaCl作为渗透压稳定剂,含0.6mol/L NaCl的PDA培养基为再生培养基。通过响应面分析法对破壁酶系统进行研究,获得最佳酶系组成为：蜗牛酶0.8%、纤维素酶0.8%、溶菌酶0.5%。
通过米曲霉原生质体灭活实验得出,用紫外线处理180S或热处理12min,米曲霉原生质体即可达到100%灭活；原生质体融合条件研究表明最适融合条件为：PEG30%,Ca2+0.02mol/L,融合时间10min,融合温度30℃。以原始菌株FS-16和诱变筛选出的突变株FU-8、FU-26、FW-111及FW-130作为亲本,采用基因组改组技术,经过三轮递推式融合后筛选得到一株能够稳定遗传的改组菌株F3-542,其酶活较原始菌株提高了61.5%,发酵周期也相对缩短了24h,达到了选育高产菌株的目的。
对改组菌株F3-542的发酵条件进行优化,首先通过单因素实验可知最佳碳源为玉米粉,最佳有机氮源为牛肉膏,最佳无机氮源为硝酸钠。最适培养条件为：初始pH值为6；接种量为10%；装液量为50mL/250mL三角瓶。通过均匀设计法对培养基配方进行进一步优化,获得它们的最佳组合(g/L)：玉米粉56,牛肉膏26,NaNO34.5,K2HP041.4,MgS041.5,FeS040.005。整体优化后,改组菌F3-542从最初酶活力为3920U/mL提高至5147.5U/mL,比最初酶活力提高了31.3%。
Original strain Aspergillus oryzae FS-16 preserved in our laboratory was treated with ultraviolet and microwaves radiation. The mutation conditions were as follows, UV for 160s and microwaves for 25s. Prescreening method was starch plate hydrolysis spot method. Rescreening method was liquid fermentation. The mutant strains with high yield, namely FU-8、FU-26 were obtained by UV mutagenesis and the mutant strains named FW-111, FW-130 were obtained by microwave irradiation
Through single factor test, the optimized conditions for protoplast preparation and regeneration were as follows:the cells being cultured for 15 h, incubation at 28℃for 2.5h to allow cell wall lysis; 0.6 mol/L NaCl as osmotic stabilizer, and PDA plate with 0.6mol/L NaCl for regeneration. Through the response surface analysis method, the optimized enzyme composition for breaking cell wall was as follows:0.8% cellulose, 0.8% snailase and 0.5% lysozyme.
The protoplast inactivation methods were determined to be ultraviolet irradiation for 180s and heat treatments for 12 min. The effects of the PEG concentration, the Ca2+ concentration, the fusion time and temperature on protoplast fusion were studied. The fusion was undergone in the fusion system of 30% PEG containing 0.02mol/L Ca2+ and processed for 10 min at 30℃
The four strains we obtained from UV and microwaves mutation and FS-16 preserved in our lab were selected as parent strains for genome shuffling. After three cycles of recursive protoplast fusion, a shuffled strain named F3-542 was selected. The productionα-amylase activity of F3-542 was increased 61.5% more than that of FS-16. At the same time, the fermentation period was relatively shortened 24h. The results of continues culture and liquid fermentation showed the strains had stable hereditary property. On the whole the breeding purpose was achieved successfully.
The primary studies on fermentation performance of the strain F3-542 was carried out.Through single factor test, the result showed that the best carbon was corn flour, the optimum nitrogen were beef extract and NaNO3; the best fermentation conditions were obtained based on the single factor test, it showed the optimal amount of inoculation was 10%, the optimum initial pH was 6 and the optimal liquid volume were 50mL culture in 250mL shake flasks.The uniform design study showed that optimal fermentation medium components (g/L) were corn flour 56,beef extract 26, NaNO3 4.5, K2HPO4 1.4, MgSO4 1.5, FeSO4 0.005. After the optimization, the activity of F3-542 was up to 5147.5U/mL, of which was increased 31.3% more than the activity before optimization.
通过单因素实验获得米曲霉原生质体制备和再生的最佳条件：菌龄15h,酶解温度28℃、裂解时间2.5h、0.6mol/L NaCl作为渗透压稳定剂,含0.6mol/L NaCl的PDA培养基为再生培养基。通过响应面分析法对破壁酶系统进行研究,获得最佳酶系组成为：蜗牛酶0.8%、纤维素酶0.8%、溶菌酶0.5%。
通过米曲霉原生质体灭活实验得出,用紫外线处理180S或热处理12min,米曲霉原生质体即可达到100%灭活；原生质体融合条件研究表明最适融合条件为：PEG30%,Ca2+0.02mol/L,融合时间10min,融合温度30℃。以原始菌株FS-16和诱变筛选出的突变株FU-8、FU-26、FW-111及FW-130作为亲本,采用基因组改组技术,经过三轮递推式融合后筛选得到一株能够稳定遗传的改组菌株F3-542,其酶活较原始菌株提高了61.5%,发酵周期也相对缩短了24h,达到了选育高产菌株的目的。
对改组菌株F3-542的发酵条件进行优化,首先通过单因素实验可知最佳碳源为玉米粉,最佳有机氮源为牛肉膏,最佳无机氮源为硝酸钠。最适培养条件为：初始pH值为6；接种量为10%；装液量为50mL/250mL三角瓶。通过均匀设计法对培养基配方进行进一步优化,获得它们的最佳组合(g/L)：玉米粉56,牛肉膏26,NaNO34.5,K2HP041.4,MgS041.5,FeS040.005。整体优化后,改组菌F3-542从最初酶活力为3920U/mL提高至5147.5U/mL,比最初酶活力提高了31.3%。
Original strain Aspergillus oryzae FS-16 preserved in our laboratory was treated with ultraviolet and microwaves radiation. The mutation conditions were as follows, UV for 160s and microwaves for 25s. Prescreening method was starch plate hydrolysis spot method. Rescreening method was liquid fermentation. The mutant strains with high yield, namely FU-8、FU-26 were obtained by UV mutagenesis and the mutant strains named FW-111, FW-130 were obtained by microwave irradiation
Through single factor test, the optimized conditions for protoplast preparation and regeneration were as follows:the cells being cultured for 15 h, incubation at 28℃for 2.5h to allow cell wall lysis; 0.6 mol/L NaCl as osmotic stabilizer, and PDA plate with 0.6mol/L NaCl for regeneration. Through the response surface analysis method, the optimized enzyme composition for breaking cell wall was as follows:0.8% cellulose, 0.8% snailase and 0.5% lysozyme.
The protoplast inactivation methods were determined to be ultraviolet irradiation for 180s and heat treatments for 12 min. The effects of the PEG concentration, the Ca2+ concentration, the fusion time and temperature on protoplast fusion were studied. The fusion was undergone in the fusion system of 30% PEG containing 0.02mol/L Ca2+ and processed for 10 min at 30℃
The four strains we obtained from UV and microwaves mutation and FS-16 preserved in our lab were selected as parent strains for genome shuffling. After three cycles of recursive protoplast fusion, a shuffled strain named F3-542 was selected. The productionα-amylase activity of F3-542 was increased 61.5% more than that of FS-16. At the same time, the fermentation period was relatively shortened 24h. The results of continues culture and liquid fermentation showed the strains had stable hereditary property. On the whole the breeding purpose was achieved successfully.
The primary studies on fermentation performance of the strain F3-542 was carried out.Through single factor test, the result showed that the best carbon was corn flour, the optimum nitrogen were beef extract and NaNO3; the best fermentation conditions were obtained based on the single factor test, it showed the optimal amount of inoculation was 10%, the optimum initial pH was 6 and the optimal liquid volume were 50mL culture in 250mL shake flasks.The uniform design study showed that optimal fermentation medium components (g/L) were corn flour 56,beef extract 26, NaNO3 4.5, K2HPO4 1.4, MgSO4 1.5, FeSO4 0.005. After the optimization, the activity of F3-542 was up to 5147.5U/mL, of which was increased 31.3% more than the activity before optimization.
引文
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