【作者】 刘焕民；

【导师】 张伟国；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 博士

【摘要】 L-缬氨酸不仅是支链氨基酸,而且是人体八种必需氨基酸之一,在生命体各种重要的生理活动中起着不可替代的作用。L-缬氨酸应用领域非常广阔,在医药、食品强化剂、饲料添加剂、调味品和农药等方面都有重要的应用。现阶段国内的L-缬氨酸发酵水平不高,全国年产量远远不能满足国内市场日益增长的需求。为了解决上述问题,本课题依据代谢工程原理,选育可以高效生产L-缬氨酸的菌种,具体结果如下:⑴用硫酸二乙酯（DES）和亚硝胍（NTG）处理出发菌株黄色短杆菌（Brevibacterium flavum） ZGH6128,经过筛选最终获得菌株NVT1103(Leu^l,α-AB^hr, 2-TA^hr,SG^r)与菌株JVHK597（Leu^-,Ile^-,Met^l,α-AB^r,2-TA^r）。在未优化的条件下,两株菌的摇瓶产酸量分别为37.6g/L和41.2g/L。⑵利用原生质融合技术进行氨基酸菌种选育的研究引起国内外技术人员的关注,本课题以菌株NV1103和JVHK597为亲本菌株,采用原生质体融合方法进行选育L-缬氨酸高产菌株。用0.5U/mL与1U/mL的青霉素钠盐溶液分别预处理双亲本菌株（NV1103与JVHK597）2.5h和3h;溶菌酶处理亲本株的最适浓度为1g/L与2g/L,分别处理9h和11h;再生培养基中渗透压稳定剂浓度为0.6mol/L;在34°C、pH10的缓冲体系中,400g/L的PEG介导25min,可以获得理想的融合率。最终选育出高产菌株NJv61,遗传标记为(Leu^-,Ile^-,Met^l, 2-TA^hr, SG^r, Glc^hr,α-AB^hr),未优化条件下,摇瓶产酸量为45.6g/L。⑶利用正交表进行实验设计确定菌株NJv61种子培养基的组成:葡萄糖24g/L,玉米浆35g/L,（NH₄）₂SO₄ 5g/L,KH₂PO₄ 0.6g/L,MgSO₄·7H₂O 0.5g/L,CaCO₃ 10g/L。对菌株发酵过程中所需的碳（氮）源、无机盐、生长因子等进行了单因素实验,在此基础上通过响应面方法确定了该菌株发酵培养基的最佳组成:葡萄糖149g/L,玉米浆19.4g/L,（NH₄）₂SO₄ 48.4g/L,KH₂PO₄ 1.5g/L,MgSO₄·7H₂O 0.6g/L,VB1 150μg/L,VH 60μg/L。在7L发酵罐中,优化条件下NJv61经过72h发酵,在发酵液中积累L-缬氨酸达到51.8g/L。⑷对L-缬氨酸产生菌筛选过程中所获得的代表菌株（ZGH6128、NVT1103、JVHK597和NJv61）,在7L发酵罐产酸过程的中后期进行取样分析,明确了该时期相关菌株细胞内与合成L-缬氨酸相关的代谢途径网络分布。与出发菌株ZGH6128相应途径上的流量相比,目的菌株NJv61在节点G6P处流向EMP途径的碳架流量有了显著的提高,同时,在HMP途径中仍然保持较大流量。在丙酮酸节点处,目的菌株流向TCA循环的代谢流量较出发菌株大大减少,使更多的碳架流进入L-缬氨酸合成途径。目的菌株中丙氨酸生物合成途径中的碳架流量较出发菌株没有明显的下降。⑸乙酰羟基酸合成酶（AHAS）是半理性化育种工作的靶酶,目的菌株NJv61对高浓度α-AB和2-TA具有抗性,研究发现该菌株的AHAS基因发生了突变,更彻底地解除L-缬氨酸反馈抑制与阻遏作用,并且酶的相对活性也有改变。本课题对AHAS基因进行了克隆,对测序的结果进行了比较分析发现,调节亚基所对应的基因区段,仅分布着整个基因突变位点总数大约10%的突变碱基,而编码催化亚基的基因区段布着的突变位点数约为整个基因所有突变位总数的90%。最后把菌株NJv61的AHAS基因与特定的表达载体相连接,导入模式菌株,经IPTG诱导,所表达的酶蛋白分子在4℃条件下不稳定,22h内该酶相对酶活迅速下降,仅保持大约20%相对活性。更多还原

【Abstract】 L-Valine is not only one of three branched-chain amino acids but also a member of eight essential amino acids which can’t be synthesized by humans themselves. It plays an important role in many kinds of physiological functions in vertebrates’bodies. The applications of L-valine are very broad: precursor of pharmaceuticals, additives to food or feedstuff for livestocks, material for pesticides, nutrition infusion for patients, health care product, and condiment and so on. Nowadays, scales of the industrial plants for L-valine in China are small, and the actual production level of L-valine prodution isn’t high. The annual production amount of L-valine can’t meet the growing demand of domestic market. In order to solve problems mentioned above, it is necessary to breed highly efficient L-valine-producing strains based on metabolic engineering principles.（1） The origin strain is strain ZGH6128 （Brevibacterium flavum） which was treated with DES and NTG in the screening process. Strain NVT1103 (Leu^l,α-AB^hr, 2-TA^hr,SG^r) and strain JVHK597（Leu^-, Ile^-, Met^l,α-AB^r, 2-TA^r） were obtained in the early stages. Under unoptimized conditions, the L-valine productions in flasks of the two strains were 37.6g/L and 41.2g/L respectively.（2） Scientists at home and abroad were drawn attention to the application of protoplast fusion technology to screen L type amino acids producing strains. Strain NV1103 and strain JVHK597 was used as parent strains during the process to beed L-valine hyper-producer by protoplast fusion technology in this thesis. Parent strains （NV1103 and JVHK597） were pretreated for 2.5 hours and 3 hours with 0.5 U/mL and 1U/mL penicillin sodium salt solution. The two parent strains were treated by lysozyme in the optimal concentration of 1g/L and 2g/L for 9 hours and 11hours repectively. The optimal concentration of osmotic pressure stabilizer in the regeneration medium is 0.6 mol/L. A good fusion rate was got when the two parent stains was mediated by PEG for 25 minutes in pH 10 buffer system at 34°C. Eventually, L-valine hyper-producer NJv61 was obtained, and genetic markers of strain NJv61 were (Leu^-, Ile^-, Met^l, 2-TA^hr, SG^r, Glc^hr,α-AB^hr). Under unoptimized conditions, the L–valine production in flasks of the strain was 45.6 g/L.（3） The composition of seed medium of strain NJv61 was fixed through orthogonal table experiment design: Glucose 24g/L, （NH₄）₂SO₄ 5g/L, KH₂PO₄ 0.6 g/L, MgSO₄·7H₂O 0.5g/L, corn-steep 35g/L, CaCO₃ 10g/L. Single-factor experiments were conducted, including carbon or nitrogen source, inorganic salts, growth factors and other necessary factors. The optimal composition of the fermentation medium was fixed by response surface methodology. The culture medium used for the fermentation of NJv61 contained: Gulcose 149g/L, （NH₄）₂SO₄ 48.4g/L, KH₂PO₄ 1.5g/L, MgSO₄·7H₂O 0.6g/L, VB1 150μg/L, biotin 60μg/L, corn-steep 19.4g/L. The L-valine production of strain NJv61 in 7L fermentor was 51.8 g/L under unoptimized conditions after 72 hours fermentation.（4） Representative strains （ZGH6128, NVT1103, JVHK597 and NJv61） were obtained in the screening process. And broth samlpes of the representative strains were analysed when these strains accumulated in the 7 fermentor during the late metaphase of the fermentation process. Metabolic flux distribution network diagram from glucose to L-valine was figured out based on results of analysis of the samples. Diagrams of frames of carbon distribution in metabolic network were compared between the initial strain ZGH6128 and the target strain NJv61. In the screened strain NJv61, Carbon flows into the EMP pathway was enhanced at the G6P node, and there was still sufficient metabolic fluxes in the HMP pathway. Compared with the original strain, metabolic fluxes into the TCA cycle were decreasing sharply at pyruvate node in order that more carbon frame fluxes flowed into the L-valine biosynthetic pathway. Compared with the starting stain, Carbon frame fluxes in the L-alanine biosynthetic pathway didn’t decline markedly in the strain NJv61.（5） Target strain NJv61 could resist the high concentration ofα-AB and 2-TA because of the mutations in acetohydroxyacid synthase gene. In cells of strain NJv61, L-valine feedback inhibition and repression effect were released drastically. Meanwhile, relative activity of the enzyme was changed. Strain NJv61’s AHAS gene was cloned and sequenced in the thesis, and the result was analysed with biological softwares. There are nearly 10% of the total uumber mutations in the whole gene distributed in the section encoding regulatory subunit; while there are 90% of mutations in the entire gene distributed in the AHAS gene segment encoding the catalytic subunit. Finally, the NJv61’s AHAS gene was connected with a given expression vector and exported into a mode strain. Induced by IPTG, the enzymatic activity of enzyme protein molecules expressed by the mode strain was unstable at 4℃, and the relative activity of the enzyme decreased rapidly in the first 22 hours, just keeping about 20% of relative activity only.更多还原