产L-丝氨酸菌株SYPS-062的鉴定及碳源对发酵的影响

采用形态学、生理生化实验和16S rDNA序列分析的方法对从自然界中筛选得到的一株能直接利用糖质原料发酵生产L-丝氨酸菌株SYPS-062的分类地位进行了研究, 确定其为谷氨酸棒杆菌(Corynebacterium glutamicum)。同时考察了碳源对菌株SYPS-062发酵产L-丝氨酸的影响, 实验结果表明, 当蔗糖浓度为60 g/L时, 菌株SYPS-062生物量和L-丝氨酸的积累均达到最大值, 分别为8.1 g/L和6.6 g/L。     

谷氨酸棒杆菌ilvE基因的敲除对相关氨基酸合成的影响

L-缬氨酸是谷氨酸棒杆菌SYPS-062发酵生产L-丝氨酸的主要副产物.为减少L-缬氨酸的积累,利用基因重组技术敲除SYPS-062转氨酶B编码基因ilvE内部的987 bp核苷酸序列,构建了ilvE基因缺失突变株SYPS-062△ilvE.研究表明,重组菌ilvE基因的缺失直接导致了分支氨基酸(Val、Ile、Leu)的合成能力的降低,影响了菌体的生长,其中Ile成为生长限制性因子,在培养基中添加分支氨基酸能明显促进其生长.重组菌培养96 h,发酵液中L-缬氨酸含量低于0.5 g/L,与出发菌株相比,其生成率降低90％.     

氨基脱氧分支酸合成酶对Corynebacterium glutamicum SYPS-062积累L-丝氨酸的影响

为阐明氨基脱氧分支酸合成酶(ADC合成酶)在Corynebacterium glutamicum SYPS-062体内积累L-丝氨酸过程中的作用,通过交叉PCR以及同源重组的方法敲除叶酸途径关键酶ADC合成酶的编码基因pabAB,构建了叶酸缺陷型菌株Corynebacterium glutamicum SYPS-062△pabAB,同时构建pabAB基因增强表达重组菌C.glutamicum SYPS-062(pJC Ⅰ-pabAB).分别考察了ADC合成酶对菌株生长的影响、对L-丝氨酸降解途径关键酶丝氨酸羟甲基转移酶(SHMT)的影响以及其对L-丝氨酸积累的影响.结果表明,与出发菌株相比,增强表达基因pabAB重组菌的ADC合成酶的酶活力提高了33%.SHMT酶的酶活力提高了30%,其最大比生长速率(μm)提高了48%,单位细胞产酸率(Yp/x)降低了36.2%;而敲除基因pabAB重组菌的ADC合成酶的酶活力降低了61%.SHMT酶的酶活力降低了20%,最大比生长速率降低了32%,单位细胞产酸率提高了12%.     

谷氨酸棒杆菌SYPS-062合成L-丝氨酸的代谢通量分析

以一株由自然界筛选获得的能够利用糖质原料直接产L-丝氨酸的谷氨酸棒杆菌Corynebacterium glutamicum SYPS-062为研究对象,考察了一碳单元循环中的辅因子—叶酸和维生素B12对菌株生长、蔗糖消耗及L-丝氨酸生成的影响,同时对处于对数生长期的菌株进行了代谢流量分析。结果发现,添加扰动因子叶酸和维生素B12对磷酸戊糖途径(HMP)碳流影响较大,碳源主要用于细胞生长及合成能量,而流向目的产物L-丝氨酸的碳流减少。同时在添加维生素B12时,增大了G3P节点的L-丝氨酸合成途径的分流比,但造成三羧酸循环(TCA)的流量不足,需要大量回补,从而限制了产物合成速率的进一步提高。     

直接利用糖质原料产L-丝氨酸谷氨酸棒杆菌glyA基因序列分析

以能够利用糖质原料产L-丝氨酸的谷氨酸棒杆菌（Corynebacterium glutamicum）SYPS-062 glyA基因为研究对象,比较cglutamicum SYPS-062与C．glutamicum模式菌株ATCC13032的glyA基因的异同。分别以SYPS-062及ATCC13032基因组为模板,利用PCR技术获得丝氨酸羟甲基转移酶编码基因glyA。核苷酸序列分析结果表明,来源于SYPS-062和ATCC13032的glyA基因片段全长均为1305bp,编码434个氨基酸,分子量为46．5kD,基因的同源性为99．54％,存在6个核苷酸的差异,引起一个氨基酸残基的突变。将获得的基因分别在大肠杆菌（Escherichia coli）BL21（DE3）中诱导表达。酶活测定结果显示,2种不同菌株来源的重组SHMT的比活力稍有差异,说明SYPS-062 glyA基因的差异对其表达产物SHMT蛋白构型及功能影响不大。提示对于C.glutamicum SYPS-062能够利用糖质原料产L-丝氨酸的机制解析应进一步从glyA基因的转录水平、翻译水平及胞内SHMT辅酶的供给情况等方面进行深入研究探讨。     

谷氨酸棒杆菌SYPS-062 L-丝氨酸脱水酶活性分析及其基因敲除对L-丝氨酸积累的影响

以谷氨酸棒杆菌(Corynebacterium glutamicum) SYPS-062基因组DNA为模板,扩增得到L-丝氨酸脱水酶(L-SerDH)的编码基因sdaA。将其克隆到表达载体pET-28a(+),并在E.coli BL21(DE3)中诱导表达,对纯化的L-SerDH进行了酶活测定,并与来自C.glutamicum ATCC13032的重组L-SerDH进行了比较,结果显示,两种不同菌株来源的重组L-SerDH降解L-丝氨酸的酶比活力差异并不显著。在此基础上敲除菌株SYPS-062 的sdaA基因,探讨该基因对C.glutamicum SYPS-062生长及产酸的影响。通过构建自杀型重组质粒pK18mobsacB-△sdaA,电击转入C.glutamicum SYPS-062中,以同源重组的方式获得了sdaA基因缺失突变株,并用PCR方法对突变株C.glutamicum SYPS-062△sdaA进行了验证。与出发菌株相比,突变菌株生长缓慢,单位菌体L-丝氨酸的产量(Y_P/X)提高了15.13%。     

Pgk和ilvN基因对谷氨酸棒杆菌产L-丝氨酸的影响

为增加谷氨酸棒杆菌A36的L-丝氨酸合成途径的碳流,首先过表达磷酸甘油酸激酶(pgk),以增加前体物质3-磷酸甘油酸的积累,但经发酵分析发现其对菌株A36的L-丝氨酸产量无显著影响。进一步敲除副产物L-缬氨酸合成途径的乙酰羟酸合酶(AHAS)基因ilvN,敲除该基因后L-缬氨酸只有微量积累,但重组菌并未形成营养缺陷型菌株,L-丝氨酸的产量反而下降,分析发现L-缬氨酸的存在在一定程度上有助于L-丝氨酸的生成。在培养基中分别添加不同质量浓度的L-缬氨酸,在L-缬氨酸添加量为750 mg/L时,重组菌L-丝氨酸产量达到34.19 g/L,糖酸转化率为0.34 g/g,生产强度为0.28 g/(L·h),相比出发菌株A36分别提高了11.8%、13.3%和12.0%。     

不同浓度有机物对雷公藤不定根生长和次生代谢产物含量的影响

以NT为基本培养基,雷公藤(Tripterygium wilfordii)不定根为材料,研究了肌醇、VB1、烟酸、VB6、甘氨酸、叶酸、生物素等有机物质对雷公藤不定根生长及其次生代谢产物雷公藤甲素和总生物碱含量的影响。结果表明:NT培养基中原有浓度的肌醇、VB1含量即可使雷公藤不定根生长量、雷公藤甲素含量、总生物碱含量及产量达到最大值。在添加的其他有机物中,添加1 mg/L烟酸、1 mg/L VB6和5 mg/L甘氨酸适合不定根的生长;添加0.5 mg/L烟酸、0.5 mg/L生物素、1 mg/L VB6、1 mg/L甘氨酸和1 mg/L叶酸适合雷公藤甲素的积累;添加0.5 mg/L甘氨酸、1 mg/L VB6、1 mg/L叶酸和1 mg/L生物素则适合不定根中雷公藤总生物碱的合成。     

L-缬氨酸产生菌的选育及其发酵培养基的优化

以黄色短杆菌(Brevibacterium flavum)NJ-237为出发菌株,通过梯度传代适应性培养及同浓度药物平板富集培养的方式,逐步提高菌体的抗药物性能,获得了1株耐高糖和耐高浓度α-氨基丁酸(-αAB)的菌株NJ-2372。在单因素实验的基础上,利用响应面分析法对影响该菌株L-缬氨酸(L-Val)产量的3个重要因素玉米浆、生物素(VH)、硫胺素(VB1)的添加量进行优化。结果表明:当玉米浆、VH、VB1最佳添加量分别为11 g/L、35μg/L和101μg/L时,摇瓶发酵72 h,L-Val摇瓶发酵产量达到52.9 g/L。     

甲基营养型L-丝氨酸产生菌的选育

以甲基营养型假单胞菌J-12为出发菌株,经硫酸二乙酯（DES）和亚硝基胍（NTG）诱变处理,D-丝氨酸结构类似物平板和高浓度甘氨酸平板定向筛选,获得1株三-丝氨酸高产菌N-13,其发酵液中L-丝氨酸产量较出发菌株提高97．9％。在含有20g/L甘氨酸和7mL／L甲醇的培养基中,L-丝氨酸积累可达4．81g／L。     

Characterization,modification, and overexpression of 3-phosphoglycerate dehydrogenase in Corynebacterium glutamicum for enhancing l-serine production

The direct fermentative production of l-serine from renewable biomass using Corynebacterium glutamicum is attracting increasing attention. In this study, wild-type C. glutamicum SYPS-062 produced up to 6.65 ± 0.23 g/L l-serine; to further improve l-serine production, the serA gene was cloned, and the C-terminal domain of 3-phosphoglycerate dehydrogenase (PGDH) from this strain was truncated. When expressed in Escherichia coli, the resultant mutein SerAΔ197 showed a specific PGDH activity of 1.092 ± 0.05 U/mg protein, representing a decrease of 25.87 % from that encoded by serA, and was no longer sensitive to high concentrations of l-serine. When serA Δ591 was overexpressed in C. glutamicum SYPS-062, the activity of PGDH in C. glutamicum pJC1-tac-serA Δ591 increased by 47.72 %, and the resultant strain C. glutamicum pJC1-tac-serA Δ591 could accumulate 7.69 ± 0.22 g/L l-serine. Furthermore, when serA Δ591 was overexpressed in C. glutamicum SYPS-062ΔsdaA, the resultant strain could accumulate 8.84 ± 0.23 g/L l-serine at 102 h, and the yield of l-serine on cells (Y p/x) improved by 60 % when compared with that noted in the control. These results demonstrate that l-serine production in C. glutamicum SYPS-062 could be improved by overexpressing a C-terminal truncation of PGDH in combination with other genetic modifications.     

Effects of pyruvate kinase on the growth of Corynebacterium glutamicum and L-serine accumulation

Pyruvate kinase (PYK) is an important enzyme in the intermediary metabolism and has attracted much attention as a target for metabolic engineering of Corynebacterium glutamicum. Genome sequencing revealed that the 308 residue of PYK was mutated from methionine in model strain C. glutamicum ATCC14067 to isoleucine in L-serine-producing strain C. glutamicum SYPS-062. Consequently, a significantly lower PYK activity (77%) was noted in C. glutamicum SYPS-062, when compared with that in C. glutamicum ATCC14067. To confirm the role of this point mutation, pyk in both C. glutamicum SYPS-062 and C. glutamicum SYPS-062-33aΔSSAA was reversely mutated to restore the PYK enzyme activity, which led to a 33.1% and 28.8% decrease in L-serine titer, respectively. This is the first report to show that the (Met-308→Ile) mutation site of pyk is closely associated with its activity and apparently affected L-serine production. Furthermore, pyk was deleted in strain C. glutamicum SYPS-062-33aΔSSAA, and the resulting strain did not show alteration in growth rate and presented a 12% increase in L-serine production.     

Reduced folate supply as a key to enhanced L-serine production by Corynebacterium glutamicum

The amino acid L-serine is required for pharmaceutical purposes, and the availability of a sugar-based microbial process for its production is desirable. However, a number of intracellular utilization routes prevent overproduction of L-serine, with the essential serine hydroxymethyltransferase (SHMT) (glyA) probably occupying a key position. We found that constructs of Corynebacterium glutamicum strains where chromosomal glyA expression is dependent on Ptac and lacIQ are unstable, acquiring mutations in lacIQ, for instance. To overcome the inconvenient glyA expression control, we instead considered controlling SHMT activity by the availability of 5,6,7,8-tetrahydrofolate (THF). The pabAB and pabC genes of THF synthesis were identified and deleted in C. glutamicum, and the resulting strains were shown to require folate or 4-aminobenzoate for growth. Whereas the C. glutamicum DeltasdaA strain (pserACB) accumulates only traces of L-serine, with the C. glutamicum DeltapabABCDeltasdaA strain (pserACB), L-serine accumulation and growth responded in a dose-dependent manner to an external folate supply. At 0.1 mM folate, 81 mM L-serine accumulated. In a 20-liter controlled fed-batch culture, a 345 mM L-serine accumulation was achieved. Thus, an efficient and highly competitive process for microbial l-serine production is available.     

Abnormal concentrations of B vitamins and amino acids in plasma and B vitamins in bile of rabbits with aflatoxicosis

The dosages of aflatoxin B1 (AFB1) required to produce significant changes in concentrations of B vitamins in plasma and bile and of amino acids in plasma of rabbits were determined. Folate increased by 98% in plasma, whereas concentration of thiamine, vitamin B6, and biotin decreased by more than 50%. In bile, choline and biotin increased 14- and 18-fold, respectively, whereas folate and niacin decreased by more than 50%. All amino acids in plasma increased between 76 and 155%. The dosages of AFB1 required to induce these changes were usually between 12.5 and 37.5 microgram/kg of body weight per day. Except for changes in biliary concentrations of pantothenic acid, folic acid, and biotin, lower threshold dosages of aflatoxin were required to produce weight loss and anorexia (5.0 and 8.5 microgram of AFB1/kg per day, respectively) than for changes in vitamins and amino acids (approximately 25 to 50 microgram of AFB1/kg per day). The data indicated that AFB1 interfered with the metabolism of B vitamins and amino acids in rabbits.     

Abnormal concentrations of B vitamins and amino acids in plasma and B vitamins in bile of rabbits with aflatoxicosis.

The dosages of aflatoxin B1 (AFB1) required to produce significant changes in concentrations of B vitamins in plasma and bile and of amino acids in plasma of rabbits were determined. Folate increased by 98% in plasma, whereas concentration of thiamine, vitamin B6, and biotin decreased by more than 50%. In bile, choline and biotin increased 14- and 18-fold, respectively, whereas folate and niacin decreased by more than 50%. All amino acids in plasma increased between 76 and 155%. The dosages of AFB1 required to induce these changes were usually between 12.5 and 37.5 microgram/kg of body weight per day. Except for changes in biliary concentrations of pantothenic acid, folic acid, and biotin, lower threshold dosages of aflatoxin were required to produce weight loss and anorexia (5.0 and 8.5 microgram of AFB1/kg per day, respectively) than for changes in vitamins and amino acids (approximately 25 to 50 microgram of AFB1/kg per day). The data indicated that AFB1 interfered with the metabolism of B vitamins and amino acids in rabbits.     

Mechanism of Folate Transport in Lactobacillus casei: Evidence for a Component Shared with the Thiamine and Biotin Transport Systems

Lactobacillus casei cells have been shown previously to utilize two separate binding proteins for the transport of folate and thiamine. Folate transport, however, was found to be strongly inhibited by thiamine in spite of the fact that the folate-binding protein has no measurable affinity for thiamine. This inhibition, which did not fluctuate with intracellular adenosine triphosphate levels, occurred only in cells containing functional transport systems for both vitamins and was noncompetitive with folate but competitive with respect to the level of folate-binding protein. Folate uptake in cells containing optimally induced transport systems for both vitamins was inhibited by thiamine (1 to 10 muM) to a maximum of 45%; the latter value increased to 77% in cells that contained a progressively diminished folate transport system and a normal thiamine system. Cells preloaded with thiamine could transport folate at a normal rate, indicating that the inhibition resulted from the entry of thiamine rather than from its presence in the cell. In a similar fashion, folate (1 to 10 muM) did not interfere with the binding of thiamine to its transport protein, but inhibited thiamine transport (to a maximum of 25%). Competition also extended to biotin, whose transport was strongly inhibited (58% and 73%, respectively) by the simultaneous uptake of either folate or thiamine; biotin, however, had only a minimal effect on either folate or thiamine transport. The nicotinate transport system was unaffected by co-transport with folate, thiamine, or biotin. These results are consistent with the hypothesis that the folate, thiamine, and biotin transport systems of L. casei each function via a specific binding protein, and that they require, in addition, a common component present in limiting amounts per cell. The latter may be a protein required for the coupling of energy to these transport processes.