Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine production by ethionine-resistant mutants of Serratia marcescens.

Ethionine reduced both the growth rate and the final growth level of Serratia marcescens Sr41. Growth inhibition was completely reversed by methionine. Strain D-315, defective in homoserine dehydrogenase I, was more sensitive to ethionine-mediated growth inhibition than was the wild-type strain. Ethionine-resistant mutants were isolated from cultures of strain D-316, which was derived from strain D-315 as a threonine deaminase-deficient mutant. Of 60 resistant colonies, 7 excreted threonine on minimal agar plates. One threonine-excreting strain, ETr17, was highly resistant to ethionine and, moreover, insensitive to methionine-mediated growth inhibition, whereas the parent strain was sensitive. When cultured in minimal medium with or without excess methionine, strain ETr17 had a higher homoserine dehydrogenase level than did strain D-316. The homoserine dehydrogenase activity was not inhibited by threonine or methionine. Transductional analysis revealed that the ethionine-resistant (etr-1) mutation carried by strain ETr17 was located in the metBM-argE region and caused the derepressed synthesis of homoserine dehydrogenase II. Strain ETr17 had a higher aspartokinase level than did the parent strain. By transductional cross with the argE+ marker, the etr-1 mutation was transferred into strain D-562 which was derived from D-505, a strain defective in aspartokinases I and III. The constructed strain had a higher aspartokinase level than did strain D-505 in medium with or without excess methionine, indicating that the etr-1 mutation led to the derepressed synthesis of aspartokinase II. Strain ETr17 produced about 8 mg of threonine per ml of medium containing sucrose and urea.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Hydrogenase activity in Brevibacterium flavum

The activity of hydrogenase was assayed in the intact cells and subcellular fractions of Brevibacterium flavum. The organism was shown to have the membrane-bound form of hydrogenase. The soluble NAD+-reducing hydrogenase was not found. Oxygen inhibited the hydrogenase activity, and its action was reversible. Molecular hydrogen activated the hydrogenase of B. flavum, which was shown to be a constitutive enzyme.     

Improvement of l-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes

Brevibacterium flavum ATCC14067 was engineered for l-valine production by overexpression of different ilv genes; the ilvEBN^rC genes from B. flavum NV128 provided the best candidate for l-valine production. In traditional fermentation, l-valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the l-valine production and conversion efficiency based on the optimum temperatures of l-valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and l-valine biosynthesis enzymes activity were obtained at high temperature, and the maximum l-valine production, conversion efficiency, and specific l-valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g⁻¹ h⁻¹, respectively, at 37°C in 48 h fermentation. The strategy for enhancing l-valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.     

Production of l-Tryptophan by Azaserine-resistant Mutants of Brevibacterium flavum

Azaserine-resistant mutants derived from a 5-fluorotryptophan-resistant, l-tryptophan-producing mutant of Brevibacterium flavum, accumulated 10.3 g/liter of l-tryptophan at maximum. The production increased to 11.4 g/liter when l-serine was added. In the mutant, only anthranilate synthase among enzymes of the tryptophan-specific bio synthetic pathway increased in activity to a 2-fold higher level than that in the parent strain, No. 187. Sensitivity of anthranilate synthase to the feedback inhibition was not altered by the mutation. Activity of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first common enzyme for aromatic amino acid biosynthesis, also increased 2.7-fold and was less sensitive to the feedback inhibition by phenylalanine and tyrosine. Tryptophan transport activity in strain A-100 was similar as that in the parent. Azaserine inhibited anthranilate synthase activity by 50% at 0.075 mm. The inhibition was of a mixed type with respect to both the two substrates. Anthranilate synthase of strain A-100 was inhibited in a similar manner to that of the parent.     

产生L-异亮氨酸的黄色短杆菌的代谢途径分析

目的:代谢工程要解决的主要问题是改变某些途径中的碳架物质流量或改变碳架物质流在不同途径中的流量分布,其目标就是修饰初级代谢,将碳架物质流导入目的产物的载流途径,以获得产物的最大转化率。方法:利用途径分析方法对黄色短杆菌生产L-异亮氨酸的途径进行了分析。结果:建立了9种基础模型,确定L-异亮氨酸理论最高摩尔产率是1;确定了黄色短杆菌生产L-异亮氨酸的最佳途径的通量分布,并以此为依据进行发酵溶氧控制优化,溶氧分阶段控制发酵生产L-异亮氨酸比溶氧恒定控制方式发酵的产率提高了8.2%。结论:根据途径分析结果,通过改变发酵过程有关参数,可使目的产物产率得到提高。     

Production of L-Isoleucine by AHV Resistant Mutants of Brevibacterium flavum

The growth of Brevibacterium flavum No. 2247A was inhibited by α-amino-β-hydroxy-valeric acid (AHV), and the inhibition was partially reversed by L-isoleucine.

AHV resistant strain ARI-129, which was isolated on a medium supplemented with 2 mg/ml of AHV, produced 11 g/liter of L-isoleucine.

No difference was observed in threonine dehydratase between No. 2247A and ARI–129. Homoserine dehydrogenase from ARI–129 was insensitive to the feedback inhibition by L-isoleucine and L-threonine.

O-Methyl-L-threonine resistant mutant, strain AORI–126, which was derived from ARI–129, produced 14.5 g/liter of L-isoleucine. Specific activity of threonine dehydratase from AORI–126 increased about two-fold higher than those from No. 2247A and ARI–129, whereas degree of inhibition of the enzyme by L-isoleucine was the same among three strains.

Among auxotrophic mutants derived from ARI–129, adenine and lysine auxotrophs produced more L-isoleucine than the parent did.

In the adenine auxotroph, L-isoleucine production was markedly reduced by the addition of excess adenine.     

Production of Lysine by Pyruvate Dehydrogenase Mutants of Brevibacterium flavum

Mutants with low pyruvate dehydrogenase (PD) activities were derived from a pyruvate kinase-deficient lysine-producing mutant of Brevibacterium flavum, No. 22. They were selected as prototrophic revertants of the acetate auxotrophs of strain No. 22. Among them strain KD-11 produced 55g/liter of lysine as its HCI salt when cultured for 72 hr in a medium containing lOOg/liter of glucose, soybean-meal hydrolysate and methionine. The lysine yield of strain KD-11 was the highest ever reported (55%). The mutant required a higher concentration of methionine for maximum production and gave a smaller amount of cell mass in cultivation than its parent. PD activity of strain No. 22 was stimulated by cysteine, stabilized by glycerol, and gave apparent Kms of 89, 22, 380, 83 μM for pyruvate, coenzyme A, 3-acetylpyridine adenine dinucleotide, and NAD, respectively, under standard conditions. The apparent Km for NAD of PD from strain KD-11 was 10-times higher than that from No. 22. When the concentration of NAD was low, the cell extracts of strain KD-11 showed low PD activity. The specific activity of phosphoenolpyruvate carboxylase of strain KD-11 was slightly higher than that of strain No. 22, while the inhibition by aspartate of the former enzyme was weaker than that of the latter.     

Regulation of lysine biosynthesis in Brevibacterium flavum

L-lysine synthesis pathway enzyme activities: β-aspartate kinase (EC.2.7.2.4), diaminopimelate decarboxylase (EC.4.1.1.20) for two L-lysine producing strains Brevibacterium flavum 22LD and RC-115 were studied. It has been found that β-aspartate kinase and diaminopimelate decarboxylase in the Br. flavum RC-115 are less sensitive to feed-back inhibition by lysine and threonine. It is supposed that desensitized β-aspartate kinase in the Br. flavum RC-115 can be determined by genetical changes of the regulatory properties of the β-aspartate kinase. Auxotrophity in the locus of homoserine dehydrogenase was tested and no homoserine dehydrogenase (EC.1.1.1.3) activity was found in either strain. The combination of these both types of mutation supplemented by the lack of catabolic repression in the RC-115 strain makes it an active lysine producer in the medium with high carbohydrates content.     

黄色短杆菌产L-组氨酸菌株的诱变育种

以黄色短杆菌为出发菌,采用诱变育种的方法选育得到一株能高产L-组氨酸的突变菌株。在加有150g·L-1葡萄 糖;35g·L-1硫酸铵;10g·L-1蛋白胨的发酵培养基中培养72h,产L-组氨酸128.28mg·L-1。     

Enhancement of methionine production by methionine analogue ethionine resistant mutants of Brevibacterium heali

In order to improve the methionine yield of the isolate B. heali, attempts were made to isolate mutants resistant to the methionine analogue DL-ethionine after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NTG). The minimum inhibitory concentration (MIC) of ethionine for B. heali was found to be 2 mM. After mutagenesis and screening, five mutants resistant to 50 mM of ethionine were isolated. The yield of the best ethionine resistant mutant, B. heali Br EthR, was 13 mg/l of methionine medium under optimum cultivation conditions.     

L－异亮氨酸产生菌选育的研究

以黄色短杆菌（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍｆｌａｖｕｍ）ＡＴＣＣ１４０６７为出发菌株,经硫酸二乙酯（ＤＥＳ）、紫外线（ＵＶ）和亚硝基胍（ＮＴＧ）逐级诱变处理,α－氨基－β－羟基戊酸（ＡＨＶ）、Ｓ－２－氨基乙基－Ｌ－半胱氨酸（ＡＥＣ）、磺胺胍（ＳＧ）、乙硫氨酸（Ｅｔｈ）、α－氨基丁酸（α－ＡＢ）、异亮氨酸氧肟酸（ＩｌｅＨｘ）等氨基酸结构类似物及琥珀酸为碳源平板定向筛选,获得一株Ｌ－异亮氨酸高产菌ＺＱ－４（ＡＨＶ~γ、ＡＥＣ~γ、ＳＡＭ~γ、ＳＧ~γ、Ｅｔｈ~γ、α－ＡＢ~γ、ＩｌｅＨｘ~γ）在含１３．５％葡萄糖培养基中,摇瓶发酵７２ｈ、Ｌ－异亮氨酸积累可达２．８－３．０％。     

产精氨酸的黄色短杆菌菌种保藏方法的研究

采用甘油防冻营养液对产精氨酸的黄色短杆菌（Brevibacterium flawm）变异株AN78在普通冰箱冷冻室（-18℃～-20℃）进行冷冻保藏试验,4年中分别进行了AN78菌株的产L-精氨酸试验,在500mL摇瓶试验中产精氨酸30-35g／L;保藏2年的菌种在20L发酵罐试验中产精氨酸63．1g／L,转化率22．8％,发酵周期81h。试验结果好于以前的报道。该方法可作为氨基酸生产行业中一种简便有效的菌种冷冻保藏方法。     

L－异亮氨酸产生菌选育的研究

以黄色短杆菌（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍｆｌａｖｕｍ）ＡＴＣＣ１４０６７为出发菌株,经硫酸二乙酯（ＤＥＳ）、紫外线（ＵＶ）和亚硝基胍（ＮＴＧ）逐级诱变处理,α－氨基－β－羟基戊酸（ＡＨＶ）、Ｓ－２－氨基乙基－Ｌ－半胱氨酸（ＡＥＣ）、磺胺胍（ＳＧ）、乙硫氨酸（Ｅｔｈ）、α－氨基丁酸（α－ＡＢ）、异亮氨酸氧肟酸（ＩｌｅＨｘ）等氨基酸结构类似物及琥珀酸为碳源平板定向筛选,获得一株Ｌ－异亮氨酸高产菌ＺＱ－４（ＡＨＶ~γ、ＡＥＣ~γ、ＳＡＭ~γ、ＳＧ~γ、Ｅｔｈ~γ、α－ＡＢ~γ、ＩｌｅＨｘ~γ）在含１３．５％葡萄糖培养基中,摇瓶发酵７２ｈ、Ｌ－异亮氨酸积累可达２．８－３．０％。     

L-缬氨酸的发酵工艺研究

目的:以黄色短杆菌XV0505为生产菌株,探讨发酵培养基和发酵控制条件对L-缬氨酸的产量和糖酸转化率的影响。方法:应用单因素实验确定发酵的工艺条件;利用纸层析-色斑洗脱比色法测定发酵液中L-缬氨酸的含量。结果:在最优发酵条件下,通过10L罐流加发酵72h,产酸量可达53.4g/L,糖酸转化率为26.7%,分别比补料分批发酵提高11.9%和3.5%。结论:环境因子和发酵控制工艺对发酵生产L-缬氨酸具有重要影响。