一株AEC^hrL—赖氨酸产生菌

从乳糖发酵短杆菌Brevibacterium Lactofermentum2645菌株出发选育L-赖氨酸产生菌,经分离复壮后,用硫酸二乙酯(DES)诱变处理,由添加S-(2-氨基乙基)—L—半胱氨酸(AEC)和L-苏氨酸的药物平板定向筛选,最后获得了一株L—赖氨酸高产菌E_(16-2)(AHV~(hr)SAM~gAEC~(hr))。进而通过正交试验、工艺条件试验最终获得了该菌株的最佳发酵条件,并考查了在此条件下的L-赖氨酸发酵过程,结果表明该菌具有生长、耗糖、产酸速度快、发酵周期短的明显特点。     

L－赖氨酸快速发酵新菌种及工艺研究

以我室选育的赖氨酸生产菌Ｓ－２１－２４为出发菌株,经硫酸二乙酯（ＤＥＳ）和紫外线（Ｕ．Ｖ．）的复合处理,选育到一株代谢速率高,发酵周期短的新菌株ＦＴＳ－１。对该菌的发酵条件作了研究,选择了最佳培养条件,该菌在５Ｌ发酵罐中发酵４８小时产酸８０ｇ／Ｌ以上,７２小时产酸可达１１０ｇ／Ｌ。     

L－赖氨酸快速发酵新菌种及工艺研究

以我室选育的赖氨酸生产菌Ｓ－２１－２４为出发菌株,经硫酸二乙酯（ＤＥＳ）和紫外线（Ｕ．Ｖ．）的复合处理,选育到一株代谢速率高,发酵周期短的新菌株ＦＴＳ－１。对该菌的发酵条件作了研究,选择了最佳培养条件,该菌在５Ｌ发酵罐中发酵４８小时产酸８０ｇ／Ｌ以上,７２小时产酸可达１１０ｇ／Ｌ。     

阿维菌素高产菌株的选育Ⅰ

目的：全面了解阿维菌产生菌S.avermitilis的生长特性并获得阿维菌素的高产菌株。方法：以S.avermitilis Y20为出发菌株,考察该菌株的菌落特征和发酵特性,分别利用紫外线（UV）、亚硝酸（HNO3）及亚硝基胍（NTG）并结合L－异亮氨酸（L－Ⅱe）诱导等手段对出发菌株Y20进行诱变处理。结果：获得高产阿维菌素变异株H336,发酵单位达到852μg/ml。结论：与出发菌株相比,突变株阿维菌素的产量稳定,发酵单位提高了10倍以上。     

以耐赖氨酸为标记的金霉素产生菌抗噬菌体变株的选育

本文以金霉素产生菌Ｆ－３０３作为出发菌株,通过ＵＶ＋ＥＭＳ复合处理,双层法不同梯度耐赖氨酸作标记,进行抗噬菌体的金霉素菌株选育,获得Ｆｓ－４８－１４菌株。该菌株不但表达了抗噬菌体的特性,而且产素效价超过Ｆ－３０３。通过无机盐正交条件试验,取得抗性变株最佳的发酵培养基配方。     

L-色氨酸生产菌的选育及其发酵条件的研究

以代谢控制发酵理论为指导 ,对L -色氨酸产生菌的定向选育、摇瓶发酵条件、30L发酵罐发酵条件进行了研究。以谷氨酸棒杆菌Tx5 - 32 (Phe- +Tyr- )为出发菌株 ,经硫酸二乙酯 (DES)多次诱变处理 ,定向选育出一株L -色氨酸产生菌TQ2 2 2 3(Phe- +Tyr- +5 -MTr+5 -FTr+SGr+CINr)。以摇瓶分批发酵最优条件为基础 ,对菌株TQ2 2 2 3进行了 30L发酵罐分批发酵试验 ,该菌株发酵 6 4h ,产L -色氨酸 7.2 8g·L- 1 。     

L-赖氨酸产生菌FH128菌株的研究(Ⅰ)FH128菌株的选育及摇瓶发酵

以赖氨酸产生菌Ａ１１１（ＨＳ－、ＡＥＣｒ）为出发株,经化学诱变剂ＭＮＮＧ（Ｎ－甲基－Ｎ′－硝基－Ｎ－亚硝基胍）及单氟醋酸处理获得单氟醋酸抗性突变株Ｆ７９,摇瓶发酵产Ｌ－赖氨酸盐酸盐７．０％～７．５％,对糖转化率３８％～４０％,分别比Ａ１１１株提高约２５％及２０％。然后,再以Ｆ７９菌为亲株经ＭＮＮＧ及噻唑丙氨酸处理获得噻唑丙氨酸抗性突变株ＦＨ１２８,在适宜的培养条件下,摇瓶发酵产Ｌ－赖氨酸盐酸盐８．５％～９．５％,最高产酸率１１％,对糖转化率４５％～５０％。在１６Ｌ自控发酵罐发酵,产Ｌ－赖氨酸盐酸盐１２％～１４％,对糖转化率４０％～４５％;在２０～１００ｍ３发酵罐发酵,产酸率为８．５％～９．５％,对糖转化率４０％～４２％,提取总收率８０％～８５％,成品（饲料级Ｌ－赖氨酸盐酸盐）质量符合国家标准（ＧＢ８２４５－８７）。ＦＨ１２８菌株遗传性能稳定,营养要求粗放,工艺较简单,便于工业化,二年前已应用于工业生产。     

基因组改组技术选育耐酸性琥珀酸放线杆菌

以琥珀酸产生菌Actinobacillus succinogenes CGMCC 1593为出发菌,分别经过紫外线-甲基磺酸乙酯(UV-EMS)和紫外线-硫酸二乙酯(UV-DES)诱变处理,得到7株耐酸性有所提高的突变株.以此作为候选菌库,经3轮原生质体递进融合,筛选获得4株可以在pH 5.6下生长的改组菌株.其中改组菌株F3-21在pH 5.6的完全液体培养基中生长的OD值是原始菌的7倍,在pH 5.2条件下仍能生长;其摇瓶发酵48h琥珀酸产量较原始菌株提高48%.在5L发酵罐中进行分批发酵,当控制pH在较低值(5.6～6.0)时,F3-21厌氧发酵48h积累琥珀酸38.1g/L,较出发菌株提高了45%;当控制pH在6.5～7.0时,F3-21厌氧发酵32h积累琥珀酸40.7g/L.F3-21在5L发酵罐中进行补料分批发酵,厌氧发酵72h,产琥珀酸达67.4g/L.结果说明基因组改组技术能够改进琥珀酸放线菌的耐酸性能及其琥珀酸的产量.     

L—赖氨酸产生菌FH128菌株的研究：（Ⅰ）FH菌株的选育及摇瓶发酵

以赖氨酸产生菌Ａ１１１（ＨＳ＾－,ＡＥＣ＾ｒ）为出发株,经化学诱变剂ＭＮＮＧ（Ｎ－’－硝基－Ｎ－亚硝基胍）及单氟醋酸处理获得单氟生突变株Ｆ７９,摇瓶发酵产Ｌ－赖氨酸盐酸盐７．０％－７．５％,对糖转化率３８％－４０％,分别比Ａ１１１株提高红２５％及２０％。     

甜菜糖蜜发酵生产L—赖氨酸通过鉴定

甘肃省科学院生物工程中心承担的“甜菜糖蜜发酵生产L—赖氨酸课题于7月27日在兰州通过鉴定。M1083菌株是适合于甜菜糖蜜为碳源的高丝氨酸、亮氨酸双重缺陷兼AEC抗性的L—赖氨酸产生菌。     

Intracellular pool of free amino acids in a wild strain of Corynebacterium glutamicum and its lysine-producing mutants

The intracellular content of free amino acids was measured in the wild-type strain of Corynebacterium glutamicum 13032 and its lysine producing mutants 410 and 133, resistant to the combined effect of threonine and S-2-aminoethyl cysteine, a lysine analog. After 18- and 48-hour cultivation of all strains the major components of the amino acid pool were glutamic acid, alanine and lysine, and those of the cell-free supernatant were alanine and lysine. After 18-hour cultivation the lysine content in mutants was 2-3 times higher than in the wild-type strain. After 48-hour cultivation the lysine content in mutants remained unchanged and in the wild-type strain increased. After 18- and 48-hour cultivation the lysine content in the supernatant of mutants was 15 and 33 times higher than in that of the parental strain. These findings are compared with the activities of aspartokinase from Cor. glutamicum 13032, 410 and 133.     

Characteristics of lysine transport in a wild type strain and lysine-producing mutant of Corynebacterium glutamicum

An active transport system high specific for 1-lysine was found in the cells of the wild strain of Corynebacterium glutamicum, Km being about 10 microM. Accumulation of lysine was higher, if the cells were cultivated on a medium containing glucose. The cells of the homoserine-deficient lysine producer have no alterations in the lysine transport. The lysine transport was also studied in three lysine producing analog resistant mutants (two mutants are resistant to aminoethylcysteine and one to lysine hydroxamate). The key enzyme of the lysine biosynthesis, aspartate kinase, is insensitive to the feedback inhibition by the mixture of lysine and threonine in all the mutant studied; at the same time the cells of these mutants grown on a glucose-containing medium above mentioned alterations are suggested to provide the resistance to the lysine analog.     

Studies on Mechanisms for Lysine Production by Pyruvate Kinase-Deficient Mutants of Brevibacterium flavurn

Methionine-insensitive revertants with normal homoserine dehydrogenase (HD) derived from Brevibacterium flavum mutant No. 1-231, a lysine producer with S-(2-aminoethyl)-l-cysteine (AEC) resistance, methionine sensitivity, a low HD level and a pyruvate kinase (PK) defect, were still AEC-resistant and PK-deficient similar to No. 1-231. But they did not produce more lysine than the original strain, No. 15-8, from which strain No. 1-231 was derived. A high lysine producing mutant, No. 22, which was derived from strain No. 1-231, selected by sensitivity to β-fluoropyruvate (FP), and was defective in HD, produced more lysine than HD-defective mutants which were derived by two-step mutation from strain No. 1-231, selected by homoserine auxotrophy. Strain No. 22 did not show FP sensitivity under the conditions tested. Among various lysine-biosynthetic enzymes examined, it had a higher level of aspartate-β-semialdehyde dehydrogenase than did its parent and the latter HD-defective mutants. Strain No. 22 produced 50 g/liter of lysine as the HC1 salt when cultured for 72 hr in a medium containing soybean-meal hydrolysate, methionine and 100 g/liter of glucose.     

Strains of Corynebacterium glutamicum with Different Lysine Productivities May Have Different Lysine Excretion Systems

The lysine excretion systems of three different lysine-producing strains of Corynebacterium glutamicum were characterized in intact cells. Two strains (DG 52-5 and MH 20-22B) are lysine producers of different efficiency. They were bred by classical mutagenesis and have a feedback-resistant aspartate kinase. The third strain (KK 25) was constructed from the wild type by introducing the feedback-resistant aspartate kinase gene of strain MH 20-22B into its genome. The three strains were shown to possess different excretion systems. Export in strain KK 25 is much slower than in the two mutants. The differences between the two lysine-producing strains are more subtle. K(m) and V(max) are similar, but pH dependence and membrane potential dependence reveal differences in the intrinsic properties of the carrier system.     

Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes

The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B.␣lactofermentum ATCC 13869. The hom- and thrB- disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine. Received: 23 January 1996 / Received last revision: 28 July 1996 / Accepted: 5 August 1996     

Regulation of alpha-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from alpha-aminoadipate into penicillin biosynthesis.

The activity and regulation of alpha-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for alpha-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of alpha-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest alpha-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5-1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM alpha-aminoadipate, highest penicillin production was observed in those strains whose alpha-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of alpha-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of alpha-aminoadipate, and hence penicillin.     

L-Methionine production by double auxotrophic mutants of a ethionine resistant strain of Brevibacterium heali

A number of lysine plus threonine double auxotrophs have been isolated from a ethionine resistant methionine producing strain of Brevibacterium heali previously isolated from soil by mutagenesis with N-methyl N′-nitro-N-nitrosoguanidine in two steps. This strain excreted L-methionine in sufficient amounts. For the three potent mutants tested, the medium of ALFOLDI was judged to be the best. Biotin and ammonium nitrate were found to be optimal at 5 μg/l and at a 40 mM level, respectively. With such an optimal dose, the strain BhLT 27 yielded 25.5 g/l methionine in a flask culture containing methionine-analogue ethionine at a minimal inhibitory concentration.     

Threonine production by dihydrodipicolinate synthase-defective mutants of Brevibacterium flavum

A novel type of threonine-producing strains, dihydrodipicolinate synthase (DPS)-defective mutants of Brevibacterium flavum, was isolated as alpha-amino-beta-hydroxyvaleric acid (AHV)-resistant producers. The third selection markers used were a strong lysine inhibition of threonine production and a lower production of lysine than that of threonine in those derived from strains with feedback-sensitive and-resistant aspartokinase (AK), respectively. The maximum threonine production by these DPS-defective mutants was 13.7 g/l at the optimum concentration of DL-diaminopimelic acid (DAP) in a medium containing 100 g/l of glucose, comparable to that by the previously reported conventional producers with feedback-resistant homoserine dehydrogenase (HD(R)). The DPS-defective mutants with feedback-sensitive AK showed a slow but substantial growth in the absence of DAP and their growth was markedly stimulated by DAP, while those with feedback-resistant AK grew well in the absence of DAP and their growth was not promoted by DAP more than that of the parent strain. DPS-defective mutants with HD(R) were derived from an HD(R) mutant producing 10 g/l of L-threonine and selected as AHV-resistant mutants with a higher productivity. The maximum production was 16 g/l.     

Effect of Hydroxylysine on the Biosynthesis of Lysine in Streptococcus faecalis

We were able to show that two lysine-independent mutants of Streptococcus faecalis ATCC 8043 contained the enzymes for the usual bacterial pathway for lysine biosynthesis. Because of this synthetic capacity, one mutant, the Lys(+)OHLys(s) strain, could not grow in the presence of hydroxylysine without a lysine supplement. Both lysine and hydroxylysine inhibited the first enzyme of the pathway, aspartokinase. Unlike the Escherichia coli enzyme, S. faecalis dihydrodipicolinic acid synthetase was not inhibited by either lysine or hydroxylysine. Both amino acids caused the repression of dihydrodipicolinic acid synthetase and diaminopimelic acid decarboxylase. Failure of Lys(+)OHLys(s) strain to grow in hydroxylysine-supplemented medium was caused by the mimicking of lysine control by hydroxylysine. Because hydroxylysine could not completely substitute for lysine and lysine could not be synthesized, the organism did not grow. We tested three lysine analogues and found that they prevented lysine-depletion lysis in the Lsy(-)OHLys(s) strain, as did hydroxylysine. Each analogue seemed to support cell wall mucopeptide synthesis, although ornithine did not. Preliminary data indicated that these analogues like hydroxylysine, have growth-inhibitory action on the Lys(+)OHLys(s) strain, but not the Lys(+)OHLys(r) strain. The nature of the specificity of the lysine-adding enzyme for cell wall mucopeptide synthesis is discussed.     

用基因组重排技术选育赖氨酸高产菌株

摘要：【目的】以北京棒杆菌（Corynebacterium pekinense）1为研究对象,选育赖氨酸高产菌株,并探索赖氨酸产生菌基因组重排育种的基本规律。【方法】利用基因组重排技术选育赖氨酸高产菌株。【结果】通过四轮基因组重排成功选育出了5株遗传稳定的高产赖氨酸菌株,其中1株重排菌株赖氨酸产量达到16.95 g/dL,比原始菌株Corynebacterium pekinense 1赖氨酸产量提高了37.14%,比亲本菌株赖氨酸产量提高了17.46％~31.19%。【结论】首次采用基因组重排技术改良赖氨酸产生菌,成功选育出了5株产量较稳定的高产赖氨酸菌株,具有潜在的应用价值。