Production of l-Threonine by Analog-resistant Mutants

Mutants resistant to α-amino-β-hydroxyvaleri0c acid (AHV) were derived from various bacteria which belong to Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Bacillus by mutational treatment with N-methyl-N′-nitro-N-nitrosoguanidine(NTG), and screened for their ability to produce l-threonine. A number of l-threonine producers were obtained from each group of bacteria. Among them, the mutants derived from C. glutamicum KY9159(Met^?) were further mutagenized with NTG to derive thialysine(S-Lys)-resistant mutants. An AHV-resistant mutant, KY10484 was proved to be much more sensitive to the growth inhibition by thialysine than the parent strain, KY9159. From KY10484, a number of AHV- and thialysine-resistant mutants were derived. Approximately a half of these mutants were found to produce more l-threonine than KY10484. Among these mutants, KY10440 (Met^?, AHV^R, s-Lys^R) was used to investigate the cultural conditions for l-threonine production. The growth of KY10440 decreased largely with addition of l-homoserine, a threonine precursor. l-Asparagine, l-cystine, l-glutamine or l-arginine partially reversed the inhibitory effect of l-homoserine. Addition of these amino acids at low level led to increase l-threonine production. The amount of l-threonine accumulation reached to a level of 14mg/ml with a medium containing 10% glucose and to a level of 10 mg/ml with a medium containing 5% molasses (as glucose).

Another AHV- and thialysine-resistant mutant, KY10251 which was also derived from KY9159 was found to produce both 9 mg/ml of l-threonine and 5.5 mg/ml of l-lysine in a culture broth.     

Histidine Production by Auxotrophic Histidine Analog-resistant Mutants of Corynebacterinm glutamicum

Corynebacterium glutamicum mutants carrying both auxotrophy and histidine analog-resistance were derived by a mutagenic treatment, and their histidine productivity was compared with that of a triazolealanine (TRA)-resistant histidine producer, C. glutamicum KY-10260. As a result, a leucine auxotrophic TRA-resistant mutant, Rα-88 was selected out of 164 auxotrophic derivatives of KY-10260. It produced histidine at a distinctly higher concentration than the parent strain under every condition tested. The concentration reached 11 mg/ml or 5.8% (w/w) of the initial sugar. Addition of an excessive amount of leucine to the medium inhibited the histidine production together with the by-production of valine by this mutant. Thiazolealanine-resistant mutants derived from a tyrosine auxotroph, a phenylalanine auxotroph and a tryptophan auxotroph gave the same or lower production in comparison with KY-10260.     

l-Isoleucine Production by Analog-resistant Mutants Derived from Threonine-producing Strain of Corynebacterium glutamicum

A thiaisoleucine-resistant mutant, ASAT–372, derived from a threonine producer of Corynebacterium glutamicum, KY 10501, produced 5 mg/ml each of l-isoleucine and l-threonine. l-Isoleucine productivity of ASAT–372 was improved stepwise, with concurrent decrease in threonine production, by successively endowing it with resistivity to such substances as ethionine, 4-azaleucine and α-aminobutyric acid. The mutant strain finally selected, RAM–83, produced 9.7 mg/ml of l-isoleucine with a medium containing 10% (as sugar) molasses.

l-Isoleucine production was significantly affected by the concentration of ammonium sulfate in the fermentation medium. At 4% ammonium sulfate l-isoleucine production was enhanced whereas l-threonine production was suppressed. At 2% ammonium sulfate l-threonine production was stimulated while l-isoleucine production decreased.     

L-Tyrosine Production by Analog-resistant Mutants Derived from a Phenylalanine Auxotroph of Corynebacterium glutamicum

Mutants resistant to various phenylalanine- or tyrosine-analogs were isolated from a phenylalanine auxotroph of Corynebacterium glutamicum KY 10233 by treatment with N- methyl-N′-nitro-N-nitrose guanidine (NTG) and screened for L-tyrosine production. A mutant, 98–Tx–71, which is resistant to 3-aminotyrosine, p-aminophenylalanine, p-fluoro-phenylalanine, and tyrosine hydroxamate was found to produce L-tyrosine at a concentration of 13.5 mg/ml in the cane molasses medium containing 10% of sugar calculated as glucose. A tyrosine-sensitive mutant, pr–20 which was derived from 98–Tx–71 produced L-tyrosine at a concentration of 17.6 mg/ml. L-Tyrosine formation in the strain pr–20 was found to be still inhibited by L-phenylalanine though it was not inhibited by L-tyrosine. The L-tyrosine formation in the mutant was repressed neither by L-phenylalanine nor by L-tyrosine.     

Production of l-Threonine by Auxotrophic Mutants of Escherichia coli

Two auxotrophic mutants of Escherichia coli have been shown to accumulate significant amounts of l-threonine in the culture medium. One mutant, 13071, is deficient in α,ε-diaminopimelic acid (DAP), and the other, 13070, is deficient in both DAP and methionine.     

l-Serine Production Improved by Analogue-resistant Mutants of a Methanol-utilizing Bacterium

In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.     

Production of l-Lysine by Fluoropyruvate-sensitive Mutants of Brevibacterium lactofermentum

Better producers of l-lysine were obtained by derivation of fluoropyruvate(FP)-sensitive mutants from Brevibacterium lactofermentum AJ3990. The coexistence of FP and excess biotin synergistically stimulated l-lysine formation by washed cells. FP inhibited 50% of growth and pyruvate dehydrogenase (PDH) activity of AJ3990 at 0.04 mm and 1 mm, respectively. Therefore, the synergistic effect of FP and excess biotin seems to be due to the optimization of the PDH/pyruvate carboxylase activity ratio in l-lysine biosynthesis. This was confirmed by the derivation of FP-sensitive mutants which have the optimal level of PDH activity for l-lysine production. The best producer, AJ11204, had about 27% PDH activity as compared with the parental strain and accumulated 70 g of l-lysine per liter with a conversion yield of 50% from glucose in the presence of excess biotin.     

拟南芥光形态突变体的筛选与基因鉴定

以哥伦比亚(Columbia)野生型拟南芥(Arabidopsis thaliana)为实验材料,用含有激活标记双元质粒pCB260的农杆菌浸花进行转化,构建拟南芥T-DNA插入突变体库.通过突变体的筛选和表型分析,获得了两株光形态突变体,子叶下胚轴伸长的光抑制效应减弱.通过TAIL-PCR(thermal asymmetric interlaced-PCR)技术,成功扩增出突变植株T-DNA插入位点侧翼序列,经NCBI序列比对,T-DNA分别插在CRY1第一和第三外显子部位.突变体的表型分析及PCR鉴定结果表明,T-DNA插入CRY1并影响到突变植株的光形态建成.     

L-Tyrosine Production by Analog-resistant Prototrophic Mutants of Glutamic Acid Producing Bacteria

p-Fluorophenylalanine (PFP) and m-fluorophenylalanine were the most effective inhibitors on the growth of Corynebacterium glutamicum ATCC 13032 among the analogs of phenylalanine and tyrosine tested. Their inhibitory effects were released by L-phenylalanine, and slightly by L-tyrosine and L-tryptophan. 3-Aminotyrosine (3AT), p-aminophenylalanine, o-fluorophenylalanine, and β-2-thienylalanine were weak inhibitors.

Resistant mutants of C. glutamicum isolated on the medium containing both PFP and 3AT or PFP and L-tyrosine were found to accumulate both L-tyrosine and L-phenylalanine, while resistant mutants isolated on the medium containing only PFP were found to produce only L-phenylalanine. Resistant mutants from other glutamic acid producing bacteria isolated on the medium containing both PFP and 3AT or both PFP and L-tyrosine were found to accumulate L-tyrosine and L-phenylalanine.     

拟南芥干旱突变体远红外成像技术的筛选和特性鉴定

利用化学诱变剂甲基磺酸乙酯(EMS)对模式植物拟南芥(Arabidopsis thaliana)进行化学诱变获得突变体筛选群体。在干旱胁迫下,以叶片的温度差异为筛选指标,利用远红外成像技术进行突变体的筛选,获得了对干旱不敏感突变体dri1(drought-insensitive 1)和敏感突变体drs1(drought-sensitive 1)。实验结果表明dri1和drs1为单基因隐性突变,气孔密度同野生型无差异,而叶片温度、气孔开度和叶片失水率则有明显改变。在MS培养基上的种子萌发实验表明在ABA、甘露醇和NaCl胁迫下dri1萌发率要比野生型高,而drs1则比野生型低。对突变基因的研究有待进一步进行。     

拟南芥乙烯突变体在干旱胁迫下的形态学差异

为了揭示乙烯在植物与环境相互作用过程中的生物学功能,以拟南芥（Arabidopsis thaliana）的ein2-5、ein3-1、EIN3ox、EIL1ox 4种乙烯突变体与Col-0野生型为材料,对比研究它们在干旱胁迫条件下的生长和形态学变化。研究发现,干旱胁迫导致莲座叶直径、叶片面积、花序、水势等指标发生显著变化,同时不同突变体的形态适应特点呈现显著差异。这些结果表明,乙烯积极参与了植物形态塑造过程,与植物的抗旱性具有紧密相关性。     

拟南芥耐低钾突变体的筛选及遗传分析

利用乙酰甲基磺酸（ＥＭＳ）诱变方法,以幼苗根在重力作用下的弯曲生长为指标、筛选得到了拟南芥（Ａrabidopsis thaliana)耐低钾突变体。经过对突变体杂交后代的遗传分析证明,其中两株突变体的耐低钾性状为隐性单基因突变所致。鉴定、分离与植物耐低钾性状连锁的基因将有可能与对培育钾高效作物品种有重要意义。     

Effect of ABA on the UV-B-Induced Ethylene Evolution by the etr and ctr Mutants of Arabidopsis thaliana

The responses of 14-day-old Arabidopsis thaliana (L.) Heynh. plants to UV-B irradiation (280–320 nm) and ABA treatment were investigated. Wild-type plants as well as ethylene-insensitive etr1-1 and ctr1-1 mutants were used. Theetr1-1 mutant considerably differed from the ctr1-1 one in the fresh weight production after UV-B treatment (29.5 kJ/m²). The irradiated etr1-1 plants fell well behind the nonirradiated ones during the first two days after stress, but by the 8th day, their weight attained 70% of control plant weight. In contrast, Ctr1-1 mutant weight comprised 70% of control level after two days of stress but, by the 8th day, it was only 56% of the weight of control plants. In wild-type and ctr1-1 plants, ABA, in the 8 × 10^–6 to 2 × 10^–4 M concentration range, increased the difference between the weights of nonirradiated and irradiated plants, but in etr1-1 plants, ABA decreased this difference. The etr1-1, ctr1-1, and wild-type plants were very similar in the dynamics of ethylene evolution after UV-B treatment (7.4 kJ/m²). In wild-type, etr1-1, and ctr1-1 plants, ABA, in a concentration-dependent manner, inhibited UV-B-induced ethylene evolution to the same extent. The results obtained show that ABA exerted an opposite effect on UV-B-dependent growth in the plants with active (wild type and ctr1-1) and blocked (etr1-1) ethylene signal pathway, whereas the inhibition of ethylene synthesis by ABA was not related to ethylene signal transmission.     

拟南芥叶片数目变化突变体对营养生长时相转变的影响

拟南芥(Arabidopsis thaliana)的营养生长可以分为2个阶段: 幼龄期与成熟期。由幼龄期向成熟期的转变(VPC)与叶片的形态学特征、茎顶端分生组织(SAM)形状、远轴面表皮毛的出现以及SPL家族转录因子表达水平的变化相关。研究表明, 造成这种转变的信号来源于叶原基。该研究利用2种莲座叶数目改变了的突变体和对野生型切除叶片的方法, 分析了叶片数目对VPC的影响。结果表明, 莲座叶数目的减少推迟了VPC的发生; 而莲座叶数目增多突变体amp1-1并未使VPC的发生提前, 推测叶源信号的产生受到了光合作用的影响。     

Isolation and Genetic Analysis of Arabidopsis Mutants with Low-K+ Tolerance

Ethyl methane-sulfonate (EMS)-mutagenized Arabidopsis M2 populations were screened in low-K+ medium using the root-bending assay. Forty-two putative low-K+-tolerant ( lkt ) mutants were selected from 150?000 tested M2 seedlings, and two of these mutants maintained their low-K+-tolerant phenotype in their M3 generations, respectively. Genetic analysis showed that either one of these two mutants has a monogenic recessive mutation in a nuclear gene, and that the two mutations in two independent mutants are allelic to each other.     

拟南芥野生型与隐花素突变体光调节的蛋白质鉴定与聚类分析

光是控制植物生长发育十分重要的环境因子之一.隐花素是植物的蓝光受体,在植 物中调节多种光形态建成,包括抑制下胚轴的伸长、子叶的伸展和调节植物的开花时间 等,但隐花素依赖蓝光调节光形态建成的分子机制尚不清楚.本文采用比较蛋白质组学 方法研究了在持续蓝光和红光下生长的拟南芥隐花素双突变体cry1cry2和野生型幼苗的全蛋白图谱.采用基质辅助激光解吸飞行时间串联质谱(MALDI-TOF-TOF)进行肽质谱指纹图谱分析.在cry1cry2和野生型中鉴定了71个差异蛋白点.这些差异蛋白质反应光的变化可以形成6类,结果表明,光调节隐花素是通过控制许多相关基因的表达而实现的,为进一步研究拟南芥隐花素的光反应机制提供一些有用的信息.研究表明,蛋白质表达图谱可用于研究各种突变体在不同光照条件下光应答之间的关系.     

Utilization of Hydrocarbons and Vitamin B12 Production by Bacillus badius

The accumulation of vitamin B₁₂ by Bacillus badins grown on hydrocarbon was investigated. The bacterium could assimilate n-alkanes of C₁₁–C₁₈, ethanol, fumarate, α-ketoglutarate and malate. n-Alkanes of C₁₆–C₁₈, were the best for vitamin B₁₂ production. The bacterium utilized well all of the nitrogen sources tested. Above all, ammonium dihydrogen phosphate was the best for the bacteria] growth and vitamin B₁₂ production. Addition of organic nutrients such as malt extract and meat extract, and addition of metal ions such as ferrous and cobalt promoted the growth and vitamin B₁₂ production. Interestingly, vitamin B₁₂ was produced mostly in the supernatant. The cyanoform of the corrinoid predominantly formed in the supernatant would confirm the identity with cobalamin.     

Fermentative Production of l-Leucine with Auxotrophic Mutants of Corynebaeterium glutamicum

An auxotrophic mutant of Corynebaeterium glutamicum was found to accumulate a large amount of l-leucine in the culture medium. The nutritional requirement of the mutant is rather complex but it’s growth was most remarkably stimulated by l-phenylalanine. Acetate (1.5~3.0%) or pyruvate (3%) stimulated the l-leucine production. By a further mutagenic treatment, 329 mutants earring some defect in addition to phenylalanine auxotrophy were derived from the mutant No. 190. Among them, a histidine auxotrophic derivative produced twice as much l-leucine as the parent strain, i.e., the level of l-leucine production by this derivative reached 16 mg/ml in a medium containing 12% glucose, 1 % (NH₄)₂SO₄ and 2.5% CH₃COONH₄ as carbon and nitrogen sources. Some other auxotrophic markers such as isoleucine- (or threonine-), threonine-, purine(s)-, homoserine-, or methionine- auxotrophy also improved the L-leucine production by No, 190.     

Feedback-resistant Phosphoribosyl-ATP Pyrophosphorylase in l-Histidine-producing Mutants of Corynehacterium glutamicum

Phosphoribosyl-ATP pyrophosphorylase of two l-histidine-producers of Corynebacterium glutamicum, each selected as a 2-thiazolealanine (TA)-resistant and a 1,2,4-triazole-3-alanine (TRA)-resistant, was found to be 100-fold resistant to l-histidine-inhibition in comparison with wild-type enzyme. It was also resistant to the inhibition by TA, but still as sensitive as the wild-type enzyme to the inhibition by α-methylhistidine. Formation of the pyrophosphorylase in these mutants was not significantly derepressed. However, two-fold derepression was noted with a further improved l-histidine producer KY-10522, a derivative of the above TRA-resistant. KY-10522 is an improved strain in l-histidine-productivity through the additions of resistance markers including increased resistance to TRA. Phosphoribosyl-ATP pyrophosphorylase of KY-10522 was found to be resistant to the feedback inhibition, like it’s parent strain.