Production of L-Tryptophan by Sulfonamide-resistant Mutants

Sulfaguanidine-resistant mutant S-225, derived from a tryptophan-producing 5-fluoro-tryptophan-resistant mutant of Brevibacterium flavum, accumulated 19 g/liter of L-tryptophan at maximum when cultured for 72 hr in a medium containing 13% glucose as carbon source, 1.8-fold higher than did the parent. Strain S-225 accumulated 17 g/liter of L-tryptophan in a medium containing 10% sucrose as carbon source (17% yield based on the sugar). It also accumulated 450 mg/liter of chorismate, an intermediate common to the biosyntheses of tryptophan and p-aminobenzoate. The accumulation was 1.7-fold higher than that by the parent, suggesting that the intracellular concentration of chorismate was increased through acquisition of the sulfaguanidine resistance. Sulfaguanidine-resistant mutants were also derived from a tryptophan-producing mutant of Corynebacterium glutamicum. The mutants showed 2.2-fold higher maximum tryptophan production than did the parent.     

Citric acid production from sugar cane molasses by 2-deoxyglucose-resistant mutant strain ofAspergillus niger

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q _s (g per L per h) and specific substrate consumption rate,q _s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y _p/x), volume productivity (Q _p) and specific product yields (q _p), the mutant strain is significantly (p≤0.05) improved over the parental strain.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

Bimutation breeding of Aspergillus niger strain for enhancing β-mannanase production by solid-state fermentation

A parent strain Aspergillus niger LW-1 was mutated by the compound mutagenesis of vacuum microwave (VMW) and ethyl methane sulfonate (EMS). A mutant strain, designated as A. niger E-30, with high- and stable-yield β-mannanase was obtained through a series of screening. The β-mannanase activity of the mutant strain E-30, cultivated on the basic fermentation medium at 32 °C for 96 h, reached 36,675 U/g dried koji, being 1.98-fold higher than that (18,501 U/g dried koji) of the parent strain LW-1. The purified E-30 β-mannanase, a glycoprotein with a carbohydrate content of 19.6%, had an apparent molecular weight of about 42.0 kDa by SDS–PAGE. Its optimal pH and temperature were 3.5 and 65 °C, respectively. It was highly stable at a pH range of 3.5–7.0 and at a temperature of 60 °C and below. The kinetic parameters K_m and V_max, toward locust bean gum and at pH 4.8 and 50 °C, were 3.68 mg/mL and 1067.5 U/mg, respectively. The β-mannanase activity was not significantly affected by an array of metal ions and EDTA, but strongly inhibited by Ag⁺ and Hg²⁺. In addition, the hydrolytic conditions of konjak glucomannan using the purified E-30 β-mannanase were optimized as follows: konjak gum solution 240 g/L (dissolved in deionized water), hydrolytic temperature 50 °C, β-mannanase dosage 120 U/g konjak gum, and hydrolytic time 8 h.     

Isolation of a mutant strain of Pseudomonas sp ATCC 31461 exhibiting elevated polysaccharide production

A mutant strain of the bacterium Pseudomonas sp. ATCC 31461 that exhibited elevated production of the polysaccharide gellan on glucose or corn syrup as a carbon source was isolated. Gellan production by the mutant strain was about twofold higher than its parent strain on glucose or corn syrup after 48 h of growth, and about 1.4-fold higher after 72 h. An increase in biomass production was not correlated with enhanced gellan synthesis by the mutant strain. The increased gellan production by the mutant strain on either carbon source resulted in an increase in its culture medium viscosity and the viscosity of the isolated polysaccharide produced by glucose-grown cells. No differences in the glucuronic acid content of the polysaccharides produced by the mutant and parent strains were observed. Journal of Industrial Microbiology & Biotechnology (2002) 29, 185–188 doi:10.1038/sj.jim.7000278 Received 13 February 2002/ Accepted in revised form 20 May 2002     

Alternation of the Dissimilation Pathway for Glycerol and Amplification of Glycerol Dehydrogenase in Mutant Strains of Cellulomonas sp. NT3060

Mutant strain ME544, which is able to grow on glycerol slowly, was derived from glycerol-negative mutant strain G011, which is a derivative strain of Cellulomonas sp. NT3060 and is defective in both the enzyme activities of glycerol kinase and glycerol 3-phosphate dehydrogenase. The mutant strain still lacked both the enzyme activities involved in the dissimilation of glycerol and had the same level of glycerol dehydrogenase activity as the parent strain. Dihydroxyacetone kinase activity in mutant strain ME544 was inducibly formed, reaching 4-fold the level in mutant strain G011 in glycerol medium. Thus, the mutant strain seemed to dissimilate glycerol by means of glycerol dehydrogenase followed by an increase in dihydroxyacetone kinase. Subsequently, a mutant strain, GP1807, which was resistant to the inhibition of growth on glycerol by 1,2-propanediol, was derived from mutant strain ME544. Glycerol dehydrogenase activity of the mutant strain was amplified about 6-fold compared to that of the wild type strain.     

Isolation of Advantageous Mutants of Pseudomonas chlororaphis B23 for the Enzymatic Production of Acrylamide

Advantageous mutants of Pseudomons chlororaphis B23 for the enzymatic production of acrylamide were isolated. A mucilage polysaccharide non-producing mutant, Am-3, was precipitated completely by brief centrifugation, in contrast with the parent strain. A mutant, AM-324, derived from Am-3 exhibited about 3.8-fold higher nitrile hydratase activity than that of the parent strain. These mutants are promising for acrylamide production on an industrial scale.     

ε-聚赖氨酸高产菌株选育及分批发酵的研究

以北里孢菌(Kitasatospora sp.)PL6-3为出发菌株,经硫酸二乙酯诱变,获得遗传性能稳定的AECr突变株MY5-36。MY5-36摇瓶发酵产ε-聚赖氨酸达1.17 g/L,是出发株PL6-3的3倍;5 L发酵罐批式发酵产酸达7.72 g/L,较出发菌株提高了7倍以上。与PL6-3相比,MY5-36菌株形态发生了很大变化,菌丝和孢子颜色都发生了改变。     

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.     

The Effect of the Plant Growth Stimulant Bactozole on Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Tolerant Mutant M-71 under Different Nitrogen Supply Conditions

The effect of the plant growth stimulant bactozole on the growth of Rhizobium leguminosarum bv. viciae 250a and its nitrogen-tolerant mutant M-71 and the synthesis of extracellular carbohydrates was studied. At a low content of nitrate (6 mM) in the medium, all three bactozole concentrations tested (0.001, 0.01, and 0.1%) exerted similar stimulating effects on the growth of the parent strain 250a (about 1.5-fold) and the synthesis of extracellular carbohydrates (about 2-fold). At a high content of nitrate (20 mM) in the medium, when the growth of the parent strain and the synthesis of extracellular carbohydrates were inhibited, bactozole at all three concentrations exerted only a growth-stimulating effect. At the same time, mutant M-71 showed better growth at higher concentrations of bactozole, whereas the ability of the mutant to synthesize extracellular carbohydrates decreased with increasing bactozole concentration. The cell biomass of the mutant accumulated at 20 mM nitrate was 1.8–2.5 times greater than it was at 6 mM nitrate. Bactozole enhanced the symbiosis of legume plants with both parent and mutant strains, raising the mass of plants and enhancing nodulation and the nitrogen-fixing activity of root nodules. The symbiotic parameters of mutant M-71 were better (irrespective of whether bactozole was present or not) when its inoculum was grown at a high nitrogen content (20 mM nitrate), whereas the respective parameters of the parent strain were better when it was grown at 6 mM nitrate. The inference is made that the better physiological characteristics of the mutant in the high-nitrate medium are due to its higher nitrate reductase activity (as compared with the parent strain) in both the free-living state and in legume nodules.     

集胞藻PCC 6803高产精氨酸藻株的紫外诱变选育

精氨酸在医药和食品工业上具有广泛用途。集胞藻PCC 6803是单细胞蓝藻, 能利用工业废气(主要成分是氮氧化物NO_x)与水反应生成的硝酸盐和亚硝酸盐合成氨基酸等化合物, 因而选育高产精氨酸藻株, 不仅能提高精氨酸产量, 而且能去除工业废气中的NO_x, 具有潜在的应用前景。研究在集胞藻PCC 6803中利用紫外诱变, 筛选抗0.8 g/L D-精氨酸和抗0.2 g/L 6-氮尿嘧啶的突变株, 选育到了一株精氨酸产量显著提高的突变株#13807-111-55, 它每OD₇₃₀值细胞的胞外精氨酸产量相比出发株提高了62.3倍, 达到(0.76±0.1) mg/(L·OD₇₃₀), 总精氨酸产量相比出发株提高了6.0倍, 达到(0.82±0.08) mg/(L·OD₇₃₀)。该突变株每OD₇₃₀值细胞的胞外精氨酸产量明显高于胞内, 表明该突变藻株是精氨酸分泌型, 因而具有潜在的应用前景。     

Purification and characterization of glucoamylase from a higher yielding mutant of Aspergillus candidus Link var. aureus

Summary A higher yielding mutant of Aspergillus candidus Link var. aureus has been isolated using the chemical mutagen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) at 5 g/ml concentration of the mutagen. This mutant produces 88 to 111 U/ml of glucoamylase compared with 40 to 50 U/ml produced by the parent strain. The enzyme from this mutant has been purified to homogeneity using hydrophobic interaction chromatography and a DEAE-cellulose column with over 65% recovery of the enzyme activity and 21-fold purification. The mutant protein is very similar to the enzyme from the parent strain in that it cochromatographs with the parent enzyme on phenylglycyl-Sepharose, norleucyl-Sepharose, Sepharose-CL-6B, Concanavalin A-Sepharose, DEAE-cellulose and DEAE-Sephadex-A-50. The two proteins exhibit similar electrophoretic behavior and have similar molecular weights and amino acid composition. However, the specific activity of the purified mutant protein is 1125 U/mg compared with 560 U/mg for the parent enzyme. Further genetic analysis of the mutant is needed to explain these observations.     

Enhanced acetoin production by Bacillus amyloliquefaciens through improved acetoin tolerance

Acetoin production by Bacillus amyloliquefaciens was used as a model of product feedback to develop a strategy to enhance the production of acetoin. To enhance the resistance of B. amyloliquefaciens to acetoin, an acetoin-tolerant mutant E-11 was screened by using adaptive evolution with acetoin stress as the selection pressure. When compared with the parent FMME044, the mutant E-11 exhibited superior fermentation performance as follows: (1) the mutant E-11 exhibited increased tolerance to high concentration of acetoin, and the specific growth rate was 265.2% higher than that of the parent FMME044 in medium containing 80 g/L acetoin; (2) acetoin production by the mutant E-11 reached 71.5 g/L at 44 h when cultured in a 7-L fermentor with 173 g/L glucose, and the acetoin concentration and productivity of the mutant E-11 were 39.6% and 14.4% higher than those of the parent FMME044, respectively; (3) the unsaturated fatty acid contents in the mutant E-11 were 64.8%, 37.8%, and 18.4% higher than those in the parent FMME044 when cultured in 0, 40, and 60 g/L acetoin, whereas the saturated fatty acid contents in the mutant E-11 were 9.5%, 13.9%, and 14.1% lower than those in the parent FMME044, respectively.     

Overproduction of a mosquitocidal chloramphenicol-resistant Lysinibacillus sphaericus mutant obtained through UV irradiation

A highly active mosquitocidal mutant UV-chloramphenicol-resistant mutant (UCR-146) of Lysinibacillus sphaericus 2362 was isolated by UV irradiation and selected through resistance to chloramphenicol which was inhibitory for growth of the parent strain. The growth of UCR-146 in NYSMCL at different concentrations of chloramphenicol (20–100 µg/ml) showed high mosquitocidal activity against Culex pipiens larvae with the optimum concentration of 35µg/ml. At this level, LC₅₀ of UCR-146 was decreased about five times from that of L. sphaericus 2362. Comparative efficiency of mosquitocidal activity of UCR-146 and L. sphaericus 2362 within 28 days of consecutive growth exhibited notable stability of both cultures through seven cycles of growth in their optimal media. UCR-146 was grown in industrial by-products media for production of the binary toxin under economic conditions. Offal medium at 2% showed the highest mosquitocidal activity of UCR-146 with LC₅₀ of 0.53 PPM which was 16.6, 13, 12.3 and 3.4 times less than that produced with L. sphaericus 2362 grown in NYSM, L. sphaericus 2362 grown in offal, UCR-146 grown in NYSM and UCR-146 grown in NYSMCL, respectively. Hence, the mosquitocidal activity of L. sphaericus 2362 increased several times through ultraviolet (UV) irradiation followed by chloramphenicol resistance selection and then growth in 2% offal medium. Finally, this procedure for selecting UV-chloramphenicol-resistant mutant and the medium used could potentially be a simple and cost effective approach for obtaining and producing a highly active mosquitocidal mutant.     

OHIO-1 β-lactamase resistant to mechanism-based inactivators

Abstract Spontaneous mutants of OHIO-1 β-lactamase, an SHV-1 family enzyme, resistant to inactivation by clavulanic acid, sulbactam and tazobactam, have been isolated. The resistant mutant (M4) was inhibited by 100 μg/ml ampicillin plus 32 μg/ml clavulanic acid compared to ≤2 μg/ml clavulanic acid required for the parent strain. The pI of the mutant beta-lactamase was 7.0, identical to the parent enzyme. Kinetic parameters showed that the M4 enzyme had an increased V_max/K_m ratio for all beta-lactam substrates compared to the parent enzyme. The apparent K _i for clavulanic acid, sulbactam and tazobactam was 15.1, 182 and 18 μM, respectively, up to 70-fold higher than the parent enzyme. Partial nucleotide sequencing revealed that the mutant enzyme had a predicted methionine₆₉→ isoleucine₆₉ substitution accounting for the observed changes in substrate specificity.     

Enhancement of pyruvate production by osmotic-tolerant mutant of Torulopsis glabrata

Pyruvate production by Torulopsis glabrata was used as a model to study the mechanism of product inhibition and the strategy for enhancing pyruvate production. It was found that the concentration of cell growth and pyruvate deceased with the increase of NaCl and sorbitol concentrations. To enhance the osmotic stress resistance of the strain, an NaCl-tolerant mutant RS23 was screened and selected through a pH-controlled continuous culture with 70 g/L NaCl as the selective criterion. Compared with the parent strain, mutant RS23 could grow well on the medium containing 70 g/L NaCl or 0.6 mol/L sorbitol. Pyruvate concentration by the mutant strain RS23 reached 94.3 g/L at 82 h (yield on glucose 0.635 g/g) in a 7-l fermentor with 150 g/L glucose as carbon source. Pyruvate concentration and yield of mutant RS23 were 41.1% and 11.1% higher than those of the parent strain, respectively. The strategy for enhancing pyruvate production by increasing osmotic stress resistance may provide an alternative approach to enhance organic acids production with yeast.     

Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Summary The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lowerK _m but similarV _max compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.     

Increased carotenoid production in Xanthophyllomyces dendrorhous G276 using plant extracts

The red yeast Xanthophyllomyces dendrorhous (previously named Phaffia rhodozyma) produces astaxanthin pigment among many carotenoids. The mutant X. dendrorhous G276 was isolated by chemical mutagenesis. The mutant produced about 2.0 mg of carotenoid per g of yeast cell dry weight and 8.0 mg/L of carotenoid after 5 days batch culture with YM media; in comparison, the parent strain produced 0.66 mg/g of yeast cell dry weight and a carotenoid concentration of 4.5 mg/L. We characterized the utilization of carbon sources by the mutant strain and screened various edible plant extracts to enhance the carotenoid production. The addition of Perilla frutescens (final concentration, 5%) or Allium fistulosum extracts (final concentration, 1%) enhanced the pigment production to about 32 mg/L. In a batch fermentor, addition of Perilla frutescens extract reduced the cultivation time by two days compared to control (no extract), which usually required five-day incubation to fully produce astaxanthin. The results suggest that plant extracts such as Perilla frutescens can effectively enhance astaxanthin production.     

复合诱变法选育香兰素高转化率菌株

通过紫外线照射和He-Ne激光修复对香兰素生产菌链霉菌Streptomyces sp.L1936进行复合诱变,得到香兰素高产菌株Streptomyces sp.L1936-8。结果表明:出发菌株经过诱变处理后,脱乙酰酶酶活提高了75%,香兰素氧化酶酶活降低了37.5%,香兰素产量提高了33%。