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     

Production of indole-3-acetic acid by mutant strains of Ustilago maydis (maize smut / huitlacoche)

Production of indole-3-acetic acid (IAA) by four strains of the maize pathogen Ustilago maydis was analyzed. The fungus induces gall formation on its host plant and IAA production by  U. maydis may be required as a pathogenicity or virulence factor. The study included the FB2 wild-type strain and the 103, 130FZ and 130FT mutants. Results show that treatment with clofibric acid, alone or in combination with UV light, can be used to obtain  U. maydis strains with defective IAA production in vitro, as quantified with the Salkowski reagent and by HPLC. The strain with the lowest production was 130FT, and its peak IAA level represented only 16% of the highest value obtained for the FB2 wild-type strain (124 μg/ml). Received: 11 April 1996 / Received last revision: 5 September 1997 / Accepted: 11 September 1997     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

Characterization of salt-tolerant mutant for enhancement of l-threonine production in Escherichia coli

 Escherichia coli strain HS3, metabolically engineered to have Met^–, AHV^r, Ile^L and AEC^r characteristics, produced 58.0 g/l of l-threonine, but it was neither salt-tolerant nor osmotolerant; and the growth and threonine production of the strain were severely inhibited both by the addition of NaCl with a concentration higher than 2% and by the presence of glucose with a concentration higher than 10%. Therefore, salt-tolerant mutants were isolated. The salt-tolerant mutants, HS454 and HS528 which were derived from strain HS3, were both tolerant to salt (2%) and hyperproductive. The growth and l-threonine production by the mutant strain HS454 were almost unaffected by a glucose concentration lower than 10%, but gradually reduced with increasing glucose concentration, up to 15%. However, the mutant strain HS528 showed slightly enhanced growth and l-threonine production with increasing glucose concentration, up to 10–12.5%. Strains HS454 and HS528 produced 69.8 g/l and 74.0 g/l of l-threonine, respectively in a 5-l jar fermentor. Received: 21 January 2000 / Received revision: 31 March 2000 / Accepted: 5 May 2000     

Production of exopolysaccharide by Pseudomonas sp. ATCC 31461 (Pseudomonas elodea ) using whey as fermentation substrate

An improved strain of Pseudomonas sp. ATCC 31461 (Pseudomonas elodea), capable of producing broth viscosities of 11 000 and 4700 mPa s (cP) when grown in enriched whey permeate and enriched sweet whey broths respectively, was isolated. The isolation was by serial transfers of the parent on lactose-rich and sweet whey broths. Maximum viscosities and biopolymer production were observed in 25% (v/v) whey concentration. In whey concentrations of 50% (v/v) or greater, residual glucose was detected in the broth and biopolymer production was low. This strain is capable of totally utilising the lactose in up to 50% (v/v) whey in 64 h. Enzyme activities suggest that the transport of lactose in P. elodea is by the permease system as opposed to the phosphotransferase system. The location of β-galactosidase is mainly intracellular. The improved strain is able to utilise lactose better than the parent and produce 1.6 times more intracellular β-galactosidase activity compared to the parent. Received: 3 May 1996 / Received revision: 8 August 1996 / Accepted: 10 August 1996     

Screening of spontaneous and induced mutants in Streptomyces avermitilis enhances avermectin production

Because of the loss of productivity in industrial strains (as a consequence of genetic instability), the selection of spontaneous and induced mutants in Streptomyces might generate enhanced producers of bioactive compounds. In this work, a spontaneously high producing mutant of Streptomyces avermitilis, strain 267/2H, was isolated. This mutant produced 8.2 times more avermectin B₁ than the wild type and it was treated with methyl methanesulphonate (MMS) in order to obtain better avermectin producers. One mutant, strain IPT-85, produced about 16 times more avermectin than the wild-type strain ATCC 31267 and twice as much as the parental strain 267/2H. Reversion studies showed that avermectin production by the IPT-85 mutant was unstable and required constant selection to maintain high levels of avermectin B₁ production. Upon a second MMS treatment of IPT-85, a new avermectin-aglycone-producing mutant, strain IPT 85-62, was isolated. Received: 2 March 1999 / Received revision: 16 June 1999 / Accepted: 27 June 1999     

Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

 Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10^-5–10^-4 g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II. Received: 22 December 1995/Received revision: 26 February 1996/Accepted: 17 March 1996     

Cytochrome c peroxidase from a methylotrophic yeast: physiological role and isolation

Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth⁺) in the absence of catalase activity. These Mth⁺ gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type strain. In contrast to the parental C-105 strain, H₂O₂ does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor strains is strongly correlated with increased levels of the alternative H₂O₂-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized. Received: 9 December 1996 / Received revision: 5 May 1997 / Accepted: 25 May 1997     

Isolation of mutants of Penicillium purpurogenum resistant to catabolite repression

 The strain Penicillium purpurogenum P-26 was subjected to UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment and mutants were isolated capable of synthesizing cellulase under the conditions of a high concentration of glucose. Initially mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated on Walseth’s cellulose/agar plates containing 15–45 mM 2-deoxy-D-glucose. These mutants were again screened for resistance to catabolite repression by glycerol or glucose on Walseth’s cellulose/agar plates containing 50 g/l glycerol or 50 g/l glucose respectively. Four mutants with different sizes of clearing zone on Walseth’s cellulose/agar plates containing 50 g/l glucose were selected for flask culture. Among them, the mutant NTUV-45-4 showed better carboxymethylcellulase activity in flask culture containing 1% Avicel plus 3% glucose than did the parental strain. Received: 9 October 1995/Received revision: 27 November 1995/Accepted: 8 January 1996     

A physical assay for meiotic recombination in Coprinus cinereus

We have used the polymerase chain reaction (PCR) method to monitor meiotic recombination in the basidiomycete Coprinus cinereus. We used DNA-mediated transformation to recover strains with modifications of the trp1 locus. The modifications were designed to introduce unique PCR priming sites separated by a homologous 2.4 kb region in which crossing over could occur. We showed that exchange occurred in this region at the frequency expected for a typical region of this genome (2.4 kb should correspond to a genetic length of 0.08 cM). We also detected products resulting from crossing over in DNAs extracted from cells in meiotic prophase. The assay should be useful for monitoring exchange in mutants that cannot complete meiosis. Received: 5 September 1996 / Accepted: 1 December 1996     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996     

Use of immobilized bacteria to treat industrial wastewater containing a chlorinated pyridinol

 Pseudomonas sp. strain M285 immobilized on diatomaceous earth beads was used to remove 3,5,6-trichloro-2-pyridinol (TCP) from industrial wastewater. Batch studies showed that immobilized Pseudomonas sp. strain M285 mineralized [2,6-¹⁴C]TCP rapidly; about 75% of the initial radioactivity was recovered as ¹⁴CO₂. Transformation of TCP was inhibited by high concentrations of salt, and addition of osmoprotectants (proline and betaine at 1 mM) did not reduce the adverse effect of salt. TCP-containing wastewater (60–140 mg/l) was passed through columns containing immobilized Pseudomonas sp. strain M285 at increasing flow rates and increasing TCP concentrations; TCP removal of 80%–100% was achieved. Addition of nutrients, such as glucose and yeast extract, retarded TCP degradation. Growing cell cultures were found to be better inocula for immobilization than resting cells. Received: 5 February 1996 / Received last revision: 12 August 1996 / Accepted: 24 August 1996     

Breeding of starch-utilizing and itaconic-acid-producing koji molds by interspecific protoplast fusion between Aspergillus terreus and Aspergillus usamii

Interspecific protoplast fusion between␣Aspergillus terreus, an itaconic acid producer, and A.␣usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment produced prototrophic fusants with a fusion frequency of 10⁻⁵−10⁻⁴. The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants, F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity from soluble starch was five times as high as that of A. terreus and 70 % of that of A. terreus from glucose (120 mg/ml). Received: 28 June 1996 / Received revision: 3 September 1996 / Accepted: 29 September 1996     

Application of factorial and Doehlert designs for optimization of pectate lyase production by a recombinant Escherichia coli

 The expression of a recombinant pectate lyase from Bacteroides thetaiotaomicron strain 217 was studied in Escherichia coli strain HB101(pBT4). First, two sets of complete 2⁴ factorial designs were used to evaluate the influences of casamino acids, glucose, magnesium, calcium, tetracycline, ampicillin, tryptophan and MOPS buffer on pectate lyase production in a basal medium. While casamino acids, glucose and magnesium were found to be the prevalent factors, the presence of tetracycline, ampicillin and MOPS buffer were necessary for the reproducibility of the process, probably by increasing the plasmid stability. Secondly, application of the Doehlert design, a response-surface methodology, allowed a good prediction of pectate lyase production according to the variation in glucose and magnesium concentrations. This optimization strategy allowed the production of biomass and recombinant pectate lyase respectively to be increased from 0.2 g l^-1 to 1.9 g l^-1 (dry weight) and from 10 units ml^-1 to 210 units ml^-1 within 24 h at 30°C in shake flasks. Received: 26 July 1995/Received revision: 22 January 1996/Accepted: 29 January 1996     

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S

A CH₃OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH₃OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants (>ca. 700 mg glucose equivalent/l) were >11–22 times higher than those produced by the wild-type strain (<ca. 60 mg glucose equivalent/l). The GC-MS analysis showed that all the mutants accumulated glucose, erythrose, threose and a disaccharide-like compound in the media. Received: 25 August 1999 / Received revision: 15 March 2000 / Accepted: 24 March 2000     

Pyrazine production by Bacillus subtilis in solid-state fermentation on soybeans

2,5-Dimethylpyrazine (2,5-DMP) and tetramethylpyrazine (TTMP) were produced using Bacillus subtilis IFO 3013 grown on soybeans. Solid-state cultivations were carried out either in 100-ml bottles or in a fixed-bed column reactor, both systems being at 27 °C. Optimization studies showed that the best way to produce the two above aroma compounds involved two separate processes. 2,5-DMP was obtained using soybeans enriched with 75 g threonine/kg initial dry weight (i.d.w.), giving 0.85 g metabolite/kg i.d.w. after 6 days. TTMP production involved addition of 90 g/kg i.d.w. acetoin to soybeans, and 2.5 g/kg i.d.w. was recovered after 14 days. These results demonstrated the suitability of solid-state cultivation for production of high-added-value compounds. Received: 30 September 1996 / Received revision: 23 December 1996 / Accepted: 30 December 1996     

Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides

A total of 182 Lactobacillus strains were screened for production of extracellular polysaccharides (EPS) by a new method: growth in liquid media with high sugar concentrations. Sixty EPS-positive strains were identified; 17 strains produced more than 100 mg/l soluble EPS. Sucrose was an excellent substrate for abundant EPS synthesis. The ability to produce glucans appears to be widespread in the genus Lactobacillus. The monosaccharide composition of EPS produced by Lactobacillus reuteri strain LB 121 varied with the growth conditions (solid compared to liquid medium) and the sugar substrates (sucrose or raffinose) supplied in the medium. Strain LB 121 produced both a glucan and a fructan on sucrose, but only a fructan on raffinose. This is the first report of fructan production by a Lactobacillus species. EPS production increased with increasing sucrose concentrations and involved extracellular sucrase-type enzymes. Received: 20 March 1998 / Received revision: 12 August 1998 / Accepted: 12 August 1998     

Selective isolation of Aspergillus niger mutants with enhanced glucose oxidase production

After mutagenization and selection, mutant Aspergillus niger strains resistant to certain agents were obtained. Seven of the mutants showed increased extracellular glucose oxidase (GOD), the level for individual cases ranged widely from 8.8 to over 138.5% in comparison with the parental strain. Studies of the relationship between method of selection and frequency of mutation showed that the highest frequency of positive mutations (15.8% and 17.3%) was obtained from mutants resistant to ethidium bromide (1 mmol 1^-1) and sodium gluconate (45%), respectively. The time course of growth and enzyme production by the most active mutant AM-11 showed intra- and extracellular GOD activities to have increased about 2.2- and 2.4-fold, respectively, compared with the parental strain.     

Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors

Cultivation conditions for the extracellular production of a hybrid β-glucanase from Bacillus were established by using Escherichiacoli JM109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid β-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of β-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of β-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of β-glucanase [E. coli JM109(pLF3)] was tested on the fermenter scale. Received: 1 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood

Trametes versicolor was shown to produce extracellular laccase during surface cultivation on glucose, wheat straw and beech wood. Growth on both wheat straw and beech wood led to an increase as high as 3.5-fold in extracellular laccase activity, in comparison with growth on glucose. The corresponding yields in fungal biomass reached only about 20% of the value obtained on glucose. Manganese peroxidase activity␣appeared during growth on wheat straw and beech wood. Mycelia grown on glucose, wheat straw and beech wood also showed intracellular laccase activities, monitored with 2,6-dimethoxyphenol, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), 4-hydroxy-3,5-dimethoxybenzaldehyde azine (syringaldazine) and 3,4-dihydroxyphenylalanine (l-DOPA). Assaying intracellular laccase with 2,6-dimethoxyphenol, syringaldazine and l-DOPA showed the maximum oxidation rates to be at pH values different from those producing maximum oxidation rates with extracellular laccase. In each case most of the total laccase activity was recovered from the culture filtrates. Growth on wheat straw and beech wood led to increased values for both extra- and intracellular laccase activities, based on fungal dry weight, in comparison with growth on glucose. Received: 18 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996