The de novo production of drosophilin A (tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (tetrachloro-1,4-dimethoxybenzene) by ligninolytic basidiomycetes

Ligninolytic basidiomycetes were screened for their ability to produce the tetrachlorinated hydroquinone metabolites drosophilin A (DA, tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (DAME, tetrachloro-1,4-dimethoxybenzene). Five fungal strains produced these metabolites in detectable amounts, including strains from Bjerkandera and Peniophora, which are genera not previously known for DA or DAME production. Phellinus fastuosus ATCC26.125 had the highest and most reliable production of DA and DAME in peptone medium, respectively 15–60 μM and 4–40 μM. This fungus was used to study culture conditions that could increase DAME production. A fourfold increase in DAME production was found after the addition of hydroquinone to growing cultures of P. fastuosus. Therefore, hydroquinone is postulated to be a possible biosynthetic precursor of DAME in the fungus. Antagonising P. fastuosus by adding filter-sterilised culture fluid of a competing fungus, Phlebia radiata, increased DAME production significantly by tenfold. This result suggests that DAME production is elicited by compounds present in the culture fluid of P. radiata, indicating that DAME has an antibiotic function in P. fastuosus. Received: 17 September 1996 / Received revision: 7 February 1997 / Accepted: 15 February 1997     

Evaluation of Candida acidothermophilum in ethanol production from lignocellulosic biomass

A Saccharomyces-cerevisiae-based simultaneous saccharification and fermentation (SSF) of lignocellulosic biomass is limited to an operating temperature of about 37 °C, and even a small increase in temperature can have a deleterious effect. This points to a need for a more thermotolerant yeast. To this end, S. cerevisiae D₅A and a thermotolerant yeast, Candida acidothermophilum, were tested at 37 °C, 40 °C, and 42 °C using dilute-acid-pretreated poplar as substrate. At 40 °C, C. acidothermophilum produced 80% of the theoretical ethanol yield, which was higher than the yield from S.cerevisiae D₅A at either 37 °C or 40 °C. At 42 °C, C. acidothermophilum showed a slight drop in performance. On the basis of preliminary estimates, SSF with C. acidothermophilum at 40 °C can reduce cellulase costs by about 16%. Proportionately greater savings can be realized at higher temperatures if such a high-temperature SSF is feasible. This demonstrates the advantage of using thermophilic or thermotolerant yeasts. Received: 20 February 1997 / Received revision: 24 June 1997 / Accepted: 4 July 1997     

Effects of phenolic compounds on the growth and the fatty acid composition of Lactobacillus plantarum

The effects of different phenolic compound concentrations on the fatty acid composition of Lactobacillus plantarum isolated from traditional home-made olive brines were determined. Increasing amounts of caffeic and ferulic acids induced a gradual increase in the amounts of myristic, palmitoleic, stearic and 9,10-methylenehexadecanoic (C17Δ, where Δ represents the cyclopropane group) acid with a concomitant decrease of lactobacillic acid (C₁₉Δ). On the other hand, the addition of tannins induced an increase in the C₁₉Δ level at the expense of vaccenic acid content. The presence of acidic phenols and tannins also affected bacterial growth, inducing the most obvious effect with tannin at 1 g l⁻¹. Received: 1 July 1997 / Received revision: 9 September 1997 / Accepted: 15 September 1997     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products

A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin. The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and V _max values toward phosphoric-acid-swollen cellulose as substrate, except that their K _m values were about fourfold higher than that of the native enzyme. Received: 4 August 1997 / Received revision: 13 October 1997 / Accepted: 31 October 1997     

Growth and nodulation competitiveness of Sinorhizobium meliloti L1 (RecA−) is less than that of its isogenic strain L33 (RecA+) but comparable to that of two S. meliloti wild-type isolates

Gnotobiotic systems were used to assess the competitive abilities of bioluminescent Sinorhizobium meliloti strains L1 (RecA⁻) and L33 (RecA⁺) for growth and host plant nodulation in the presence of a reconstructed S. meliloti population. Three wild-type strains belonging to infective subgroups of a natural S. meliloti population were chosen as competitors in microcosm studies. Whereas the RecA⁺ strain L33 dominated the reconstructed population with respect to growth and alfalfa nodulation, the competitiveness of the RecA⁻ strain L1 was reduced compared to that of one of the field strains, but comparable to that of the other field isolates. This result indicates that strain L1, despite its recA mutation, has the potential to compete successfully with a resident S. meliloti population after environmental release. Received: 4 November 1996 / Received revision: 9 January 1997 / Accepted: 17 January 1997     

Secretion, purification, and characterisation of barley α-amylase produced by heterologous gene expression in Aspergillus niger

Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence. Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997     

Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)

We have used a plasmid containing the argB gene to transform an Aspergillus nidulansargB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ∼3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A. nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi. Received: 22 August 1997 / Accepted: 20 November 1997