Enzymatic hydrolysis of biomass from wood.

Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood.

High-titer production of astaxanthin by the semi-industrial fermentation of Xanthophyllomyces dendrorhous.

An improved semi-industrial process for astaxanthin production by fermentation of Xanthophyllomyces dendrorhous has been developed. The culture medium was designed at the flask scale, reaching an astaxanthin cellular content of 3.0 mgg(-1) cell weight and a volumetric yield of 119 mgL(-1) broth. Astaxanthin production in flask was significantly improved by white light (4.0 mgg(-1) and 221 mgL(-1)), and by ultraviolet light (4.4 mgg(-1) and 235 mgL(-1)). The scale-up to 10- and 800-L fermentors was developed by feeding with glucose. Representative data for illuminated fermentation processes are presented and discussed at the 10-L scale, where 420 mgL(-1) (4.7 mgg(-1)) astaxanthin were produced, and the 800-L scale, with productivities of 350 mgL(-1) (4.1 mgg(-1)) astaxanthin. The purity of the astaxanthin in the broth was about 84%, with accumulation of the following carotenoids other than astaxanthin: 4% beta-carotene, 4% canthaxanthin, 5% HDCO, 1% zeaxanthin and 2% phoenicoxanthin. This technology can be easily scaled-up to an industrial application for the production of this xanthophyll widely demanded nowadays.

Why did the Fleming strain fail in penicillin industry?

Penicillin, discovered 75 years ago by Sir Alexander Fleming in Penicillium notatum, laid the foundations of modern antibiotic chemotherapy. Early work was carried out on the original Fleming strain, but it was later replaced by overproducing strains of Penicillium chrysogenum, which became the industrial penicillin producers. We show how a C(1357)-->T (A394V) change in the gene encoding PahA in P. chrysogenum may help to explain the drawback of P. notatum. PahA is a cytochrome P450 enzyme involved in the catabolism of phenylacetic acid (PA; a precursor of penicillin G). We expressed the pahA gene from P. notatum in P. chrysogenum obtaining transformants able to metabolize PA (P. chrysogenum does not), and observing penicillin production levels about fivefold lower than that of the parental strain. Our data thus show that a loss of function in P. chrysogenum PahA is directly related to penicillin overproduction, and support the historic choice of P. chrysogenum as the industrial producer of penicillin.

The nagA gene of Penicillium chrysogenum encoding beta-N-acetylglucosaminidase.

We purified the beta-N-acetylglucosaminidase from the filamentous fungus Penicillium chrysogenum and its N-terminal sequence was determined, showing the presence of a mixture of two proteins (P1 and P2). A genomic DNA fragment was cloned by using degenerated oligonucleotides from the Nt sequences. The nucleotide sequence showed the presence of an ORF (nagA gene) lacking introns, with a length of 1791 bp, and coding for a protein of 66.5 kDa showing similarity to acetylglucosaminidases. The NagA deduced protein includes P1 and P2 as incomplete forms of the mature protein, and contains putative features for protein maturation: an 18-amino acid signal peptide, a KEX2 processing site, and four glycosylation motifs. The sequence just after the signal peptide corresponds to P2 and that after the KEX2 site to P1. The nagA transcript has a size of about 2.1 kb and is present until the end of the fermentation process for penicillin production. NagA is one of the most largely represented proteins in P. chrysogenum, increasing along the fermentation process. The suitability of the nagA promoter (PnagA) for gene expression in fungi was demonstrated by expressing the bleomycin resistance gene (ble(R)) from Streptoalloteichus hindustanus in P. chrysogenum.

The tylosin biosynthetic cluster from Streptomyces fradiae: genetic organization of the left region.

The genetic organization of the left edge (tyIEDHFJ region) of the tylosin biosynthetic gene cluster from Streptomyces fradiae has been determined. Sequence analysis of a 12.9 kb region has revealed the presence of 11 ORFs, 10 of them belonging to the biosynthetic cluster. The putative functions of the proteins encoded by these genes are as follows: peptidase (ORF1, ddcA), tylosin resistance determinant (ORF2, tlrB), glycosyltransferase (ORF3, tylN), methyltransferase (ORF4, tylE), ketoreductase (ORF5, tylD), ferredoxin (ORF6, tylH2), cytochrome P450 (ORF7, tylH1), methyltransferase (ORF8, tylF), epimerase (ORF9, tylJ), acyl-CoA oxidase (ORF10, tylP) and receptor of regulatory factors (ORF11, tylQ). The functional identification of the genes in the proposed tylosin biosynthetic pathway has been deduced by database searches and previous genetic complementation studies performed with tylosin idiotrophic mutants blocked at various stages in tylosin biosynthesis. The tlrB gene has been shown to be useful as a tylosin resistance marker in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor and the effect of tylF on macrocin depletion has been confirmed. A pathway for the biosynthesis of 6-deoxy-D-allose, the unmethylated mycinose precursor, involving the genes tylD, tylJ and tylN is proposed.

D-amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217.

The complete nucleotide sequence of the DAO1 gene encoding D-amino-acid oxidase (DAAO) in the yeast Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217 has been determined. The primary structure of DAAO was deduced from the nucleotide sequence of a cDNA clone that covered the entire amino acid coding sequence. Comparison of cDNA and genomic sequences of DAO1 revealed the presence of five introns. Because this is the first gene of strain ATCC 26217 that has been cloned so far, the nucleotide sequences of these introns were compared to those from other fungi. Upstream of the structural gene there was a stretch of C + T-rich DNA similar to that found in the promoter region of a number of yeast genes. The cDNA gene, which encoded a protein of 368 amino acids (molecular mass 40 kDa), was overexpressed in Escherichia coli under the control of the strong lipoprotein promoter. Interestingly, a significant fraction (13-62%) of the total DAAO activity was recovered in its apoenzyme form, the percentage depending on the culture conditions. This fact allowed a rapid purification of the recombinant DAAO by affinity chromatography. The high level of expression achieved in E. coli and the possibility of modifying its catalytic properties by protein engineering provide a new model for the study of this enzyme.