From accurate genome sequence to biotechnological application: The thermophile Mycolicibacterium hassiacum as experimental model.

Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc2 155 being the most common one. In this work, we focus on the use of a thermophilic mycobacterium, Mycolicibacterium hassiacum, which grows optimally above 50°C, as an emerging experimental model valid to extend our general knowledge of mycobacterial biology as well as for application purposes. To that end, accurate genomic sequences are key for gene mining, the study of pathogenicity or lack thereof and the potential for gene transfer. The combination of long- and short-massive sequencing technologies is strictly necessary to remove biases caused by errors specific to long-reads technology. By doing so in M. hassiacum, we obtained from the curated genome clues regarding the genetic manipulation potential of this microorganism from the presence of insertion sequences, CRISPR-Cas, type VII ESX secretion systems, as well as lack of plasmids. Finally, as a proof of concept of the applicability of M. hassiacum as a laboratory and industrial model, we used this high-quality genome of M. hassiacum to successfully knockout a gene involved in the use of phytosterols as source of carbon and energy, using an improved gene cassette for thermostable selection and a transformation protocol at high temperature.

Scale-Up of Phytosterols Bioconversion into Androstenedione.

Phytosterols, coming as a by-product of vegetable oils or wood pulp, contain the cyclopentanoperhydrophenanthrene nucleus and can be bioconverted into steroid intermediates by removing the C17 side chain. This chapter shows the scale-up, from flask to bioreactor, of phytosterols bioconversion into 4-androstene-3,17-dione (androstenedione; AD) using Mycolicibacterium neoaurum B-3805. Due to the fact that phytosterols and AD are nearly insoluble in water, two-phase systems and the use of chemically modified cyclodextrins have been described as methods to solve it. Here, we use a water-oil two-phase system that allows the bioconversion of up to 20 g/L of phytosterols into AD in 5 L and 20 L bioreactors.

Strain Improvement Program of Streptomyces roseosporus for Daptomycin Production.

Daptomycin is a cyclic lipopeptide antibiotic with potent activity against gram-positive bacteria. It has a calcium-dependent mechanism of action that disrupts multiple features of the bacterial membrane function. This antibiotic is highly demanded due to its effectiveness against to microorganisms resistant to other antibiotics, including vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant S. aureus (MRSA). Daptomycin is produced by fermentation of Streptomyces roseosporus, currently identified as Streptomyces filamentosus. However, low fermentation yields and high production costs are reported. This chapter describes a method of strain improvement involving random mutagenesis, rational screening by bioassay, and flask fermentation. The ultimate objective is to select mutants of S. roseosporus overproducing daptomycin in order to design a more cost-effective daptomycin production.

Lycopene Production by Mated Fermentation of Blakeslea trispora.

Lycopene is a carotenoid mainly present in red-colored fruits and vegetables. Its value in the pharmaceutical and food industry is linked to its benefits for the human health, including properties against cancer and cardiovascular diseases, and its use as a food colorant. Lycopene can be produced either by synthetic or natural means, but there is a preference for the second, since it is considered a more eco-friendly and less harmful process. Among natural methods for obtaining lycopene, microbial fermentation is a good alternative to extraction from plants that naturally contain lycopene, since it implies obtaining higher and more specific amounts of this carotenoid. This chapter describes lycopene production by fermentation of the fungus Blakeslea trispora, a naturally carotenoid producer, at 30 L scale. This procedure involves separated growth of the two sexual mating types of B. trispora during the vegetative stages and the use of a lycopene cyclase inhibitor to achieve lycopene accumulation during the production stage.

Scale-Up of Phytosterols Bioconversion into Androstenedione.

Phytosterols, generated as a by-product of vegetable oils or wood pulp, contain the cyclopentane-perhydro-phenanthrene nucleus, and can be converted into steroid intermediates by removing the C17 side chain. This chapter shows the scale-up, from flask to fermentor, of the phytosterols bioconversion into 4-androstene-3,17-dione (androstenedione; AD) with Mycobacterium neoaurum B-3805. Due to the fact that phytosterols and AD are nearly insoluble in water, two-phase systems and the use of chemically modified cyclodextrins have been described as methods to solve it. Here we use a water-oil two-phase system that allows for the bioconversion of up to 20 g/L of phytosterols into AD in 20 L fermentor.

Metabolic engineering of Mucor circinelloides for zeaxanthin production.

Mucor circinelloides is a ß-carotene producing zygomycete amenable to metabolic engineering using molecular tools. The crtS gene of the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous encodes the enzymatic activities ß-carotene hydroxylase and ketolase, allowing this yeast to produce the xanthophyll called astaxanthin. Here we describe the fermentation of X. dendrorhous in astaxanthin producing conditions to purify mRNA for the cloning of the cDNA from the crtS gene by RT-PCR. Further construction of an expression plasmid and transformation of M. circinelloides protoplasts allow the heterologous expression of the crtS cDNA in M. circinelloides to obtain ß-cryptoxanthin and zeaxanthin overproducing transformants. These two xanthophylls are hydroxylated compounds from ß-carotene. These results show that the crtS gene is involved in the conversion of ß-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.

Xanthophyllomyces dendrorhous for the industrial production of astaxanthin.

Astaxanthin is a red xanthophyll (oxygenated carotenoid) with large importance in the aquaculture, pharmaceutical, and food industries. The green alga Haematococcus pluvialis and the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous are currently known as the main microorganisms useful for astaxanthin production at the industrial scale. The improvement of astaxanthin titer by microbial fermentation is a requirement to be competitive with the synthetic manufacture by chemical procedures, which at present is the major source in the market. In this review, we show how the isolation of new strains of X. dendrorhous from the environment, the selection of mutants by the classical methods of random mutation and screening, and the rational metabolic engineering, have provided improved strains with higher astaxanthin productivity. To reduce production costs and enhance competitiveness from an industrial point of view, low-cost raw materials from industrial and agricultural origin have been adopted to get the maximal astaxanthin productivity. Finally, fermentation parameters have been studied in depth, both at flask and fermenter scales, to get maximal astaxanthin titers of 4.7 mg/g dry cell matter (420 mg/l) when X. dendrorhous was fermented under continuous white light. The industrial scale-up of this biotechnological process will provide a cost-effective method, alternative to synthetic astaxanthin, for the commercial exploitation of the expensive astaxanthin (about $2,500 per kilogram of pure astaxanthin).

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.

The NADP-dependent glutamate dehydrogenase gene from the astaxanthin producer Xanthophyllomyces dendrorhous: use of Its promoter for controlled gene expression.

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase (GDH) activity from Xanthophyllomyces dendrorhous has been cloned and characterized, and its promoter used for controlled gene expression in this red-pigmented heterobasidiomycetous yeast. We determined the nucleotide sequence of a 4701 bp DNA genomic fragment, showing an open reading frame of 1871 bp interrupted by five introns with fungal consensus splice-site junctions. The predicted protein (455 amino acids; 49 kDa) revealed high identity to GDHs, especially to those from the fungi Cryptococcus neoformans (70%), Sclerotinia sclerotiorum (66%), and several species of Aspergillus (66-67%). Gene phylogenies support the grouping of X. dendrorhous GDH close to those from the majority of the filamentous fungi. The promoter region of the gdhA gene (PgdhA) contains a TATA-like box and two large pyrimidine stretches. The use of PgdhA for gene expression was validated by electrotransformation of X. dendrorhous using an in-frame fusion with the hygromycin resistance gene (hygR) as a reporter. X. dendrorhous transformants were able to grow in YEME complex medium and in Czapek minimal medium supplemented with 50 microg/ml hygromycin, but gene expression in Czapek medium was repressed when using ammonium acetate as a nitrogen source. PgdhA is a valuable tool for controlled gene expression in Basidiomycetes.

Binding of the PTA1 transcriptional activator to the divergent promoter region of the first two genes of the penicillin pathway in different Penicillium species.

The aim of this work is to establish the correlation between the transcriptional activator PTA1 and the expression of the penicillin genes in different penicillin-producing strains. The level of expression of the first two genes of the penicillin pathway was clearly higher in Penicillium chrysogenum than in Penicillium notatum and Penicillium nalgiovense. The divergent promoter pcbAB-pcbC region contains binding sequences for several transcriptional factors that are conserved in P. notatum and P. chrysogenum, but not in P. nalgiovense. Binding of the purified P. chrysogenum transcriptional activator PTA1 to the palindromic heptamer TTAGTAA took place when the P. chrysogenum 35 bp DNA fragment containing the heptamer was used as a probe, but not when the sequence occurring in P. nalgiovense was used. P. nalgiovense protein fractions purified by heparin agarose chromatography did not bind to the 35-bp DNA fragment either from P. nalgiovense or P. chrysogenum, although some degree of binding was observed when crude extracts were used. This finding may explain the low expression of pcbC in P. nalgiovense. All the P. chrysogenum strains, including the industrial strain E1, showed the same nucleotide sequence, including the consensus PTA1 binding site.

Engineering the halophilic bacterium Halomonas elongata to produce beta-carotene.

Engineering halophilic bacteria to produce carotenoids is a subject of great scientific and commercial interest, as carotenoids are desirable products used as additives and colorants in the food industry, with beta-carotene the most prominent. With this target, we expressed the beta-carotene biosynthetic genes crtE, crtY, crtI, and crtB from Pantoea agglomerans and the cDNA encoding isopentenyl pyrophosphate isomerase from Haematococcus pluvialis in the halophilic bacterium Halomonas elongata obtaining a strain able to produce practically pure beta-carotene. Reverse transcription-polymerase chain reaction analysis showed crtY, crtI, and crtB heterologous expression in a selected exconjugant of H. elongata. Biosynthesis of beta-carotene was dependent on NaCl concentration in the culture medium, with the highest production (560 microg per g of dry weight) in 2% NaCl. On the contrary, no beta-carotene was detected in 15% NaCl. Successful construction of the beta-carotene biosynthetic pathway in H. elongata opens the possibility of engineering halophilic bacteria for carotenoid production.

The crtS gene of Xanthophyllomyces dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the conversion of beta-carotene into astaxanthin and other xanthophylls.

The conversion of beta-carotene into xanthophylls is a subject of great scientific and industrial interest. We cloned the crtS gene involved in astaxanthin biosynthesis from two astaxanthin producing strains of Xanthophyllomyces dendrorhous: VKPM Y2410, an astaxanthin overproducing strain, and the wild type ATCC 24203. In both cases, the ORF has a length of 3166 bp, including 17 introns, and codes for a protein of 62.6 kDa with similarity to cytochrome-P450 hydroxylases. crtS gene sequences from strains VKPM Y2410, ATCC 24203, ATCC 96594, and ATCC 96815 show several nucleotide changes, but none of them causes any amino acid substitution, except a G2268 insertion in the 13th exon of ATCC 96815 which causes a change in the reading frame. A G1470 --> A change in the 5' splicing region of intron 8 was also found in ATCC 96815. Both point mutations explain astaxanthin idiotrophy and beta-carotene accumulation in ATCC 96815. Mutants accumulating precursors of the astaxanthin biosynthetic pathway were selected from the parental strain VKPM Y2410 (red) showing different colors depending on the compound accumulated. Two of them were blocked in the biosynthesis of astaxanthin, M6 (orange; 1% astaxanthin, 71 times more beta-carotene) and M7 (orange; 1% astaxanthin, 58 times more beta-carotene, 135% canthaxanthin), whereas the rest produced lower levels of astaxanthin (5-66%) than the parental strain. When the crtS gene was expressed in M7, canthaxanthin accumulation disappeared and astaxanthin production was partially restored. Moreover, astaxanthin biosynthesis was restored when X. dendrorhous ATCC 96815 was transformed with the crtS gene. The crtS gene was heterologously expressed in Mucor circinelloides conferring to this fungus an improved capacity to synthesize beta-cryptoxanthin and zeaxanthin, two hydroxylated compounds from beta-carotene. These results show that the crtS gene is involved in the conversion of beta-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.

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 gamma-actin encoding gene from the beta-carotene producer Blakeslea trispora.

We determined the nucleotide sequence of a 4599-bp DNA genomic fragment including the gamma-actin encoding gene from Blakeslea trispora, showing an open reading frame of 1561 bp interrupted by four introns with fungal consensus splice-site junctions. The untranslated regions of the actA gene contain a consensus TATA box, a CCAAT motif, a large pyrimidine stretch, and the polyadenylation sequence AATAAA. The predicted protein (375 amino acids) revealed high identity to gamma-actins from fungi (>90%), and gene phylogenies support the grouping of B. trispora actin close to those from the majority of the filamentous fungi. actA transcript (1.4 kb) level in beta-carotene producing conditions was faintly higher than carRA (1.9 kb) and slightly lower than carB (1.8 kb) beta-carotene biosynthetic genes. The use of the actA promoter (PactA) for heterologous gene expression was ascertained by the transformation of gene fusions with the bleomycin resistance gene (bleR) from Streptoalloteichus hindustanus and the geneticin resistance marker (aphI) from Tn903, into Escherichia coli and Acremonium chrysogenum.

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.

Strain improvement for cephalosporin production by Acremonium chrysogenum using geneticin as a suitable transformation marker.

An Acremonium chrysogenum strain improvement program based on the transformation with cephalosporin biosynthetic genes was carried out to enhance cephalosporin C production. Best results were obtained with cefEF and cefG genes, selecting transformants with increased cephalosporin C production and lower accumulation of biosynthetic intermediates. Phleomycin resistant transformants, designated B1 and C1, showed a single copy random integration event, higher levels of cefEF transcript and, according to immunoblotting analyses, higher amounts of deacetylcephalosporin C acetyltransferase (DAC-AT) protein than their parental strains. Moreover, DAC-AT activity was higher in the transformants. Plasmids carrying geneticin resistance markers based on the nptII gene from Tn5 and the aphI gene from Tn903 were constructed to transform again B1 and C1, showing that the cassette Pgdh-nptII-trpC was able to confer geneticin resistance to A. chrysogenum and demonstrating that geneticin is a helpful selection marker.