微生物学技术

Generally two selection markers are required to obtain homozygous mutations in a diploid background, one for each gene copy that is interrupted. In this chapter is described a method that allows the double gene deletions of the two copies of a gene from a diploid organism, a wild-type strain of the Xant ...

Approaches for improving astaxanthin yields in Xanthophyllomyces dendrorhous include optimization of fermentation conditions and generation of hyperproducing mutants through random mutagenesis using chemical or physical means. A key limitation of classical mutag ...

Double-stranded RNA (dsRNA) molecules are widely found in yeasts and filamentous fungi. It has been suggested that may play important roles in the evolution of eukaryote genomes and may be a valuable tool in yeast typing. The characterization of these extrachromosomal genetic elements is u ...

Astaxanthin is a xanthophyll pigment of high economic value for its use as a feeding component in aquaculture. Xanthophyllomyces dendrorhous is a basidiomycetous fungi able to synthesize astaxanthin as its major carotenoid, and the only known yeast species bearing the capability to pr ...

For many years, benefits and disadvantages of pigments production either by microalgae or yeasts have been under analysis. In this contribution we shall deal with Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) and Haematococcus pluvialis, which are known as major pr ...

Cellular targeting of biosynthetic pathway enzymes is an invaluable technique in metabolic engineering to modify metabolic fluxes towards metabolite of interest. Especially, recombinant carotenoid biosynthesis in yeasts should be balanced with a precursor pathway prese ...

Plasmids introduced in Mucor circinelloides (and most transformable Mucorales) tend to replicate autonomously, and hardly ever integrate in the genome. This is critical if we want to express exogenous genes, because plasmids are easily lost during vegetative growth, and the ratio of pl ...

The classical strain breeding method based on random mutation and selection cannot avoid introducing detrimental or unnecessary mutations into the genome. A methodology that overcomes the limitations of the classical method is “genome breeding.” In this approach, biotechno-lo ...

A bench-scale protocol for the purification of plasmid DNA (pDNA) produced in Escherichia coli is described. The method is specifically designed to prepare pDNA vectors for gene therapy and DNA vaccination applications. The method comprises alkaline lysis, concentration with iso-p ...

Streptomyces clavuligerus is used as model to illustrate the determination and quantification of antimicrobial metabolites with different biological activities produced by cultures of this strain. S. clavuligerus produces an array of compounds with different structures. ...

Actinomycetes produce many bioactive compounds with clinical, veterinary, or agricultural applications. Many of these natural products contain sugars attached to the corresponding agly-cons, which usually participate in the molecular recognition of the cellular target. T ...

Actinomycetes are among the most fascinating microorganisms. Their developmental life cycle with its morphological and physiological differentiation and the rich repertoire of secondary metabolites (about 70-80% of bioactive secondary metabolites are being produced by ...

The ascomycete Ashbya gossypii, a filamentous fungus, is a natural overproducer of vitamin B2 and is currently exploited for the industrial production of this vitamin. Classical mutagenesis and selection of mutants showing improved production capacities have been routinely app ...

Carotenoids are high-value natural products utilized by several industrial sectors. Some filamentous fungi such as Phycomyces blakesleeanus, Mucor circinelloides, and Blakeslea trispora possess the ability to synthesise carotenoids. In this chapter, procedures for comp ...

Carotenoids are chemical compounds that are in an increasing demand in the market because of their applications in the food, feed, cosmetics, and pharmaceutical industries. To date, most of the β-carotene is manufactured by chemical processes, but because it is used as feed and food additives, t ...

A pilot-plant process for the production of a recombinant sweet-tasting protein in the filamentous fungus Aspergillus awamori is described. Each step of the scale-up and downstream processes (concentration, diafiltration and ion-exchange chromatography) for the production ...

The Gram-positive bacterium Bacillus megaterium is frequently employed for the industrial production of exoenzymes including amylases, proteinases, and penicillin acylases, for instance. Here, we describe the utilization of the excellent protein secretion capacity of this ...

This chapter introduces 13C tracer techniques combined with labeling measurement by mass spectrometry (MS) for the quantification of metabolic pathway fluxes. As an example, the relative contribution of (1) de novo synthesis from glucose and (2) synthesis from building blocks conta ...

A strain improvement procedure is outlined for the isolation of mutant strains from the bacterium Pseudomonas sp. ATCC 31461 that are capable of enhanced production of the polysaccharide gellan compared to its parent strain. With gellan having a number of industrial applications, the is ...

In this chapter are described the applications of the Morganella morganii phoC gene, encoding a molecular class A nonspecific phosphatase, as a reporter both in prokaryotic (Escherichia coli) and in eukaryotic (Saccharomyces cerevisiae) systems. The activity of PhoC can be detected by ...