**1. Introduction**

Terpenes are valuable natural substrates commonly used in the production of fine chemicals. Turpentine, obtained from biomass and also as a side product of the softwood industry, is rich in monoterpenes, such as α-pinene and β-pinene, which are widely used as raw materials in the synthesis of flavors, fragrances, and pharmaceutical compounds [1,2]. Oxidative transformation of abundant and low-priced monoterpenes holds considerable potential for the production of a wide variety of different terpenoid derivatives which are difficult and often costly to obtain directly from plant species. Oxygenated monoterpenes are the main flavor and fragrance impact molecules of essential oils. Many of them have beneficial effects on health [3–5], including anti-inflammatory activity [6]. However, their high volatility, poor solubility in aqueous media, and natural toxicity to microorganisms still limit the use of these compounds in applied biocatalysis. These limitations can be overcome by slow/gradual addition of terpene into the reaction medium, use of biphasic systems containing biocompatible organic solvents or a co-solvent, and in situ product removal techniques [7–9].

β-Pinene is commercially available at US\$ 36 per kg, whereas the price of its oxygenated derivative, *trans*-pinocarveol, can be as high as US\$ 134,000 per kg [10,11]. There are data regarding the antimicrobial properties of β-pinene, used either as a purified chemical or as a constituent of essential oils [12,13]. Some studies have also demonstrated that β-pinene and *trans*-pinocarveol, contained in the oils, show various biological effects [14–18].

Some microbial isolates have been found capable of transforming β-pinene into α-terpineol [19]. However, the rate and direction of the conversion are often inadequate for the process to be economically viable [20].

Strain developers have searched for improved strains among random survivors of mutagenesis. Even though the outcome of classical mutagenesis is difficult to predict, and the selection of mutants is always phenotypic, it still remains a fact that many producer strains with enhanced productivity currently used in industrial processes have been generated by random mutagenesis [21–23]. In contrast to genetic engineering methods, classical mutagenesis facilitates obtaining highly efficient mutants in an easy and rapid way without specialized knowledge about microbe genomes. To obtain the required mutants, experimenters have enriched cell populations by culturing them in special environmental conditions, toxic to most cell types but less toxic or non-toxic to a desired minority of cells [24–28]. Induced mutation has been successfully used in a number of microbial processes (other than biotransformation) employed in the production of useful end products [27–29].

Organic-solvent-tolerant mutants of bacteria can be selected using mutagen treatments or genetically engineered from solvent-sensitive parental strains [30–32]. Microbial tolerance to organic solvents can be improved by transforming cloned genes which encode various proteins involved in this tolerance, located in the cytoplasm or the inner or outer membrane [33,34]. Overexpression of genes which confer tolerance to specific organic solvents results in enhanced tolerances, which can be put to practical use [35].

Mutants resistant to a particular type of abiotic stress were better adapted to other stress conditions, as expressed by their enzymatic activities. The ability of one stress condition to provide protection against other stresses is referred to as cross-protection [36]. Adaptation processes are used in many branches of industrial biotechnology, including biotransformation of chemical compounds [37–39], biofuel production [40,41], and polymer synthesis [42].

In a previous paper, we demonstrated that the psychrotrophic fungus *Chrysosporium pannorum* A-1 showed promise for the biotransformation of (1S)-(−)-α-pinene because it could be used at 20◦C. This is an advantage in bioprocess involving volatile terpenes. Thus far, it has also given the best yields of verbenol and verbenone (722 and 176 mg/L, respectively) among the microorganisms studied [43].

In the present experiments, mutagenesis (UV irradiation and *N*-methyl-*N* -nitro-*N*-nitrosoguanidine (NTG)) and adaptation to a toxic substrate (β-pinene) were used to select mutants of the psychrotrophic fungus *C. pannorum* A-1 characterized by an improved efficiency of biotransformation of β-pinene to the main product *trans*-pinocarveol.
