Indian Journal of Biotechnology Vol 2, July 2003, pp 293-301 Autolysis ofZYXWVUTSRQPONMLKJIHGFEDCBA Penicillium chrysogenum-A Holistic Approach Istvan Pocsi", Tunde Pusztahelyi, Laszlo Saw and Tamas Emri Department of Microbiology and Biotechnology, Faculty of Science, University of Debrecen, Egyetem ter 1, H-4010 Debrecen, Hungary Received 20 January 2003; accepted 20 February 2003 Despite of its biotechnological significance, the autolysis of filamentous fungi is a poorly studied and understood area of fungal physiology. The autolysis of 13-lactam producing fungus, Penicillium chrysogenum shares some simi- larities with the apoptosis of higher eukaryotes. For example, the biosynthesis and processing of age-related hydro- lases were highly regulated in carbon-depleted cultures. The in vivo inhibition of autolytic chitinase activity hindered considerably the disintegration of pelleted structures that are typical of the exponential growth phase. In the absence of conidiation, round-ended "yeast-like" hyphal fragments were the dominant surviving morphological forms, which were characterised with decreasing total respiration, increasing cyanide-resistant respiration, intracellular accumu- lation of reactive oxygen species (ROS) and declining viability in the autolytic and post-autolytic phases of growth. The term "ageing" was used to describe these physiological changes, and the surviving fragments may undergo oxi- dative-stress induced programmed cell death. Although variations in oxygen tension and extracellular ROS concen- trations are key elements in the initiation of morphological changes, the genomic expression programmes of fungi governing morphological transitions including autolysis are likely to be activated by different kinds of environmental stress and signal transduction pathways. The glutathione (GSH) and ROS metabolisms of P. chrysogenum were in- fluenced by many extrinsic and intrinsic factors in each growth phase studied. As a consequence, no firm correlation was found between the GSHIglutathione disulphide (GSSG) redox status, the intracellular ROS levels and the ob- served morphological and physiological characteristics of the cells. Keywords: autolysis, apoptosis, ageing, fragmentation, vacuolation, chitinase, respiration Introduction The autolysis of industrially important filamentous fungi has been recently reviewed by White et al (2002). Fungal autolysis as a dynamic process of cell death influences numerous biotechnological processes including secondary metabolite and heterologous protein productions. Although the papers published in this field are mainly either physiology or morphology oriented, these approaches are aiming at the same phenomenon using different experimental tools. Both intrinsic (differentiation, ageing and cell death pro- grammes of the micro-organism) and extrinsic (changes in the culture media, physical stress) effec- tors have significant, mostly inseparable impact on fungal autolysis observable in fermentation processes * Author for correspondence: Tel: 36-52-512900, ext 2061; Fax: 36-52-533677 E-mail: pocsi@tigris.klte.hu Abbreviations: AOX: alternative oxidase; GOX: glucose oxidase; GPx: glutathione peroxidase; GR: glutathione reductase; GSH: glutathione; GSSG: glutathione disulphide; HexNAc-ase: N- acetyl-I3-D-hexosaminidase; ROS: reactive oxygen species; SOD: superoxide dismutase. (White et al, 2002). Physiological differentiation along the growing and stationary phase hyphae in- cluding progressing vacuolation in the older com- partments and weakening of cell wall due to the ac- tion of autolytic hydrolases influences the 'resistance of mycelia to mechanical forces introduced by agita- tion (Paul et al, 1994; Harvey et al, 1998; Ji.isten et al, 1998). On the contrary, the agitation intensity influ- ences considerably both the vacuolation (Ji.isten et al, 1998) and hydrolase production (Harvey et al, 1998). Undoubtedly, a holistic approach is needed to get a deeper understanding of the underlying mechanism of the interdependent morphological and physiological events leading to the disintegration of hyphae in sub- merged cultures in the absence of conidiation (White et al, 2002). In the last decade, the authors characterized the autolysis of P. chrysogenum NCAIM 00237, a high ~- lactam producing filamentous fungus, both physio- logically and morphologically. Because of their inter- est in the intrinsic cell death and ageing programmes of the microorganism, in the first place autolysis was triggered by the depletion of carbon source in the ab-