Penicillium and Acremonium - Genetic Engineering of Acremonium chrysogenum , the Cephalosporin C Producer Youjia Hu Additional information is available at the end of the chapter http://dx.doi.org/10.5772/55471 1. Introduction Acremonium chrysogenum , belongs to Filamentous fungi, is an important industrial microor‐ ganism. One of its metabolites, cephalosporin C (CPC), during fermentation is the major resource for production of 7-amino cephalosporanic acid (7-ACA), an important intermediate for the manufacture of many first-line anti-infectious cephalosporin-antibiotics, in industry. Cephalosporins belong to the family of beta-lactam antibiotics. Comparing the first-discovered penicillin, cephalosporins have obvious advantages since they are more stable to penicillinase and are more effective to many penicillin-resistant strains. The incidence of adverse effects for cephalosporins is also lower than that for penicillins and other anti-infectious agents. Thus, cephalosporins are among the most-widely used anti-infectious drugs clinically. In China, the research on cephalosporins started from the 1960s, and cefoxitin was first developed in 1970. In the past 30 years, cephalosporin-antibiotics are one the most developed medicines on the domestic market. They accounts for more than 40% of the anti-infectious drug market share. As the major resource for manufacturing 7-ACA, the production and cost of CPC is of the utmost importance in the cephalosporin-antibiotics market. The Ministry of Science and Technology of China has listed the fermentation of CPC as the major scientific and technical project in the past 30 years due to the continuous demand of strain improvement for the CPC- producing Acremonium chrysogenum . Because of the limitation of traditional techniques on strain improvement for A. chrysogenum , along with the ubiquitous applications of molecular biology, genetic engineering has become a powerful tool to manipulate the antibiotic producing strain and to obtain a high-yielding mutant strain. This paper will summarize the most recent developments on genetic manipulation of A. chrysogenum Biosynthesis of CPC The industrialization of CPC fermentation has been established tens of years ago with the breakthrough in key technologies including fermentation yield, fermentation regulation and preparation and purification. Nevertheless, there has been a lot of publications, recently on the improvement of CPC-producing strain by traditional methods, such as UV [ 1 ] or NTG [2 ]mutagenesis, and optimization of fermentation process [ 3 ], as well. However, most of the latest strain breeding techniques are at the molecular level, and the most important approach has been the research on the biosynthesis of the target metabolite. The biosynthesis of CPC during the fermentation of A. chrysogenum has been well investigated. There are two gene clusters on the chromosome that are involved in the biosynthesis of CPC. The “early” cluster consists of pcbAB-pcbC and cefD1-cefD2 . The pcbAB-pcbC encode two enzymes responsible for the first two steps in CPC biosynthesis [ 4 ]. While the cefD1-cefD2 encode proteins that epimerize isopenicillin N (IPN) to penicillin N [ 5 ]. The “late” cluster consists of cefEF and cefG genes, which encode enzymes responsible for the last two steps [ 6]. The biosynthesis pathway of CPC is illustrated in figure 1. The ACV synthase, encoded by the pcbAB gene, condenses 3 precursors L- α -aminoadipic acid, L-cysteine, L-valine to the ACV tripeptide. The ACV is then cyclized into IPN by IPN synthase encoded by pcbC gene. The step from IPN to penicillin N is catalyzed by a two-component epimerization system encoded by cefD1-cefD2 . The cefEF encodes a unique bi-functional enzyme, deacetyloxy-cephalosporin C (DAOC) synthase-hydroxylase which successively transforms penicillin N into DAOC and deacetyl-cephalosporin C (DAC). The last step in CPC biosynthesis is catalyzed by a DAC- acetyltransferase (DAC-AT) which is encoded by cefG . The crystal structure of DAC-AT has been published [ 7 ]. It has been shown that DAC-AT belongs to α/β hydrolase family according to the formation of DAC-enzyme complex [ 7 ]. Among these, pcbAB , cefEF and cefG were considered as the rate-limiting steps in CPC biosynthesis [8]. In recent years, some other regulatory proteins, which have been found to be important in CPC biosynthesis, as well as their coding genes have been discovered. For example, Ac veA , a homologue of veA from Aspergillus , regulates the transcription of all 6 major CPC biosynthesis genes including pcbAB , pcbC , cefD1 , cefD2 , cefEF and cefG . Disruption of Ac veA leads to a dramatic reduction of CPC yield. A cefP gene located in the early cluster of CPC biosynthesis cluster has just been characterized. This gene encodes a transmembrane protein anchored in a peroxisome. It regulates the epimerization of IPN to penicillin N catalyzed by CefD1-CefD2 two-component enzyme complex in peroxisome. The cefP disruptant accumulated IPN and lost CPC production [ 10]. To compensate for the disruption of cefP , both cefP and cefR need to be introduced simultane‐ ously. The CefR is the repressor of CefT, and stimulates the transcription of cefEF . A mutant A. chrysogenum without cefR showed delayed transcription of cefEF and accumulation of penicillin N resulted in reduction of CPC yield

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