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TitleMolecular Biotechnolgy of Fungal beta-Lactam Antibiotics and Related Peptide Synthetases: -/-
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Regulation of Cephalosporin Biosynthesis

Esther K. Schmitt 1 · Birgit Hoff 2 · Ulrich Kück 2 (✉)
1 Novartis Pharma AG, NPU, 4002 Basel, Switzerland
2 Ruhr-Universität Bochum, Lehrstuhl für Allgemeine und Molekulare Botanik,

44780 Bochum, Germany
[email protected]

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 Precursors and Competing Pathways . . . . . . . . . . . . . . . . . . . . . 3
2.1 L-a-Aminoadipic Acid (L-a-AAA) Marks a Biosynthesis Branch Point . . . 3
2.2 L-Valine as a Metabolic Signal . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.3 Non-Conventional Biosynthesis of L-Cysteine . . . . . . . . . . . . . . . . . 5

3 Biosynthesis of Cephalosporin . . . . . . . . . . . . . . . . . . . . . . . . . 7
3.1 General b-Lactam Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . 8
3.1.1 Cellular Localization and Structure of IPNS . . . . . . . . . . . . . . . . . . 10
3.2 Cephalosporin Specific Biosynthesis . . . . . . . . . . . . . . . . . . . . . . 11
3.2.1 Final Reaction of Cephalosporin Biosynthesis . . . . . . . . . . . . . . . . 12

4 Structural Genes of Cephalosporin Biosynthesis . . . . . . . . . . . . . . . 13
4.1 “Early” Cephalosporin Genes . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.2 “Late” Cephalosporin Genes . . . . . . . . . . . . . . . . . . . . . . . . . . 17

5 Multiple Layers of Control . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.1 Transcript Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
5.2 Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5.3 Correlation Between Secondary Metabolism and Morphogenesis . . . . . . 20

6 Transcription Factors as Activators and Repressors
of Cephalosporin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . 22

6.1 PACC – pH-Dependent Transcriptional Control . . . . . . . . . . . . . . . . 22
6.2 CRE1 – A Glucose Repressor Protein . . . . . . . . . . . . . . . . . . . . . . 24
6.3 CPCR1 – Cephalosporin C Regulator 1 . . . . . . . . . . . . . . . . . . . . 26
6.4 Comparison of Cephalosporin and Penicillin Biosynthesis Regulation . . . 30

7 Molecular Differences in Production Strains . . . . . . . . . . . . . . . . . 30

8 Examples of Molecular Engineering of A. chrysogenum . . . . . . . . . . . 33
8.1 Genetic Tools for Molecular Engineering . . . . . . . . . . . . . . . . . . . 33
8.2 Optimization of Cephalosporin C Biosynthesis . . . . . . . . . . . . . . . . 35

9 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Adv Biochem Engin/Biotechnol (2004) 88: 1– 43
DOI 10.1007/b99256
© Springer-Verlag Berlin Heidelberg 2004

Page 2

Abstract The filamentous fungus Acremonium chrysogenum is the natural producer of the
b-lactam antibiotic cephalosporin C and is as such used worldwide in major biotechnical ap-
plications. Albeit its profound industrial importance, there is still a limited understanding
about the molecular mechanisms regulating cephalosporin biosynthesis in this fungus. This
review focuses on various regulatory levels of cephalosporin biosynthesis. In addition to pre-
cursor and antibiotic biosynthesis, molecular genetic characteristics of cephalosporin
biosynthesis genes and the knowledge of multiple layers of their regulatory expressional
control, as well as the function of activators or repressors on cephalosporin biosynthesis are
jointly being surveyed. Furthermore, this review summarizes (i) molecular features, which
distinguish strains with different production levels and (ii) examples of molecular engi-
neering approaches to A. chrysogenum.

Keywords Acremonium chrysogenum · Cephalosporin · Gene regulation ·
Transcription factors · Genetic engineering

1
Introduction

Cephalosporin C and its semisynthetic derivatives are very potent and wide-
ly used b-lactam antibiotics of general and applied interest. However,
the knowledge of the molecular regulation of b-lactam biosynthesis in the
corresponding host is still limited. In the case of cephalosporin biosynthesis,
even the total number of involved biosynthesis genes is not known and has yet
to be identified. Cephalosporin is exclusively produced by Acremonium
chrysogenum (syn. Cephalosporium acremonium), but compared to other fil-
amentous fungi, genetic manipulation of this fungus is rather difficult. Acre-
monium chrysogenum belongs to the Deuteromycetes, which lack a sexual cy-
cle and are thus not accessible for any conventional genetic analysis. In
addition, this fungus produces only very few conidiospores, which in other
biotechnically relevant fungi are the preferred cells for DNA-mediated trans-
formations.

In 1945, A. chrysogenum was first isolated from Sardinian coastal seawater
by Prof. Brotzu. Brotzu was also the first to describe the antibiotic effect of ex-
tracts generated from this fungus and, some years later, the structure of the ac-
tive compound was determined [1]. Cephalosporin C was shown to be active
against Gram-positive as well as Gram-negative bacteria. Today, A. chryso-
genum is cultured worldwide to yield approximately 2500 tons of cephalosporin
derivatives. Semisynthetic derivatives are mainly used as broad-spectrum an-
tibiotics for the treatment of bacterial infections.

In biotechnical applications, intensive strain improvement programs re-
sulted in production strains that yield a significantly higher titer of the an-
tibiotic than wild-type strains. Approximately 40 years of mutation and se-
lection cycles separate today’s industrial strains from the genetic potential of
the original isolates. For basic as well as for applied research, the comparison
of wild-type and production strains is of specific interest when differences of

2 E. K. Schmitt et al.

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Received: April 2004

264 H. von Döhren

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