1. Introduction
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
Because of the limitation of traditional techniques on strain improvement for
2. 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
The biosynthesis pathway of CPC is illustrated in figure 1. The ACV synthase, encoded by the
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
A
A
3. Techniques for molecular breeding
To introduce exogenous DNA into
Considering the significant improvement after introduction of
A lot of basic research was done to facilitate the genomic DNA extraction [19] and endogenous promoter capture [20] from the chromosome of
The last step in CPC biosynthesis, DAC transformed into CPC catalyzed by DAC acetyltransferase, was further investigated, as many reports have demonstrated that this is the rate-limiting step while DAC acetyltransferase coding gene,
4. Molecular breeding of Acremonium chrysogenum
Among the three rate-limiting enzymes, PcbAB is relatively difficult to manipulate due to its larger coding gene. Thus, researchers focus on
The fermentation process of
The earliest report on genetic modification for
Although controlled by the same bi-directional promoter, the transcription levels of
There is another report on the introduction of
Using molecular breeding technology, some CPC derivatives can be directly produced by engineering
Besides the introduction of exogenous genes, disruption and/or silencing of the endogenous genes is also a common strategy for genetic breeding of a certain strain. The recently developed RNA interference (RNAi) technique can be used as an alternative to silence the transcription of target genes instead of homologous recombination. RNAi in
There is another interesting research for the molecular breeding of
It is worth noting that all of the above genetic breeding reports were on the background of an
5. Industrialization research on molecular breeding of A. chrysogenum
Our research is focused on the molecular breeding of
We then applied this achievement to a CPC industrial strain. Although we didn’t obtain a mutant that doubled the CPC yield, we did obtain an engineering strain whose CPC yield was increased by 20%, which has a promising industrialized potential.
We also tried the RNAi technique in the high-yield strain. A plasmid vector containing
The fermentation product of
Whether two-step or one-step, fermentation of CPC is the prerequisite followed by enzymatic biotransformation
A CPC acylase gene was designed according to the codon bias of
6. Perspectives
As a novel tool for strain improvement, genome shuffling is of widespread concern in the field of industrial microbiology since it was first reported [41]. This has been applied in Bacteria and Streptomyces, and the yield of a lot of metabolites got a substantial increase by genome shuffling. However, genome shuffling in Filamentous fungi is rare, maybe due to the undeveloped genetic manipulation system. In 2009, the cellulase production in
The biosynthesis of CPC in
Besides its use in studying the mechanism of disease development, the application of comparative proteomics in antibiotic-producing microorganisms also showed promise. For instance, 345 different proteins were identified as critical during the conversion from primary to secondary metabolism in
Thus, we believe that the molecular breeding of
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