Study on genetic engineering of Acremonium chrysogenum, the cephalosporin C producer.

Acremonium chrysogenum is an important filamentous fungus which produces cephalosporin C in industry. This review summarized the study on genetic engineering of Acremonium chrysogenum, including biosynthesis and regulation for fermentation of cephalosporin C, molecular techniques, molecular breeding and transcriptomics of Acremonium chrysogenum. We believe with all the techniques available and full genomic sequence, the industrial strain of Acremonium chrysogenum can be genetically modified to better serve the pharmaceutical industry.

Introduction

Acremonium chrysogenum, belongs to Filamentous fungi, is an important industrial microorganism. One of its metabolites, cephalosporin C (CPC), is the major resource for production of 7-amino cephalosporanic acid (7-ACA), an important intermediate for manufacturing of many first-line anti-infectious cephalosporins-antibiotics, in industry.

Cephalosporins belong to the family of beta-lactam antibiotics. Compared with the first-discovered penicillin, cephalosporins have obvious advantages since they are more resistant 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 in clinic.

In China, the research & development of cephalosporins started from 1960s, and cefoxitin was first developed in 1970. In the past 40 years, cephalosporins-antibiotic is one of the most developed medicines in Chinese market. It counts for more than 40% of the anti-infectious market share.

As the major resource for manufacturing 7-ACA, the production and cost of CPC is of the utmost importance for the cephalosporins-antibiotic market. The Ministry of Science and Technology of China has listed the fermentation of CPC as the key scientific and technical projects during the past 30 years due to the continuous demanding of strain improvement for 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 high-yield mutant strain.

This year, we will celebrate the 90th birthday of Dr. Arnold Demain, the leader of β-lactam antibiotics research and development [1]. This review will focus on the study of genetic engineering of A. chrysogenum in our lab and other groups to memorize the achievements of Dr. Demain's contribution in this field.

Biosynthesis of CPC

The industrialization of CPC fermentation has been established years ago with the breakthrough in key technologies including fermentation yield, fermentation regulation and preparation & purification. Dr. Arnold Demain is truly a pioneer and a founder in discovering the biosynthesis of CPC [2], [3] such as stimulation by methionine [4] and its precursors [5]. Nevertheless, there are still a lot of recent publications on improvement of CPC-producing strain by traditional methods, such as UV [6] or NTG [7] mutagenesis, and optimization of fermentation process [8]. More recently, glycerol was found can be used in the fermentation broth [9], [10] replacing methionine that Dr. Arnold Demain used decades ago, to stimulate the biosynthesis of CPC. Researchers also tried solid-state and submerged fermentation of CPC [11] besides the traditional production. However, most of the latest strain breeding techniques are at molecular level. And the most important approach is the research on biosynthesis of the target metabolite.

Demain's group has studied on the role of methionine in the fermentation of CPC [12]. Methionine can stimulation the production of CPC in the crude media, and the addition of methionine was still kept in current A. chrysogenum fermentation. They found that methionine does not repress or inhibit cysteine metabolism. Although methionine can upregulate the transcriptions of pcbAB, pcbC and cefEF, and stimulates the mycelial fragmentation, the mechanism behind the fact still needs to be investigated [13], [14].

The biosynthesis of CPC during the fermentation of A. chrysogenum has been well elucidated now. There are two gene clusters on the chromosome that involved in the biosynthesis of CPC. The “early” cluster consists of pcbAB-pcbC and cefD1-cefD2. pcbAB-pcbC encode two enzymes responsible for the first two steps in CPC biosynthesis [15]. cefD1-cefD2 encode proteins that epimerize IPN to penicillin N [16]. The “late” cluster consists of cefEF and cefG, which encode enzymes responsible for the last two steps [17].

The biosynthesis pathway of CPC is illustrated in Fig. 1. ACV synthase, encoded by pcbAB, condense 3 precursors including l-α-aminoadipic acid, l-cysteine, l-valine to ACV tripeptide. A comprehensively written review on this enzyme was published by Zhang & Demain in 1991 [18]. In Streptomyces clavuligerus, Lysine ε-aminotransferase (LAT) is also within the early gene cluster for cephamycin C biosynthesis. Since cephamycin C shares most of the biosynthetic genes with CPC biosynthesis pathway, it has been studied extensively by Demain's group [19], [20], [21].

Fig. 1

The biosynthesis pathway of CPC.

ACV is then cyclized into isopenicillin N (IPN) by IPN synthase encoded by pcbC. Conversion from IPN to penicillin N was catalyzed by a two-component epimerization system encoded by cefD1-cefD2. 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 DAC-acetyltransferase (DAC-AT) which was encoded by cefG. The crystal structure of DAC-AT has been published. It was found that DAC-AT belongs to α/β hydrolase family based on the formation of DAC-enzyme complex [22]. Among these enzymological steps, pcbAB, cefEF and cefG were considered as the rate-limiting factors in CPC biosynthesis [23].

Recent years, some other regulatory proteins, which have been found important in CPC biosynthesis, as well as their coding genes were discovered. For example, AcveA, a homologue of veA from Aspergillus, regulates the transcription of all six major CPC biosynthesis genes including pcbAB, pcbC, cefD1, cefD2, cefEF and cefG. Disruption of AcveA leads to a dramatic reduction of CPC yield [24]. We also tried overexpression of AcveA and the production of CPC was increased, as expected, by 22.7%. The transcriptions of pcbC, cefEF and cefG were all upreglated [25].

A cefP gene located in the early cluster of CPC biosynthesis cluster was just characterized. This gene encodes a transmembrane protein anchored on peroxisome. It regulates the epimerization of IPN to penicillin N catalyzed by CefD1-CefD2 two-component enzyme complex in peroxisome. cefP disruptant accumulated IPN and lost CPC production [26]. To compensate the disruption of cefP, cefP and cefR are necessary to be introduced simultaneously. CefR is the repressor of CefT, and stimulate the transcription of cefEF. The cefR knock out mutant showed a delayed transcription of cefEF and accumulation of penicillin N results in reduction of CPC yield [27].

A cefM gene was also found downstream of cefD1. Disruption of cefM accumulates penicillin N but no CPC production at all [28]. It is suggested that CefM may involve in the translocation of penicillin N from peroxisome to cytoplasm. Without cefM, cells are unable to transport penicillin N which is epimerized in peroxisome into cytoplasm, where CPC is synthesized.

Genetic engineering of Acremonium chrysogenum

Acremonium chrysogenum belongs to the family of Filamentous fungi. The techniques for genetic engineering are somehow hard to develop due to its complicate structure of cell wall and the special life cycle. Our lab has started the study of A. chrysogenum at molecular level based on some published results from host, transformation, homologous recombination and selection marker of A. chrysogenum [29], [30].

To introduce exogenous DNA into A. chrysogenum, a traditional PEG mediated protoplast transformation method is commonly used [31]. Since we are focusing on high-yield, or industrial strains, which usually have a stronger restriction-modification system than wide-type strain, the traditional transformation method is not efficient enough for introduction of exogenous genes.

Agrobacterium tumefaciens mediated transformation has been widely used in plant genetic engineering, and in some of the Filamentus fungi including Penicillium chrysogenum and Aspergillus nidulans as well. We have developed an adapted A. tumefaciens mediated transformation protocol for A. chrysogenum, which has a higher transformation efficiency than the PEG mediated method [32], and more importantly, this protocol can also be applied in A. chrysogenum high-yield strain. This is the first report of A. tumefaciens mediated A. chrysogenum transformation.

A lot of basic research was done to facilitate the genomic DNA extraction [33] and endogenous promoter capture [34] from the chromosome of A. chrysogenum. A notable progress is the cloning of pcbAB-pcbC bi-directional promoter from the chromosome of A. chrysogenum [35]. This enables convenient manipulation of A. chrysogenum by introduction of multiple genes afterwards.

Two inducible endogenous promoter Pmir1 [36] and Pxyl1 [37] were identified to be suitable for conditional expression in A. chrysogenum. These approaches not only provide the efficient tools for genetic engineering of A. chrysogenum, but also make it possible for selection marker recycling, given the truth that only a few selection markers are applicable in this Filamentus fungus.

The exogenous promoter Smxyl from Sordaria macrospore can also be used to construct the selection marker free recombinant mutant by using AcKu70 knock out strain as the recipient, and one step FLP/FRT recombinant system was successfully introduced to facilitate the genetic engineering of A. chrysogenum. This process can insert the exogenous gene into the genome of the CPC-producing strain without addition of the antibiotic-resistant gene [38].

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 A. chrysogenum was first published in 2007 [39]. The latest report was silencing of pcbC gene in Penicillium chrysogenum and cefEF gene in A. chrysogenum by RNAi [40]. These reports demonstrated the feasibility of RNAi technique in the Filamentous fungi.

We also tried the RNAi technique in A. chrysogenum. A plasmid vector containing cefG double strand RNA transcription unit was constructed and transformed into CPC-producing A. chrysogenum. The cefG transcription level in the transformants was measured by quantitative RT-PCR. The cefG transcription level decreases to 20% in two mutant strains. Their CPC yield was also found to decrease by 34.6% and 28.8%, respectively [41]. This result demonstrated the feasibility of RNAi application in A. chrysogenum and possible, industrial strain. Moreover, this is important for metabolic pathway reconstitution and novel CPC derivatives fermentation in A. chrysogenum.

By using the techniques that have been developed, researchers are able to study gene function in vivo to elucidate their impact on CPC production in A. chrysogenum. A glutathione reductase coding gene glrA was knocked out in A. chrysogenum, and the activity of glutathione reductase was disrupted leading to the accumulation of glutathione disulfide. This mutant is sensitive to oxygen stress and produces non-detectable cephalosporins. Overexpression of glrA can restore the glutathione reductase activity and the ratio of GSH/GSSR. The growth and CPC production can also be recovered by addition of methionine [42]. Similarly, a thioredoxin reductase-encoding gene ActrxR1 was also knocked out and spore germination and hyphal growth were inhibited. However, addition of DL-methionine can restore the delay and CPC production was enhanced, as well as the transcription of pcbC, cefEF and cefG [43].

The fungal nitrogen regulatory GATA zinc-finger protein AcareA was disrupted in A. chrysogenum and the mutation causes the reduction of CPC production. Meanwhile, transcriptions of CPC biosynthetic genes including pcbAB, cefD2, cefEF and cefG were downregulated. AcareA binds not only to the promoter region of nitrate reductase gene niaD-niiA, but also to the bidirectional promoter of pcbAB-pcbC [44].

Impaired growth mutant of A. chrysogenum was analyzed and a partial deleted putative septation protein encoding gene AcsepH was identified. Absence of AcsepH leads to reduction of septation and formation of multinucleate cells in A. chrysogenum. Also, conidia and CPC production was significantly decreased along with delay and decreased transcriptions of pcbC, cefD1, cefD2 and cefEF [45].

Acatg1 is a homolog of eukryotic serine/threonine kinase involved in autophagy. Disruption of Acatg1 in A. chrysogenum significantly reduces the formation of conidia, similar to disruption of AcsepH. However, CPC production was surprisingly enhanced in the Acatg1 disruption mutant and PcbC expression was kept at high level all through the fermentation process [46].

Our lab cloned a putative thiazole biosynthesis gene Acthi and found knock out of Acthi in A. chrysogenum reduced the intracellular concentration of thiamine and CPC biosynthesis precursor amino acids, while the formation of conidia was also decreased. Overexpression of Acthi can restore the reduction, upregulate the transcriptions of CPC biosynthetic genes and increase the production of CPC [47].

Omics study of Acremonium chrysogenum

Recent years, researchers have started genetic engineering of A. chrysogenum at the omics level by comparing the high yield strain with the low yield strain. Scientists from Russia used qPCR to detect the transcriptional level of CPC biosynthesis related genes in a high-producing strain RNCM F-4081D and the wide-type strain ATCC 11550 [48]. Results revealed that regulatory genes cefM and cefR were upregulated in the CPC high-producing strain. All biosynthetic genes including pcbAB, pcbC, cefD1, cefD2, cefEF and cefG had significantly higher transcriptional levels in RNCM F-4081D strain, which produces 100-fold more CPC than the wide-type strain. Although these results are anticipated, the balanced transcriptions of the biosynthetic genes and regulatory genes could give us some ideas to improve CPC production in A. chrysogenum.

We have studied the proteomics [49] and transcriptomics of an industrial CPC-producing strain (high-yield, HY) and the wide-type (WT) strain. According to comparative proteomics results, several significantly differentially expressed proteins including Acthi were identified. Comparative transcriptomics study revealed more information. A total of 4329 genes were identified that have significantly differential transcription levels in which 1737 were upregulated and 2592 were downregulated in HY strain [50]. These differentially expressed genes belong to the pathways of carbohydrate metabolism and energy, defense and virulence mechanisms, CPC biosynthetic precursor amino acids, oxidative stress defenses, and other secondary metabolites.

We also looked at the transcriptional level of some key genes involved in the primary metabolism in A. chrysogenum, which haven't been systemically studied as well as its correlation with CPC production to date. Glycolysis and gluconeogenesis, TCA cycle and Glyoxylate cycle, Pentose phosphate pathway and GABA shunt, and serine, lysine, cysteine, valine biosynthesis were studied and five important intermediates including glucose 6-phosphate, 3-phosphoglycerate, pyruvate, malate and succinate were identified. The involved metabolic pathways showed shifted flows which are favorable for switching to CPC production in HY strain [51]. These are very important information for future metabolic engineering of the CPC-producing A. chrysogenum.

Without the genomic sequence, omics study gave us the overall profile of the CPC-producing A. chrysogenum. Not surprisingly, this important industrial strain was sequenced recently, and the full genomic sequence was publicly revealed [52]. The sequence showed it has about 28.6 Mb in length, and approximately 8901 coding regions. However, this is shorter than the estimation of 32.7 Mb before [53]. Also the sequenced strain is the wide-type ATCC 11550. Lots of gaps are still open in the sequence. This also drew our attentions because there are several genes identified in our transcriptome study were not found in the published sequence, including Acthi.

The published genome also contains the mitochondrial sequence, which was published by other researchers elsewhere. This 27 Kb circular molecule contains 19 coding regions and 26 tRNA genes. Interestingly, all genes are located on one strand and may transcribed in the same direction [54]. Publication of both the chromosomal and mitochondrial sequences, as well as the transcriptome and proteome study will boost the genetic engineering of this important Filamentus fungus.

Molecular breeding of Acremonium chrysogenum

Among the three rate-limiting enzyme, PcbAB is relatively difficult to manipulate due to its larger coding region. Thus, researchers focus on cefEF and cefG for molecular breeding of A. chrysogenum. Besides, extra copy numbers of cefT could increase the yield of CPC in A. chrysogenum [55]. And, overexpression of cefP and cefR in A. chrysogenum can decrease the accumulation of penicillin N and promote the yield of CPC by about 50% [27].

The fermentation process of A. chrysogenum is an extreme oxygen-consuming procedure. All the rate-limiting enzymes are oxygen-requiring enzymes. The Vitreoscilla Hemoglobin (VHb) is very attractable since it's capable of oxygen transmission in oxygen-limiting environment. A recombinant strain bearing VHb can significantly improve the usage of oxygen during the fermentation process and increase the product yield, which has been proved in Aspergillus [56]. Introduction of vgb, the coding gene for VHb, into A. chrysogenum can also maintain a higher specific growth rate and specific production rate resulted in a 4–5 fold higher yield of the mutant strain [57]. Actually, there are many industrial A. chrysogenum strains that have recombinant vgb.

The earliest report on genetic modification for A. chrysogenum was published in 1989, when researchers from Eli Lilly Co. introduced extra copy of cefEF-cefG fragment into A. chrysogenum resulted in a 15%–40% higher producing strain [58]. This is the first evidence that molecular breeding can be a powerful tool in strain improvement of A. chrysogenum.

Although controlled by the same bi-directional promoter, the transcription levels of cefEF and cefG showed a huge difference according to RT-PCR. The transcription of cefG is much lower than that of cefEF. This leads to the accumulation of DAC in the metabolites since they can not be efficiently transformed into CPC. As a matter of fact, CPC/DAC ratio is a quality control parameter in the industrial production of CPC fermentation. Thus, introduction of extra copy numbers of cefG produced an engineering strain whose CPC yield is 3-fold-higher than the parental strain [59].

There is another report on introduction of cefT into A. chrysogenum, and the resulting mutant doubled the CPC yield [60]. This could attribute to the enhancement of CefT, the efflux pump protein, so that the feedback inhibition in vivo triggered by the fermentation product was attenuated, resulted in an elevated product yield.

Using molecular breeding technology, some CPC derivatives can be directly produced by engineering A. chrysogenum fermentation. For example, by disruption of cefEF and introduction of cefE originated from Streptomyces clavuligerus, a novel DAOC producing strain was obtained. Followed by two enzymatic transformations, the industrial important 7-ADCA can be produced [61]. By introduction of two coding genes simultaneously into A. chrysogenum for the enzymes used in industrial production of 7-ACA by immobilized enzymatic transformation, the engineering strain can produce 7-ACA by fermentation [62].

Only one enzyme (DAOC/DAC synthase) is responsible for synthesis of deacetylcephalosporin C (DAC) from penicillin N. The coding gene for DAOC/DAC synthase is cefEF in A. chrysogenum. However, in Streptomyces clavuligerus (cephamycin C producer), there are two enzymes responsible for this two step conversion encoded by cefE and cefF. Introduction of cefF (coding for DAOC hydroxylase) into A. chrysogenum can significantly decrease the DAOC/CPC ratio given that DAOC is a major impurity in the fermentation of CPC production [63].

There is another interesting research for the molecular breeding of A. chrysogenum in a different way. As we mentioned above, CefD1-CefD2 is a two-component enzyme complex that transform IPN into penicillin N by an epimerization system located in peroxisome. cefD1-cefD2 block mutant lacking this epimerization system accumulated a large amount of IPN to more than 650 μg/mL, almost the total relative CPC yield. The unstable IPN, which never has been purified before, could be purified by several steps of chromatography in this mutant [64]. Characterization of its half-life and stability under variety conditions can greatly help us for the investigation of IPN.

Genome shuffling [65] is a powerful tool for strain improvement especially when lacking the genomic sequence of the target strain. We have proved that this technique is also effective in A. chrysogenum. By genetic engineering, we have constructed a strain of A. chrysogenum that can directly produce 7-ACA by fermentation, however, the yield is still low [66]. With the help of genome shuffling, the yield of 7-ACA has been increased by 6.5 fold by two round of screening [67]. This makes it potential possible to manufacture 7-ACA, the important β-lactam intermediate, by one-step A. chrysogenum fermentation instead of the current two-step process which consist of fermentation of A. chrysogenum plus the enzymatic transformation of CPC [68].

It's worth noting that all of the above genetic breeding reports were on the background of an A. chrysogenum wide type strain, whose CPC yield is only around 1 mg/mL, far less than the industrial production level, which is around 15–20 mg/mL in flask and 35–40 mg/mL in fermentor. Although some great achievements were obtained in improvement of A. chrysogenum fermentation and modification of metabolic products, those achievements are still far away from application in industry.

Genetic engineering of industrial Acremonium chrysogenum

Our research is focusing on molecular breeding of A. chrysogenum high-yield and/or industrial strains. We introduced different combinations of cefG/cefEF/cefT/vgb genes into CPC high-producing strain and found that an extra copy of cefG has significant positive effect on CPC fermentation level. Since random integration was occurred in A. chrysogenum, different transformants with cefG introduction showed different elevated levels, with 100% promotion at most. An extra copy of vgb gene that had been under directed evolution and had elevated bioactivity [69] also displayed a significant improvement up to 30% more of CPC yield. Meanwhile, introduction of cefEF and cefT has no obvious effect on CPC production in high-yield strain [70]. This revealed the apparent discrepancy between the genetic background of wide-type strain and high-yield strain, and also suggested that endogenous cefEF and cefT may already achieve high bioactivity after several rounds of mutagenesis breeding that a high-yield strain usually undertaken.

We then applied this achievement to a CPC industrial strain. Although we couldn't double the CPC yield, we did obtain an engineering strain whose CPC yield was increased by 20%, which has a promising industrialization potential. See Table 1 for a complete list of genes that have been manipulated in A. chrysogenum (low or high producers) and their influence on CPC yield.

Table 1

Manipulated genes in CPC low/high producers and their influence on yield.

Gene	Manipulation	Producer	CPC yield	Reference
AcveA	Knock out	low	decrease	[24]
AcveA	overexpression	low	increase	[25]
cefP	Knock out	low	None	[26]
cefR	Knock out	low	decrease	[27]
cefP/cefR	overexpression	low	increase	[27]
cefM	Knock out	Low	decrease	[28]
cefEF	Knock down	low	decrease	[40]
cefG	Knock down	low	decrease	[41]
glrA	Knock out	low	none	[42]
ActrxR1	Knock out	low	delay	[43]
AcareA	Knock out	low	decrease	[44]
AcsepH	Knock out	low	decrease	[45]
Acatg1	Knock out	low	increase	[46]
Acthi	Knock down	low/high	decrease	[47]
Acthi	overexpression	low/high	increase	[47]
cefT	overexpression	low	increase	[55][60]
vgb	overexpression	low	increase	[57]
cefEF-cefG	overexpression	high	increase	[58]
cefG	overexpression	low	increase	[59]
cefG/vgb	overexpression	low/high	increase	[70]

The fermentation product of A. chrysogenum, CPC, is the major resource for industrial manufacturing of 7-ACA, the important intermediate of a large variety of cephalosporins antibiotics. A traditional producing route of 7-ACA is the chemical semi-biosynthesis. Up to date, the more environmental-friendly biotransformation has been widely used in industry. Two step transformation using DAO (d-amino acid oxidase) and GL-7-ACA acylase dominates the market for decades [71], more recently one step transformation from CPC to 7-ACA by CPC acylase has become available [72], although, the substrate specificity of CPC acylase still need improved [73].

Whether two-step or one-step, fermentation of CPC is the prerequisite followed by enzymatic biotransformation in vitro. We are thinking of introducing CPC acylase gene into A. chrysogenum to construct the engineering strain that can produce 7-ACA directly by fermentation, a breakthrough in the production of 7-ACA.

A CPC acylase gene was designed according to the codon bias of A. chrysogenum and introduced into an industrial strain. Our result showed that this CPC acylase was expressed in A. chrysogenum with bioactivity. The recombinant acylase can transform the original product CPC into 7-ACA in vivo, which makes the engineering strain capable of direct fermentation of 7-ACA. Based on enzymological profiles of CPC acylase in vitro, we performed a preliminary optimization of medium composition and culture condition. 7-ACA yield was increased significantly in this engineering strain with as least 30% of the fermented CPC was transformed into 7-ACA [74]. We believe this in vivo conversion can be more effective if a stronger transcription cassette can be introduced and copy number can be increased. With the incorporation of traditional breeding technology, in vivo conversion will be realized in industry.

Perspectives

The biosynthesis of CPC in A. chrysogenum has been investigated thoroughly. However, the mechanism of its regulation as well as the precursor biosynthesis in primary metabolism is still unclear [75]. Full sequence of A. chrysogenum has been published, plus there are more than 10 species belongs to Filamentous fungi have been sequenced [76]. However, gaps need to be filled, and all genes need to be annotated and studied to understand their in vivo functions. With the information of the genes that have been published and studied, we can manipulate the genome of A. chrysogenum more efficiently. CRISPR/Cas9 is a powerful tool for genome editing in animals [77] and mammalian cells [78] since its discovery. Currently, this technique has been used in microorganisms including E. coli [79], Streptomyces [80] and Yeast [81]. Although Filamentous fungi is relative difficult to genetically modify, we are trying this system in A. chrysogenum in our lab. There are also some publications on genome editing of Filamentous fungi such as Aspergillus [82], [83], [84], Pyricularia [85], Trichoderma [86] and Penicillium [87] in the recent two years. Thus, we believe it is possible to edit the genome of A. chrysogenum by the newly developed system.

By identifying differentially expressed proteins during CPC fermentation in wide-type strain and high-yield strain, a comprehensive regulation system of A. chrysogenum fermentation may be proposed based on the popular theory of metabolic engineering and systems biology [88] because omics study has shown to be a powerful tool to study the metabolic pathway from a systemic scope. A study on Penicillin chrysogenum revealed that 950 proteins involved in precursor biosynthesis, stress response and pentose phosphate pathway were found to be related to the fermentation yield in three penicillin-producing strains [89]. We have identified several differentially expressed proteins that are associated with several metabolic pathways, especially those involved in primary metabolism of A. chrysogenum. Study of these genes along with their involved pathways is in progress and can help us better understand how the primary metabolism will influence the CPC biosynthesis. And we believe this should incorporate metabolomics study [90] that helps us explain from the transcription and expression to metabolism.

Dr. Demain opened up a new era fifty years ago. Now, with the help of modern molecular techniques and more powerful tools, we believe that genetic modified Acremonium chrysogenum will play an important role in the industrial production of CPC and its derivatives.