Heterologous Protein Production in<i>Acremonium chrysogenum</i>: Expression of Bacterial Cephalosporin C Acylase and Human Thrombomodulin Genes

Honda, Goichi; Matsuda, Akio; Zushi, M; Yamamoto, Shuji; Komatsu, Kenichi

doi:10.1271/bbb.61.948

Cited by 8 publications

(4 citation statements)

References 5 publications

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“…Southern blot analysis detected single copies of the foreign gene in two transformants. Here, we detected multiple integrations of the heterologous gene, similar to previous comparable studies in A. chrysogenum [ 29 , 68 ]. Secondly, Western blot analysis of the two fungal transformants showed no α-subunit, which, however, was detectable in transgenic E. coli strains [ 74 ].…”

Section: Discussionsupporting

confidence: 90%

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A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum

Lin

Lambertz

Dahlmann

et al. 2022

JoF

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The pharmaceutical industry has developed various highly effective semi-synthetic cephalosporins, which are generated by modifying the side chains of the core molecule 7-aminocephalosporanic acid (7-ACA). In industrial productions, the 7-ACA nucleus is obtained in vitro from cephalosporin C (CPC) by chemical or enzymatic processes, which are waste intensive and associated with high production costs. Here, we used a transgenic in vivo approach to express bacterial genes for cephalosporin C acylase (CCA) in the CPC producer Acremonium chrysogenum. Western blot and mass spectrometry analyses verified that the heterologous enzymes are processed into α- and β-subunits in the fungal cell. Extensive HPLC analysis detected substrates and products of CCAs in both fungal mycelia and culture supernatants, with the highest amount of 7-ACA found in the latter. Using different incubation times, temperatures, and pH values, we explored the optimal conditions for the active bacterial acylase to convert CPC into 7-ACA in the culture supernatant. We calculated that the best transgenic fungal strains exhibit a one-step conversion rate of the bacterial acylase of 30%. Our findings can be considered a remarkable contribution to supporting future pharmaceutical manufacturing processes with reduced production costs.

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Section: Discussionsupporting

confidence: 90%

“…Full processing of another acylase was previously reported by Honda et al [ 68 ]. There, both the α- and β-subunits of the wild-type acylase II from Pseudomonas sp.…”

Section: Discussionmentioning

confidence: 59%

A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum

Lin

Lambertz

Dahlmann

et al. 2022

JoF

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“…While glycosylation patterns of fungi are different from those of mammalian proteins (Archer and Pederby, 1997), a small number of human proteins have been successfully expressed and produced. Among these are human mucus proteinase inhibitor (Mikosch et al, 1996), human interleukin-6 (Broekhuijesen et al, 1993), human lactoferrin (Ward et al, 1995), human thrombomodulin (Honda et al, 1997), human tissue plasminogen activator (Wiebe et al, 2001), and humanized antibodies (Ward et al, 2004). However in neither case there was an attempt to evaluate the glycosylation pattern of the recombinant protein.…”

Section: Introductionmentioning

confidence: 99%

Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Chill

Trinh

Azadi

et al. 2008

Biotech & Bioengineering

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Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.

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“…Most common is the two-enzyme process that uses D-amino acid oxidase and CA to produce 7-ACA in sequential reactions (1). In addition, because most of the CAs studied thus far have a substrate preference of GL-7-ACA over CPC, great efforts have been made to obtain CA with high CPC affinity by screening for novel CA enzymes or by protein engineering to increase their CPC hydrolysis activity (10,12,13,27,28). The CA from Pseudomonas sp.…”

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confidence: 99%

Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

Zhang

Liu

Zheng

et al. 2005

Appl Environ Microbiol

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In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151␣F and Q50␤N, showed two-to threefold-increased catalytic efficiency (k cat /K m ) compared to wild-type CA130. Their K m values were decreased by ca. 50%, and the k cat values increased to 14.4 and 16.9 s ؊1 , respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121␤A and K198␤A had a 30 to 58% longer half-life than wild-type CA130, and K198␤A and D286␤A showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50␤N/K198␤A was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130.

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Heterologous Protein Production inAcremonium chrysogenum: Expression of Bacterial Cephalosporin C Acylase and Human Thrombomodulin Genes

Cited by 8 publications

References 5 publications

A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum

A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum

Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Improving the Activity and Stability of GL-7-ACA Acylase CA130 by Site-Directed Mutagenesis

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