Environmentally safe production of 7-ACA by recombinant Acremonium chrysogenum.
7-Amino cephalosporanic acid (7-ACA), which is currently obtained by chemical deacylation from cephalosporin C (CPC), is a major intermediate for industrial production of β-lactam antibiotics. 7-ACA can also be produced from CPC by enzymatic route including two-step and one-step procedures. In our research, an ecs gene coding for CPC acylase was synthesized and cloned into pET-28a(+) to construct an E. coli expression plasmid pYG232. E. coli BL21(DE3) bearing pYG232 was induced by IPTG and successfully expressed the recombinant ECS (88.9 kDa). Under the optimal conditions: 0.5 mg/ml purified ECS protein, 5 mg/ml CPC, 100 mM Tris-Cl (pH 9.6), supplement with 7 mM Zn(2+), slightly shaking for 6 h at 25°C, the transformation productivity was 54.4%. Then, ecs was cloned downstream of an A. chrysogenum endogenous promotor, PpcbC, to construct pYG233 for expression in A. chrysogenum. pYG233 was introduced into a CPC high-producer via integrative transformation of protoplasts. Two independent bleomycin-resistant transformants were investigated by PCR, Southern blotting, quantitative RT-PCR, western blotting, and fermentation. Although these two transformants both have one copy of integrated ecs, they showed different expression level of ECS protein and 7-ACA production. When concentration of CaCO(3) was reduced to 50 mM, ZnSO(4) was increased to 7 mM, CuSO(4) was eliminated from the fermentation media, and the pH was adjusted to 8.0 at day 4 during fermentation, 7-ACA production of one of the transformants could reach 1701 μg/ml, indicated that more than 30% of CPC produced by this high-producer have been transformed into 7-ACA directly in vivo. This is the highest 7-ACA production by direct fermentation ever reported.
Liu
Yan
Y
State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 1111 Zhongshan Road (North No. 1), Shanghai, 200437, China.
Gong
Guihua
G
Zhu
Chunbao
C
Zhu
Baoquan
B
Hu
Youjia
Y
D000164:Acremonium / Q000235:genetics / Q000378:metabolism*; D015139:Blotting, Southern; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D002511:Cephalosporins / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011523:Protoplasts; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014170:Transformation, Genetic
D002511:Cephalosporins; D003470:Culture Media; C030735:7-aminocephalosporanic acid; D002265:Carboxylic Ester Hydrolases; C019861:cephalosporin-C deacetylase
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