New insights into the isopenicillin N transport in Penicillium chrysogenum
New insights into the isopenicillin N transport in Penicillium chrysogenum
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DOI：
10.1016/j.ymben.2014.01.004
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发表时间：
2014-03-01
影响因子：
8.4
通讯作者：
Ullan, R. V.
中科院分区：
文献类型：
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作者：
Fernandez-Aguado, M.;Martin, J. F.;Ullan, R. V.
在产黄青霉中，β-内酰胺生物合成途径是分开的。这一事实迫使青霉素中间体分子跨细胞膜的运输过程发生。这种分子运输的许多方面仍然不清楚，但应该涉及跨膜转运蛋白。在目前的工作中，对 P. chrysogenum 的主要促进子型二级转运蛋白（PenM）进行了深入研究。 penM 靶向沉默导致 penM 表达水平降低，导致沉默转化体（尤其是 SilM-35）中青霉素产量减少。相反，高效启动子的 penM 过表达增加了青霉素的产量和生物合成基因的表达。此外，当沉默菌株SilM-35在补充6-氨基青霉烷酸的青霉素生产条件下培养时，观察到与6-氨基青霉烷酸利用率成比例的苄基青霉素产量的增加。通过这种现象，可以得出结论，由于penM沉默，苄青霉素转运保持完整，但过氧化物酶体异青霉素N的转运结果受到影响。作为通过表达荧光重组PenM-DsRecl蛋白获得的最终结果，确定PenM天然位于P.chrysogenum过氧化物酶体中。总之，我们的实验结果表明，PenM 最有可能通过异青霉素 N 穿过 P. chrysogenum 过氧化物酶体膜从细胞质转移到过氧化物酶体腔来参与青霉素的生产。 (C) 2014 年国际代谢工程学会。由爱思唯尔公司出版。保留所有权利。
In Penicillium chrysogenum the beta-lactam biosynthetic pathway is compartmentalized. This fact forces the occurrence of transport processes of penicillin-intermediate molecules across cell membranes. Many aspects around this molecular traffic remain obscure but are supposed to involve transmembrane transporter proteins. In the present work, an in-depth study has been developed on a Major Facilitator-type secondary transporter from P. chrysogenum named as PenM. The reduction of penM expression level reached by penM targeted silencing, leads to a decrease in benzylpenicillin production in silenced transformants, especially in SilM-35. On the contrary, the penM overexpression from a high efficiency promoter increases the benzylpenicillin production and the expression of the biosynthetic genes. Moreover, when the silenced strain SilM-35 is cultured under penicillin production conditions with 6-aminopenicillanic acid supplementation, an increase in the benzylpenicillin production proportional to the 6-aminopenicillanic acid availability is observed. By this phenomenon, it can be concluded that due to the penM silencing the benzylpenicillin transport remains intact but the peroxisomal isopenicillin N import results affected. As a culminating result, obtained by the expression of the fluorescent recombinant PenM-DsRecl protein, it was determined that PenM is naturally located in P. chrysogenum peroxisomes. In summary, our experimental results suggest that PenM is involved in penicillin production most likely through the translocation of isopenicillin N from the cytosol to the peroxisomal lumen across P. chrysogenum peroxisomal membrane. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.