title: Autodisplay of an archaeal γ-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (-) vince lactam.
abstract: At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter Xcc_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of Xcc_Est as an anchoring motif for surface display of γ-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of γ-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated Xcc_Est could efficiently transport γ-lactamase to the cell surface. Compared with the free enzyme, the displayed γ-lactamase exhibited optimum temperature of 30 °C other than 90 °C, with a substantial decrease of 60 °C. Under the preparation system, the engineered E. coli with autodisplayed γ-lactamase converted 100 g racemic vince lactam to produce 49.2 g (-) vince lactam at 30 °C within 4 h. By using chiral HPLC, the ee value of the produced (-) vince lactam was determined to be 99.5 %. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed γ-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (-) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China, wangjj@im.ac.cn.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Wang
authors.forename: Guogang
authors.initials: G
authors.lastname: Zhao
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Zhang
authors.forename: Qiulin
authors.initials: Q
authors.lastname: Liang
authors.forename: Cong
authors.initials: C
authors.lastname: Min
authors.forename: Sheng
authors.initials: S
authors.lastname: Wu
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000998:Antiviral Agents / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D015153:Blotting, Western; D061505:Cell Surface Display Techniques; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005434:Flow Cytometry; D007769:Lactams / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048229:Sulfolobus solfataricus / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D016959:Xanthomonas campestris / Q000235:genetics
chemical_list: D000998:Antiviral Agents; D019843:Archaeal Proteins; D001426:Bacterial Proteins; D007769:Lactams; D011993:Recombinant Fusion Proteins; D000581:Amidohydrolases; C401943:gamma-lactamase