Metalloadsorption by Escherichia coli Cells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

Abstract

Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB153-YMT and LamB153-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. colicells to bind Cd2+ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.

Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins

“…Enhanced Cd 2+ accumulation by genetically engineered E. coli with surface-expressed metal-binding peptides have been reported by others (Sousa et al, 1996(Sousa et al, , 1998Xu and Lee, 1999). One of the most successful is the LamB fusion system, which has been used to anchor MTs and short metal-binding peptides onto the surface of E. coli (Kotrba et al, 1999;Sousa et al, 1998). However, the Cd 2+ binding capability of cells expressing EC20 is almost twice the amount obtained using the LamB system .…”

Enhanced arsenic accumulation by engineered yeast cells expressing Arabidopsis thaliana phytochelatin synthase

“…Attempts to develop useful biosorbents by increasing the heavy metal content of plants, algae or microorganism via genetic engineering have been made in recent years (Ma et al, 2001;Maitani et al, 1996;Sar and D'Souza, 2001). Although significant improvements in heavy metal accumulation have been observed (Li et al, 2000;Pazirandeh et al, 1995) by the overexpression of metal-chelating peptides such as metallothioneins (Sousa et al, 1998) or synthetic phytochelatins (Bae et al, 2000(Bae et al, , 2001, the ability of these peptides in arsenite binding has never been confirmed. Naturally occurring phytochelatins are particularly attractive alternatives as they are not subject to extensive proteolysis because of the g-carboxyamide bond between Glu and Cys, and they are known to bind to arsenic with high affinity (Sauge-Merle et al, 2003).…”

“…Bioremediation using microbes is often considered as a cost effective and efficient way of removing heavy metals (Mejare and Bulow, 2001). Several attempts have been made to enhance the intracellular heavy metal content of microorganisms by expressing metal-chelating peptides such as metallothioneins (MT) (Ma et al, 2001;Sar and D'Souza, 2001;Say et al, 2003;Sousa et al, 1996Sousa et al, , 1998, however, they generally lacked specificity and affinity for As. Recently, it was shown that specific arsenic accumulation in E. coli can be achieved by expressing an arsenic-binding protein arsR (Kostal et al, 2004).…”

Biotechnological applications for surface‐engineered bacteria

“…There have been several reports on surface display of yeast and mammalian MTs in recombinant E. coli cells [148–150]. Sousa et al [149] reported on a 15–20‐fold increase in Cd accumulation for E. coli cells displaying yeast (CUP1) and mammalian (HMT‐1A) MTs anchored to the OMP LamB, and similar reports have been published by others [148,150]. The use of laboratory strains of E. coli might, however, not be suitable for in situ soil remediation and other more suitable strains have therefore been investigated [31,54].…”