Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Directed Evolution of <i>Mycobacterium tuberculosis</i> β-Lactamase Reveals Gatekeeper Residue That Regulates Antibiotic Resistance and Catalytic Efficiency

<div><p>Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium <i>Mycobacterium tuberculosis,</i> which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A β-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to β-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F) that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a ‘gatekeeper’ residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.</p></div

Structural basis for enhanced BlaC-mediated resistance.

<p>(a) Active sites of wt BlaC (top), BlaC(I105F) (middle), or structural alignment of both (bottom). (b) Structural alignment of wt BlaC (yellow), BlaC(I105F) (cyan), and TEM-1 Bla (magenta). Arrow indicates aromatic residues of BlaC(I105F), and TEM-1 Bla.</p

Heterospecific expression of <i>M. tuberculosis</i> BlaC in <i>E. coli</i>.

<p>(a) Serially diluted wt or Δ<i>tatC</i> cells expressing ssTorA-Bla or ssTorA-BlaC chimeras were spotted on Amp. (b) Western blot analysis of cytoplasmic (cyt) and periplasmic (per) fractions prepared from wt cells expressing ssTorA-BlaC, full-length BlaC, or BlaC lacking a signal peptide (ΔspBlaC). Arrow indicates BlaC. Samples prepared from an equivalent number of cells were loaded in each lane. Blots were probed with anti-FLAG antibodies.</p

Chlamydia trachomatis Secretion of an Immunodominant Hypothetical Protein (CT795) into Host Cell Cytoplasm ▿

The Chlamydia-specific hypothetical protein CT795 was dominantly recognized by human antisera produced during C. trachomatis infection but not by animal antisera raised against dead chlamydia organisms. The immundominant region recognized by the human antibodies was mapped to the N-terminal fragment T22-S69. The endogenous CT795 was detected in the cytoplasm of host cells during C. trachomatis infection and was highly enriched in the host cytosolic fraction but absent in the purified chlamydia organisms, suggesting that CT795 is synthesized and secreted into host cell cytoplasm without incorporation into the organisms. All C. trachomatis serovars tested secreted CT795. A predicted signal peptide of CT795 directed the mature PhoA to cross Escherichia coli inner membranes. The secretion of CT795 in Chlamydia-infected cells was inhibited by a C16 compound targeting signal peptidase I, but not by a C1 compound known to block the type III secretion pathway. These results suggest that CT795, like CPAF (a Chlamydia-secreted virulence factor), is secreted into the host cell cytoplasm via a sec-dependent mechanism and not by a type III secretion pathway. The above characterizations of CT795 have provided important information for further understanding the potential roles of CT795 in C. trachomatis pathogenesis