Cell Surface Display System For Bacillus Thuringiensis: A Novel Development Using Cell Wall-Bound Autolysins

Bacillus thuringiensis is an important agricultural strain that have been widely exploited for a variety of applications. It is also generally regarded as an attractive host for developing a cell surface display system, particularly a multifunctional system that would extend its agricultural or biotechnological applications. In the present study, a putative autolysin mbG was used as the functional anchor protein for development of a novel surface display system in B. thuringiensis vegetative cells, followed by the multiple optimization analyses on the feasibilities and efficiencies of various full-length or truncated mbG anchors, and further applied to surface display two different heterologous proteins onto B. thuringiensis BMB171 cells using the optimum mbG anchor. Thus, the study could provide the fundament for development of multifunctional application systems.Analyses on several genomes of B. thuringiensis showed that some sequences encoding the putative cell wall-associated proteins. These proteins are implicated in the activities of the cell wall-binding. By designing the specific primers, PCRs were performed to amplify three peptidoglycan hydrolyase (autolysin) genes from B. thuringiensis wild-type strain YBT-1520, which were designated as mbA, mbG and mbP. The surface display efficiency of these encoded proteins were compared by FACS analysis, the results indicated that under the control of themselves promoters, mbG had the largest display efficiency, as much as 47.5%. Amino acid sequence analysis showed that mbG had no obvious secretion signal sequence and transmembrane segment sequence, with some distinctive features. Thus, we employed mbG as the major study subject.The further structural predicted analysis indicated that mbG was a putative endo-β-N-acetylglucosaminidase, included two domains: a cell wall anchoring activity N-terminal domain with two tandemly aligned LysM and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. The result also showed that the fusion protein had a low expression and display ability.Low-level constitutive expression of mbG was elevated by introducing a sporulation-independent promoter of cry3Aa. Truncated mbG domains with separate N-terminus (nmbG), C-terminus (cmbG), LysM₁, or LysM₂ were further compared for their cell-wall binding efficiencies. The N-terminus nmbG contributed to cell-wall binding, while LysM₁ was the active domain. Two tandemly repeated nmbGs exhibited the highest binding activity, while the activity of three repeated nmbGs was decreased, which indicated that the length of the fusion protein determined transmembrane transport.A putative laccase gene was cloned from Shigella dysenteriae W202 and expressed in Escherichia coli as a soluble fusion protein with high yield. The purified product (Wlac) was characterized as the CueO-like laccase from E. coli, a monomer of molecular mass 55 kDa, with a maximum activity of 24.4 U/mg (K_m=0.086) and a pH optimum of 2.5, in a standard assay using ABTS (2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate)) as the substrate. Activity was stable at 0 to 25℃but inhibited above 40℃. Purified Wlac was completely inhibited by 200 mmol EDTA and partially by 32 mmol SDS, 50 mmol NaN₃ and 60 mmol thioglycolic acid. Activity was stimulated by Cu²⁺, other metal ions had only slight or negative effects. Two mutated variants, WlacS and WlacD, were obtained by substituting Glu 106 with Phe 106, and adding a deletion of anα-helix domain (from Leu 351 to Gly 378). WlacS had a 2.2-fold (52.9 U/mg) and WlacD a 3.5-fold (85.1 U/mg) higher enzyme activity than the wild-type laccase and WlacD showed greater thermostability at higher temperatures. Sce VMA intein-associated fusion proteins maintained～80% of total enzyme activity.WlacD was successfully displayed onto the surface of B. thuringiensis target cells using the optimum (nmbG)₂ anchor, without radically altering its catalytic activity. After preparing antiserum using the purified laccase, Western blot analysis indicated that WlacD had located on the cell surface. The measurement of whole cell laccase activity showed the engineering strain's laccase activity was 15.3 U/mL, and it could be used to dispose sewerage and so on.For another, we obtained the mutant strain YBT-1520e losting partial endogenesis plasmids from B. thuringiensis YBT-1520, using the methods of gradual increasing temperature and adding low dosage SDS. YBT-1520e could synthesize parasporal crystal proteins, elevate the transform efficiency, and with a plasmid stability up to 100%.