In directed evolution, a high-throughput screening system is often a prerequisite for sampling the enzyme variants. When the target enzyme is expressed intracellularly, for example when Escherichia coli is used as the host, chemical or enzymatic disruption of cell membrane is often required in many cases, which can be tedious, time-consuming, and costly. In this study, a set of heat-inducible autolytic vectors were constructed to solve this problem, in which the SRRz lysis gene cassette from bacteriophage λ was placed downstream of heat-inducible promoters, λ cI857/p_Rpromoter and its mutant, cI857/p_R(M). The artificial autolytic units were inserted into the backbone of pUC18 (away from the multiple cloning sites). For the wild promoter, cI857/p_R, the SRRz lysis cassette was expressed by temperature up-shift from 28° to 38°C, and the lysis efficiency of transformed bacterial cells was found to be consistent and could reach 96.3% as measured by the reporter β-galactosidase assay. In order to obtain a higher cell growth rate, the mutant promoter cI857/p_R(M) was utilized to allow bacteria growth at 35°C and lysis at 42°C. However, this heat-inducible system showed significant inconsistency in terms of lysis efficiency. Bacillus subtilis 168 lipase A gene was further in-serted into the multiple cloning sites of the autolytic vector containing cI857/p_R, and 93.7% of the expressed lipase activity was found in the culture medium upon heat induction, demon-strating the utility of the vector for expression and rapid extracellular assay of heterologous enzymes.

Introduction

Aiming to search protein variants with improved or even novel activity by screening or selection from a large pool of protein variants (1,2,3), directed evolution has been widely used in engineering enzymes since it emerged in the past decade (4). Screening, where all the variants in a certain library are generally assayed individually, is commonly used in directed evolution, as selection in most cases is unattainable. A quick and high-throughput screening strategy is thus significant for many directed evolution efforts, especially when Escherichia coli is used as the expression host, since most heterologous proteins or enzymes are expressed intracellularly, while the cell membrane is not permeable to most substrates. Here we set out to develop a set of autolysis vectors for large-scale screening (as needed in, for example, directed evolution) that require only a physical signal, heat.

Many previous efforts in self-disruptive E. coli systems utilizing the autolysis mechanism were based on the modification of the E. coli genome (5), controlled expression by a chemical inducer (i.e., isopropyl-β-D-thiogalac-topyranoside; IPTG) of bacterial phage lysis genes, such as the SRRz genes of phage λ, gene E of φX 174, gene e and gene t of phage T4 (6,7,8,9), or the joint action of additional osmotic, chemical, or enzymatic treatment (10), which may be time-consuming and costly for screening. We report here two heat-inducible autolytic vectors in which the lysis gene cassette, SRRz from bacte-riophage λ was placed downstream of two heat-inducible promoters, λ cI857/p_R, and a mutant form of the promoter, cI857/p_R(M) (8,11,12). The cell lysis profiles for these two autolytic vectors were quantitatively analyzed. The wild cI857/p_R promoter is reported to be stringently repressed at temperatures lower than 30°C and inducible by a temperature shift to 38°C (13,14). The mutant promoter cI857/p_R(M) (with a T→C mutation in the cI857/p_R promoter) was also chosen, since it has an extended heat stability allowing bacterial growth around 37°C and thus affords a faster cell growth profile (8,11,12). These two promoters have been used in preparing empty bacterial cell envelopes or “bacterial ghosts” in conjunction with the gene E of φX 174 (8,11,12), which provides the basis for the construction of heat-inducible autolytic vectors in our study. The mechanism for cell lysis induced by SRRz genes is known (6,8). The S gene product causes lesions in the cytoplasmic membrane through which the R and Rz gene products degrade the murein.

Materials and Methods

Construction of Heat-Inducible Vectors

The autolytic genes used in our study was the SRRz cassette of bacterio-phage λ (6,8). The SRRz gene cassette was placed under the transcriptional control of heat-inducible prompters and upstream of the strong rrnB terminator. A 1082-bp DNA fragment containing the promoter λ cI857/p_R was generated by PCR using DeepVent_R® polymerase (New England Biolabs, Ipswich, MA, USA) with λ phage DNA (cI857 Sam7; Promega, Madison, WI, USA) as the template and primers cI857/p_RFor and cI857/p_RRev (see Table 1, the under-lined nucleotides indicate the SapI, EcdRI, and XhoI sites, respectively). For the mutant promoter, cI857/p_R(M), site-directed mutagenesis was performed by introducing a T→C base mutation into the cI857/p_R promoter with primers cI857/p_RFor and cI857/p_RRev(M) (Table 1, the underlined nucleotides in the latter indicates the XhoI site, and the boxed nucleotide represents the site mutation) and with λ phage DNA as template. The 246-bp rrnB terminator was amplified by PCR using ExTaq™ polymerase (Takara, Dalian, China) with the genomic DNA of E. coli DH5-α (Dingguo, Beijing, China) as the template and with primers rrnBFor and rrnBRev (Table 1, the underlined nucleotides indicate the EcoRI, NcoI, and AflIII sites, respectively). The cI857/p_R promoter was first ligated with the rrnB terminator (doubly digested with EcoRI and AflIII) and then inserted between the SapI and AflIII sites of pUC18 vector (Takara), yielding the plasmid pUC18-cI857/p_R-rrnB.

Table 1. Primers Used in This Study (Click to enlarge)

The SRRz cassette was amplified by PCR from the λ DNA (cI857 Sam7). As the cI857 Sam7 template is an S minus variant, which has a stop codon (TAG) in the S gene, site-directed mutagenesis was performed to replace the stop codon with the original one (TGG). Four primers were used, SRRz1For, SRRz1Rev, SRRz2For, and SRRz2Rev (Table 1, the underlined nucleotides indicate the XhoI and NcoI sites, respectively, and the boxed nucleotides indicate site mutations). Two PCR fragments (using SRRz1For/SRRz1Rev and SRRz2For/SRRz2Rev primer pairs, respectively) were obtained by PCR using DeepVent_R polymerase. These two fragments were combined at a 1:1 ratio to yield the full-length SRRz, via overlapping extension PCR and inserted into pUC18-cI857/p_R-rrnB to obtain pUC 18-cI857/p_R-SRR z-rrnB (between the XhoI and NcoI sites).