| Several studies have focused on how to promote ruminant roughage utilization. Applications of cellulosic decomposing strains to improve the crude fiber degradation of the roughage may be an efficient strategy. With the development of genetic engineering technology and the intense research of Lactic acid bacteria(LAB), application of the engineered LAB expressing multicomponent cellulases to degrade the roughage or to act as the active microbial agent for improving the utilization rate of livestock may become a more convenient, effective and low-cost strategy. However, the security of recombinant strains need to be specifically considered. Therefore, construction of non-antibiotic marker, effective, convenient and stabile recombinant strains has been a hot topic in the microbial genetic engineering research.To improve the repertoire of constructing the recombinant Lactobacillus coexpressing multiple cellulases and the non-antibiotic marker recombinant Lactobacillus and build the corresponding evaluation method, we firstly cloned three types of cellulase genes from cellulosic decomposing strains that were isolated by our laboratory previously, then build methods and techniques for constructing complex vector and screened the host bacteria LAB promoters and signal peptides. This will provide the technological base for the development of functional food-grade Lactobacillus. The results are as follows:1. The mature peptides of endoglucanase and β-glucosidase gene were successfully cloned from the cDNA of A. fumigatus WL002, about 1434 bp and 2517 bp, named cenA(No. KU933946) and bglW(No. KM520393), respectively. A difunctional cellulase gene, eglS3(No. KU933947), consists of an open reading frame of 1527 nucleotides and a β-glucosidase gene, bglS(No. KU921415), about 1203 bp, were cloned from the genomic DNA of B. subtilis WL001 and Streptomyces griseorubens WL003, respectively. These four genes were solubly expressed in E. coil Rosseta(DE3), respectively. Then, analysis of these recombinant proteins properties showed that CENA(pH 4.5), EGLS3(pH 6.0-6.5) and BGLS(pH 6.0) exhibited the maximum cellulase activity under the acidic condition, while BGLW(pH 9.0) in the alkaline condition. Therefore, the first three genes were selected to construct the Lactobacillus coexpression vector of multiple cellulase genes.2. A simply, rapid method based on prolonged overlap extension PCR for simultaneously assembling multiple DNA fragments into a vector at any desired position was described. Using this method, three fragments could be simultaneously and precisely inserted in a vector in only 2–3 days. Furthermore, the acceptable transformation efficiency was determined through the tested host strains; more than 95% of the colonies were positive transformants. Therefore, this method is less labor intensive and time-consuming and significantly improved the efficiency of constructing complex vector.3. To improve the expression level of foreign cellulase genes in the Lactobacillus coexpression plasmid and the extracellular secretion amount of recombinant enzymes, six constitutive promoters were investigated for their capability to drive the expression of cellulase reporter gene(celL15) in Lactobacillus reuter XNY by comparing the endoglucanase activity. Three promoters were obtained, in which Paldo proved to be the most effective promoter in Lactobacillus reuter, followed by Ppgm and PermB. The total cellulase activity produced by Paldo, Ppgm and PermB were approximately 1.5-2 fold as that of P32, which was commonly used in construct the Lactobacillus expression vector. In addition, five reported LAB signal peptides were used to construct Lactobacillus expression vectors for screening the signal peptides by assay the endoglucanase activity of extracellular and intracellular recombinant proteins. The results showed that three signal peptides(SPlp0373, SPamyLand SPusp45) appeared as promising candidates for directing secretion. Thereafter, the secretory efficiency of the three signal peptides were further compared using β-glucosidase, and the results showed that SPlp0373 was the best performing with different reporter proteins. Therefore, SPlp0373 should have more wide application potential in secretory expression vectors of Lactobacillus.4. Then, we successfully construct a Lactobacillus coexpression plasmid(pLEM415-cenA-eglS3-bglS) of three cellulase genes and obtained an engineered Lactobacillus, called XNY/pLEM415-cenA-eglS3-bglS. It is capable of simultaneously expressing an endoglucanase, a difunctional cellulase and a beta-glucosidase under the conventional fermental condition, and the extracellular recombinant proteins had the high endoglucanase activity(1.42±0.13 U/mL/OD600), exoglucanase activity(0.42±0.06 U/mL/OD600), β-glucosidase activity(0.71±0.09 U/mL/OD600) and filter paper activity(0.72±0.07 U/mL/OD600) at 45-50 °C and pH 5.5-6.0, which indicated that the engineered Lactobacillus had a capability for degradation of feed crude fiber. Subsequently, XNY/pLEM415-cenA-eglS3-bglS was carried out the SSF using wheat straw or maize straw, and the degradation rate of crude fiber was 12.6% and 9.7%, respectively, which suggested that the XNY/pLEM415-cenA-eglS3-bglS has a potential for fermentation of coarse fodder or active microbial agents of livestock.5. To improve the recombinant technology of Lactobacillus and obtain non-antibiotic recombinant Lactobacillus, a encoding gene of phenylalanyl-tRNA synthetase alpha subunit, pheS(No.KF915808), was for the first time amplified from the genomic DNA of L. reuteri XNY, about 1047 bp. A point mutant(GCC312GGC) of the pheS gene was proved to be a promising negative selection marker in Lactobacillus in the presence of 25 mM p-Cl-Phe. Meanwhile, we described a recombination approach for the creation of non-antibiotic Lactobacillus mutations applying a counterselection cassette generated by a negative selection marker(pheSMn) and a positive selection marker(erythromycin resistance gene, em). In this method, the counterselection cassette was site-directed integrated into the genome of Lactobacillus by homologous recombination, and a mutation was firstly generated and was sensitive to p-Cl-Phe. Subsequently, the counterselection cassette was replaced by the expression cassette of target gene by homologous recombination, and the non-antibiotic recombinant strain with p-Cl-Phe resistance could be obtained. Using this approach, a non-antibiotic marker Lactobacillus reuter mutation, XNY-celL15, was obtained by site-directed integrating a cellulase gene into the genome of wild type L. reuter XNY. The XNY-celL15 exhibited an endoglucanase activity of 0.81 U/mL/OD600. This recombination approach will provide technological base for the development of functional food-grade Lactobacillus. |
PDF Full Text Request