| Microorganisms are an important source of natural products.With the rapid developments of synthetic biology and the advancement of genome sequencing technology,more and more microbial genomes have been sequenced.It has been found that huge number of biosynthetic gene clusters in the microbial genome have not been characterized and utilized yet,similarly many microorganisms are difficult to genetically manipulate or cannot be cultured in the laboratory growth conditions.Heterologous expression means transferring of native biosynthetic gene clusters from original host into a heterologous host strain having clear genetic background and easy to manipulate.Heterologous expression is an important strategy to study biosynthetic gene clusters.However,microbial secondary metabolites gene clusters are generally larger than 20 kb in size and are difficult to obtain by PCR or de novo synthesis.The traditional method of obtaining biosynthetic gene clusters was to construct genomic libraries and screen the target gene cluster or directly clone the target biosynthetic gene cluster by using the yeast recombination system,but these methods were time consuming and laborious,which cannot satisfy the high-throughput gene cluster cloning.In 2012,Fu et al.found that full-length RecE and RecT proteins increased the recombination efficiency between two linear DNA fragments and capture the target DNA fragment directly from the genome.At present,RecET direct cloning technology has been widely used in studies of secondary metabolite gene cluster's heterologous expression.Whereas heterologous expression of secondary metabolites often require modification of the expression vector for example addition a gene transfer element,inserting a positive regulatory gene and knock out a negative regulatory gene etc.Traditional DNA recombination methods are based on restriction enzyme digestion and ligation,which are difficult to accomplish these genetic modifications due to restriction of the cleavage site availability and DNA molecule size.In 1998,Zhang et al.used Red?? protein to efficiently recombine linear and circular DNA molecules in E.coli and established a DNA recombination engineering that is not restricted by DNA size and availability of restriction sites,they named it as Recombineering.The technology can accurately and efficiently perform for gene insertion,gene knockout,point mutation and module replacement on DNA molecules such as plasmids,cosmids and bacterial artificial chromosomes,and is especially suitable for genetic transformation of natural product gene clusters.In current study,the RecET DNA direct cloning system and the Red?? DNA modification system were integrated into an E.coli host,and the high-throughput direct cloning,modification and heterologous expression of biosynthetic pathways were established by standardization of cloning vectors and DNA transfer elements.In this technology platform,we constructed an easy-to-operate direct cloning vector for different size gene clusters and different DNA sequence features,while conjugal transfer and transposable cassettes,site-specific recombination cassettes,and broad-host multicopy replicon can be also be added according to downstream transfer process.All the cassettes has been standardized so that it can be rapidly inserted into all of the above cloning vectors by Redap recombination,one vector can be applied to different genetic manipulation methods such as bacterial conjugal transfer,transposition and site-specific recombination.The technology platform enables seamless docking of biosynthetic pathways for cloning,transfer and heterologous expression.It takes only one week to complete the cloning of biosynthetic pathways and the insertion of gene transfer elements,greatly simplifying the DNA cloning,modification,transfer and heterologous expression.Using this technology platform,we cloned and heterologously expressed the secondary metabolite gene clusters from genomes of Streptomyces sp.ATCC 27952,S.spinosa NRRL1 8395 and Streptomyces avermitilis DSM 46492.The secondary metabolite gene clusters in soil and marine metagenomics are difficult to obtain by direct cloning due to low concentration.De novo synthesis is an effective method for the cloning of gene clusters in metagenomics,but this method requires high DNA assembly efficiency,In this study,a method for synthesizing gene clusters was established by combining oligonucleotide annealing in vitro and RecET-mediated homologous recombination between linear DNA molecules.The strategy of this method is:(1)assemble ten 60 nt oligos that overlap each other by 20 bp resulting into a 420 bp double-stranded DNA fragment;(2)assemble ten 420bp fragment that overlap each other by 40 bp and resulted into a 4020 bp fragment;(3)the 4020 bp fragments with a 40 bp homologous sequence was assembled into the full length gene cluster.We used this method to de novo synthesis of the 12.7 kb non-ribosomal polypeptide gene cluster patellamide A which found in the sphagnum symbiotic cyanobacteria,and heterologous expression the gene cluster in E.coli.The gene cluster cloning,modification and heterologous expression technology platform based on Red/ET homologous recombination constructed in this paper provides convenience for the development and utilization of microbial genome. |
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