Heterologous Production Of Daptomycin Via Synthetic Biology

Synthetic biology is an emerging field of biological research in the21st Century,it can bring direct benefits by purposefully transform existing organisms, helping tosolve certain major issues that human faced, and it will light shine for solving energy,human health, and the environment. Synthetic biology has great potentials inpromoting future developments in renewable chemicals, biofuels, pharmaceuticalintermediates, health care products and new tools for environment protection.Weexpect to use parts, modules or devices of synthetic biology to artificially design,assemble and optimize biosynthetic pathways，generating and assembling functionalmodular components，developing novel applications on processes，and achieving thegoal products effectively. Synthetic biology is in rapid development period, but thereare some technical bottlenecks need to be addressed, mainly are DNA synthetictechnology and transplantation for large fragments or genome. Although the Ventergroup reported that they had been successfully synthesized1.08Mb genome, in theleading level in the world, synthetic biology is still in an early developmental stage inChina.We have not master the key technologies, in terms of cost and synthetic rate,the current synthetic technology is still far from meeting the needs of syntheticbiology. Currently, the large fragments of DNA synthesis over100kb has not beenreported, which restricts the development of China’s synthetic biology. Great effortsare needed for the further research.Natural compounds derived from biological secondary metabolites in the body,have many important physiological activities. Currently, about half of the drugs arenatural compounds or derivatives, such as antibiotics (erythromycin), antimalarialdrugs (artemisinin), antitumor drugs (paclitaxel). Synthetic biology has provided the new production pattern for the natural products development. In fully understandingof biosynthetic mechanism of natural products,we can re-design and re-construct thebiosynthetic pathways in chassis cells, to realize efficient biological synthesis oftarget compounds. Daptomycin, a novel cyclic lipopeptide antibiotic from Streptomy-ces roseosporus provides rapid bactericidal activity against gram-positive pathogensin vitro. Its unique chemical structure and mode of action make it difficult to generatebacterial resistance to it, and the side effects are small. Daptomycin is biosynthesizedby a nonribosomal peptide synthetase (NRPS). A128kb region of S. roseosporusGC-rich DNA contains the daptomycin biosynthetic gene cluster.The purpose of this paper is to establish key technologies of synthetic biology,such as large DNA synthesis and DNA assembly. We choose daptomycin as targetproduct,through synthesis and assembly of128kb daptomycin biosynthetic cluster aswell as transplantation and genetic manipulation of large DNA fragments, to achievethe de novo synthesis and heterologous expression of daptomycin. By detecting thecorrect expression of daptomycin, we test and verify the techniques of DNA synthesisand assembly. Finally,we mastered the techniques of DNA synthesis and assembly formore than100kb length DNA fragments, and it will provide the foundation forsynthetic biology research and development. Main achievements are as follows:1、 We have designed and divided daptomycin biosynthetic gene cluster into severalDNA fragments, the designed cassettes were generally1,080bp with80-bpoverlaps to adjacent cassettes.About20cassettes were chemically synthesized bycompanies. In addition, we established a high-fidelity PCR amplification methodfor GC-rich, large DNA fragments. We combined PCR additive7-deaza-dGTPwith Touch down PCR procedure, successfully amplified the remaining108DNA fragments, and reduced the cost of DNA synthesis.2、 We chose in vitro recombination system--Gibson isothermal One-step methodand in vivo yeast homologous recombination system to assemble daptomycingene cluster. We optimized and mastered these two most advanced and efficientmethods, and by comparison of the two methods, we found that in vitrorecombination system does not meet the high GC content DNA assembly because of the high GC content and more complex of daptomycin biosyntheticgene template, but in vivo yeast homologous recombination system is so efficient,accurate that we determined to use in vivo yeast homologous recombinationsystem to assemble daptomycin biosynthetic gene cluster.3、 We have synthesized a128kilo-base pair daptomycin biosynthetic gene cluster.We developed a hierarchical strategy to assemble the gene cluster in2stages bytransformation and homologous recombination in yeast from1281kb cassettes.In the first stage,10cassettes and a pRS414/424vector were recombined in yeastAH109cells by PEG/LiAc and the recombinant vector was transferred to E.coli,plasmid DNA was then isolated from individual E.coli clones and digested toscreen for cells containing a vector with an assembled10-kb insert. MultiplexPCR was performed on selected yeast clones. In the last stage, the pooled10-kbassemblies were digested with SpeI and a BAC-YAC cloning vector pYES1Lwere transformed into yeast to complete the final128-kb assembly, afternutritional deficiencies screening, PCR identification, pulsed-field gelelectrophoresis identification and sequenced,we eventually obtained the entirelycorrect128kb daptomycin gene cluster.4、 Based on pBeloBAC11plasmid, we constructed a E.coli and Streptomyces BACshuttle vector pBTIBAC11using red homologous recombination method. Wefurther cloned128kb daptomycin gene cluster into pBTIBAC11vector PmeI site,then it was conjugated to S.lividans TK24,128kb daptomycin gene cluster wassite-specific integrated into S.lividans TK24chromosomes by attP/int sites. Andthe product daptomycin in fermentation cultures was detected by HPLC analysis.After verifying daptomycin’s molecular weight and biological activity, weobtained the functional daptomycin consistent with natural daptomycin.5、 In addition, we evaluated the activities of daptomycin against Bacillus anthracisAP422and to discuss its probalble mechanism of action. The rapid bactericidalactivities and novel mode of action make daptomycin serve as an appealingalternate therapy for Bacillus anthracis infections and warrant furtherinvestigation. In summary, we mastered the techniques of DNA synthesis and assembly formore than100kb large DNA fragments, and it will provide the foundation forsynthetic biology research and development.