Construction And Applications Of Burkholderiales Chassis For Natural Products

The numerous and structurally diverse microbial metabolites are of paramount significance in human health and biological control.In particular,the natural products(NPs)with complex structures as well as their derivatives have become the pivotal sources of novel antibiotics and pharmaceuticals for treatment of cancers and other indications.However,the ubiquitous outbreak of multidrug-resistant pathogens suggests an unprecedented demand for new-to-nature drug and/or lead compound discovery.In recent years,a growing number of studies have reported the identification of plentiful of novel NPs with intriguing skeletons and high-value bioactivity derived from Gram-negative bacteria,particularly proteobacteria.These findings highlight the vast biosynthesis potential for producing broader spectrum antibacterial NPs from these microbe,complementing the extensively studied Gram-positive bacteria like Actinobacteria.Nowadays,the advancement in microbial pan-genome sequencing and bioinformatics has facilitated the prediction and analysis of biosynthetic gene clusters(BGCs)responsible for previously unidentified NPs in microorganisms.In general,the in-depth exploration of those promising microbes mainly relies on two methods in genome mining:The first one is to activate the expression of targeted cryptic BGCs by modifying promoters and regulatory mechanisms in native strains.The other method involves heterologous expression of the desired BGCs in suitable chassis strains after cloning and modification,which is helpful to activate cryptic BGCs from genetically inaccessible strains or failed to be activated in native metabolic context.Nonetheless,the major bottleneck is insufficient chassis strains avalable for multifarious BGCs from all kingdoms of microorganisms,especially those from Gram-negative bacteria.The Gram-negative β-proteobacteria class Burkholderiales order Schlegelella brevitalea DSM 7029,previously known as Polyangium brachysporum K481-B101 or Burkholderiales strain DSM 7029,has demonstrated its potential as an efficient heterologous host,has been successfully applied in the heterologous production of several proteobacteria-derived non-ribosomal peptides/polyketides.In-depth study reported that the DSM 7029 wild type(WT),yet,undergoes severe cell autolysis during the early stages of liquid culturing(after 24 hours),hindering its further biotechnological applications.Herein,we aimed to address the problem of low yield in NP heterologous expression owing to the early-stage cell autolysis.Meanwhile,we also prioritized eight intriguing bacterial BGCs for heterologous expression.The results are as follows:(1)Construction and phenotype characterization of rational genomereduced Burkholderiales strain DSM 7029 mutantsGiven that the DSM 7029 undergoes severe cell autolysis in the early stage of liquid culturing,which ultimately affects the final biomass of DSM 7029 as well as heterologous expression yields of target compounds,this study performed rational streamlinearization of genome based on the efficient genetic operating system established previously aiming to alleviate the cell autolysis,increase the biomass during fermentation,and enhance the biotechnological applications of the Burkholderia DSM 7029 after determining the disposable genomic regions by the combination of genomics,transcriptomes,metabolisms,as well as several bioinformatic analytic tools.As expected,DT series genome-reduced mutants(DT1-DT7)obtained by combinatorial depletion of transposons,IS sequence elements,prophage regions and other unessential genes exhibited alleviated cell autolysis,shorten cell size,increased biomass.Based on this,the DT8-DT10 mutant strains were stepwisely constructed by inactivating three endogenous secondary metabolites pathways(BGCs 5,6,and 7).Contrastly,the DC series genome-reduced mutant strains(DC 1-DC7)generated by sequential deletion of multiple large endogenous BGCs displayed significant attenuation of cell growth.Additionally,this study also suggested that the moderate genome reduction could improve the uptake and carry efficiency of exogenous DNA.(2)Utilizing DSM 7029 rational genome-reduced chassis strains to optimize yield of heterologous natural productsThis study performed heterologous expression and relative production comparison of six known natural prodcuts biosynthetic pathways from Gram-negative proteobacteria in DSM 7029 genome-reduced chassis DT6～DT10,DSM 7029 wild type and two widely-used Gram-negative chassis(Escherichia coli and Pseudomonas peutida).The six natural products mentioned above are the NRPs rhizomide A and holrhizin A,the NRP/trans-AT PK hybrid rhizoxins from Paraburkholderia rhizoxinica HKI 454,the NRP icosalide A1 from Burkholderia gladioli ATCC 10248,and the NRP/PK hybrid epothilones derived from Sorangium cellulosum So ce90,as well as myxochelins,a NRP siderophore from Myxococcus xanthus DK 1622.The six corresponding BGCs of those NPs were modified,and site-specifically integrated into abovementioned host strains via phiC31-mediated attB/attP site-specific integration for heterologous expression.The relative yield comparison suggested that DT6-DT10 genome-reduced DSM 7029 mutant strains with optimized cell growth,alleviated cell autolysis and simplified metabolic backgrounds are more effective in biosynthesizing heterologous NPs,exhibiting improved biosynthetic robustness.(3)Utilizing DSM 7029 rational genome-reduced chassis strains to discover cryptic natural product from proteobacteriaThis study try to activate a cryptic BGC chm from Chitinimonas koreensis DSM 17726 by virtue of rational genome-reduced DSM 7029 chassis strains DT6-DT10 as heterologous hosts.After successfully cloning,modifying and integrating the BGC chm into the genomes of DSM 7029 WT,DT6-DT10 genome-reduced mutants and another two commonly-used Gram-negative chassis strains via phiC31-mediated attB/attP sitespecific integration.The LC-MS analysis gave the hint that DT6 to DT10 genomereduced mutants harboring the chm gene cluster exhibited a series of obvious differential peaks compared to the negative blank control.Yet,only two minor differential peaks were detected in the DSM 7029 WT while E.coli and P.putida were unable to activate the expression of chm BGC.Heterologous yields comparison showed that chitinimides series NPs produced in the genome-reduced DT6-DT10 was higher than those of DSM 7029 WT and native producer C.koreensis DSM 17726.The chitinimides A-D were purified and identified by NMR in succession,and the structures of the chitinimides E-H were deduced by HRESIMS/MS analysis.Additionally,the preliminary biosynthesis was proposed and biological activity evaluation of chitinimides was also conducted in this study,which warrant further investigation.(4)Heterologous expression-drived genome mining of bacterial cryptic natural productsGram-negative proteobacteria(e.g.,Myxobacteria)and Gram-positive bacteria(e.g.,Streptomyces spp.)are well-known in producing structurally diverse NPs with valuable bioactivities.The genome mining strategies on the basis of BGCs cloning,modification and systematic heterologous expression of BGCs to excavate abundant cryptic microbial NPs and novel derivatives of known NPs have been extensively studied in last two decades.In this study,a series of diversiform BGCs sharing low similarity with known clusters,or containing self-resistence genes and/or intriguing post-translational modification enzyme coding genes were prioritized from Cytophagales spp.,Myxobacteria and Streptomyces spp.via bioinformatic analysis of the bacterial genomes from the public database and in-house database within the Helmholz Institute for Pharmaceutical research Saarland(HIPS).Utilizing diverse BGC capture technologies,e.g.,RecET direct cloning.Gibson assembly,cosmid-based stitching,together with Red/ET recombineering enabled promoter engineering and transfer element integration as well as heterologous expression,eight cryptic BGCs were studied herein.Of which,the PKS/NRPS BGC mxpt,responsible for the biosynthesis of structurally elucidated myxopentacins(unpublished)was successfully expressed in the heterologous host M.xanthus DK1622,leading to the production of novel derivatives.Additionally,SAV17,a cryptic BGC derived from Streptomyces sp.,was successfully activated in the heterologous expression host S.albus Del14,resulting in identification of novel benzoxazole NPs.In terms of the heterologous expression of other six cryptic BGCs involved in this study,corresponding products have not been detected for the time being.In summary,the efficient heterologous expression,as one of the most accredited strategies,has been applied in the cryptic BGCs activation and the yield optimization of desired compounds,which largely depends on the suitablility of applied hosts.This study expanded the panel of preferable hosts for highly efficient heterologous expression of proteobacteria-derived NPs by constructing a series of rational genomereduced S.brevitalea DSM 7029 chassis strains.This study for the first time optimizedβ-proteobacterial strain as an efficient chassis cell and also provided a strategic reference in constructing and optimizing non-model microbial chassis strains for the activation of broader cryptic BGCs,which indicated that removing transposons,genomic islands,prophages,and other non-essential chromosomal regions based on the combinatorial bioinformatic analysis and multi-omics could facilitate phenotype optimization of the chassis strain,thereafter,improving the production yields of heterologous natural products and the availability in biotechnology.Besides,the M.xanthus DK1622 series strains and S.albus Del14 with simplified metabolic background used as heterologous hosts in the fifth chapter have also been harnessed as suitable hosts to activate two cryptic BGCs from diverse bacteria,while more prioritized BGCs remained silent so far.Accordingly,broadening the panel of appropriate bacterial heterologous expression chassis cells is of great importance in untapping the cryptic NP biosynthetic potential from bacteria.