Heterologous Production and Yield Improvement of Epothilones in

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Heterologous production and yields improvement of epothilones in the Burkholderiales strain DSM 7029 Xiaoying Bian, Biao Tang, Yucong Yu, Qiang Tu, Frank Gross, Hailong Wang, Aiying Li, Jun Fu, Yuemao Shen, Yue-Zhong Li, A. Francis Stewart, Guoping Zhao, Xiaoming Ding, Rolf Müller, and Youming Zhang ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 3, 2017

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ACS Chemical Biology

Heterologous production and yields improvement of epothilones in the Burkholderiales strain DSM 7029 ∥

Xiaoying Bian,†,‡,# Biao Tang,§,# Yucong Yu,§ Qiang Tu,†‡ Frank Gross, Hailong Wang,† Aiying Li,† Jun Fu†, , Yuemao Shen,† Yue-zhong Li,† A. Francis Stewart, ∥

∥

Guoping Zhao,§,⊥ Xiaoming Ding,*,§ Rolf Müller,*,‡ , and Youming Zhang*,† † Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Qingdao, China.

‡ Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research, Helmholtz Centre for Infection Research and Saarland University, 66123 Saarbrücken, Germany.

§ Collaborative Innovation Center for Genetics and Development, State Key Laboratory of Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan University, Shanghai, China ∥Genomics,

Biotechnology Center, Technische Universität Dresden, Dresden, Germany

⊥CAS Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

# These authors contributed equally to this work.

*Correspondence and requests for materials should be addressed to X.D. (email: [email protected]), R.M. (email: [email protected]) or Y.Z. (email: [email protected])

ACS Paragon Plus Environment


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Abstract Cloning and heterologous expression of microbial natural product biosynthetic gene clusters in a suitable host has proven to be a feasible approach to improve yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products, but only limited choices of heterologous hosts have been exploited. We describe here that the Burkholderiales strain DSM 7029 could be a potential heterologous host for functional expression of myxobacterial secondary metabolites. By the newly established electroporation procedure, the 56-kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilone A, B, C and D was detected despite low yields. The following medium optimization, introduction of the exogenous methylmalony-CoA biosynthetic pathway and overexpression of rare tRNA genes resulted in the total yields of epothilones increasing approximately 75-fold to 306.98 µg L-1. These results showed that the strain DSM 7029 has the potential to produce epothilones with reasonable titers and might be a broadly applicable host for heterologous expression of other myxobacterial PKS/NRPS expediting the process of genome mining. Keywords: Burkholderiales strain DSM 7029, heterologous expression, epothilone, medium optimization, methylmalony-CoA


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Bacteria are an important source for novel natural products for drug discovery. The majority of bacterial secondary metabolites isolated so far are polyketides (PKs) and nonribosomally made peptides (NRPs), or hybrids thereof, which are biosynthesized by large molecular multienzyme assembly lines composed of polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs).1-3 Numerous genome-sequencing projects revealed that a large proportion of secondary metabolite biosynthetic gene clusters presented in bacterial genomes are cryptic or ‘silent’.4, 5 To access the unexploited majority of natural products, heterologous expression of the cryptic biosynthetic gene cluster in combination with comparative metabolic profiling has proven to be a feasible approach to mine those secondary metabolites in microorganisms or metagenomic DNA libraries.6-9 However, the application of heterologous expression strategy for genome mining was impeded by difficulties in handling large gene clusters or searching for suitable expression hosts. The newly developed linear plus linear homologous

recombination

(LLHR)

in

Escherichia

coli,

transformation-associated

recombination (TAR) cloning in Saccharomyces cerevisiae, and RNA-guided Cas9 nuclease-assisted targeted cloning (CATCH) can rapidly clone large natural product biosynthetic gene clusters,10-12 and thus seem to circumvent the former genetic problem. However, an amenable host for expression of any secondary metabolite gene cluster is still far from reality and thus improvements towards this goal are urgently required to increase chances of success in the genome mining strategy. Myxobacteria are a prolific source of novel bioactive secondary metabolites.13-15 Especially the genus Sorangium is a rich source of natural products with unique structures and mode of actions. For example, the marketed anticancer pharmaceutical IXEMPRATM (ixabepilone) is derived from epothilone B which is produced by Sorangium cellulosum.16, 17 Most of the myxobacteria, however, are notoriously difficult to manipulate genetically, and grow relatively slowly. To investigate the biosynthetic potential of myxobacteria, heterologous expression of their natural product biosynthetic gene clusters is a workable alternative to the traditional ‘homologous expression’ strategy for genome mining in those strains.6, 9 To date, a dozen myxobacterial secondary metabolites have been heterologously produced in several



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different hosts. Most of the examples with higher yields than the wildtype strain were achieved in related host strains, particularly myxobacteria such as Myxococcus xanthus DK1622 and thermophilic myxobacterial strains.18, 19 However, as myxobacteria are generally difficult to handle, other GC-rich (non-related) organisms such as genus Streptomyces and Pseudomonas have been explored as alternative host strains.9 In general, the product yields obtained to date have been relatively low. However, the PKS/NRPS hybrid compounds myxochromides S were produced in Pseudomonas putida at much higher yields compared to the natural producer Stigmatella aurantiaca DW4/3–1.20 P. putida cannot provide polyketide extender units of the methylmalonyl-CoA type in a detectable level, which precludes the expression of many polyketides. Consequently, this strain has been engineered to allow the strains to produce methylmalonyl-CoA yielding a production host capable to generate low amounts of myxothiazol, a methylmalonyl-CoA dependent natural product.21 Thus, it is obviously necessary to continue to search the heterologous host strains including such that have the capacity for methylmanlonyl-CoA generation. The epothilones are potent anticancer drugs that bind to tubulin and inhibit the disassembly of microtubules in a manner similar to paclitaxel.22 Epothilones were originally identified as secondary metabolites produced by the soil-dwelling myxobacterium Sorangium cellulosum. 23, 24

The major metabolites of this bacterium are epothilones A (1) and B (2), whereas

epothilones C (3) and D (4) are the early biosynthetic precursors of epothilones A and B, respectively. The biosynthetic pathway of epothilone involves a hybrid PKS/NRPS assembly line with incorporation of propionyl-CoA, malonyl-CoA, (2S)-methylmalonyl-CoA, S-adenosylmethionine (SAM) and L-cysteine to form the 16-membered macrolactones epothilones C and D. A P450 enzyme EpoK converts epothilones C and D to obtain epothilones A and B (Figure 1).17, 25-28 Because the original producer is difficult to handle genetically and grows very slowly (with a doubling time of 16 h), several attempts have been made to transfer the epothilone biosynthetic gene cluster into advantageous hosts for production optimization or engineering for new derivatives. The heterologous expression was achieved in Escherichia coli, streptomycetes (Streptomyces coelicolor and S. venezuelae) and


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in the myxobacterium M. xanthus,25, 29-34 but yields did not compare to those in a traditionally optimized natural host.35 In addition, the modification of the underlying myxobacterial PKS/NRPS megasynthetases followed by heterologous expression in M. xanthus led to generation of unnatural epothilone derivate for further evaluation.36-39 [Polyangium] brachysporum DSM 7029 (=K481-B101=ATCC53080) was isolated from a soil sample in Greece more than twenty years ago.40, 41 Phylogenetic analysis of the 16S ribosomal rRNA gene and complete genome sequence of strain DSM 7029 suggested that “Polyangium brachysporum” was misclassified and should be located in the Burkholderiales, but couldn’t be assigned to a genus.42,

43

This strain also mentioned as Burkholderia

K481-B101 or DSM 7029 in other publications.44-46 Here we use Burkholderiales strain DSM 7029 to represent this species. It grows fast with a doubling time of one hour, in contrast to, for example, many species of myxobacteria such as M. xanthus (approximately 5 h) or Sorangium species (approximately 16 h).47 Moreover, it can form single colonies on solid agar plates (Figure 2a), leading to colonies that are visible in two days.47 In addition to the easiness with which it can be cultured, strain DSM 7029 is capable of producing useful NRPS-PKS hybrid molecules, glidobactins,42, 45 from its own genetic and metabolic arsenal. Recently, the complete genome sequence of strain DSM 7029 was published exhibiting an average GC content 67.51% close to myxobacteria. Preliminary genome scanning showed the presence of a putative 4’-phosphopantetheinyl transferase (PPTase) (GenBank: AKJ29876.1) required for the formation of PKs and NRPs in its chromosome.

43, 48

The above mentioned

characteristics of strain DSM 7029 show its potential to be a candidate host for the expression of myxobacterial natural products. Herein, we describe the successful production of the myxobacterial NRPS/PKS secondary metabolite epothilones in strain DSM 7029. Besides, introduction of the exogenous (2S)-methylmalonyl-CoA biosynthetic pathway and overexpression of rare tRNA genes lead to yield improvement of epothilones. RESULTS AND DISCUSSION

Transformation of large-size DNA into Burkholderiales strain DSM 7029 by



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electroporation When strain DSM 7029 was cultured in liquid CYC medium at 30℃ according to the DSMZ cultivation conditions, it showed an unexpected growth curve during which the OD600 reached the highest value (1.2) after 28 hours, and then started to decrease until an OD600 value of roughly 0.4 was reached. Culturing was continued at this level for several days (Figure 2b). We then analyzed the effect of addition of glycerol to CYC medium to form CYCG medium, and found strain DSM 7029 to grow well and exhibiting a classic bacterial growth curve (Figure 2). The highest OD600 value can be reached (around 3) after 1 day cultivation and be kept for at least another 3 days (Figure 2). The addition of glycerol may provide more carbon source for sustaining normal growth of DSM 7029 strain. This medium optimization can increase bacterial cell density and be beneficial for the following genetic manipulation. Strain DSM 7029 was seldomly used in genetic engineering approaches and there are only few tools available. Previously only a single method of transforming strain DSM 7029 has been described that it based on conjugation.42 However, conjugation methods in general are laborious and time consuming. In this investigation, we used one-step electroporation to provide an efficient and effective method for transforming DSM 7029 with heterologous genetic elements such as a large sized PKS/NRPS gene cluster. The use of electroporation instead of conjugation in this method allows rapid transformation of DSM 7029 cells, with the uptake of DNA molecules occurring extremely quickly. Extended culture times are not needed, as with conjugation, nor is it necessary to separate donor and recipient cells following transformation. Generally, the bacterial cells were cultured in CYCG medium for 4-5 hours and washed using deionized water twice. Next, approximately 1 µg of plasmid DNA was added into the cell suspension in an electroporation cuvette (1mm) for electroporation using an Eppendorf electroporator at 1350V. The cells were incubated to recover for 2-3 hours prior to plating on CYCG agar containing suitable antibiotics and incubated at 30 ℃ for 48 hours to allow colonies to grow and become visible (detailed electroporation procedure see method


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section). Heterologous Production of epothilones in DSM 7029 and preliminary yield improvement by medium optimization

The epothilone gene cluster under the control of constitutive Tn5-kan promoter was engineered into plasmid p15A-epo-IR-Tps-bsd-oriT-IR-kan to carry a MycoMar transposase (Tps) and inverted sequences (IR) as described before.32 After transforming strain DSM 7029 using this plasmid by performing the electroporation procedure as described in the materials and methods section, transposase expression occurred and let to integration of the 56-kb engineered epothilone gene cluster randomly into the chromosome of DSM 7029.32 Stable integrated recombinants were selected on CYCG plates with kanamycin. More than 100 colonies were found on kanamycin plates per transformation. Compared to the conventional conjugation methods, this electroporation transformation method is more efficient, simpler and faster and is not dependent on a helper strain. The resulting resistant colonies were checked by colony PCR reaction to verify integration of the biosynthetic gene cluster. All the checked mutants contained the epothilone gene cluster. Several resulting mutants DSM 7029/epo were cultivated followed by compound extraction and analysis. All mutants produced detectable amounts of epothilones A, B, C and D as shown by high performance liquid

chromatography-high

resolution

mass

spectrometry

analysis

including

the

fragmentation of target ions (HPLC-HRMS/MS), but the yields of epothilones were quite low. In the best producer (mutant G32), the yield of epothilone C was highest but merely ~ 2.3 µg L-1, the other epothilone derivates were produced at levels less than 1 µg L-1 and the total yields of epothilones were less than 4 µg L-1 (Figure 3, Table 1). These yields were far lower than those in a phylogenetically closely related host myxobacterium M. xanthus,31, 34 but still higher than the yields after hetereologous expression of the gene cluster in the classical secondary metabolite proficient producer Streptomyces venezuelae (Supplementary Table 2).30 The successful production of epotholines in strain DSM 7029 shows that the Tn5-kan promoter is functional in this strain DSM 7029 which also can produce some (2S)-methylmalonyl-CoA as an important extender unit for the biosynthesis of polyketides



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such as epothilones. The relative higher production of epothilones A and C in strain DSM 7029 system is probably due to the higher intracellular concentration of malonyl-CoA pool compared to (2S)-methylmalonyl-CoA as the production ratio of epothilones A to B (or epothilones C to D) has been speculated to be influenced by the intracellular concentrations of malonyl-CoA and (2S)-methylmalonyl-CoA pools.

31,

49,

50

Thus, the intracellular

concentration of malonyl-CoA in wild-type strain DSM 7029 would be higher than those of (2S)-methylmalonyl-CoA. To optimize yields of epothilones, primary consideration was addition of biosynthetic precursors or substrates into the medium. Thus, 50 mg L-1 sodium acetate, 100 mg L-1 sodium propionate, 100 mg L-1 methylmalonic acid, 2.5 mg L-1 cysteine, 5 mg L-1 serine and 0.1 % (v/v) trace element solution was added to the fermentation medium. CaCl2·2H2O was replaced by MgCl2·6H2O in the improved fermentation medium. This simple medium optimization enabled the total yields of epothilones in G32 to increase remarkably (approximately 15-fold) relative to the starting medium and reached 63.00 µg L-1 (Table 1), the yield of dominant epothiline C was 35.52 µg L-1 . Comparison of the yields of epothilones using Tn5-kan and Ptet promoters

To investigate if the tetracycline promoter Ptet, a versatile tetracycline-based regulatory system which has been widely used in a handful of host for functional expression of multiple natural products,51-56 also can drive the large epothilone gene cluster in strain DSM 7029, we replaced the Tn5-kan cassette in the plasmid p15A-epo-IR-Tps-bsd-oriT-IR-kan with a genta-tetR-Ptet cassette (genta, gentamycin resistance gene; tetR, encoding the tetracycline repressor

protein

TetR;

Ptet,

tetracycline

promoter

tetO)

to

form

p15A-epo-IR-Tps-bsd-oriT-IR-genta-Ptet in which the epothilone gene cluster was under the control of the tetracycline inducible promoter. The prospective plasmid was transformed into strain DSM 7029 to build integration mutants DSM 7029/Ptet-epo. The total yields of

epothilones were 55.58 µg L-1 in the best mutant G51-1 and comparable to that in G32 (Table 1), which indicated that Ptet promoter in strain DSM 7029 is functional and the activity of Ptet was similar to the Tn5-kan promoter because of the comparable yields of epothilones. The result provides more choices of promoters for heterologous expression of large natural


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product biosynthetic pathways in this strain and suggests that the Ptet promoter is indeed a versatile promoter for heterologous expression. Production improvement of epothilones by introduction of methylmalony-CoA biosynthetic pathway and overexpression of rare tRNA genes in strain DSM 7029

Since (2S)-methymalonyl-CoA is an important extender unit for epothilone biosynthesis, we were interested in the question whether it was the main factor limiting the production of epothilones. We sequenced the genome of DSM 702943 and found that this strain lacks enzyme propionyl-CoA carboxylase (PCC) which generally presents in many natural product proficient for catalyzing the carboxylation of propinoyl-CoA to efficiently form (2S)-methylmalonyl-CoA . Therefore, we cloned the two versions of accA1-pccB and pccA-pccB genes encoding PCC from the Streptomyces coelicolor A3(2) genome57,

58

,

respectively. Comparing the tRNA usage of strain DSM 7029 with M. xanthus DK1622 and Sorangium cellulosum So0157-2 by using the tRNAScan program,59-61 four rare tRNA genes (Arg anti-GCG, Arg anti-TCG, Gln anti-CTG and Glu anti-CTC) were identified for DSM 7029 and next added to optimize the translation of epothilone gene cluster. The (2S)-methylmalonyl-CoA biosynthetic genes and/or rare tRNA genes were transposed into the genome containing epothilone gene cluster, respectively. The yields of epothilones increased significantly after integration of single- or combined- tRNA gene, accA1-pccB or pccA-pccB genes into mutant G32 (Table 1). Introduction of rare tRNA genes (G32-t) made the total yields of epthilones raise to 229.37 µg L-1 (3.6-fold), which indicated supplementary of rare tRNAs usage in the heterologous host is certainly important for robust heterologous expression and production improvement. The addition of accA1-pccB (G32-ap) and pccA-pccB (G32-pp) also led to the total yields elevate to 192.24 µg L-1 (3.5-fold) and 281.53 µg L-1 (4.4-fold), respectively. Combined import of rare tRNA genes and accA1-pccB genes (G32-apt) continuous improved the total yields, to 306.98 µg L-1 (4.87-fold), and the yields of epothilones A, B, C and D were 14.37 µg L-1, 4.74 µg L-1, 195.02 µg L-1 and 92.85 µg L-1 (Table 1), respectively. However, the yields in G32 with pccA-pccB-tRNAs (G32-ppt, 240.36 µg L-1) was lower than that in G32-pp, the possible reason would be that different integration



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positions impact the expression efficiencies of epothilone biosynthetic genes.33 The accA1-pccB-tRNAs cassette also greatly enhanced the epothilone production (3.9-fold) in G51-1 from 55.58 µg L-1 to 218.26 µg L-1 (G51-1-apt, Table1). With these manipulations, the yields of epothilones in this host boosted approximately 75-fold, the yields were comparable to those in the phylogenetically closely related myxobacterium M. xanthus in the shake flask fermentation condition,32, 34 but were higher than those in other heterologous hosts such as Streptomyces, Escherichia coli and Pseudomonas (Supplementary Table 2). In addition, the growth rate and incubation characteristics (simple nutritional requirement and easy culture) of DSM 7029 are superior to M. xanthus. Our efforts regarding discovery and engineering of the amenable host Burkholderiales strain DSM 7029 enabled the production of epothilones in this host and to improve the titer to more than 300 µg L-1. Thus, this strain might become a robust heterologous host for epothilone production and even have the potential for industrial application in the future. CONCLUSION In this report, we firstly established a simple transformation system by convenient and efficient electroporation which can introduce large-size plasmid into strain DSM 7029. The biosynthetic gene cluster could be integrated into the bacterial chromosome by random transposition. Two kinds of promoters (Tn5-kan and Ptet) are functional in this strain. With these considerations, epothilones were successfully produced in strain DSM 7029, and the production was improved by 75-fold by medium optimization and host melioration. In summary, the Burkholderiales strain DSM 7029 shows potential to be used as a decent and genetically easily tractable host for heterologous expression of epothilones. Subsequent work to further improve the yields could focus on host improvement by genome reduction and tuning expression of epothilone biosynthetic genes. Besides, this strain might also be a feasible host for genome mining by heterologous expression of PKSs, NRPSs, and hybrid PKS/NRPS derived from microorganisms or metagenomic DNA libraries. MATERIALS AND METHODS

Bacterial strains and culture condition


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Burkholderia K481-B101 (DSM 7029) was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig) and grew on CYC medium (3.00 g L-1 bactotryptone, 1.36 g L-1 CaCl2·2H2O, 1.00 g L-1 yeast extract) at 30 °C. Ten mL of 50% glycerol (v/v) was added into 1 L CYC medium to form CYCG medium for robust electroporation and cultivation. For fermentation medium, besides CaCl2·2H2O was replaced by MgCl2·6H2O, 50 mg L-1 sodium acetate, 100 mg L-1 sodium propionate, 100 mg L-1 methylmalonic acid, 2.5 mg L-1 cysteine, 5 mg L-1 serine and 0.1 % (v/v) trace element solution were also added into CYCG medium. The plate was prepared by supplementation of 15g L-1 agar. Appropriate antibiotics were added when needed (kanamycin, 50 µg mL-1; gentamicin, 5 µg mL-1, apramycin 50 µg mL-1). Transformation process by electroporation The

detailed

protocol

of

electroporation

(modified

according

to

the

patent

PCT/IB2010/055923)47 was as follows: 1.

Pick single colonies of strain DSM 7029 from a CYCG plate and inoculate in 1.0 mL CYCG medium. Incubate at 30 °C with shaking (900 rpm) for two days.

2.

Make a hole in the lid of a 2.0 mL Eppendorf tube and inoculate about 100 µL of overnight culture into 1.8 mL CYCG. (Starting OD600 = 0.24)

3.

Incubate the tube in a heating block at 30 °C with shaking (900 rpm) for about 4-5 hours.

4.

Spin down the cells at 9,000 rpm speed for 1 min in an Eppendorf centrifuge at room temperature.

5.

Discard the supernatant.

6.

Resuspend the cells in 1 mL of ddH2O.

7.

Spin down the cells at 9,400 rpm speed for 1 min and discard the supernatant.

8.

Resuspend the cells in 1 mL of ddH2O.

9.

Spin down the cells at 9,400 rpm speed for 1 min.

10. Discard the supernatant using a 1 mL pipette leaving around 20−30 µL of solution. 11. Add DNA (about 1 µg), mix by pipetting up and down and place mixture into an electroporation cuvette (1 mm).



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12. Using an Eppendorf electroporator to electroporate the cells at 1350V. 13. Add 1.0 mL of CYCG medium into the cuvette to resuspend the cells and transfer back into the eppendorf tube. 14. Incubate at 30°C for 2-3 hours with shaking for recovery. 15. Plate part of cells on CYCG agar plates containing suitable antibiotic like kanamycin (50 µg mL-1). 16. Incubate the plates at 30°C for 2−4 days. Construction of p15A-epo-IR-Tps-bsd-oriT-IR-genta-Ptet To construct the expression plasmid with epo gene cluster under control of Ptet promoter, a genta-tetR-Ptet cassette flanked with homology arms was used to replace the Tn5-kan resistance marker in p15A-epo-IR-Tps-bsd-oriT-IR-kan by Red/ET recombineering in E. coli GB2005-red.10 The primers used for amplification of genta-tetR-Ptet were gentPtEp5 and gentPtEp3 (Supplementary Table 1). Sequences used as homology arms for recombineering are underlined. The template for this cassette was plasmid pR6K-Tps-Genta.52 The resulting plasmid was checked by gentamicin selection and restriction digestion analysis and then transformed into the strain DSM 7029. Construction of plasmids for expression of the methylmalony-CoA pathway and rare tRNA genes

The genes accA1, pccA and pccB were cloned from the genome of S. coelicolor A3(2) by PCR. The oligonucleotides used for amplification of accA1 were accA1-F and accA1-R (Supplementary Table 1). The primers used for amplification of pccA were pccA-F and pccA-R (Supplementary Table 1). The oligonucleotides used for amplification of pccB were pccB-F and pccB-R (Supplementary Table 1). The length of gene accA1, pccA and pccB were 1773bp, 1845bp and 1593bp. The tRNAs region sequence (Supplementary Table SI) was synthesized by General Biosystems (Anhui, China). The three genes and the tRNAs were under the Tn5-kan

promoter.

All

the

genes

were

ligated

to

transposition

vector

p15A-Apra-IR-Tps-bsd-oriT-IR by using In-Fusion Cloning (ClonExpress MultiS One Step Cloning Kit, Vazyme) to form corresponding transposition plasmids containing tRNAs gene,


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accA1-pccB, pccA-pccB, accA1-pccB-tRNAs, and pccA-pccB-tRNAs, respectively. All of them were transformed into mutant G32 or G51-1, respectively, for the succeeding fermentation and LC-MS analysis. Fermentation, extraction and epothilones analysis of extracts The single colonies were picked into 3 mL medium supplemented with suitable antibiotics and shaking at 30°C, 200 rpm for 2 days. Then 2 mL culture was transferred to a 250 mL conical flask containing 50 mL medium and shaking for 2 days. At last, 25 mL culture was transferred to 500 mL fermentation medium containing 1% XAD-16 resins in 2L-flask for shaking at 30°C, 200 rpm for 4 days. After 4 days cultured the resins were collected by Standard Sieves (100 meshes) and analyzed with 25 mL methanol twice at 30°C, 200 rpm for 24 hours. 100 µL methanol extract was filtrate by 0.22 µm filter membrane and was diluted using 900 µL acetonitrile. The supernatant was analyzed by HPLC-HRMS after centrifuged at 12,000 rpm for 5 minutes. For initial qualitative analysis, the HRMS was performed on an Accela UPLC-system (Thermo-Fisher) coupled to a linear trap-FT-Orbitrap combination (LTQ-Orbitrap), operating in positive ionization mode. Mass spectra were acquired in centroid mode ranging from 200 – 2000 m/z at a resolution of R = 30000. The crude extracts were separated on a gradient starting at 5% acetonitrile and running to 95% at 9min on a Waters BEH RP-C18 column (50×2 mm;1.7 µm particle diameter, flow rate 0.6 mL min-1). The quantitative analysis of epothilones was performed by UHPLC-MS/MS, Shimadzu 30A series, API 4000, AB Sciex Instruments. The parameters of the column were 2.1×50 mm, 2.5 µm particle diameter and the flow rate was 0.8 mL/min. The injection volume was 5.00 µL each time. 0.1 ng mL-1, 0.2 ng mL-1, 0.5 ng mL-1, 1 ng mL-1, 10 ng mL-1, 50 ng mL-1, 100 ng mL-1 and 500 ng mL-1 standard samples were also tested to make the standard curve. The characteristic peaks (m/z) of epothilone A were 494.500 and 306.200; epothilone B were 508.400 and 320.300; epothilone C were 478.400 and 290.200; epothilone D were 492.400 and 304.200.



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Acknowledgements This work was supported by funding to Y.Z. from the Recruitment Program of Global Experts (1000 plan) and Shandong Innovation and Transformation of Achievements Grant (2014ZZCX02601), funding to X.D. from the National key research development program of China (2016YFA0500600) and the National Basic Research Program of China (973 Program No. 2012CB721102), funding to J.F. from the International S&T Cooperation Program of China (ISTCP 2015DFE32850), funding to X.B. from the Qilu Youth Scholar Startup Funding of SDU, National Natural Science Foundation of China (31500033 and 31670098). Work in R.M’s laboratory is supported by BMBF and DFG. Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The tables of primers and synthesized DNA sequences used in this study as well as the yields of epothilones in different heterologous hosts are included. Competing interests The authors declare that they have no competing interest.

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Figures

Figure 1. Simplified biosynthetic pathway (EpoABCDEK) for epothilones A-D (1-4) formation from the primary metabolites acetyl-CoA, malonyl-CoA, (2S)-methylmalonyl-CoA, S-adenosylmethionine (SAM) and L-cysteine. EpoK converts epothilones C and D to epothilones A and B, respectively.

Figure 2. Single colonies incubated for two days (a) of the Burkholderiales strain DSM 7029 on the CYCG plate, and growth curve (b) of strain DSM 7029 in liquid CYC (♦) and CYCG media (■).



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Figure 3. HPLC−HRMS analyses of heterologous epothilone production in Burkholderiales strain DSM 7029 in CYC medium. Shown are base peak chromatograms at m/z = 494.257 ([M+H]+ of epothilone A (1)), 508.273 ([M+H]+ of epothilone B (2)), 478.262 ([M+H]+ of epothilone C (3)), and 492.278 ([M+H]+ of epothilone D (4)). Methanolic extracts from Burkholderiales strain DSM 7029 wild type (a) and Burkholderiales strain DSM 7029 mutants G32 harboring epothilone gene cluster under the control of Tn5-kan promoter (b). The yield of epothilone C (3) was only 2.3 µg L-1.


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Table 1. The yields of epothilones in Burkholderiales strain DSM 7029 mutants Yields of epothilones (µg L-1)a

DSM 7029 mutants

A (1)

B (2)

C (3)

D (4)

Total

Tn5-kan-epo

Heterologous Production and Yield Improvement of Epothilones in

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