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 Research Article

Engineered Production of Tryprostatins in E. coli through Reconstitution of a Partial ftm Biosynthetic Gene Cluster from Aspergillus sp.

Gopitkumar R Shah,1,# Shane R. Wesener,1,# and Yi-Qiang Cheng1,2*

1Department of Biological Sciences, University of Wisconsin–Milwaukee, USA
2UNT System College of Pharmacy, University of North Texas Health Science Center, USA
#Present Address: G.R. Shah, Medical College of Wisconsin, USA
#S.R. Wesener, Bryant & Stratton College, USA

*Corresponding author:  Dr. Yi-Qiang Cheng, UNT System College of Pharmacy, University of North Texas Health Science Center3500 Camp Bowie Boulevard, Fort Worth, Texas 76107, USA, Tel: (817) 735-0165;
Fax: (817) 735-2603; 
E-mail: yiqiang.cheng@unthsc.edu

Submitted: 10-13-2014 Accepted: 12-15-2014 Published: 12-18-2014

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Article


Abstract

Tryprostatin A and B are indole alkaloid-based fungal products that inhibit mammalian cell cycle at the G2/M phase. They are biosynthetic intermediates of fumitremorgins produced by a complex pathway involving a nonribosomal peptide synthetase (FtmA), a prenyltransferase (FtmB), a cytochrome P450 hydroxylase (FtmC), an O-methyltransferase (FtmD), and several additional enzymes. A partial fumitremorgin biosynthetic gene cluster (ftmABCD) from Aspergillus sp. was reconstituted in Escherichia coli BL21(DE3) cells, with or without the co-expression of an Sfp-type phosphopantetheinyltransferase gene (Cv_sfp) from Chromobacterium violaceum No. 968. Several recombinant E. coli strains produced tryprostatin B up to 106 mg/l or tryprostatin A up to 76 mg/l in the fermentation broth under aerobic condition, providing an effective way to prepare those pharmaceutically important natural products biologically.

Keywords: ftm Biosynthetic Gene Cluster; Heterologous Production; Tryprostatins

Abbreviations
A: Adenylation Domain;
BCRP: Breast Cancer Resistance Protein;
C: Condensation Domain;
IPTG: Isopropyl-β-D-Thiogalactopyranoside;
LC-MS: Liquid Chromatography-Mass Spectrometry;
MAP2: Microtubule Associated Protein 2;
MCS: Multiple Cloning Site;
MDR: Multidrug Resistance;
NRPS: Nonribosomal Peptide Synthetase;
PCP: Peptidyl Carrier Protein Domain;
PPTase: Phosphopantetheinyltransferase;
TPS-A: Tryprostatin A;
TPS-B: Tryprostatin B;
RT-PCR: Reverse Transcription-Polymerase Chain Reaction

Introduction

Tryprostatin A (TPS-A) and tryprostatin B (TPS-B) are anticancer natural products containing an isoprenylated diketopiperazine indole (Figure 1a), first isolated as mammalian cell cycle inhibitors from the fermentation broth of marine fungus Aspergillus fumigatus BM939 [1-3]. They are biosynthetic intermediates of fumitremorgins [3, 4]. Cui et al. showed that TPS-A, TPS-B and related demethoxyfumitremorgin C inhibit cell cycle progression of mouse tsFT210 cells at the G2/M phase with minimum inhibitory  concentrations in the low μM range [1]. Usui et al. demonstrated that TPS-A specifically blocks MAP2 (microtubule associated protein 2)-dependent assembly of microtubules [5]. Furthermore, both TPS-A and fumitremorgin C were reported to be potent inhibitors of breast cancer resistance protein (BCRP), a member of the ABC transporter family, which has been associated with multidrug resistance (MDR) of various cancers [6-8].

 bio tech 10.1

Figure 1. Schematic depiction of heterologous production of tryprostatins in recombinant E. coli strains. (a) The biosynthetic pathway of tryprostatins and fumitremorgins. (b) The ftm gene cluster in the genome of A. fumigatus BM939. (c) Source of a Sfp-type phosphopantetheinyltransferase gene from Chromobacterium violaceum No. 968 [15]. (d) Scheme of a representative recombinant E. coli BL21(DE3) cell (GS07Plus strain) that harbors all three gene expression constructs.

The tryprostatin and fumitremorgin biosynthetic pathway (Figure 1a) is encoded by the fumitremorgin biosynthetic cluster (ftm) which contains nine genes (Figure 1b). The same orthologous gene cluster has been identified in three A. fumigatus isolates (Af293, A1163 and BM939) and in Neosartorya fischeri NRRL 181 [4]. The biosynthesis of tryprostatins and fumitremorgins was proposed to begin with the condensation of a tryptophan (L-Trp) and a proline (L-Pro) to form brevianamide F. This reaction is catalyzed by FtmA, a dimodular nonribosomal peptide synthetase (NRPS) with a domain organization of A-PCP-C-A-PCP-A, where A stands for adenylation domain, PCP for peptidyl carrier protein domain, and C for condensation domain. This reaction was proven by heterologous expression of ftmA in A. nidulans and identification of brevianamide F as the biosynthetic product [9]. Brevianamide F is subsequently converted to TPS-B by a renyltransferase,FtmB, as demonstrated by in vitro assays [10]. TPS-B undergoes hydroxylation at C-6 position of the indole ring catalyzed by a cytochrome P450 hydroxylase, FtmC, and followed by methylation catalyzed by an O-methyltransferase, FtmD, to produce TPS-A [4, 11]. Further biosynthesis leads to several fumitremorgins and verruculogen [4].

Although the ftm gene cluster was first identified in the genome of Af293 [10], it was thought to be not expressed in Af293 because no fumitremorgins could be detected in this strain [9]. However, a recent study showed by RT-PCR that all ftm genes are expressed almost equally well in both Af293 and BM939 strains [12]. Furthermore, a point mutation was found in ftmD in the genome of Af293 to cause an arginine to leucine substitution at position 202 of FtmD, resulting in a dramatic decrease of the catalytic efficiency of FtmD. This mutated form of FtmD appeared not functioning under physiological conditions in Af293 to produce any detectable levels of TPS-A or any downstream metabolites [12]. 

 

TPS-A and TPS-B are produced at merely 0.4 mg/l by the native A. fumigatus BM939 strain in shaker flasks under Inlaboratory conditions [1]. Maiya et al. heterologously co-expressed ftmA and ftmB, under the control of a strong promoter, PalcA, in a naïve host A. nidulans, and obtained TPS-B at an impressive titer of 250 mg/l [13]. However, to date no study has been reported for the overproduction of TPS-A. Here we report that, through reconstitution of a partial ftm gene cluster (four genes, ftmABCD, on two compatible plasmids) from Aspergillus sp., we obtained recombinant E. coli strains that produce TPS-B up to 106 mg/l and TPS-A up to 76 mg/l in shaker flask fermentation, providing an effective way to prepare those pharmaceutically important natural products. 

Materials and Methods

General microbiology and molecular biological manipulations

Bacterial and fungal strains, and plasmids used in this study are listed in Table 1. Bacterial culture conditions and general molecular biological manipulations were performed according to standard protocols [14], or according to manufacturer’s manuals. Chemicals and biochemicals were purchased from Fisher Scientific Inc. (Pittsburgh, OH), and enzymes from New England BioLabs (Ipswich, MA), unless otherwise indicated. YPD medium (1% yeast extract, 2% peptone, 2% dextrose) was used to grow Aspergillus sp. cultures, from which all fungal genomic DNA sample were prepared with a Fungi/Yeast Genomic DNA Isolation Kit (Norgen Bioteck Co., Ontario, Canada) and all fungal total RNA samples were prepared with an RNeasy Mini Kit (Qiagen, Valencia, CA) after mycelia having been frozen in nitrogen and ground into fine powder. 

Construction of gene expression constructs

The Duet vectors (Novagen, Madison, WI) were used for the partial ftm gene cluster reconstitution to allow for multiple vector combinations and expression of one or two genes on each vector when necessary. All cloned genes are arranged under the control of the same inducible T7 promoter. PCR primers used for gene amplification are listed in Table 2. The 1.4-kb intron-less ftmB gene was amplified by PCR using the high fidelity Phusion DNA polymerase and the primer set AscI-ftmB-F/NotI-ftmB-R from the genomic DNA of A. nidulans PSM An(355)-8, which was engineered to overproduce TPS-B by Maiya et al. [13], and was cloned into the MCS1 of pCDFDuet-1 vector at the AscI/NotI sites to create pCDFDuet- ftmB. The 6.6-kb intron-less ftmA gene was amplified similarly by PCR using the primer set KpnI-ftmA-F/PspXI-ftmA- R, and cloned into the MCS2 of pCDFDute-ftmB at the KpnI/XhoI sites to create pCDFDuet-ftmAB. The 1.7-kb cDNA of ftmC gene was amplified by reverse transcription (RT)- PCR using an OneStep RT-PCR Kit (Qiagen) and the primer set EcoRI-ftmC-F/HindIII-ftmC-R from the purified RNA sample of A. fumigatus BM939, and was cloned into the MCS1 of pCOLADuet-1 vector at the EcoRI/HindIII sites to create pCOLADuet-ftmC. The 1.0-kb cDNA of ftmD gene was amplified similarly by RT-PCR using the primer set NdeI-ftmD-F/ KpnI-ftmD-R, and cloned into the MCS2 of pCOLADuet-ftmC at the NdeI/KpnI sites to create pCOLADuet-ftmCD. To convert the apo-form of PCP domains on FtmA to its holo-form, the 0.7-kb Cv_sfp gene encoding an Sfp-type phosphopantetheinyltransferase (PPTase) from Chromobacterium violaceum No. 968 (Figure 1c) was excised from pCDFDuet-sfp [15] by NcoI/ HindIII digestion and subcloned into the same sites on pACYCDuet- 1 vector to create pACYCDuet-Cv_sfp. Every cloned gene was re-sequenced to ensure DNA sequence fidelity.


Table 1. Strains and plasmids used in the study.

 bio tech  table 10.1

FGSC, Fungal Genetic Stock Center, Kansas City, MO, USA; IPOD, International Patent Organism Depositary, Tsukuba, Japan; Smr, streptomycin resistant; Kanr, kanamycin resistant; Chlr, chloramphenicol resistant.

Table 2. Primers used for gene amplification and detection of gene expression by RT-PCR.

bio tech  table 10.2

Bacterial fermentation and quantification of tryprostatin production

To measure tryprostatin production, recombinant E. coli strains were first fermented aerobically in shaker flasks for 4 d at 37°C under constant agitation (200 rpm) in LB medium supplemented with appropriate antibiotics individually or in combination (streptomycin was added at 50 μg/ml to select for pCDFDuet-1 based plasmid, kanamycin at 50 μg/ml to select for pCOLADuet-1 based plasmid, and chloramphenicol at 34 μg/ml to select for pACYCDuet-1 based plasmid). Gene expression was induced by adding isopropyl-β-D-thiogalactopyranoside  (IPTG; from Sigma-Aldrich, St. Louis, MO) to a final concentration of 0.5 mM when bacterial culture reached an optical density of 0.4 at 600 nm (OD600). For production of tryprostatins under anaerobic condition, bacterial culture was grown still in tightly capped glass bottles with occasional shaking in a Coy anaerobic chamber (Grass Lake, MI) for 5 d at room temperature. Sampling, extraction, detection and quantification of TPS-B and TPS-A by liquid chromatography-mass spectrometry (LC-MS) on an Agilent 1100 Series LC-MSD Trap SL equipped with an Eclipse XDB-C18 column (Agilent, 5 μm particle size, 2.1 x 50 mm), was performed according to previously reported methods with minor modifications [13]. The solvent systems consist of buffer A (water with 0.1% formic acid) and buffer B (acetonitrile with 0.1% formic acid). The LC program included a linear gradient from 40% buffer B to 100% buffer B in 10 min, an isocratic elution in 100% buffer B for 2 min, followed by a linear return to 40% buffer B in 1 min. Flow rate was set at 0.5 ml/min and UV spectrum was monitored at 226 nm. The inert nitrogen gas flow rate, nebulizer pressure and drying temperature for the MS system were 10 l/ min, 30 psi and 350°C, respectively. MS was scanned in positive mode in a range of 100 to 1,000 m/z; the target compounds, TPS-A (381.1 + 0.1 m/z for [M + H]+) and TPS-B (351.7 + 0.2 m/z for [M + H]+), were eluted at 3.7-4.6 min or 5.1-5.4 min, respectively, under those conditions. Standard curves were generated by plotting the concentrations (ranging from 1 to 10 mg/l) of authentic compounds (TPS-A purchased from Santa Cruz Biotechnology, Santa Cruz, CA; TPS-B was a gift from James Cook, University of Wisconsin-Milwaukee) as a function of ion signal peak areas detected by LC-MS in triplicates (Figure 2). The standard curves showed a good linear correlation between the varying concentrations of injected analytes and peak areas of the extracted ion signal of tryprostatins.

bio tech 10.2

Figure 2. Standard curves for the quantification of TPS-A (a) and TPS-B (b) accumulation in the fermentation broths by LC-MS.

Analysis of gene expression by semi-quantitative RT-PCR Cultivation of recombinant E. coli strains in LB growth medium supplemented with appropriate antibiotics. Extraction and purification of RNA samples, and RT-PCR detection of the
expression level of each gene were performed according to previously described protocols [16]. Primers used in RT-PCR are listed in Table 2. RNA samples that had not been subjected to reverse transcription were used as negative controls; a positive control was set to amplify the 16S rRNA gene.

Results and Discussion

Generation of a serial of recombinant E. coli strains Aimed at developing a bacterium-based fermentation platform for large-scale production of TPS-A and TPS-B, two empty vectors (pCDFDuet-1 and pCOLADuet-1, as controls) and five expression constructs (pCDFDuet-ftmB, pCDFDuet- ftmAB, pCOLADuet-ftmC, pCOLADuet-ftmCD, and pACYCDuet- Cv_sfp) were transformed individually or in combination into E. coli BL21(DE3) cells to generate a series of 10 recombinant strains listed in Table 1. Those strains were designed to not only test the production of TPS-B or TPS-A under both aerobic and anaerobic fermentation conditions, but also to confirm the assigned function of FtmABCD enzymes (Figure 1a).

Aerobic production of TPS-A and TPS-B

Under aerobic fermentation conditions, the control strains GS01 (harboring pCDFDuet-1 vector) and GS04 (harboring pCOLADuet-1 vector), and the intermediate strains GS02 (harboring pCDFDuet-ftmB), GS05 (harboring pCOLADuet-ftmC), GS06 (harboring pCOLADuet-ftmCD) or GS08 (harboring pACYCDuet- Cv_sfp) did not produce any TPS-B or TPS-A, as expected. The target strains GS03 (harboring pCDFDuet-ftmAB) and GS03Plus (GS03 with pACYCDuet-Cv_sfp) accumulated 98.1-106.0 mg/l of TPS-B in the bacterial broth after 4 d of fermentation, and the target strains GS7 (harboring both pCDFDuet-ftmAB and pCOLADuet-ftmCD) and GS07Plus (GS07 with pACYCDuet-Cv_sfp) accumulated 8.2-9.0 mg/l of TPS-B and 70.1-76.0 mg/l of TPS-A in the bacterial broth after 4 d of fermentation (Table 3). Those observations confirmed that ftmAB genes are necessary and sufficient to make TPS-B, and likewise, ftmABCD genes to make TPS-A. The quantitative results demonstrated that reconstruction of a partial ftm gene cluster in E. coli could produce impressive amounts of target products TPS-B and TPS-A. Production of secondary metabolites in a heterologous host such as E. coli drastically reduces the cost, time and effort in downstream natural product purification process [17]. Furthermore, the observed insignificant difference of compound accumulation levels between strains without and with pYCACDuet-Cv_sfp (GC03 vs. GC03Plus, GC07 vs. GS07Plus) prompted us to postulate that the PCP domains on FtmA could be fully phosphopantetheinylated in E. coli BL21(DE3) by one of its three endogenous PPTases, namely AcpS involved in the biosynthesis of fatty acids, EntD involved in the biosynthesis of enterobactin siderophore, and AcpT involved in the biosynthesis of O-antigens [18]. EntD is the most likely candidate for the PCP phosphopantetheinylation because it is known to be able to modify the carrier proteins of heterologous NRPS proteins [19].


Anaerobic production of TPS-A and TPS-B

We were also interested in assessing the production of TPS-A and TPS-B under anaerobic fermentation conditions. Recombinant strains (GS03, GS03Plus, GS07, and GS07Plus) were fermented in LB medium supplemented with appropriate antibiotics in strictly anaerobic conditions in a Coy anaerobic chamber for 5 d at room temperature with occasional manual agitation. All four strains were found to accumulate 18.1-20.0 mg/l of TPS-B in the fermentation broth; but neither GS07 nor GS07Plus strain produced detectable level of TPS-A (Table 3).

Table 3. Levels of TPS-B and TPS-A accumulation in the fermentation broths of recombinant E. coli strains. Data are the mean values of results from triplicate experiments, with calculated standard deviation of the mean provided.

 bio tech  table 10.3

N/A, not anticipated/not applied; N/D, not detected. 

RT-PCR detection of gene expression 

To examine whether ftmABCD genes were adequately expressed in a representative GS07 strain under either aerobic or anaerobic conditions, aliquots of the culture were collected at 0 h (pre-IPTG induction) and 24 h post-IPTG induction. Total bacterial RNA was extracted, cleaned and subjected to semi-quantitative RT-PCR. It was found that, prior to IPTG induction, none of the four genes was expressed under either aerobic or anaerobic fermentation conditions, whereas all four genes were expressed at 24 h post-IPTG induction under both aerobic and anaerobic conditions (Figure 3). Adequately induced gene expression under aerobic conditions is consistent with the observed accumulation of both TPS-A and TPS-B in the fermentation broth (Table 3). However, under anaerobic conditions the situations are not so straightforward. The induced gene expression is consistent with the observed TPS-B accumulation in the anaerobically fermented broth, whereas it is incongruent with the lack of TPS-A accumulation in the anaerobically fermented broth (Table 3). This incongruity is likely due to the fact that conversion from TPS-B to desmethyltryprostatin A, a TPS-A precursor, requires an oxygen-dependent P450 hydroxylase (FtmC) (Figure 1a). Although ftmC was overexpressed, the resultant P450 enzyme could not catalyze the biochemical reaction in the absence of oxygen.

 bio tech 10.3

Figure 3. Analysis of the ftm gene expression in GS07 strain by RTPCR. (a) Pre-induction (0 h) under aerobic conditions. (b) 24 h post- IPTG induction under aerobic conditions. (c) Pre-induction (0 h) under anaerobic conditions. (d) 24 h post-IPTG induction under anaerobic conditions. 16S rRNA gene amplification was included as a positive control.


Summary

Since the regulation of tryprostatins or fumitremorgins biosynthesis in Aspergillus sp. is totally unknown, yield improvement could be achieved either through classical iterative strain improvement processes, or through increasing the copy number of biosynthetic gene cluster, or through heterologous biosynthesis as demonstrated in this work. In fact, heterologous biosynthesis has emerged as effective route to mass production of natural products and intermediates [20, 21]. Although the production of TPS-B up to 106 mg/l by a recombinant E. coli strain (GS03) demonstrated in this work is less than the previously reported 250 mg/l of TPS-B production in a recombinant A. nidulans strain [13], it provides an alternative source for cost effective, large-scale production of TPS-B, because E. coli strain is more amenable for large-scale fermentation. Second, we engineered the first recombinant bacterial strains (GS07 and GS07Plus) to produce TPS-A up to 76.0 mg/l. Third, this work sets up an excellent platform for combinatorial biosynthesis and structural derivatization of tryprostatin analogues with potentially enhanced anticancer activities. 

Acknowledgements

This work was supported in part by an Idea Award from the US Department of Defense Breast Cancer Research Program (BC073985), and by a grant from the US National Institutes of Health/National Cancer Institute (CA152212).

 

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Cite this articleShah GR et al. Engineered Production of Tryprostatins in E. coli through Reconstitution of a Partial ftm Biosynthetic Gene Cluster from Aspergillus sp. J J Biotech Bioeng. 2014, 2(1): 009.

 

 

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