Bioassay-Guided Evolution of Glycosylated Macrolide Antibiotics in Escherichia coli

Macrolide antibiotics such as erythromycin are clinically important polyketide natural products. We have engineered a recombinant strain of Escherichia coli that produces small but measurable quantities of the bioactive macrolide 6-deoxyerythromycin D. Bioassay-guided evolution of this strain led to the identification of an antibiotic-overproducing mutation in the mycarose biosynthesis and transfer pathway that was detectable via a colony-based screening assay. This high-throughput assay was then used to evolve second-generation mutants capable of enhanced precursor-directed biosynthesis of macrolide antibiotics. The availability of a screen for macrolide biosynthesis in E. coli offers a fundamentally new approach in dissecting modular megasynthase mechanisms as well as engineering antibiotics with novel pharmacological properties.


Introduction
Polyketides are a diverse and clinically important class of natural products which exhibit anti-infective, antitumor, immunosuppressive, and cholesterol-lowering properties, among others [1]. The modular architecture of polyketide synthases provides an attractive scaffold for biosynthetic engineering [2]. However, many natural products from this family require post-polyketide synthase modifications, including glycosylation, alkylation, and oxidation/reduction, to be fully active [3]. For example, glycosylation plays a critical role in the activity of macrolide antibacterial agents, such as erythromycin. Reconstitution of glycosylation pathways from soil bacteria into heterologous hosts requires the horizontal transfer of genes that encode nucleoside diphosphate (NDP) sugar biosynthesis, including aminosugars and deoxysugars, as well as appropriate glycosyl transferases capable of ligating these activated sugars to acceptor substrates. Because of these challenges, there are very few examples of reconstitution of glycosylation pathways in heterologous hosts [4][5][6]. In Escherichia coli, for example, glycosylation efficiency is low [7].
Earlier work from our laboratory led to the reconstitution of the deoxyerythronolide B synthase (DEBS) pathway in E. coli, resulting in substantial production of the aglycone 6deoxyerythronolide B (6dEB; ; 200 mg/l) [8]. The antibiotic erythromycin D, however, bears two deoxysugars, desosamine and mycarose, both of which are crucial for high antibacterial activity. Here, we describe the coexpression of both the glycosylation pathways and DEBS, resulting in successful production of biologically active 6-deoxyerythromycin D (6d-EryD) in E. coli BAP1 ( Figure 1A). The reconstitution of erythromycin biosynthesis in E. coli provides a unique opportunity for a genetics-led approach to biosynthetic engineering. As a first step toward realizing this potential, an activity-based screening assay was developed. Initial applications of this high-throughput assay are also described.
Once TDP-D-desosamine (E) is synthesized in vivo, it must be transferred to the acceptor substrate by an appropriate glycosyl transferase. Glycosyl transferases play important roles in a variety of biological processes, including cell wall biosynthesis, signal transduction, and macrolide biosynthesis [21]. Two desosaminyl transferase genes, eryCIII and desVII, that have been functionally expressed in E. coli [22][23][24], were evaluated. EryCIII, a desosaminyl transferase from Saccharopolyspora erythraea [22,23], catalyzes the attachment of desosamine to a-mycarosyl-erythronolide B (aMEB). The activity of this highly selective transferase increases dramatically in the presence of EryCII [23]. DesVII from S. venezuelae [25] also requires a chaperone protein, DesVIII, for full activity [24], but unlike EryCIII, it displays broad substrate tolerance for both aglycones [25] as well as TDP-sugar substrates [26]. However, since EryCII/EryCIII are more efficient in accepting the substrate of interest, aMEB, than DesVII/DesVIII (data not shown), eryCII and eryCIII were combined with the above desosamine biosynthetic genes on a single expression plasmid, pHL74 (Cm R ) or, alternatively, pHL50 (Kan R ) ( Figure  1B and Table 1).

Reconstitution of TDP-Mycarose Biosynthesis and Transfer
Mycarose is a common 2,6-deoxysugar found in polyketide compounds and contributes significantly to the high antibacterial activity of erythromycin. For example, desosaminyl clarithronolide, which lacks a mycarose substituent, has less than 2% of the activity of erythromycin D against Bacillus subtilis (unpublished data). Therefore, to synthesize fully active erythromycin analogs, we reconstituted an optimal set of TDP-L-mycarose biosynthetic genes from the homologous erythromycin and tylosin biosynthetic gene clusters ( Figure 1C).

Author Summary
The antibacterial activity of erythromycin, an important polyketide antibiotic precursor, requires the transfer of two unusual sugars called mycarose and desosamine (both glycosyl groups), onto the nonsugar part of the glycoside molecule (macrocyclic aglycone). We reconstituted the biosynthetic pathways of both sugars in Escherichia coli to yield the 6-deoxyerythromycin D antibiotic. By engineering a recombinant strain of E. coli that produces the bioactive macrolide 6-deoxyerythromycin D from propionate, we have developed a fundamentally new tool for enhancing the efficiency of biosynthetic engineering of this class of antibiotics. Initially, this recombinant strain produced barely enough antibiotic activity to establish an activity-based screening assay. We therefore used the assay to screen for antibiotic overproducers. After three rounds of screening, we identified E. coli cells that overproduced the 6-deoxyerythromycin D antibiotic with significant modifications in the mycarose biosynthetic pathway. We used the same activitybased screening system to evolve E. coli mutants capable of more efficient precursor-directed biosynthesis. As the first example of bioassay-guided evolution of an antibiotic pathway in E. coli, these results open the door for harnessing the power of genetics for mechanistic investigations into polyketide synthases and also for biosynthetic engineering.

Efficiency of Deoxysugar Biosynthesis and Transfer in E. coli
To assess the metabolic capacity of the desosamine biosynthesis and transfer pathway in E. coli, BL21(DE3)/ pHL50 was grown in the absence of antibiotics. The IPTGinduced culture was fed with aMEB (isolated from a mutant strain of S. erythraea), and the time course of erythromycin D accumulation was monitored by a B. subtilis inhibition assay using an authentic sample of erythromycin D as a reference ( Figure S1A). Upon induction with 10 lM IPTG and the addition of 100 mg/l aMEB at 20 8C, approximately 25% conversion to erythromycin D was observed ( Figure 2). Consistent with earlier observations [22], the biosynthetic efficiency doubled when GroEL/GroES were coexpressed with the synthetic desosamine operon.
The cumulative efficiency of the mycarose and desosamine pathways was evaluated in shake-flask studies with E. coli BL21(DE3)/pHL71/pHL50, using 100 mg/l 6dEB substrate. After 72 h, the culture supernatant showed activity comparable to 3 mg/l erythromycin D ( Figure 2 and Figure S1B). Because 6-deoxyerythromycins are less active than the corresponding erythromycins [30,31], the biosynthetic efficiency of the two-sugar pathway was judged to be .10 mg/l.

Macrolide Resistance and Export
Macrolide antibiotics inhibit bacterial cell growth by binding to the exit tunnel of the 50S ribosomal subunit [32]. A common resistance mechanism involves ribosome methylation, which prevents macrolide binding to the ribosome by introducing steric hindrance in the antibiotic binding pocket. Although the heterologous expression of the methylase gene ermE [33] in E. coli BL21(DE3) renders the host more resistant to erythromycin A in liquid culture (unpublished data), on semisolid plate media the host is not inhibited by endogenously produced erythromycin in the absence of ermE. Correspondingly, BL21(DE3)/pHL50 is naturally resistant to aMEB up to 400 mg/l, and ermE was not deemed necessary in our system.
It is known that endogenous multidrug pumps such as MacAB [34] and AcrAB [35,36] in E. coli are efficient at exporting macrolide antibiotics bearing the mycarose sugar [34,37]. To test whether aMEB is secreted by BL21/pHL80/ pHL50, we cospotted E. coli strains BL21(DE3)/pHL80/pHL50 (pHL80 is a Strep R analog of the mycarose plasmid pHL71) and BL21(DE3)/pHL50 in close proximity on a Petri plate containing 6dEB. It was anticipated that aMEB secreted by the former strain would be converted into 6d-EryD by the latter strain. A dramatic increase in antibiotic activity was observed around BL21(DE3)/pHL50 ( Figure S2). This is consistent with the observations that monoglycosylation is efficient, whereas diglycosylation is inefficient ( Figure 2). It also suggests that, while endogenous multidrug resistance mechanisms in E. coli enable this host to synthesize erythromycin without self-destruction, they also contribute to biosynthetic inefficiency by prematurely exporting the mycarosylated precursor.

Production of 6d-EryD in E. coli
To synthesize a bioactive erythromycin in E. coli, the host strain BAP1 (engineered for the phosphopantetheinyl modification of DEBS as well as propionyl-CoA biosynthesis [38]) was cotransformed with plasmids pBP144 (Carb R , encoding the DEBS1 and the pccAB genes), pBP130 (Kan R , encoding for DEBS2 and DEBS3), pHL74 (a Cm R analog of the desosamine plasmid pHL50), and pHL80 (a Strep R analog of the mycarose plasmid pHL71). The resulting strain produced low levels of 6d-EryD, detectable by mass spectrometry, but estimated to be very low. This poor productivity is consistent with an earlier report of , 1 mg/l glycosylated macrolide production in E. coli [7]. Indeed, it was not possible to observe bioactivity from single colonies of this transformant on a Petri plate. However, as described below, an activity-based screening assay was developed to enhance the productivity of this progenitor strain of E. coli.

Bioactivity-Based Screening Assay for 6d-EryD Overproducers
Although single colonies of E. coli BAP1/pBP144/pBP130/ pHL80/pHL74 were unable to synthesize adequate antibiotic to generate a halo in a B. subtilis overlay assay, small patches (;0.5 cm 2 ) of individual colonies revealed detectable growth inhibition in an equivalent assay. We therefore tested several independent transformants via this method and isolated single colonies of the best two producers by restreaking, followed by repeated bioassays on small patches derived from individual colonies. After three rounds of screening, individual colonies showed a readily observable signal in a B. subtilis overlay assay ( Figure 3A). Two overproducers, mutant A and mutant B, produced 6d-EryD, comparable to 2 mg/l erythromycin D in shake flask experiments ( Figure 3B). Consider-  ing the effect of 6-hydroxyl group on the activity of erythromycin, we estimated a titer of 5-10 mg/l 6d-EryD production in shake-flask experiments.

Analysis of 6d-EryD Overproducers
To obtain preliminary insights into the mechanistic basis for 6d-EryD overproduction in the above mutants, we first compared the stability of each plasmid of a representative overproducer and the wild-type strain. Although the overproducer showed marginally improved stability (;23), this difference could not explain the considerable increase in antibiotic productivity. Therefore, we purified each plasmid from a mutant cell line and retransformed E. coli BAP1 cells along with the other three wild-type plasmids. Only the mutant plasmid pHL80* was sufficient to reconstitute the overproducer phenotype, as judged by single-colony assays ( Figure S3). Restriction analysis of pHL80* showed no differences relative to pHL80 that would be suggestive of a subtle mutation. However, comparative protein expression analysis of BL21(DE3)/pHL80* versus BL21(DE3)/pHL80 showed major differences after 5 h of induction with 0.5 mM IPTG at 30 8C (Figure 4). The mutant pHL80* revealed more balanced expression of the mycarose biosynthetic and transfer enzymes compared to pHL80, which selectively overexpressed the ketoreductases EryBII and EryBIV. Further analysis revealed that the copy number of pHL80* is significantly lower than pHL80 (15%-20%; Figure 4B). Other investigators have also observed that lower-copy-number plasmids can enhance the production of natural products in bacteria [39]. Presumably, the lower copy number of pHL80* can reduce the overall burden of heterologous protein expression in the host cell, although further investigations are warranted in this regard.

Bioassay-Guided Evolution of Precursor-Directed Biosynthesis of Antibiotics
Because the plasmid pHL80* significantly enhanced macrolide antibiotic biosynthesis in E. coli, we hypothesized it would also improve the productivity of other related antibiotic-producing systems. We therefore introduced pHL80* and pHL74 into BAP1/pBP130/pBP175, which contains a deletion of the loading didomain and module 1 of DEBS. The resulting strain is inherently incapable of polyketide production, but does so in the presence of a variety of exogenously introduced thioester substrates [40]. This method has been used to prepare a wide range of new macrolide antibiotics with promising biological activities [41,42]. As predicted, colonies of BAP1/pBP130/pBP175/pHL80*/pHL74 supplemented with 100 lM (2S,3R)-2-methyl-3-hydroxy-pentanoyl-SNAc (NDK) produced 6d-EryD, whereas no signal was observed with the control strain harboring wild-type plasmids (BAP1/pBP130/pBP175/pHL80/pHL74) in the B. subtilis inhibition assay. This result demonstrated the utility of pHL80* as a general toolkit for improving glycosylated macrolide biosynthesis in E. coli.
Single colonies of E. coli BAP1/pBP130/pBP175/pHL80*/ pHL74 were grown for 48-60 h in the presence of 25 lM, 100 lM, 300 lM, or 1 mM NDK. At substrate concentrations below 100 lM, halo sizes increased with increasing NDK concentrations, whereas no further increase in signal was observed at [NDK] . 100 lM (unpublished data). Therefore, we screened colonies at a subsaturating NDK concentration of 25 lM, and isolated mutants (Mutant C, Mutant D) that exhibited a significantly larger halo size ( Figure 5A). Shakeflask comparisons of Mutant C, Mutant D, and wild-type BAP1/pBP130/pBP175/pHL80*/pHL74 revealed that Mutant C and Mutant D are more effective than wild-type in converting NDK to 6d-EryD ( Figure 5B and 5C). Although the mechanistic basis for this phenotype is under investigation, this example illustrates the potential for directed evolution in macrolide biosynthetic engineering.
In conclusion, we have reconstituted the 6d-EryD biosynthetic pathway in E. coli, and used it to develop an activitybased screen for macrolide biosynthesis. Our results represent the first example of the bioassay-guided evolution of an antibiotic pathway in a heterologous host, thereby opening the door for harnessing the power of genetics for understanding and manipulating polyketide biosynthesis.

9-A A A A A A C A T A T G A A T G G G A T C A G T G A T T -
Genes encoding tylAI, tylCIII, and tylCVII were amplified from genomic DNA of S. fradiae using following primers with restriction sites (underlined) by PCR: tylAI, forward, 59-AAAAAACATATGAAC-GACCGTCCCCGCCGC-39, reverse, 59-AAAA AACTCGAGT-C A C T G T G C C C G G C T G T C -3 9; t y l C I I I , f o r w a r d , 5 9-AAAAAACATATGCCCGCTGTTCCCCGAGAG-39, reverse, 59-AAAAAAACTAGTTACGACGTCGAGCCGGGG-39; and tylCVII, forward, 59-AAAAAACATATGATCATCACCGAGACCAGGGTC-39, reverse, 59-AAAAAACTCGAGCATGGCCGGATAGGCC À39.
Conversion of aMEB into erythromycin D. BL21/pHL50 (200 ml) was grown at 37 8C to an OD 600 ¼ 0.6. The culture was chilled on ice for 10 min and spun down at 4,000g for 15 min. After washing with LB, the cell pellet was resuspended in 10 ml of fresh LB without any antibiotic. To this culture, 100 mg/l aMEB and IPTG was added, and the cell culture was incubated at 18 8C or 20 8C for 72 h.
Conversion of 6dEB into 6d-EryD. BL21(DE3) cells were cotransformed with pHL50 and pHL71 and grown in the presence of kanamycin (50 lg/ml) and chloramphenicol (34 lg/ml) at 37 8C. A 100ml LB culture was shaken at 200 rpm at 37 8C until OD 600 ¼ 0.6. The culture was chilled on ice for 10 min and cells were harvested by spinning 10 min at 4,000g. Cells were washed once with 100 ml of icecold LB, resuspended in 5 ml of LB without antibiotics, and induced with 10 lM IPTG in the presence of 100 lg/ml 6dEB at 20 8C.
B. subtilis inhibition assays. To detect or quantify a glycosylated macrolide in the spent culture medium of an E. coli strain, a test sample of the 0.2 lm filtered culture medium was added to a freshly inoculated culture of B. subtilis. No exogenous antibiotics were used during the growth of the E. coli culture. The growth rate of B. subtilis, calculated by measuring OD 600 as a function of time, was used to estimate the amount of macrolide antibiotic. To detect macrolides produced by single E. coli colonies, LB plates were prepared with 0.5 mg/ml of sodium propionate or other substrates, such as 6dEB or aMEB, added at appropriate concentrations. A sterilized cellophane disk soaked with water was placed on top of the LB plate. The test strain of E. coli was plated on the cellophane disk at an appropriate cell density. After 2-3 d at 30 8C, the cellophane disk was removed from the plate, and 2.5 ml of a soft agar overlay containing 0.1% B. subtilis culture was added to each plate. After overnight growth at 30 8C, halos arising due to growth inhibition of B. subtilis were visualized around individual colonies.
Biosynthesis of 6d-EryD. E. coli BAP1 cells were cotransformed with pBP130 (Carb R ), pBP144 (Kan R ), pHL80 (Sm R ), and pHL74 (Cm R ). Cells were grown in 1 l LB with antibiotics until OD 600 ¼ 0.6, and concentrated in 50 ml of LB in shake-flask as above without any antibiotics, and induced with 2.5 g/l sodium propionate and 0.1 mM IPTG at 20 8C or 30 8C.
Analyses of Mutant A. To isolate individual plasmids from the overproducer Mutant A, a 10-ml LB culture was grown overnight with kanamycin (50 mg/l), carbenicillin (100 mg/l), chloramphenicol (34 mg/l), and streptomycin (50 mg/l). The cells were centrifuged, and plasmids were purified by ethanol precipitation. The purified plasmid mixture (1 ll) was transformed to XL1-Blue and spread on LB plates with each of the four antibiotics present individually. The antibiotic resistance profiles of selected colonies from each plate were screened, and individual plasmids were purified from colonies bearing only one plasmid.
To test the stability of each plasmid, mutant A was grown in LB with all antibiotics (kanamycin, carbenicillin, chloramphenicol, and streptomycin), and diluted 10 6 -fold. 100-ll aliquots were spread on plates containing each antibiotic individually. By comparing the number of colonies on each plate to a control plate with no antibiotic, the stability of each plasmid was assessed.
To evaluate protein expression levels in BL21(DE3)/pHL80* and BL21(DE3)/pHL80, 100-ml LB cultures with 50 lg/ml streptomycin were incubated at 37 8C until OD 600 ¼ 0.6. The culture was induced with 0.5 mM IPTG at 30 8C, and allowed to incubate for 5 h. Cells were harvested by centrifugation at 4,000g, and lysed by sonication. Ni-NTA affinity purification was used for further enrichment of proteins expressed by plasmid-borne genes.
To analyze the relative copy number of pHL80 and pHL80* in E. coli, 5-ml LB cultures were grown with 50 lg/ml streptomycin at 37 8C. After 12 h, cells were harvested, and DNA was extracted using QIAprep Spin Miniprep Kit (DNA was eluted after 2 min of incubation with 200 ll of 70 8C H 2 O; QIAGEN, http://www.qiagen. com). The amount of DNA was calculated based on the absorbance at 260 nm, and the cell density was calculated by serial dilution. The relative copy number of a plasmid was measured as the amount of plasmid DNA per cell.