The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

Summary

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

Abstract

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.

Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling >50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.

Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.

Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

Introduction

Erythromycin A is a polyketide antibiotic produced by the Gram-positive soil bacterium Saccharopolyspora erythraea, and current production has been incrementally improved to ~10 g/L through decades of traditional mutagenesis and screening protocols and more recently through process optimization schemes ^1-6. Mutagenesis and screening strategies are common in antibiotic natural product development as a result of difficulties in culturing and/or genetically manipulating native production hosts and because of the readily available antibiotic activity or improved growth phenotypes to aid selection. In the case of erythromycin A, S. erythraea is limited by a slow growth profile and the lack of more direct genetic manipulation techniques (relative to organisms like E. coli), thus, hampering rapid improvements in production and the biosynthesis of new derivatives. Having recognized the production issues and unlocked diversification possibilities with compounds like erythromycin A, the research community began to pursue the idea of heterologous biosynthesis (Figure 1) ⁷. These efforts coincided with available sequence information for the erythromycin A gene cluster ^8-11. It should be emphasized that the number of sequenced complex natural product gene clusters has greatly expanded ^12-16, providing the impetus for continued efforts in heterologous biosynthesis to access encoded medicinal potential. To do so, heterologous reconstitution requires that the new host meet the needs of the specific biosynthetic pathway. E. coli provides technical convenience, a wide-spanning set of molecular biology techniques, and metabolic and process engineering strategies for product development. Yet, when compared to native production hosts, E. coli does not exhibit the same level of complex natural product production. It was therefore unknown whether E. coli could serve as a viable heterologous option for complex natural product biosynthesis. However, it was assumed that E. coli would be an ideal host organism if heterologous biosynthesis could be accomplished.

With this goal in mind, initial efforts began to produce the polyketide aglycone 6-deoxyerythronolide B (6dEB) through E. coli. However, native E. coli metabolism could not provide appreciable levels of the propionyl-CoA and (2S)-methylmalonyl-CoA precursors needed to support 6dEB biosynthesis nor could the new host post-translationally modify the deoxyerythronolide B synthase (DEBS) enzymes. To remedy these issues, a metabolic pathway composed of native and heterologous enzymes was built into E. coli such that exogenously fed propionate was converted intracellularly to propionyl-CoA and then (2S)-methylmalonyl-CoA; during the engineering to complete this pathway, an sfp gene was placed into the chromosome of E. coli BL21(DE3) to produce a new strain termed BAP1. The Sfp enzyme is a phosphopantetheinyl transferase capable of attaching the 4′-phosphopantetheine cofactor to the DEBS enzymes ^17,18. The three DEBS genes (each ~10 kb) were then placed on two separately selectable expression vectors containing inducible T7 promoters. After a key adjustment of post-induction temperature (to 22 °C), the DEBS genes were coordinately expressed within BAP1 in an active state capable of generating 6dEB ¹⁹.

The pursuit of full erythromycin A biosynthesis then began using an analogous gene cluster from Micromonospora megalomicea or a hybrid pathway composed of genes from S. erythraea, S. fradiae, and S. venezuelae which produced the intermediates erythromycin C and 6-deoxyerythromycin D, respectively ^20-22. Recently, our group has extended these efforts by producing erythromycin A (the most clinically-relevant form of erythromycin) through E. coli. In contrast to previous work, our strategy coordinately expressed the 20 original S. erythraea genes needed for polyketide biosynthesis, deoxysugar biosynthesis and attachment, additional tailoring, and self-resistance (Figure 2). In total, 26 (native and heterologous) genes were engineered to allow E. coli to produce erythromycin A at 4 mg/L ^23,24. This result established complete production of a complex polyketide natural product using E. coli and serves as a basis to leverage this new production option or pursue new ones.

Protocol

The text below is specific for the erythromycin A antibiotic, but the steps are designed to be generally applicable to other natural products as candidates for heterologous biosynthesis. 1. Erythromycin A Genetic Cluster Transfer Design PCR primers to amplify all of the genes associated with the erythromycin A cluster within the S. erythraea chromosome. This step is specific to those natural product candidates whose genetic sequences have been determined. Alternatively,…

Representative Results

The desired outcome of this approach is the production of a fully bioactive natural product from the E. coli heterologous host. This is best represented by the LC-MS results used to confirm and quantify production (Figure 6) and the antibacterial bioassay used to confirm final activity (Figure 7). In the overall scheme of heterologous biosynthesis, this result defines success. Once accomplished, research efforts then turn to optimization (both at the cellular and process s…

Discussion

Critical steps in heterologous biosynthesis are encountered at each of the three procedural points in the process: 1) genetic transfer; 2) biosynthetic reconstitution; and 3) product analysis. A problem at any stage will derail the ultimate objective of establishing heterologous biosynthesis. Perhaps the most challenging aspect of the process is establishing reconstituted biosynthesis, since this is absolutely required to allow successful analysis. However, reconstitution is dependent upon careful design and transfer of …

Materials

Name of the reagent	Company	Catalogue number	Comments (optional)
PCR machine	Eppendorf	Mastercycler personal
Dimethyl sulfoxide (DMSO)	Fisher	BP231
Electroporator	BioRad	Micropulser
IPTG	Fisher	BP1620
Sodium propionate	Sigma	P1880
L-arabinose	Sigma	A3256
Refrigerated Shaker	Thermo Scientific	MaxQ 4000
Microfuge	Eppendorf	Centrifuge 5415D
pGro7	Takara	Chaperone Plasmid Set (3340)
pET21, pET28, pCDF-Duet-1	EMD Chemicals	69742-3, 69864, 71340
LC-MS	Applied Biosystems	3200 Q-Trap
Ethyl acetate	Sigma	270989
Methanol	Sigma	322415
Vacuum centrifuge	Eppendorf	Concentrator 5301
Rotary Evaporator	Buchi	R-200