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

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.     

Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

Metabolic and pathway engineering to influence native and altered erythromycin production through E. coli

The heterologous production of the complex antibiotic erythromycin through Escherichia coli provides a unique challenge in metabolic engineering. In addition to introducing the 19 foreign genes needed for heterologous biosynthesis, E. coli metabolism must be engineered to provide the propionyl-CoA and (2S)-methylmalonyl-CoA substrates required to allow erythromycin formation. In this work, three different pathways to propionyl-CoA were compared in the context of supporting E. coli erythromycin biosynthesis. The comparison revealed that alternative citramalate and threonine metabolic pathways (both starting from exogenous glycerol) were capable of supporting final compound formation equal to a proven pathway reliant upon exogenous propionate. Furthermore, two pathways to (2S)-methylmalonyl-CoA were compared in the production of a novel benzyl-erythromycin analog. A pathway dependent upon exogenous methylmalonate improved selectivity and facilitated antibiotic assessment of this new analog.     

Improved heterologous erythromycin A production through expression plasmid re‐design

The production of complex compounds from technically convenient microorganisms is an emerging route to the chemical diversity found in the surrounding environment. In this study, the antibiotic compound erythromycin A is produced from Escherichia coli as an alternative to native production through the soil bacterium Saccharopolyspora erythraea. By doing so, there is an opportunity to apply and refine engineering strategies for the manipulation of the erythromycin biosynthetic pathway and for the overproduction of this and other complex natural compounds. Previously, E. coli‐derived production was enabled by the introduction of the entire erythromycin pathway (20 genes total) using separately selectable expression plasmids which demonstrated negative effects on final biosynthesis through metabolic burden and plasmid instability. In this study, improvements to final production were made by altering the design of the expression plasmids needed for biosynthetic pathway introduction. Specifically, the total number of genes and plasmids was pruned to reduce both metabolic burden and plasmid instability. Further, a comparison was conducted between species‐specific (E. coli vs. S. coelicolor) protein chaperonins. Results indicate improvements in growth and plasmid retention metrics. The newly designed expression platform also increased erythromycin A production levels 5‐fold. In conclusion, the steps outlined in this report were designed to upgrade the E. coli erythromycin A production system, led to improved final compound titers, and suggest additional forms of pathway engineering to further improve results from heterologous production attempts. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:862–869, 2013     

Computational analysis of phenotypic space in heterologous polyketide biosynthesis—Applications to Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae

Polyketides represent a class of natural product small molecules with an impressive range of medicinal activities. In order to improve access to therapeutic polyketide compounds, heterologous metabolic engineering has been applied to transfer polyketide genetic pathways from often fastidious native hosts to more industrially-amenable heterologous hosts such as Escherichia coli, Saccharomyces cerevisiae, or Streptomyces coelicolor. Efforts thus far have resulted in titers either inferior to the native host and significantly below the theoretical yield, emphasizing the need to computationally investigate and engineer the interaction between native and heterologous metabolism for the improved production of heterologous polyketide compounds. In this work, we applied flux balance analysis on genome-scale models to simulate cellular metabolism and 6-deoxyerythronolide B (the cyclized polyketide precursor to erythromycin) production in three common heterologous hosts (E. coli, Bacillus subtilis, and S. cerevisiae) under a variety of carbon-source and medium compositions. We then undertook minimization of metabolic adjustment optimization to identify single and double gene-knockouts that resulted in increased polyketide production while maintaining cellular growth. For the production of 6-deoxyerythronolide B, the results suggest B. subtilis and E. coli are better heterologous hosts when compared to S. cerevisiae and that several single and multiple gene-knockout mutants are computationally predicted to improve specific production, in some cases, over 25-fold.     

Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering

Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product.     

Deoxysugar pathway interchange for erythromycin analogues heterologously produced through Escherichia coli

The overall erythromycin biosynthetic pathway can be sub-divided into macrocyclic polyketide formation and polyketide tailoring to produce the final bioactive molecule. In this study, the native deoxysugar tailoring reactions were exchanged for the purpose of demonstrating the production of alternative final erythromycin compounds. Both the d-desosamine and l-mycarose deoxysugar pathways were replaced with the alternative d-mycaminose and d-olivose pathways to produce new erythromycin analogues through the Escherichia coli heterologous system. Both analogues exhibited bioactivity against multiple antibiotic-resistant Bacillus subtilis strains. Besides demonstrating an intrinsic flexibility for the biosynthetic system to accommodate alternative tailoring pathways, the results offer an initial attempt to leverage the E. coli platform for erythromycin analogue production.     

Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

Metabolically engineered Escherichia coli for efficient production of glycosylated natural products

Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP‐4‐keto‐6‐deoxyglucose resulting in increased production of the heterologous sugars TDP‐L‐mycarose and TDP‐d ‐desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6‐deoxyerythronolide B (6‐dEB) or 3‐α‐mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60‐ and 25‐fold more erythromycin D (EryD) than the original strain K207‐3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.     

Computational identification of gene over-expression targets for metabolic engineering of taxadiene production

Taxadiene is the first dedicated intermediate in the biosynthetic pathway of the anticancer compound Taxol. Recent studies have taken advantage of heterologous hosts to produce taxadiene and other isoprenoid compounds, and such ventures now offer research opportunities that take advantage of the engineering tools associated with the surrogate host. In this study, metabolic engineering was applied in the context of over-expression targets predicted to improve taxadiene production. Identified targets included genes both within and outside of the isoprenoid precursor pathway. These targets were then tested for experimental over-expression in a heterologous Escherichia coli host designed to support isoprenoid biosynthesis. Results confirmed the computationally predicted improvements and indicated a synergy between targets within the expected isoprenoid precursor pathway and those outside this pathway. The presented algorithm is broadly applicable to other host systems and/or product choices.     

Probing the heterologous metabolism supporting 6‐deoxyerythronolide B biosynthesis in Escherichia coli

Heterologous biosynthesis offers a new way to capture the medicinal properties presented by complex natural products. In this study, production of 6‐deoxyerythronolide B (6dEB), the polyketide precursor to the antibiotic erythromycin, was used to probe the heterologous pathways needed for Escherichia coli‐derived biosynthesis. More specifically, the heterologous proteins responsible for 6dEB production were varied by adjusting their respective gene dosage levels. In this way, heterologous components required for posttranslational modification, 6dEB biosynthesis, and substrate provision were adjusted in expression levels to observe the relative effect each has on final heterologous biosynthesis. The results indicate that both the biosynthetic and substrate provision heterologous proteins impact 6dEB formation to a greater extent when compared with posttranslational modification and suggest these components for future protein and metabolic engineering.     

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l -carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.     

Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli

Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.     

Metabolic engineering of Escherichia coli for the production of benzoic acid from glucose

Benzoic acid (BA) is an important platform aromatic compound in chemical industry and is widely used as food preservatives in its salt forms. Yet, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the de novo production of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. First, three different natural BA biosynthetic pathways originated from plants and one synthetically designed pathway were systemically assessed for BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately established in E. coli by expressing the genes encoding the necessary enzymes and screened heterologous enzymes under optimal plasmid configurations. BA production was further optimized by applying several metabolic engineering strategies to the engineered E. coli strains harboring each metabolic pathway, which included enhancement of the precursor availability, removal of competitive reactions, transporter engineering, and reduction of byproduct formation. Lastly, fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway were conducted, which resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics of high industrial interest.     

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli

Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize polyketides are collectively referred to as polyketide synthases (PKSs). Because many of the natural hosts that produce polyketides are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become an important goal toward the engineered biosynthesis of polyketides and analogues. Here, we summarize the recent progresses in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established E. coli as a powerful host for producing polyketides.     

Engineered EryF hydroxylase improving heterologous polyketide erythronolide B production in Escherichia coli

In the last two decades, the production of complex polyketides such as erythromycin and its precursor 6-deoxyerythronolide B (6-dEB) was demonstrated feasible in Escherichia coli. Although the heterologous production of polyketide skeleton 6-dEB has reached 210 mg l⁻¹ in E. coli, the yield of its post-modification products erythromycins remains to be improved. Cytochrome P450EryF catalyses the C6 hydroxylation of 6-dEB to form erythronolide B (EB), which is the initial rate-limiting modification in a multi-step pathway to convert 6-dEB into erythromycin. Here, we engineered hydroxylase EryF to improve the production of heterologous polyketide EB in E. coli. By comparative analysis of various versions of P450EryFs, we confirmed the optimal SaEryF for the biosynthesis of EB. Further mutation of SaEryF based on the crystal structure of SaEryF and homology modelling of AcEryF and AeEryF afforded the enhancement of EB production. The designed mutant of SaEryF, I379V, achieved the yield of 131 mg l⁻¹ EB, which was fourfold to that produced by wild-type SaEryF. Moreover, the combined mutagenesis of multiple residues led to further boost the EB concentration by another 41%, which laid the foundation for efficient heterologous biosynthesis of erythromycin or other complex polyketides.     

Biosynthesis of paromamine derivatives in engineered Escherichia coli by heterologous expression

Aims: Paromamine is a vital and common intermediate in the biosynthesis of 4,5 and 4,6‐disubstituted 2‐deoxystreptamine (DOS)‐containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine. Methods and Results: We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose‐6‐phosphate isomerase (pgi) of primary metabolic pathway to increase glucose‐6‐phosphate pool inside the host. Disruption was carried out by λ Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2‐deoxy‐scyllo‐inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Δpgi. Secondary metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside biosynthesis. Significance and Impact of the Study: This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial biosynthesis of 4,5‐ and 4,6‐disubtituted route of DOS‐containing aminoglycosides.     

Biosynthesis of isoprene in <Emphasis Type="Italic">Escherichia coli</Emphasis> via methylerythritol phosphate (MEP) pathway

Isoprene is an aviation fuel of high quality and an important polymer building block in the synthetic chemistry industry. In light of high oil prices, sustained availability, and environmental concerns, isoprene from renewable materials is contemplated as a substitute for petroleum-based product. Escherichia coli with advantages over other wild microorganisms, is considered as a powerful host for biofuels and chemicals. Here, we constructed a synthetic pathway of isoprene in E. coli by introducing an isoprene synthase (ispS) gene from Populus nigra, which catalyzes the conversion of dimethylallyl diphosphate (DMAPP) to isoprene. To improve the isoprene production, we overexpressed the native 1-deoxy-d-xylulose-5-phosphate (DXP) synthase gene (dxs) and DXP reductoisomerase gene (dxr) in E. coli, which catalyzed the first step and the second step of MEP pathway, respectively. The fed-batch fermentation results showed that overexpression of DXS is helpful for the improvement of isoprene production. Surprisingly, heterologous expression of dxs and dxr from Bacillus subtilis in the E. coli expressing ispS resulted in a 2.3-fold enhancement of isoprene production (from 94 to 314 mg/L). The promising results showed that dxs and dxr from B. subtilis functioned more efficiently on the enhancement of isoprene production than native ones. This could be caused by the consequence of great difference in protein structures of the two original DXSs. It could be practical to produce isoprene in E. coli via MEP pathway through metabolic engineering. This work provides an alternative way for production of isoprene by engineered E. coli via MEP pathway through metabolic engineering.