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Review
. 2011 Jan;28(1):125-51.
doi: 10.1039/c0np00037j. Epub 2010 Nov 9.

Methods and options for the heterologous production of complex natural products

Haoran Zhang  1 , Brett A Boghigian, John Armando, Blaine A Pfeifer
Affiliations
Review

Methods and options for the heterologous production of complex natural products

Haoran Zhang et al. Nat Prod Rep. 2011 Jan.

Abstract

This review will detail the motivations, experimental approaches, and growing list of successful cases associated with the heterologous production of complex natural products.

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Figures

Fig. 1
Fig. 1
Established therapeutic natural products in three major classes: (a) polyketides are represented by erythromycin, daunorubicin, and lovastatin; (b) non-ribosomal peptides are represented by vancomycin; and (c) isoprenoids are represented by Taxol.
Fig. 2
Fig. 2
(a) A comparison of the typical characteristics between native and heterologous hosts. (b) A generalized workflow for establishing natural product biosynthesis in a heterologous host. First, a native host is identified as a producer of a natural product of interest. Second, the genes responsible for producing the product are identified, isolated, and integrated within a heterologous host whether it be through an artificial replicon or within the host’s own chromosome(s). Third, expression of the heterologous genes is initiated, producing soluble protein and the product of interest. Last, this host and process are scaled up and/or further modified for large-scale production.
Fig. 3
Fig. 3
A schematic of erythromycin biosynthesis. A molecule of propionyl-CoA is primed on the loading domain of the DEBS1 enzyme. Polyketide formation requires six (2S)-methylmalonyl-CoA substrates and NADPH as a cofactor in the reductive steps on DEBS1, DEBS2, and DEBS3. The thioesterase domain of DEBS3 is responsible for cyclization and release of the polyketide from the PKS complex. Glucose-1-phosphate is used as a substrate for the EryB and EryC pathways to generate and attach the two sugars (l-mycarose and d-desosamine, respectively) to the aglycone 6dEB core. Abbreviations: AT = acyl transferase; ACP = acyl carrier protein; KS = ketosynthase; KR = ketoreductase; DH = dehydratase; ER = enoyl reductase; TE = thioesterase.
Fig. 4
Fig. 4
A schematic of epothilone biosynthesis. A molecule of acetyl-CoA is primed on the loading domain of the EpoA enzyme. The hybrid polyketide-nonribosomal peptide chain requires one molecule of cysteine on the NRPS EpoB, and one molecule of malonyl-CoA and eight molecules of (2S)-methylmalonyl-CoA substrates on EpoC, EpoD, EpoE and EpoF. The thioesterase domain on EpoF is responsible for cyclization and release of the hybrid chain from the NRPS-PKS complex. Epothilone A is generated by an EpoK-catalyzed epoxidation.
Fig. 5
Fig. 5
A schematic of artemisinin and Taxol biosynthesis. First, either the mevalonate or the non-mevalonate pathways generate the two universal C5 precursors for isoprenoid natural products, isopentenyl diphosphate and dimethylallyl diphosphate. These two can be interconverted through the action of isopentenyl diphosphate isomerase. One molecule of dimethylallyl diphosphate and one of isopentenyl diphosphate are condensed to give the C10 geranyl diphosphate by a geranyl diphosphate synthase. The C15 farnesyl diphosphate is generated from one molecule of geranyl diphosphate and one of isopentenyl diphosphate by a farnesyl diphosphate synthase. Lastly, the C20 geranylgeranyl diphosphate is generated from one molecule of farnesyl diphosphate and one of isopentenyl diphosphate by a geranylgeranyl diphosphate synthase. The first committed steps towards artemisinin and Taxol biosynthesis produce amorphadiene and taxadiene by cyclization of the C15 and C20 intermediates by an amorphadiene synthase and a taxadiene synthase, respectively. Amorphadiene and taxadiene both undergo significant oxidations on their cyclic cores to generate the final molecules. Abbrevations: IPP = isopentenyl diphosphate; DMAPP = dimethylallyl diphosphate; IDI = isopentenyl diphosphate isomerase; GPP = geranyl diphosphate; FPP = farnesyl diphosphate; GGPP = geranylgeranyl diphosphate; GPPS = geranyl diphosphate synthase; FPPS = farnesyl diphosphate synthase; GGPPS = geranylgeranyl diphosphate synthase; ADS = amorphadiene synthase; CYP = cytochrome P450; A-4,11-D H = amorpha-4,11-diene hydroxylase; AAD = artemisinic alcohol dehydrogenase; A-ALD-R = artemisinic aldehyde reductase; AAR = artemisinic alcohol reductase; DHAAD = dihydroartemisinic aldehyde dehydrogenase; T-5α-H = taxadiene 5α hydroxylase; T-5α-ol O-AT = taxdien-5α-ol O-acetyltransferase; T-13α-H = taxadiene 13α hydroxylase; T-10β-H = taxane 10β-hydroxylase; 2α-HT 2-O-BT = 2-α-hydroxytaxane 2-O-benzoyltransferase; 10-DB III-10-O-AT = 10-deacetylbaccatin III 10-O-acetyltransferase.
Fig. 6
Fig. 6
Cumulative trends in heterologous hosts used for natural product production of polyketides, nonribosomal peptides, isoprenoids, and their intermediates/derivatives. Data were generated from the literature over the time period indicated, and each data point represents a publication presenting results of a novel heterologously produced natural product (including novel methods of producing natural products previously reported; for example 6-MSA through S. cerevisiae and E. coli).,– Omitted were papers focused upon gene expression (for the purpose of recombinant protein products or enzyme biochemical characterization) or optimization of a previously produced heterologous compound. Hybrid polyketide-nonribosomal peptide compounds were included in the nonribosomal peptide category totals. Extrapolation curves to 2015 were calculated based upon trend slopes over the 5 years from 2005 to 2010. Due to the volume of data produced during the analysis, the results may not be completely comprehensive, but they serve to illustrate current and future trends within the heterologous natural product biosynthetic field. (a) Overall trends of heterologous natural product production of the three classes highlighted in this review. (b) Trends in heterologous hosts for polyketide production. (c) Trends in heterologous hosts for nonribosomal peptide production. (d) Trends in heterologous hosts for terpenoid/isoprenoid production.
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