Engineered biosynthesis of natural products in heterologous hosts

Abstract

Natural products produced by microorganisms and plants are a major resource of antibacterial and anticancer drugs as well as industrially useful compounds. However, the native producers often suffer from low productivity and titers. Here we summarize the recent applications of heterologous biosynthesis for the production of several important classes of natural products such as terpenoids, flavonoids, alkaloids, and polyketides. In addition, we will discuss the new tools and strategies at multi-scale levels including gene, pathway, genome and community levels for highly efficient heterologous biosynthesis of natural products.

Figure 1

Routes for heterologous biosynthesis of natural products. (A) Synthesis of natural products by traditional isolation from plants. (B) Synthesis and screening of small molecules by combinatorial chemistry. (C) Production of natural products by heterologous biosynthesis in microbes.

Figure 2

Schematic of various biosynthetic pathway of terpenoids. Two pathways, the MVA pathway and the DXP pathway, provide the precursors for the biosynthesis of terpenes, which are the backbones to synthesis of terpenoids, including monoterpenoids, sesquiterpenoids diterpenoids, triterpenoids and carotenoids.

Figure 3

Schematic of sesquiterpenoids biosynthesis. Farnesyl diphosphate (FPP), the 15-carbon precursor, is converted to different sesquiterpene by various sesquiterpenoid synthases (ADS, amorphadiene synthase; CsPTS, valencen synthase; PMSTS, Persicaria minor sesquiterpene synthase; PTS, patchoulol synthase; VMPSTS, Vanda mimi palmer sesquiterpene synthase; SanSyn, santalene synthase). Some of these sesquiterpenes can be further oxidized to their derivatives by various cytochrome P450 oxidases (CYP71AV8, CnVO, CYP71D51V2 and CYP71AV1). In the biosynthesis pathway of artemisinin, the well-known sesquiterpenoid, the artemisinic alcohol can be further converted to artemisinin or artemisinic acid by several enzymes (AaALDH1, artemisinic aldehyde dehydrogenase; ADH1, artemisinic alcohol dehydrogenase; DBR2, artemisinic aldehyde reductase; CPR1, reductase of CYP71AV1; CYB5, cytochrome b5 from A. annua).

Figure 4

Schematic of the paclitaxel biosynthetic pathway. Paclitaxel is synthesized by the combination of the core taxane and the side chain. The core taxane structure is synthesized by the modification of the taxadiene, which is produced by the cyclization of GGPP. The solid arrows in the pink background mean the steps with known reactions and known enzymes. The solid arrows in the light blue background mean the steps with known reactions but unknown enzymes. The dotted arrows mean the steps with unknown reactions and known enzymes. FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. TDC1, Taxadiene synthase; CYP725A2: taxane 13 alpha-hydroxylase; CYP725A1, taxane 10 beta-hydroxylase; TBT, taxoid 2a-O-benzoyl transferase; DBAT, 10-deacetylbaccatin III 10-O-acetyltransferase; PAM, phenylalanine aminomutase.

Figure 5

Schematic of the various biosynthetic pathways of carotenoids. (A) The common pathway for lycopene and β-carotene biosynthesis in bacteria and fungi. IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; CrtE, GGPP synthase; CrtB, phytoene synthase; CrtI, phytoene desaturase; CrtY, lycopene β-cyclase; (B,C,D) The astaxanthin biosynthetic pathway in carotenogenic bacteria and algae (B), red yeast X. dendrorhous (C), and plant Adonis aestivalis (D). CrtZ, β-carotene hydroxylase; CrtW, β-carotene ketolase; CrtS, P450 cytochrome monooxygenase; CrtR, cytochrome P450 reductase; CBFD, carotenoid β-ring 4-dehydrogenase; HBFD, carotenoid 4-hydroxy-β-ring 4-dehydrogenase.

Figure 6

Schematic of the various biosynthetic pathways of flavonoids. PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase; 4CL, 4-coumarate:Co1-ligase; C4H, cinnamate-4-hydroxylase; CHI, chalcone isomerase; CHR, chalcone reductase; CHS, chalcone synthase; FSI, flavone synthase I; FSII, flavone synthase II; IFS, isoflavone synthase; TAL, tyrosine ammonia lyase.

Figure 7

Schematic of the various biosynthetic pathways of alkaloids. CNMT, coclaurine-N-methyltransferase; DODC, DOPA decarboxylase; MAO, monoamine; NCS, norcoclaurine; TYR, tyrosinase; 3,4-DHPAA, 3,4-dihydroxyphenylacetaldehyde; 6-OMT, norcoclaurine 6-O-methyltransferase; 4’-OMT, 3’-hydroxy-N- methylcoclaurine 4’-O-methyltransferase.

Figure 8

Schematic of the biosynthetic pathway of erythromycin. (A) the biosynthetic assembly line for the polyketide antibiotic erythromycin. AT: acyltrasferase; ACP: acyl carrier protein; KS: ketosynthase; KR:ketoreductase; DH:dehydratse ; ER:enoylreductase; TE: thioesterase. (B) Domain engineering; (C) Domain inactivation; (D) Engineering of tailoring reactions.

Figure 9

Strategies to tune the gene expression levels in the natural product biosynthetic pathways. The expression of heterologous genes are regulated at the gene level (variation on copy numbers of genes, selection of plasmids), at the transcriptional level (promoter strength, mutation of promoter functional sites, addition of upstream activation sites, and inducible artificial promoters), and at the translational level (special sequence repeats, synthetic riboswitches, rational ribosome binding sites, random translation starting sites, tuneable intergenic region options, and artificial codons). The codon optimization of the heterologous genes is another efficient strategy to tune gene expression.

Figure 10

Strategies to enhance the pathway flux by modifying the distances of proteins and protein re-localization. In order to enhance heterologous metabolic flux, fusion of proteins with different linkers is efficient. DNA-, RNA- and protein-based scaffolds are used to assemble the heterologous enzymes to improve the sequential reactions. Re-localization of enzymes switches the subcellular position of heterologous proteins to enhance the production by either enhancing enzyme concentration or taking advantage of environmental differences. Colourful solid arrows represent the heterologous enzymes.

Figure 11

Exploration and construction of novel natural product biosynthetic pathways. (A) Heterologous expression of uncharacterized genes or proteins with potential functions to catalyze the specific reaction is applied to explore new pathways for natural product biosynthesis. (B) Heterologous expression of uncharacterized genes to explore the biosynthetic pathway for ginsenoside CK. (C) Heterologous expression of protein with similar activity to construct a novel biosynthetic pathway for Salvianic acid A. Colourful solid arrows represent the heterologous enzymes. Adapted by permission from Macmillan Publishers Ltd: [Cell Research] (ref. 58), copyright (2014). Reprinted from Metabolic Engineering, 19, Yao YF, Wang CS, Qiao J, Zhao GR., Metabolic engineering of Escherichia coli for production of salvianic acid A via an artificial biosynthetic pathway, Pages 79-87, Copyright (2013), with permission from Elsevier.

Figure 12

Strategies to optimize natural product biosynthetic pathways assisted by DNA assembly methods. A heterologous pathway can be optimized by changing different promoters to regulate the expression of different genes. DNA assembly in vivo and in vitro can be used to introduce variations either inside the genes or in regulation parts (promoters or regulators) to build a library, which can be used to select the desired strains with the heterologous pathway. Colourful solid arrows represent the heterologous enzymes.

Figure 13

Strategies of modular pathway engineering for metabolic precursor synthesis. Strategies to increase metabolic precursors include: (1) increasing the supply of the substrate, such as adding new biosynthetic pathway of precursor; (2) increasing the whole flux of the desired biosynthetic pathway, such as improving the gene expression for precursor synthesis; (3) decreasing or eliminating the flux of the branch pathways, such as deleting the branch pathways. These strategies can be achieved by manipulating the copy number of genes, changing promoters and introducing either heterologous upstream or heterologous downstream pathways. Colourful solid arrows represent the heterologous enzymes. Red arrow with two tailors means the down regulation.

Figure 14

Biosensor-directed real-time control and evolution. The promoters inhibited by the target products are used as the biosensors to control the expression of DNA mutator proteins, such as mutD5. This design can be used to generate a mutant library. Meanwhile, the same promoter also control the expression of a fluorescent protein, which is used as the reporter for phenotype selection. Colourful solid arrows represent the heterologous enzymes. Adapted by permission from Macmillan Publishers Ltd: [Nature Communication] (ref. 156), copyright (2013).

Figure 15

The SCRaMbLE system based on genome synthesis. Based on the Cre-LoxP system, the SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) is able to generate a mutant library by gene deletion, gene reversion, gene insertion, and gene translocation at a genome scale. Colourful solid arrows represent the heterologous enzymes. Reproduced with permission from ref. 175, copyright 2014 John Wiley and Sons.

Figure 16

Engineering of the microbial consortium to improve heterologous biosynthesis of complex natural products. (A) Schematic of the distribution of the long synthetic pathway for complex natural products into different strains of the consortium. (B) The artificial consortium with engineering E. coli and S. cerevisiae produce oxygenated taxanes using xylose and ethanol. Colourful solid arrows represent the heterologous enzymes. Adapted by permission from Macmillan Publishers Ltd: [Nature Biotechnology] (ref. 177), copyright (2015).