Microbial cell factories are extensively used for the biosynthesis of value-added chemicals, biopharmaceuticals, and biofuels. Microbial biosynthesis is also realistic for the production of heterologous molecules including complex natural products of plant and microbial origin. Glycosylation is a well-known post-modification method to engineer sugar-functionalized natural products. It is of particular interest to chemical biologists to increase chemical diversity of molecules. Employing the state-of-the-art systems and synthetic biology tools, a range of small to complex glycosylated natural products have been produced from microbes using a simple and sustainable fermentation approach. In this context, this review covers recent notable metabolic engineering approaches used for the biosynthesis of glycosylated plant and microbial polyketides in different microorganisms. This review article is broadly divided into two major parts. The first part is focused on the biosynthesis of glycosylated plant polyketides in prokaryotes and yeast cells, while the second part is focused on the generation of glycosylated microbial polyketides in actinomycetes.

Introduction

Sugar-functionalized natural products have fascinated scientific communities for a long time. Thus, sugar units present in biomolecules obtained from both plant and microbial sources have been studied extensively to elucidate their potential importance. Therapeutically important molecules along with nutraceuticals and cosmetically useful compounds have been particularly exploited to obtain insights into the role of sugar units in their structures.

Some molecules isolated from microbial sources such as doxorubicin, calicheamicin, staurosporine, vancomycin, amphotericin, erythromycin, and many more have been used to deduce the roles of various sugars attached to them [1,2]. Sugars are known to influence absorption, distribution, metabolism, and excretion processes in human body. They also play crucial roles in the stability, solubility, and bioavailability of these molecules. Therapeutic efficacies of some protein pharmaceuticals are adversely affected by biosynthesis, purification, storage, and delivery processes due to their physical and chemical instabilities. Glycosylation is a well-known method to enhance the overall stability of such molecules. Thus, rational engineering of sugar moieties in natural products is becoming a promising tool to generate novel molecules with better physico-chemical properties and enhanced bioactivities [3]. However, glycosylation does not always enhance biological activities of molecules. Some organisms can glycosylate exogenous molecules to detoxify compounds and excrete out of the cell or body [4]. In contrast, plants generally produce glycosylated molecules for intracellular localization, regulation of cellular homeostasis, detoxification, and metabolism [5]. Such glycosylated molecules usually have enhanced stability that can be stored for a long time.

Natural product glycosides can be generated using various approaches (Figure 1). Chemists have developed synthesis approaches/methods to produce diverse glycosylated natural products in medicinal chemistry for a long time. However, synthesizing sugar-conjugated derivatives is a tedious, time-consuming, and low-yielding process. Other approaches, including chemo-enzymatic glycodiversification and neo-glycorandomization, have also been used to produce glycosylated products [6]. Enzymatic approaches require pure enzymes, enzymatic synthesis, and purification of activated nucleotide sugars which result in lower yield. Moreover, chemo-enzymatic glycodiversification and neo-glycorandomization are difficult to be scaled up at the industrial level due to the increased cost of synthesized molecules. Thus, metabolic engineers employed recent systems/synthetic biology and genetic engineering tools to generate robust microbial cells to produce diverse sugar-conjugated natural products. A cytosolic pool of natural or non-natural nucleotide sugars is generated by introducing heterologous nucleotide diphosphate (NDP)-sugar pathway genes to modulate host central NDP-sugar biosynthesis. Uridine diphosphate glycosyltransferase (UGT)-mediated transfer of these sugar moieties to various acceptor molecules can be achieved inside cells, while most glycosylated molecules are excreted outside into the medium. Such cells can utilize sustainable carbon sources to produce a wide range of glycosylated heterologous molecules in practical quantities by a simple fermentation approach [7,8].

Different approaches for natural product glycosides production.

Figure 1.
Different approaches for natural product glycosides production.

(A) Chemical synthesis (Abbreviations: P: protecting group; L: leaving group), (B) neoglycosylation, (C) enzymatic glycosylation, (D) microbial biosynthesis using single-engineered cell [de novo biosynthesis of glycoside, biotransformation of compounds using NDP-sugar pathway engineered host strain, and biotransformation of compounds supplementing various sugars which are eventually accepted by anomeric kinase (AK) and nucleotidyltransferase (NT) to generate NDP-sugar], and (E) co-culture of two different engineered strains — one producing aglycon while another strain biotransforming the compound produced by first strain to glycoside.

Figure 1.
Different approaches for natural product glycosides production.

(A) Chemical synthesis (Abbreviations: P: protecting group; L: leaving group), (B) neoglycosylation, (C) enzymatic glycosylation, (D) microbial biosynthesis using single-engineered cell [de novo biosynthesis of glycoside, biotransformation of compounds using NDP-sugar pathway engineered host strain, and biotransformation of compounds supplementing various sugars which are eventually accepted by anomeric kinase (AK) and nucleotidyltransferase (NT) to generate NDP-sugar], and (E) co-culture of two different engineered strains — one producing aglycon while another strain biotransforming the compound produced by first strain to glycoside.

However, biosynthesis of complex glycosylated natural products from actinomycetes is limited due to insufficient availability of a pool of endogenous NDP-sugars in cells and tolerance of glycosyltransferase (GT) to donor and acceptor substrates [9]. Sugar moieties attached to metabolites produced by actinomycetes play crucial roles in target binding, specificity, and bioavailability of these molecules that can be extensively modified by endogenous sugar modification enzymes such as dehydratase, epimerase, ketoreductase, aminotransferase, and methyltransferase [3]. Thus, biological routes to develop chemical diversity through conjugation of bulky sugar moieties have fascinated chemical biologists to engineer sugars in natural products using engineered microbes developed through the application of advanced chemical biology and metabolic engineering tools. The discovery or evolution of novel highly flexible GTs with broad tolerance to donor and acceptor substrates has led to the derivatization of natural products with tailored pharmacological properties [1012].

Metabolic engineering for glycosylated plant polyketide biosynthesis

Microbial cells, such as Escherichia coli, yeast, and Streptomyces, are widely used as production hosts for food additives, therapeutics, commodity chemicals, biofuels, and cosmetics ingredients (Figure 2). Easy genetic tracking and commonly available systems/synthetic biology tools for pathway and host metabolic network engineering of these organisms have led them to be extensively used in industry and academia. Microbial cells have been harnessed with either partial or entirely synthetic pathways for the production of various molecules. These engineered pathways often utilize endogenous building blocks and cofactors for energy and reactions. Some notable examples include the production of precursors of complex molecules of plant origin such as artemisinin and taxol in Saccharomyces cerevisiae and E. coli, respectively [1315]. Several small molecules, such as plant polyketides, particularly polyphenols (flavonoids, anthocyanidins, stilbenes, chalcones, terpenoids, and small aromatic compounds), have also been extensively produced in E. coli, yeast, and Streptomyces (Table 1). Numerous post-modified derivatives of plant polyphenols have also been generated using microbial cells, majority of which are O-glycosylated and O-methylated derivatives [16]. However, only a few prenylated/geranylated, hydroxylated, C-methylated, and C-glycosylated derivatives non-natural to microbial systems from enzymatic or microbial biotransformation of plant metabolites have been reported [1621].

Engineered microbial platform host organism for the production of plant and microbial polyketide glycosides.

Figure 2.
Engineered microbial platform host organism for the production of plant and microbial polyketide glycosides.

Selected glyco-engineered polyketides are presented in the figure.

Figure 2.
Engineered microbial platform host organism for the production of plant and microbial polyketide glycosides.

Selected glyco-engineered polyketides are presented in the figure.

Table 1
Summary of production of plant and microbial polyketide glycosides from different hosts
 Starting materialReferences
Plant polyketide derivatives 
 Escherichia coli 
  Kaempferol 3-O-glucoside (astragalin) Naringenin, kaempferol [23,34
  Isoflavone O-glucosides Genistein, biochanin A, daidzein and formononetin [24
  Flavonol 3-O-rhamnosides Kaempferol, quercetin, fisetin, Myricetin, morin [33,34
  Flavonol 3-O-glucosides Kaempferol, quercetin, fisetin, Myricetin, morin [34,70
  Flavonol 3-O-galactosides Kaempferol, quercetin, fisetin, Myricetin, morin [31,33
  Flavonol 3-O-allosides Quercetin, kaempferol [29
  Quercetin 3-O-xyloside Quercetin [30,32
  Luteolin 7-O-glucuronide Luteolin [33
  Genistein methylrhamnosides Genistein [36
  Quercetin 3-O-glucuronide Quercetin [31
  Quercetin 3-O-arabinoside Quercetin [32
  Flavone 6-C-glucoside Chrysin and luteolin [19
  Flavonol 3-O-4-amino-4,6-dideoxy-d-galactoside,
3-O-3-amino-3,6-dideoxy-d-galactoside 
Kaempferol, quercetin, fisetin [50,51
  Quercetin 3-O-glucoside-7-O-rhamnoside Quercetin, quercetin 3-O-glucoside [38,40
  Quercetin 3,7-bisrhamnoside Quercetin [38
  Quercetin 3-O-glucosyl (1 → 2) xyloside Quercetin [39
  Quercetin 3-O-glucosyl (1 → 6) rhamnoside Quercetin [39
  Quercetin 3-O-(N-acetyl) quinovosamine and quercetin
3-O-(N-acetyl)xylosamine 
Quercetin [52
  Luteolin O-(N-acetyl)glucosaminuronic acid Luteolin [52
  Pelargonidin 3-O-glucoside Naringenin [53,54
  Cyanidin 3-O-glucoside Eriodictyol [5355
  Peonidin 3-O-glucoside Cyanidin [56
  Apigenin 7-O-glucoside p-Coumaric acid [61
  Anthocyanidin 3-O-glucoside Glucose [59
  Umbelliferone glucoside (Skimmin) Umbelliferone [66
  Coumarin C-glucosides Coumarin [20
  Anthraquinone O-glucoside Emodin, aloe-emodin [68
  Carvacrol, thymol, geraniol, eugenol, menthol and tyrosol
O-glucoside 
Carvacrol, thymol, geraniol, Eugenol, menthol, tyrosol [69
  8-Prenylkaempferol 3-O-rhamnoside 8-Prenylkaempferol [70
  Anhydroicaritin 3-O-rhamnoside Anhydroicaritin [70
  Indoxyl O-glucosides Tryptophan [8
 Schizosaccharomyces pombe and Saccharomyces cerevisiae 
  Vanillin Glucose [62
  Scutellarein 7-O-glucoside Scutellarein [41
  7-Hydroxycoumarin glucuronide 7-Hydroxycoumarin [67
  Pinocembrin, naringenin, chrysin, apigenin 4-Coumaric acid, cinnamic acid, malonate [42
Microbial polyketide derivatives 
 Streptomyces sp.   
  Narbomycin glycosides  [71
  Anthracycline glycosides  [73
  YC-17 glycosides  [72
  Jadomycin glycosides  [75
  Macrolactone glycosides  [76
  Juvenimicin, M-4365 and rosamicin glycosides Juvenimicin, M-4365, rosamicin [77
  Dihydrochalcomycin glycosides  [79
 Starting materialReferences
Plant polyketide derivatives 
 Escherichia coli 
  Kaempferol 3-O-glucoside (astragalin) Naringenin, kaempferol [23,34
  Isoflavone O-glucosides Genistein, biochanin A, daidzein and formononetin [24
  Flavonol 3-O-rhamnosides Kaempferol, quercetin, fisetin, Myricetin, morin [33,34
  Flavonol 3-O-glucosides Kaempferol, quercetin, fisetin, Myricetin, morin [34,70
  Flavonol 3-O-galactosides Kaempferol, quercetin, fisetin, Myricetin, morin [31,33
  Flavonol 3-O-allosides Quercetin, kaempferol [29
  Quercetin 3-O-xyloside Quercetin [30,32
  Luteolin 7-O-glucuronide Luteolin [33
  Genistein methylrhamnosides Genistein [36
  Quercetin 3-O-glucuronide Quercetin [31
  Quercetin 3-O-arabinoside Quercetin [32
  Flavone 6-C-glucoside Chrysin and luteolin [19
  Flavonol 3-O-4-amino-4,6-dideoxy-d-galactoside,
3-O-3-amino-3,6-dideoxy-d-galactoside 
Kaempferol, quercetin, fisetin [50,51
  Quercetin 3-O-glucoside-7-O-rhamnoside Quercetin, quercetin 3-O-glucoside [38,40
  Quercetin 3,7-bisrhamnoside Quercetin [38
  Quercetin 3-O-glucosyl (1 → 2) xyloside Quercetin [39
  Quercetin 3-O-glucosyl (1 → 6) rhamnoside Quercetin [39
  Quercetin 3-O-(N-acetyl) quinovosamine and quercetin
3-O-(N-acetyl)xylosamine 
Quercetin [52
  Luteolin O-(N-acetyl)glucosaminuronic acid Luteolin [52
  Pelargonidin 3-O-glucoside Naringenin [53,54
  Cyanidin 3-O-glucoside Eriodictyol [5355
  Peonidin 3-O-glucoside Cyanidin [56
  Apigenin 7-O-glucoside p-Coumaric acid [61
  Anthocyanidin 3-O-glucoside Glucose [59
  Umbelliferone glucoside (Skimmin) Umbelliferone [66
  Coumarin C-glucosides Coumarin [20
  Anthraquinone O-glucoside Emodin, aloe-emodin [68
  Carvacrol, thymol, geraniol, eugenol, menthol and tyrosol
O-glucoside 
Carvacrol, thymol, geraniol, Eugenol, menthol, tyrosol [69
  8-Prenylkaempferol 3-O-rhamnoside 8-Prenylkaempferol [70
  Anhydroicaritin 3-O-rhamnoside Anhydroicaritin [70
  Indoxyl O-glucosides Tryptophan [8
 Schizosaccharomyces pombe and Saccharomyces cerevisiae 
  Vanillin Glucose [62
  Scutellarein 7-O-glucoside Scutellarein [41
  7-Hydroxycoumarin glucuronide 7-Hydroxycoumarin [67
  Pinocembrin, naringenin, chrysin, apigenin 4-Coumaric acid, cinnamic acid, malonate [42
Microbial polyketide derivatives 
 Streptomyces sp.   
  Narbomycin glycosides  [71
  Anthracycline glycosides  [73
  YC-17 glycosides  [72
  Jadomycin glycosides  [75
  Macrolactone glycosides  [76
  Juvenimicin, M-4365 and rosamicin glycosides Juvenimicin, M-4365, rosamicin [77
  Dihydrochalcomycin glycosides  [79

For details to know about productivity yield and biosynthetic steps of plant polyketide glycosides, go through recent reviews [16,82].

Among E. coli, Streptomyces, and S. cerevisiae, E. coli is the most preferred strain for the production of glycosylated polyphenols. The primary central carbon metabolic pathway of E. coli has been exploited either for diverting the flow of carbon toward target NDP-sugar (i.e. natural to host metabolism) or for generating a pool of non-natural NDP-sugars. Simple NDP-sugars are present in E. coli. They are essential for the biosynthesis of cell wall components and other structural units. However, E. coli cells usually lack NDP-sugars that are highly modified by sugar-modifying genes. Natural NDP-sugars are generally produced in limited amount for cell growth and maintenance. Since the same molecule is utilized by heterologously expressed GTs as a donor substrate and transferred to the acceptor molecule, engineering cells with the activated nucleotide sugar pathway are essential to generate sufficient pool for biosynthesis of glycosylated natural products.

Biosynthesis of natural flavonoid glycosides

Most plant secondary metabolites produced from microbial sources published in the literature are natural glycosides specifically conjugated with glucose, galactose, glucuronic acid, rhamnose, xylose, allose, arabinose, and N-acetyl-glucosamine. Among these, glucose-conjugated derivatives (also known as glucosides) dominate over other sugar-attached molecules. This process needs enhanced UDP-glucose pool in the cell to balance cell growth and physiology while producing glucosides in the meantime. Thus, engineering glucose catabolism is essential as active glucose catabolic pathway can rapidly deplete glucose-1-phosphate, one of the shared intermediates of UDP-glucose pathway and active glucose catabolic pathways (glycolysis and pentose phosphate pathways), resulting in enhanced production of building block small molecules, energy, and cell biomass [22]. To utilize external glucose as C6-structural component of natural product glucosides, the central glucose catabolism pathway needs to be manipulated to divert the flow of glucose supplemented in the medium toward UDP-glucose, the donor substrate of UGTs.

Several attempts have been made to block glucose catabolism pathway genes for diverting the flow of carbon to UDP-glucose and producing glucosylated polyphenols from E. coli while maintaining cell growth using additional carbon sources such as glycerol and mannitol supplied in the medium. An E. coli strain deficient in glucose-6-phosphate isomerase (pgi, the very first step of glycolysis pathway that converts d-glucose-6-phosphate into d-fructose 6-phosphate) and d-glucose-6-phosphate dehydrogenase (zwf) that diverts the same precursor to 6-phosphogluconolactone of pentose phosphate pathway has been used for the enhanced pool of UDP-glucose for the production of kaempferol 3-O-glucoside (astragalin) from exogenously supplemented naringenin. Blocking of additional UDP-glucose hydrolase (ushA) gene, overexpression of heterologous UDP-glucose pathway genes, and optimization of cell growth for utilization of glycerol and mannitol as supplemental carbon source have led to the production of astragalin at 109.3 mg/l [23] (Table 1). The same background strain harboring UDP-glucose pathway genes and different GTs can produce several isoflavonoids [24] and phloretin glucosides [25]. Similar host strains have been generated to produce significantly higher amounts of molecules such as 2 g/l of d-glucaric acid, 0.8 g/l of myo-inositol [26,27], and 8.2 g/l of trehalose [28] from glucose in E. coli.

Perceiving the significance of different sugar units in the structure of natural products, E. coli cells have also been engineered to produce diverse polyphenol glycosides through derivatization of sugars (Figure 3). E. coli has been designed in such a way that the pool of specific nucleotide sugar is generated in cytosol which is then transferred to different plant polyphenols using regio-specific or flexible GTs. Biosynthetic pathway genes for TDP (thymidine diphosphate)-rhamnose and TDP-allose have been introduced into E. coli BL21 (DE3)/Δpgi strain to produce 3-O-rhamnoide and 3-O-alloside derivatives of quercetin and kaempferol [29]. Similarly, UDP-glucose pool has been diverted to UDP-xylose in E. coli BL21 (DE3)/ΔpgiΔzwfΔushA strain by using two additional genes from Micromonospora to produce quercetin 3-O-xyloside [30].

Engineered nucleotide sugar pathways for biosynthesis of different plant and microbial polyketides.

Figure 3.
Engineered nucleotide sugar pathways for biosynthesis of different plant and microbial polyketides.

The sugar pathways present in the box are UDP-sugars, while in oval-shaded circles are TDP-sugars. Most of the UDP-sugars are engineered in E. coli and yeasts to produce plant polyphenol glycosides, while TDP-sugars are engineered in Streptomyces for microbial polyketide glycoside biosynthesis.

Figure 3.
Engineered nucleotide sugar pathways for biosynthesis of different plant and microbial polyketides.

The sugar pathways present in the box are UDP-sugars, while in oval-shaded circles are TDP-sugars. Most of the UDP-sugars are engineered in E. coli and yeasts to produce plant polyphenol glycosides, while TDP-sugars are engineered in Streptomyces for microbial polyketide glycoside biosynthesis.

To enhance UDP-glucuronic acid pool, araA gene encoding UDP-4-deoxy-4-formamido-l-arabinose formyltransferase/UDP-glucuronic acid C-4″ decarboxylase has been deleted in E.coli, while UDP-glucose dehydrogenase (ugd) gene is overexpressed. This engineered strain produced practical quantities of luteolin-7-O-glucuronide and quercetin-3-O-glucuronide. Similarly, overexpression of uge encoding UDP-glucose epimerase from Oryza sativa after diverting UDP-glucose to UDP-galactose pool produced quercetin 3-O-galactoside [31]. In another approach, flavonol pentosides, such as quercetin 3-O-xyloside and quercetin 3-O-arabinoside, have been produced by overexpressing UDP-xylose and UDP-arabinose biosynthesis genes along with GTs in background strain of E. coli lacking arnA (UDP-l-Ara4N formyltransferase/UDP-GlcA C-4″-decarboxylase) gene that competes for UDP-xylose and UDP-arabinose biosynthesis intermediates [32].

Similarly, two E. coli W platforms for galactosylation and rhamnosylation have been developed for stereospecific production of quercetin glycosides. Instead of using glucose as the source of structural part of the flavonoid glycoside, sucrose was hydrolyzed to glucose 1-phosphate and fructose by sucrose phosphorylase. As described in previous studies, the glucose 1-phosphate metabolism pathway was impeded by mutating several genes such as pgm, glucose-1-phosphatase (agp), ushA, and galactose operon (galETKM) in the genome. Fructose was used as an alternative source of carbon for cell growth and energy, while glucose 1-phosphate was diverted toward UDP-glucose and finally to UDP-galactose and UDP-rhamnose. By using GTs specific to transfer galactose (F3GT) and rhamnose (RhaGT), 10 different flavonol glycosides (i.e. 3-O-galactoside and 3-O-rhamnoside derivatives of quercetin, kaempferol, myricetin, morin, and fisetin) were produced [33] (Table 1). Recently, synthetic vectors have been used to assemble multiple genes in a single vector to maintain fine-tuning of gene expression by varying promoter strength and ribosome-binding sites. Single-vector system can avoid the metabolic burden imposed by several antibiotics added into the medium as selection pressure in contrast with multi-vector systems. Several biosynthetic pathway genes required for enhanced pool of TDP-glucose and TDP-rhamnose have been assembled under individual promoters in a single vector. The newly designed system can efficiently biotransform five different flavonols (quercetin, kaempferol, fisetin, myricetin, and morin) to respective glucosides and rhamnosides in practical quantities [34]. Using the same TDP-glucose biosynthesis sugar cassette, chrysin 6-C-glucoside and luteolin 6-C-glucoside derivatives have been produced in E. coli for the first time [19]. Natural and non-natural flavonoids rhamnosides, tamarixetin glucoside, and isoflavone: genistein 7-O-methylrhamnosides have been produced by expressing a versatile rhamnosyltransferase AtUGT89C1 along with sugar O-methyltransferases [3537].

Biosynthesis of di- and bis-glycosides

Naturally occurring rare bis-glycoside derivatives have also been produced by sequential expression of two regio-specific GTs that can attach sugar moiety at different hydroxyl groups of flavonoids. In 2013, Kim et al. [38] produced quercetin 3-O-glucoside-7-O-rhamnoside and quercetin 3,7-O-bisrhamnoside in E. coli by engineering host strain harboring GTs specific to transfer rhamnose and glucose along with the UDP-rhamnose synthase gene from Arabidopsis thaliana. The same group also produced natural diglycoside derivatives of quercetin 3-O-glucosyl (1 → 2) xyloside and quercetin 3-O-glucosyl (1 → 6) rhamnoside in an engineered E. coli strain by overexpressing UDP-xylose and UDP-rhamnose biosynthesis pathway genes, respectively [39]. Sugar units were transferred by two independent GTs either overexpressed or integrated into the genome of the E. coli host strain. However, Roepke and Bozzo [40] have produced quercetin 3-O-glucoside-7-O-rhamnoside, a diglycoside by feeding quercetin 3-O-glucoside to an E. coli strain engineered to produce UDP-rhamnose and harboring a GT (Table 1).

Saccharomyces cerevisiae for flavonoid glucoside biosynthesis

Although several chemicals and microbial or plant secondary metabolites can be efficiently produced using yeasts as platform organisms, endogenous glucosidase enzymes hinder the production of glucoside derivatives using such a platform. Endogenous enzymes encoded by genes, such as EXG1, SPR1, and YIR007W, are considered to be responsible for the glucosidase activity exhibited by the S. cerevisiae strain. A recent study has shown that EXG1 is the most important gene for hydrolyzing glucosides among all three different genes. S. cerevisiae/ΔEXG1 strain lacking glucoside-hydrolyzing activity harboring Scutellaria baicalensis Georgi UGT and UDP-glucose pool with enhanced phosphoglucomutase and UTP-glucose-1-phosphate uridylyltransferase genes can produce 1200 mg/l of scutellarein 7-O-glucoside from exogenously fed scutellarein [41] (Table 1). Selected flavanones, flavones, and stilbenes have been produced in limited amounts from exogenously fed substrates in engineered Streptomyces venezuelae deficient in its native pikromycin polyketide synthase [42]

Non-natural flavonoid glycosides from engineered E. coli

Conjugating plant secondary metabolites with unusual sugar moieties biosynthesized in microbial host cells has been developed as a tool to generate novel molecules with altered physical, chemical, and biological properties. Such modifications of natural products also add chemical diversity to original natural products [43,44]. Since the pool of nucleotide sugars can be easily altered by engineering host nucleotide sugar pathway genes and introducing heterologous nucleotide sugar biosynthesis genes to produce non-natural nucleotide sugars, such sugars can be subsequently transferred to exogenously supplemented molecules by using engineered promiscuous GTs.

Most of therapeutically important microbial glycosylated secondary metabolites contain deoxy-amino-sugars in their structures [2]. These deoxy-amino-sugars can improve water solubility, basicity of the molecule, and alter the mechanism of action of molecules due to their potential to form ionic interactions under normal physiological conditions [4549]. Thus, conjugation of deoxy-amino-sugars is of particular importance to enhance pharmacological properties of natural products. Efforts have been made to conjugate microbial-derived deoxy-amino-sugars to plant secondary metabolites such as flavonoids by engineering E. coli nucleotide sugar pool to synthesize non-natural nucleotide deoxy-amino-sugar pools.

E. coli strain mutated to dam up glycolysis and pentose phosphate pathways has been further engineered to create a pool of thymidine diphosphate 4-keto 4,6-dideoxy-d-glucose (dTKDG) from glucose-1-phosphate by deleting uridylyltransferase (galU) gene. dTKDG, an intermediate of various dTDP sugars, was diverted to produce pool of dTDP-d-viosamine, dTDP 4-amino 4,6-dideoxy-d-galactose, and dTDP 3-amino-3,6-dideoxy-d-galactose sugars using sugar aminotransferases in cytosol by introducing heterologous genes from microbial sources (Figure 2). These recombinant E. coli strains could convert exogenously supplemented kaempferol, quercetin, and fistein into non-natural deoxy-amino-sugar-conjugated derivatives such as kaempferol 3-O-4-amino-4,6-dideoxy-d-galactoside, quercetin 3-O-4-amino-4,6-dideoxy-d-galactoside, fisetin 3-O-4-amino-4,6-dideoxy-d-galactoside, kaempferol 3-O-3-amino-3,6-dideoxy-d-galactoside, and quercetin 3-O-3-amino-3,6-dideoxy-d-galactoside [50,51]. Recently, a similar approach has been used to develop E. coli strains by introducing heterologous genes diverting endogenous UDP-N-acetyl-d-glucosamine to UDP-N-acetylquinovosamine, UDP-N-acetyl-d-glucosaminuronic acid, and UDP-N-acetyl-d-xylosamine (Figure 3). Two of these newly synthesized sugars were transferred to quercetin to produce non-natural quercetin 3-O-(N-acetyl) quinovosamine and quercetin 3-O-(N-acetyl) xylosamine, while N-acetyl-d-glucosaminuronic acid was conjugated to luteolin to produce luteolin O-(N-acetyl) glucosaminuronic acid [52] (Table 1).

Biosynthesis of anthocyanidin glycosides

E. coli wild-type strains engineered only for plant aglycone can produce detectable amounts of glucosylated derivatives of cyanidin and pelargonidin [53]. However, upon engineering of host strain for intracellular UDP-glucose pool and optimization of gene expression in fusion protein increased their production to 70.7 and 78.9 mg/l, respectively [54]. Cyanidin 3-O-glucoside can be produced from (+)-catechin after introducing anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (At3GT) to an E. coli strain by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters [55]. A dually modified methylated as well as glucosylated derivative, peonidin 3-O-glucoside, has also been produced in an E. coli metJ repressed strain generated by the CRISPR/dCas9 system harboring Petunia hybrida ANS (PhANS), A. thaliana anthocyanidin 3-O-glucosyltransferase (At3GT), and 3′-O-methyltransferase from Vitis vinifera or orthologous gene from fragrant cyclamen ‘Kaori-no-mai’ [56]. Anthocyanidin synthase was the major bottleneck so far for sustainable production of anthocyanin derivatives. Three different anthocyanin derivatives were biosynthesized from a simple molecule, glucose, in S. cerevisiae by exploring series of enzyme from various plant sources was reported for the first time [57].

E. coli co-culture/polyculture for polyphenol glucoside production

The biosynthetic burden due to host biosynthesis network engineering and assembly of several heterologous genes essential for the biosynthesis of molecule in a single cell is a detrimental factor for cells with impaired health and low yield of target compound. To split the metabolic burden, biosynthetic modules can be divided among strains utilizing either the same or different carbon sources [58,59]. Such strains are mixed and fermented together to produce final compounds. Recently, microbial co-culture or polyculture metabolic engineering approaches have been used to produce plant-derived polyphenol glycosides. Four recombinant E. coli strains have been cultured together as a polyculture system to produce anthocyanins [60]. The distribution of biosynthetic pathway genes among these four different strains can balance the need of polyketide building blocks and other pathways specific for indigenous cofactors, thus reducing the metabolic burden in a single cell. A similar approach of co-culture has been used for the biosynthesis of apigenin-7-O-β-d-glucoside. An E. coli engineered to produce apigenin has been cultured with another E. coli engineered to produce a pool of UDP-glucose to produce apigenin-7-O-β-d-glucoside [61].

Biosynthesis of miscellaneous plant metabolite glycosides

Besides flavonoids and anthocynidins, glucoside derivatives of other plant secondary metabolites have also been produced using engineered recombinant microbial strains. Some remarkable examples are highlighted briefly in this section (Table 1).

Vanillin, a plant-derived secondary metabolite, is one of the widely used high demand flavoring agents, with an annual world market demand of 16 000 tons. However, its current requirement is fulfilled by chemical synthesis from fossil hydrocarbons and lignin. To achieve sustainable and environment-friendly microbial production of vanillin, several engineering approaches have been used. Vanillin β-d-glucoside has been produced from glucose in two different yeasts Schizosaccharomyces pombe and S. cerevisiae after introducing several genes from various sources [62]. The production of vanillin was increased up to 500 mg/l in the same S. cerevisiae strain by in silico designing the strain using a set of stoichiometric modeling tools applied to genome-scale metabolic network of yeast [63].

Besides flavoring agents, glucoside derivatives of plant secondary metabolites used as aroma chemicals are also produced from microbes. Coumarins are well known to exhibit potent pharmacological and anticancer activities. They are also used in certain perfumes as aroma enhancers. Recently, different derivatives of coumarins have been produced using chemical, enzymatic, and microbial approaches [64,65]. Skimmin, a glucoside derivative of umbelliferone, has been produced along with other hydroxylated (aesculetin) and methylated (herniarin) derivatives [66]. Similarly, E. coli harboring engineered CGT (MiCGTb-GAGM) from Magnifera indica can produce 12 different coumarin C-glucosides by a whole-cell biotransformation approach [20]. Different glucuronide derivatives of 7-hydroxycoumarin and other drugs have been produced in engineered S. cerevisiae co-expressing human or mammalian UGTs and UDP-glucose-6-dehydrogenase (UGDH) [67].

Anthraquinone glucosides, another group of plant secondary metabolites, are also produced using engineered E. coli strain harboring Bacillus GT [68]. A similar approach of whole-cell biotransformation of several chemicals and aromatic compounds, such as carvacrol, thymol, geraniol, eugenol, vanillin, menthol, tyrosol, and other hydroxyl fatty acids (16-hydroxy palmitic acid, octanol, decanol, and hexadecanol), has been successfully carried out to produce glucoside derivatives using GTs from different plants [69]. Likewise, a recombinant E. coli BL21 (DE3) host strain harboring a flavonol rhamnosyltransferase and a UDP-rhamnose synthase from Epimedium pseudowushanense has been established recently to produce baohuoside II from 8-prenylkaempferol [70]. Indican, a glucoside derivative of indoxyl, has been recently produced in engineered E. coli that is resistant to spontaneous air oxidation due to attached glucose moiety. Such bio-based indican is equally efficient as chemically synthesized indigo to dye clothes [8].

Metabolic engineering for glycosylated microbial polyketide biosynthesis

Microorganisms produce various secondary metabolites in response to environmental conditions as a defense mechanism. Some of these molecules produced by microorganisms are toxic to themselves. Thus, microorganisms can use glycosylation as a detoxification mechanism to make these molecules less toxic to themselves; however, such glycosylated products are toxic to organisms around them. Actinomycetes are excellent sources of novel polyketides. Although there are continuous efforts to investigate and develop promising drug candidates, many natural hosts that produce polyketides are difficult to culture and manipulate. Metabolic engineering has been developed as a tool to continuously expand and facilitate the use of microbial machinery. Several actinomycetes have been engineered to produce glycodiversified secondary metabolites in the last two decades. Several extensive reviews are available on this topic [2,3]. In this section, we will discuss selected recent engineered approaches used for the production of microbial glycosylated polyketides (Table 1).

Owing to ease in growth and genetic manipulation, S. venezuelae has been extensively studied for metabolic engineering and biosynthesis of various polyketide natural products. It has been used in combinatorial biosynthesis systems to generate structurally diverse natural products. Several glycosylated analogs of narbomycin produced by engineered S. venezuelae strain containing different deoxysugar biosynthetic gene cassettes have been reported. These derivatives exhibit greater antibacterial activity than narbomycin [71]. Similarly, sugar pathway-specific genes have been heterologously expressed in S. venezuelae to synthesize four different YC-17 analogs with improved antibacterial activities [72]. Donor or acceptor substrate flexibility of GTs plays a vital role in diversifying natural product glycoconjugates. Seven out of 16 non-natural sugar-conjugated novel derivatives of anthracycline have been successfully generated from an in vivo co-cultivation via the same approach [73] (Figure 3). A strain of S. venezuelae engineered to biosynthesize aglycon has been co-expressed with a recombinant strain expressing various nucleotide deoxysugar biosynthesis genes and a substrate-flexible GT. The same group also used an avermectin-producing strain S. avermitillis to synthesize milbemycin derivatives (milbemycins A3, A4, D, B2, B3, and G) with improved insecticidal properties [74]. Jadomycin is an angucyclic natural product produced by S. venezuelae which is engineered by altering sugar moiety d-olivose instead of l-digitoxose without affecting its cytotoxicity. Because of flexible and natural O-GT, six different analogs of jadomycin B have been reported alongside [75].

Applications of a regiodivergent catalysis for position selective introduction of sugar moiety to 6-deoxyerythronolide B and oleandomycin-derived macrolactones that lead to the biosynthesis of three different macrolides have been described [76]. Total structures of tylactone-based macrolides, juvenimicin, M-4365, and rosamicin have been reconstructed in vitro, revealing the potential of polyketide biosynthetic pathway enzymes followed by in vivo glycosylation via biotransformation using a mutated strain of S. venezuelae DHS316 and their glycoconjugate diversity. These compounds exhibited strong antimicrobial activities against both Gram-positive and Gram-negative pathogens [77]. Similarly, structural diversity of herboxidine has been obtained by expressing a versatile GT and substrate-flexible cytochrome P450 in a native herboxidine producer Streptomyces chromofuscus. This modification altered its biological properties from anticancer to antibacterial [78]. A few years ago, a novel desosaminyl derivative of a hybrid macrolide dihydrochalcomycin was reported by genetic engineering of a Streptomyces sp. KCTC0041BP [79].

E. coli has been engineered to heterologous express polyketide biosynthesis enzymes to generate 6-deoxyerythronolide and finally NDP-sugar pathways are recombined to create final erythromycin compound [49]. The same group in a follow-up study has shown an alternative way of biosynthesis of novel molecules rather than isolating completely new compounds from natural sources. As a proof of concept, a variety of native deoxysugar glycosylation patterns along with flexible GT have been engineered in E. coli to glycorandomize the complex polyketide compound [49]. Erythromycin A, one of the medically important compounds, has been structurally diversified using post-modification enzymes over its aglycone again and again. Its native producer, Saccharopolyspora erythraea, has been engineered by replacing GTs from S. venezuelae to generate a cell factory to produce erythromycin D [80]. Novel derivatives of paulomycin A and B altering native sugar to l-paulomycose have been generated from the combinatorial biosynthesis approach by studying inactivation of enzymes involved in deoxysugar biosynthesis and its glycosyltransferase. However, biological activities of these compounds were found to be lower than those of original compounds [81].

Conclusion

Highlighted current examples of metabolic engineering trends in this review demonstrate the potential of pathway engineering and evolution studies for microbial production and chemical diversification of natural products by glycosylation. Using natural products as scaffolds, biosynthesis, and their derivatization with diverse sugars in an engineered microbial cell can be highly efficient and advantageous over chemical synthesis or their production in natural sources. Easy access to state-of-the-art metabolic engineering tools with rapid gene synthesis and genome sequencing has enabled chemical engineers to develop robust microbial cell factories for easy production of chemicals, commodities, and several natural products using sustainable carbon sources. Different approaches have been used to produce natural product glycosides in microbial cells by manipulating native carbon metabolism networks, introducing engineered biosynthetic pathway genes, and developing high substrate tolerance GTs by mutagenesis. The microbial production of some natural product glycosides has reached practical quantities in laboratory scale flasks and fermenters. An industrial production of these molecules still needs extensive studies for several factors of metabolic engineering. This includes pathway optimization, fine-tuning of gene expression and regulation, background host strain development with rapid growth using sustainable carbon sources, and evolutionary development of promiscuous and highly efficient enzymes.

Future directions

Recent developments on metabolic engineering and systems/synthetic biology tools enabled the production of polyphenols and polyketide glycosides using microbial systems. However, the biosynthesis of these compounds is limited and requires extensive engineering of microbial cells for industrial production. The productivity is greatly determined by the precursor metabolites during heterologous biosynthesis using synthetic pathways. Thus, the integrated use of modern systems/synthetic biology and metabolic engineering, transcriptomics, and proteomics tools could aid the design of robust strain to pool up the precursors for polyketide backbone synthesis and simultaneous sugar unit biosynthesis. The sufficient availability of cytosolic precursor metabolites could resolve the issue of limited glycoside production from microbial cells. Moreover, the glycosylation step and biosynthesis of non-natural activated nucleotide sugars in prokaryotic cells are the major hurdles on the production of non-natural sugar-conjugated polyphenols and polyketides. This issue could be addressed by identifying alternative rate-limiting and bottleneck enzymes from various sources through high-throughput techniques or evolution of enzymes for higher catalytic conversion and substrate specificity. Similarly, functional characterization and structural and mechanistic studies of polyketide biosynthetic enzymes are still in preliminary stage which lacks the insights into their reaction mechanisms in hosts that fails engineering efforts for better productivity. Hence, exploration of novel enzymes is crucial to expand the product range and develop a powerful system for high-scale production of natural product glycosides.

A combinatorial biosynthesis approach has been of a particular interest for bioactive compounds production in recent trends. Instead of setting up burden to a single cell by engineering multiple pathways, the use of co-/polyculture has been emerged as an alternative approach for polyketides and other natural product biosynthesis. What if all ideal cells are rationally designed for the production of target intermediates through computational analyses and engineering the mixed culture for the production of target compounds? The combined application of aforementioned approaches could develop a highly robust system for value-added natural product biosynthesis in near future.

Summary
  • Sugar moieties are functional groups of several microbial and plant secondary metabolites that have proven to play vital roles in their biological activities. Thus, engineering sugar units in natural products is a promising way to diversify chemical scaffolds.

  • Several plant and microbial natural product glycosides are produced using the metabolic engineering and combinatorial biosynthesis approach in industrially proven hosts. The central carbon metabolism network of host strains can be engineered to divert carbon flow toward different activated natural and non-natural NDP-sugars for GT-mediated conjugation of sugar moieties to various natural products. As a result, several natural and non-natural glycodiversified natural products can be produced from engineered microbial cells. Some newly developed glycosylated molecules exhibit potent biological activities against different pathogens, while other molecules remain to be studied.

  • Metabolic engineering of different microbial cells discussed in this review highlights newly developed platform organisms for efficient production of glycosylated polyketides of plant and microbial origin.

Abbreviations

     
  • ANS

    anthocyanidin synthase

  •  
  • At3GT

    anthocyanidin 3-O-glycosyltransferase

  •  
  • dTKDG

    thymidine diphosphate 4-keto-4,6-dideoxy-d-glucose

  •  
  • NDP-sugar

    nucleotide diphosphate sugar

  •  
  • TDP

    thymidine diphosphate

  •  
  • UDP

    uridine diphosphate

  •  
  • ugd

    UDP-glucose dehydrogenase

  •  
  • UGT/GT

    UDP-glycosyltransferase/glycosyltransferase

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) [NRF-2017R1A2A2A05000939] to Jae Kyung Sohng and [NRF-2017R1C1B5018056] to Ramesh Prasad Pandey.

Author Contribution

R.P.P. and P.P. prepared the manuscript. J.K.S. revised and added valuable comments.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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