Appl Microbiol Biotechnol (2009) 83:799–808 DOI 10.1007/s00253-009-2039-z
MINI-REVIEW
Biosynthesis and biotechnological production of flavanones: current state and perspectives Zachary L. Fowler & Mattheos A. G. Koffas
Received: 7 April 2009 / Revised: 7 May 2009 / Accepted: 7 May 2009 / Published online: 28 May 2009 # Springer-Verlag 2009
Abstract Polyphenols produced in a wide variety of flowering and fruit-bearing plants have the potential to be valuable fine chemicals for the treatment of an assortment of human maladies. One of the major constituents within this chemical class are flavonoids, among which flavanones, as the precursor to all flavonoid structures, are the most prevalent. We review the current status of flavanone production technology using microorganisms, with focus on heterologous protein expression. Such processes appear as attractive production alternatives for commercial synthesis of these high-value chemicals as traditional chemical, and plant cell cultures have significant drawbacks. Other issues of importance, including fermentation configurations and economics, are also considered. Keywords Flavanones . Fermentation . Polyphenols . Cofactor engineering
Introduction Flavonoids are a highly diverse group of plant secondary metabolites, ubiquitously found throughout the plant kingdom with many flavonoid structures displaying a variety of biochemical activities, including estrogenic, antioxidant, antiviral, antibacterial, antiobesity, and anticancer. Typically, flavonoids are broken down into six major categories: isoflavones, flavanones, flavones, flavonols, catechins, and anthocyanins. Bond saturation, hydroxylation, and ring Z. L. Fowler : M. A. G. Koffas (*) Department of Chemical and Biological Engineering, University at Buffalo, Buffalo 14260 NY, USA e-mail:
[email protected]
position are used to differentiate each class. The diverse pharmicodynamic abilities of flavonoids have drawn major attention to the molecules for personal health applications (Harborne and Williams 2000; Forkmann and Martens 2001) and also from pharmaceutical companies keen on their native nutraceutical properties or to use as starting formulations for market pharmaceuticals. While currently only being used as dietary supplements, flavonoids are being intensively investigated as treatment options to many chronic human pathological conditions, including cancer and diabetes (Zava and Duwe 1997; Potter et al. 1998; Caltagirone et al. 2000; Pouget et al. 2001; Greenwald 2004; Hou et al. 2004; Allister et al. 2005; McDougall et al. 2005; Popiolkiewicz et al. 2005). For example, using diabetic mice as model systems, the glucosylated flavanone hesperidin and the flavanone naringenin were found to be highly effective at improving lipid metabolism by altering hepatic enzyme activities while at the same time lowering blood sugar levels through the down-regulation of the hepatic GLUT2 and glucose-6phosphotase and simultaneously up-regulating the hepatic glucokinase and adipocyte GLUT4 (Ae Park et al. 2006). While this study and numerous others are promising, flavonoid availability in humans is a concerning problem due to diets, in many parts of the world, that are insufficient in the amount of fruits and vegetables ingested and therefore preventing sufficient flavonoid consumption (Birt et al. 2001). Thus, as a result of low consumption rates and to a lesser degree low flavonoid concentrations in planta, the commercialization of a large-scale production platform becomes more important for using flavonoids as nutraceutical supplements. Flavanones, the direct precursors of the vast majority of flavonoids, are synthesized from the amino acid phenylalanine or tyrosine (Fig. 1). The process begins with the
800 Fig. 1 Detailed biosynthetic steps for flavanones and the diversification of flavonoids. The three successive carbons added from malonyl-CoA by CHS are shown in green, red, and blue. The R-groups denote the hydroxylation patterns for the natural flavonoid compounds although unnatural substitutions can be performed at these positions. Abbreviations are in text or as follows: DFR dihydroflavanone reductase, LAR leucoanthocynanidin reductase, ANS anthocyanidin synthase, 3GT uridine, flavanone 3-glucoside transferase, FSI flanone synthase, CHR chalcone reductase, IFS isoflavanone synthase, FHT flavanone hydroxytransferase, FLS flavonol synthase
Appl Microbiol Biotechnol (2009) 83:799–808 OH OH
PAL/TAL HOOC
NH2
HOOC
Tyrosine
C4H
p-Coumaric Acid OH
PAL OH HOOC
HOOC
NH2
HOOC
4CL
Cinnamic Acid
Phenylalanine
R1
Mal-CoA
R1
Caffeic acid
CHS
R2
R2
CoAS
CoAS O
O
Mal-CoA
CHS
CoA CO2
H2C
Acid-CoA Complex
CoA CO2
O
COSCoA
Malonyl-CoA
R1 R1
COOH
Mal-CoA OH
HO
R2
R2
CHS
CoAS O
O
OH
O
Chalcones
O
2 CoA CO2
CHR IFS
CHI
R3 O+
HO
OH OH
R2
OH
Anthocyanidins
FHT DFR
HO
R1 O
OH O
R2
Flavanones
3GT
O
IFS
R2
LAR ANS
HO
R1
O
FHT FLS
FSI
Isoflavones R3
R3 O+
HO
OH O
OH
HO
R2
Glc
Anthocyanin 3-O-glucosides
enzyme phenylalanine/tyrosine ammonia lyase (PAL/TAL), converting these amino acid building blocks into phenylpropanoic acids. The flavanone-biosynthetic pathway also includes a cytochrome-P450 enzyme, cinnamate 4hydroxylase (C4H), which adds a 4′-hydroxyl group to the phenylalanine aromatic ring. The CoA esters are subsequently synthesized from phenylpropanoic acids by the action of phenylpropanoyl-CoA ligases, such as 4coumaryl:CoA ligase (4CL). The type III polyketide synthase chalcone synthase (CHS) then catalyzes the sequential condensation of three malonyl-CoA moieties with one CoA-ester molecule to form chalcones. This is the first committed step into flavonoid biosynthesis, as alternative type III polyketide synthases exist exhibiting high homology to CHS (>70%) and using these same precursors to form stilbenes (using three malonyl-CoA units), benzy-
R1
O
HO
R2
OH O
Flavones
OH
O OH
R2
OH O
Flavonols
lacetolactone (using only one malonyl-CoA unit), and other aromatic molecules. While the flavonoid biosynthetic enzymes were originally thought to be only plant-derived, reports have appeared demonstrating the presence of type III polyketide synthases in microorganisms (Ueda et al. 1995; Moore and Hopke 2001; Kaneko et al. 2003a, b). The final flavanone structure is formed only when chalcones are stereospecifically isomerized into (2S)-flavanones by chalcone isomerase (CHI), a reaction known to occur spontaneously in alkali environments. After generation of the flavanone, a myriad of enzymes can act to functionalize and/or alter conformations of the three-ring phenylpropanoid core resulting in the generation of over 8,000 different chemical structures. Functionalization can manifest as hydroxylations, reductions, alkylations, oxidations, and glucosylations alone or in combinations.
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This functionalization leads to many of the biologically active properties seen for flavonoids (Table 1). In this review, we will focus our attention on efforts to achieve efficient flavanone biosynthesis in microorganisms, the first and core flavonoid structure. As such, we will explore the current state of the biotechnological production of flavanones by examining both the current methodologies for attaining flavanones as well as the more recent bioengineering approaches. As such, we will place an emphasis on not only the introduction of the plant biosynthetic pathway into the host cell but also on any redesigning of its native metabolism.
Production in practice With interest rising due to their wide application potential toward a number of consumer products, developments in chemical and biotechnical methods for flavonoid synthesis have occurred. While many groups have demonstrated the feasibility of efficient production of a number of these plant-derived natural products through chemical synthesis (Furlong and Nudelman 1985; Tanaka et al. 2000; Kumazawa et al. 2001; Lim et al. 2001; Wan and Chan 2004; Tanaka et al. 2007), the use of harsh or toxic chemical solvents has limited this methodology to specialized small quantity production. More importantly, chemical synthesis of flavanones, like many other aromatic products, yields a mixture of two stereoisomers. This is a critical flaw since only the (2S)-flavanones have been shown to be biologically active (Andersen and Markham 2006). Since
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the use of flavonoids in general has been as supplements, plant extraction has been the dominant form of isolation for these diverse nutraceuticals providing a process that requires only minimal processing efforts. However, plant extraction also yields a mixture of various substituted flavonoids; thus generating an extract containing only one class, let alone a single substitution pattern, is challenging task that would require countless purification techniques adding to economical cost and environmental impacts. Alternatively, bioreactor-based systems for mass production of flavonoids from plant cell cultures have been described for a few species (Zhong et al. 1991; Kobayashi et al. 1993), but to date, economic feasibility has not been established, partly because of engineering challenges in large-scale cultivation. One major challenge is that plant cells tend to form aggregates that influence culture productivity (Hanagata et al. 1993) since cells within aggregates are not adequately exposed to the required lighting needed to induce flavonoid biosynthesis by plant tissue. For example, expression of PAL, a key enzyme in the biosynthetic pathway, is promoted in plants primarily by UV wavelengths, particularly those of the UV-B region (Wellmann 1975), while other enzymes, particularly those of the anthocyanin biosynthetic branch, appear to be regulated in part by UV and in part by the phytochromeactivating wavelengths of 700–800 nm (Meyer et al. 2002). In that respect, irradiance becomes a limiting factor to productivity not only when excluded from cells at the interior of an aggregate (Hall and Yeoman 1986) but also when in a dense cell culture, at reduced cell dosing, or
Table 1 Current and potential application of flavonoids or flavonoid based drugs Flavonoid
Current/potential applications (disease)
Reference
Flavanones and chalcones
LDL oxidation (arthrosclerosis) Cyclooxygenase inhibitory ability (cancer) Malaria chemotherapy (malaria) Inflammation response trigger (reduce inflammation) GLUT inhibitor (diabetes) Cyclooxygenase inhibitory ability (cancer) GLUT inhibitor (diabetes) Pancreatic lipase inhibitors (diabetes) Inhibitors of cell cycle control kinases (cancer) Alpha-glucosidase inhibitor (diabetes) GLUT inhibitor (diabetes) Improves cholesterol regulation (diabetes) Promote cardiovascular health (heart disease) Pancreatic lipase and glucosidase inhibitor (diabetes) Regulate adipocyte function (obesity) Improves glucose and lipid metabolism (diabetes) Modulate blood hormone levels (multiple sclerosis)
(Miranda et al. 2000; Kumar et al. 2003; Kinghorn et al. 2004; Kontogiorgis et al. 2008; Mojzis et al. 2008)
Flavone Flavonols
Isoflavonoids
Catechins and Anthocyanins
(Kinghorn et al. 2004; Kwon et al. 2007; Chemler et al. 2009) (Park and Levine 2000; Nakai et al. 2005; Hsu and Yen 2006; Kwon et al. 2007; Chemler et al. 2009) (Kim et al. 2000; Song et al. 2002; Lee 2006; Kwon et al. 2007; Chemler et al. 2009)
(Shi et al. 1998; Kim et al. 2000; Tokimitsu 2004; Wolfram et al. 2006; Sternberg et al. 2008; Tsuda 2008)
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when the vessel wall composition selectively restricts certain wavelengths (Smith and Spomer 1995). Expression of flavonoid biosynthetic genes in transgenic plants has also been investigated for production of these ubiquitous secondary metabolites. In a recent study, CHI from Saussurea medusa was transformed into Nicotiana tabacum, a non-leguminous species, and resulted in an up to 5-fold total flavonoid production increase when compared to wild-type plants (Li et al. 2006). A similar method was later used in separate studies to introduce isoflavone synthase (IFS), a cytochrome-P450 monooxgenase using flavanone precursors, into tobacco plants (Yu et al. 2000) and soybeans (Yu et al. 2003). Even though the transgenic plants successfully synthesized isoflavonoids, the amounts were low due to competing pathways leading to the biosynthesis of other flavonoid classes. This diversion of metabolic flux and issues inherent with plant cell suspension cultures (Hellwig et al. 2004) are some of the reasons why until now no such system has been established for the commercial, large-scale production of flavonoids despite a more than 10-year effort in this field. The emerging bioreactor-based systems for flavonoid production have and will continue to be focused on the heterologous biosynthesis of these natural products using well-characterized recombinant hosts, including the yeast Saccharomyces cerevisiae and the gram-negative bacterium Escherichia coli. Although other microorganisms may in time be identified as more suitable hosts, the tractability of these two hosts inclines in their favor. In actuality, much of the recent characterization and understanding of mechanisms and control within the flavonoid biosynthetic pathway has been a result of studies using recombinant protein expression. More importantly, the use of microbial factories poses several significant advantages over chemical synthesis or plant cell cultures, including rapid growth rates, ease of cultivation, and the availability of convenient genetic manipulation techniques. All these are factors that enable high-production levels to be maintained from continuous fermentative cultures. Moreover, this enzymebase production strategy enables increased product selectivity and reduced use in both toxic chemicals and energy.
Flavanone biosynthesis in microorganisms Engineering of the biosynthesis of flavanones has rapidly evolved in the last 6 years from the simple expression of the plant enzymes in a host to entail more advanced engineering strategies that improve factors such as precursor levels and expression stability. While yeast may be a more suitable expression platform, as we will discuss, a vast majority of the major biotechnological developments have been introduced into E. coli expression systems for
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different metabolic configurations (Fig. 2) and yielding a variety of production levels (Table 2). Nevertheless, it is important to consider that both systems have been developed cocurrently and have highlighted some important characteristics of phenylpropanoid biosynthesis. As more information about the specific flavonoid enzymes becomes available, achieving highly efficient production strategies will require the selection of optimal combinations in which to cluster the expressed proteins. More specifically, it has been noted already that there exists a great variability and flexibility in the types of activities phenylpropanoid biosynthetic genes have toward accepting different substrates (Jiang et al. 2005; Katsuyama et al. 2007). In fact, a recent study used the broad substrate specificity of Petroselinum crispum 4CL, Petunia X hybrida CHS, and Medicago sativa CHI to create a library of unnatural flavanones (Chemler et al. 2007). E. coli With a plethora of molecular biological techniques at hand and a history of a robust ability for recombinant expression, E. coli is traditionally the first choice as a production platform in the biotech industry. As such, it is not surprising that the first attempt to afford plant-specific flavanone biosynthesis was in E. coli. It was accomplished through the simultaneous expression of plant biosynthetic enzymes, namely PAL from Rhodotorula rubra, 4CL from Streptomyces coelicolor A3(2), and CHS from Glycyrrizha echinata using three different vector constructs with each construct varying the number and location of promoters and ribosome binding sites (RBS) for each gene. While an optimal pET expression vector construct consisting of both strong T7 promoters (PT7) and RBS sites before each gene produced the highest levels of production (Hwang et al. 2003; Kaneko et al. 2003a, b), the titers were still considerably low and thus would prohibit large-scale synthesis. Later, a similar strategy was employed to recombinantly express PAL, C4H, 4CL, and CHS from the model plant Arabidopsis thaliana (Watts et al. 2004). However, the C4H was inactive, therefore limiting production of naringenin to cultures supplemented with exogenous p-coumaric acid. In the end, a newly cloned and partially characterized TAL from Rhodobacter sphaeroides was used to generate naringenin from tyrosine-supplemented cultures resulting in production levels that reached 20.8 mg/L (Watts et al. 2004). Interestingly, the authors in both studies relied on alkali conditions for the conversion of the chalcone to the flavanone; thus, the inclusion of a chalcone isomerase could have significantly improved production titers. In fact, more recent studies have not only included the presence of a chalcone isomerase but also undergone a
Appl Microbiol Biotechnol (2009) 83:799–808 Fig. 2 Biosynthetic pathways used for engineering flavanone biosynthesis. Glucose, or another simple sugar, is metabolized into the major precursors for flavanone production. The multifunctional acetyl-CoA carboxylase requiring the action of biotin ligase is also shown. Engineered pathways discussed in the text are shown in orange while flavanone synthesis is in purple
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Extracellular
Cellular
CoaA ADP + Pi
Glucose (x)
PAL / TAL
Aromatic Amino Acids
Substrate (x)
Glucose
ATP + HCO3-
Esters
BirA
AccA AccD
Table 2 Engineered biosynthetic clusters for flavanone production in recombinant E. coli and yeast
Fatty Acids MatB
Expressed in E. coli Rhodotorula rubra PAL Streptomyces coelicolor A3(2) 4CL Glycyrrhiza echinata CHS Arabidopsis thaliana TAL, 4CL, CHS Rhodotorula rubra PAL Streptomyces coelicolor A3(2) 4CL Glycyrrhiza echinata CHS
a
Catalytic enzymes abbreviations: PAL, phenylalanine ammonia lyase; TAL, tyrosine ammonia lyase; 4CL, coumarate:coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; ACC; acetyl-coenzyme A carboxylase; BPL, biotin ligase; ACS, acetate synthase
b
Production conditions for pinocembrin (P), eriodictyol (E), and naringenin (N) vary between studies
Flavanones
Productionb (mg/L) P
+ Photorhabdus luminescens BPL + Escherichia coli ACS Petroselinum crispum 4CL Petunia X hybrida CHS Medicago sativa CHI Rhizobium trifolii MatB/MatC Expressed in S. cerevisiae Rhodosporidium toriloides PAL Arabidopsis thaliana 4CL Hypericum androsaemum CHS Petroselinum crispum 4CL Petunia X hybrida CHS, CHI Arabidopsis thaliana C4H
CHI
Malonate
Recombinant gene clustera
Pueraria lobata CHI Corynebacterium glutamicum ACC Petroselinum crispum 4CL Petunia X hybrida CHS Medicago sativa CHI Photorhabdus luminescens ACC
CHS
Chalcones
Acetate MatC
Biotin ATP
Malonyl -CoA
acs
Malonate (x)
4CL
PPi apoBCCP AMP apoBirA Propanoyl-CoA
holoBCCP
Acetyl -CoA
C4H
Coenzyme A
AccC holoBCCP*
Propanoic Acids
E
Reference N
(Hwang et al. 2003) 0.75 –
58
– –
0.45 20.8
–
60
(Watts et al. 2004) (Miyahisa et al. 2005)
(Leonard et al. 2007)
367 429
50 52
69 119 (Leonard et al. 2008)
480
50
155 (Jiang et al. 2005)
0.8
–
7 (Yan et al. 2005)
16.3
6.5
28.3
804
more extensive metabolic engineering effort to attain production-scale quantities of flavanones in E. coli. Specifically, various strategies were explored that amplify malonyl-CoA, the rate-limiting metabolite in E. coli, which serves as a flavonoid building block. The first approach was the most rational in design. Since malonyl-CoA is synthesized directly from acetyl-CoA by the enzyme acetyl-CoA carboxylase (ACC), its overexpression in E. coli was proposed to improve flavanone synthesis levels (Miyahisa et al. 2005). However, overexpression of E. coli’s native ACC has been reported to be detrimental to cell viability (Davis et al. 2000). To bypass this phenomenon, a plasmid harboring an artificial biosynthetic gene cluster that included PAL from a yeast, a cinnamate/coumarate:CoA ligase from S. coelicolor A3(2), licorice CHS, and Pueraria CHI along with a separate vector containing the two subunit genes of ACC from Corynebacterium glutamicum were expressed in the recombinant E. coli cells. The coexpression of these two vectors improved production titers to 60 mg/L of (2S)-naringenin (Miyahisa et al. 2005). In a related study, the four subunits, accABCD, of Photorhabdus luminescens ACC, were cloned with accA and accD individually controlled under separate PT7, while the transcription of the accBC operon was regulated under another PT7. Combining this strategy with the expression of P. crispum 4CL, P. hybrida CHS, and M. sativa CHI permitted flavanone synthesis to reach 196 mg/L of pinocembrin and 67 mg/L of naringenin. Since the functionality of the carboxylase domain of ACC requires biotinylation by the action of biotin ligase (BPL; encoded by birA), overexpression in concert with ACC overexpression was also investigated. Interestingly, BPL is known to recognize ACCs across species; therefore, the recombinant system was tested with the endogeneous E. coli BPL, the BPL from P. luminescens, and a chimeric BPL containing the N-terminus from E. coli and the C-terminus of P. luminescens. It was found that coexpression of ACC and BPL both from P. luminescens resulted in improved flavanone yields of up to 367 mg/L of pinocembrin (Leonard et al. 2007), although naringenin production only increased marginally to 69 mg/L. These results suggested that the protein interaction between ACC and BPL was important for optimum malonyl-CoA synthesis. The improved flavanone synthesis by coexpression of ACC and BPL also depended on biotin supplementation to the fermentation broth, a technique not viable for largescale synthesis especially considering the cost of biotin as an additive. As such, further metabolic engineering of the acetate pool was also explored to allow the conversion of inexpensive metabolites into high-value flavanones. In this case, the amplification of two acetate assimilation pathways in E. coli was explored, specifically the acetate kinase (ACK) and phosphotransacetylase (PTA) pathway and the
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acetyl-CoA synthetase (ACS) pathway (Leonard et al. 2007). While a strain harboring overexpressions for ACK and PTA, together with ACC only, resulted in only a moderate increase in flavanone synthesis, the coexpression of ACC with ACS resulted in flavanone production up to 383 mg/L of pinocembrin along with a decrease in acetate accumulation. Exogenous supplementation of acetate at a low level further improved flavanone synthesis to 429 mg/L of pinocembrin (Leonard et al. 2007). It is quite clear that the pool of intracellular malonyl-CoA is undersized for large-scale biosynthesis of flavanones from E. coli. Therefore, in order to increase malonyl-CoA levels, the culture supplementation using cheep exogenous sources of carbon was explored. For example, the identified malonate transport and assimilation pathway that forms malonyl-CoA by genes matB and matC from Rhizobium trifolii (An and Kim 1998) was proposed as a way to directly increase malonyl-CoA. Specifically, this malonate assimilation pathway was simultaneously overexpressed with the flavanone-biosynthetic genes 4CL, CHS, and CHI while supplementing with exogenous malonate. This resulted in production titers of 480 mg/L of pinocembrin and 155 mg/L of naringenin (Leonard et al. 2008). In an effort to retard the native degradation pathways of malonyl-CoA, the specific fatty acid inhibitor cerulenin was added to fermentations of E. coli strains harboring the flavanone-biosynthetic pathway. Doing so induced a dosedependent response of flavanone production with the maximum pinocembrin production of 710 mg/L accomplished when FabB/F was repressed using 0.2 mM of cerulenin (Leonard et al. 2008). Despite the near gram per liter productivity, the excessive cost of cerulenin makes this strategy ill-suited for an industrial process; therefore, an alternative strategy that focuses on re-routing native metabolic carbon flows to improve precursor and cofactor accessibility maybe more suitable for increasing production levels. Such an approach might utilize stoichiometric modeling (more commonly flux balance analysis) to identify unique and non-intuitive gene deletions within the host genotype, thereby significantly expanding the carbon flows from glucose into the metabolite pools needed for flavonoid biosynthesis. While flux balance analysis has been used to investigate the theoretical constraints of numerous metabolic systems, few examples of its practical application exist in the literature (Alper et al. 2005a, b; Fong et al. 2005). Recently, however, this approach to simultaneously balance carbon flows into the cofactor pools was employed to great success for flavanone biosynthesis (Fowler et al. 2009). It was shown that a stochastic model of E. coli metabolism, which also included the pathways for flavanone biosynthesis, could effectively identify gene deletions and overexpression targets required to increase availability
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of malonyl-CoA and other biosynthetic cofactors leading to improved production of flavanones (Fowler et al. 2009). To understand the connectivity of the metabolic network, an optimization routine termed CiED was utilized to evolve in silico strains and identify gene deletion targets leading to improved flavanone production. The authors demonstrated 3-fold increases in cellular malonyl-CoA levels in the mutant genotypes constructed from CiED predictions after which the maximum production of flavanones increased from 69 mg/L to in excess of 210 mg/L. By including overexpressions also predicted from the model, and the previously mentioned ACS, ACC, and BPL overexpressions, production levels rose to 270 mg/L of the flavanone naringenin (Fowler et al. 2009).
S. cerevisiae S. cerevisiae offers a number of distinct advantages over E. coli. Perhaps the one most important to commercial implementation is that the United States Food and Drug Administration classifies yeast as a GRAS organism, thus recognizing it as a safe production method for synthesis of consumed goods. On the other hand, as a eukaryote, yeast, has the ability to support functional expression of membrane-bound cytochrome P450 enzymes. P450 enzymes require the attachment to the eukaryotic cell’s endoplasmic reticulum (ER) membrane and a redox partner typically in the form of a P450 reductase that transports electrons from the NADPH donor to the heme-core of the P450 complex. More importantly, there are a number of flavonoid biosynthetic enzymes, including the C4H, which are of the P450 enzyme class. This feature, together with the expectation of better expression of the plant-derived enzymes due to the capability of posttranslational modifications and its phylogenetical similarity to plants, makes yeast an attractive host platform for flavonoid biosynthesis. The first time enzymes of the phenylpropanoid pathway, namely PAL and C4H, were positive expressed in S. cerevisiae was done by Ro and Douglas together with a cytochrome P450 reductase to aid the activity of the C4H monoxygenase (Ro and Douglas 2004). In this work, not only was the ability of yeast to express the flavonoid biosynthetic pathway demonstrated but the formation of metabolons or complexes between the two phenylpropanoid enzymes was also investigated. In their work, using radio-labeled substrates, the others showed that efficient carbon flux from phenylalanine to coumarate is not facilitated by the formation of metabolons as suspected, but rather by a biochemical coupling of the two enzymes (Ro and Douglas 2004). While production levels were fairly low—approximately 1.2 μmol of phenylpropanoids—the functional expression of the cytochrome P450s was permitted.
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A later study looked to enhance production levels using two engineered yeast strains along with a stronger GAL10 promoter system (Jiang et al. 2005). While only expressing a PAL from Rhodosporidium toriloides, 4CL from A. thaliana, and CHS from Hypericum androsaemum, the production levels reached nearly 7 mg/L of naringenin and 0.8 mg/L of pinocembrin. Interestingly, a deletion of PAD1 in one of the yeast strains caused a surprisingly different PAL (TAL) behavior that produced lower levels of flavanones, though with a different distribution. While the low 4CL activity toward trans-cinnamic acid may be attributed to the A. thaliana 4CLs Km value (6.3 mM), it was notably lower in the PAD1 mutant than in the parental strain, possibly due to the Pad1P decarboxylase (or another endogenous enzyme) out-competing 4CL for the transcinnamic acid (Jiang et al. 2005). In related study, the synthesis of the flavonoid naringenin from the phenylpropanoic acids was afforded in a recombinant S. cerevisiae strain expressing a P. crispum 4CL, P. hybrida CHS and CHI, and the P450 C4H from A. thaliana (Yan et al. 2005). Although this work used a similar GAL1 promoter system within a commercial yeast strain having a similar genetic background, production levels were increased 62% for naringenin and 22% for pinocembrin over the previous study. An extension of this work, also performed in yeast, involved the biosynthesis of the flavones that are derivatives of the flavanones (Leonard et al. 2005), which are known to have potent medicinal properties (Caltagirone et al. 2000).
Future challenges in flavonoid biosynthesis To achieve efficient production platforms of flavanones or flavonoids in general, improving the availability of starting materials used during the biosynthetic process is of chief concern. While many of the early studies resorted to the more practical approach of substrate feeding to improve the intracellular concentrations of the aromatic amino acids in an effort to increase production levels, the recent studies have bypassed the conversion of aromatics to the phenylpropanoic acids. This is because phenylpropanoid acids are readily available from abundant, renewable sources such as, lignin, one of the most abundant organic polymers in nature (Boerjan et al. 2003). The three typical monolignols used within the backbone of lignin are p-coumaryl, coniferyl, and sinapyl alcohols (Martone et al. 2009). A number of recent studies have investigated a variety of fungal enzymes linked with the degradation of plant matter to identify esterases and laccases that can be used to carry out the bioconversion of lignin (Bergbauer and Eggert 1994; Leonowicz et al. 2001; Benoit et al. 2006; Latha et al. 2007; Guglielmetti et al. 2008) to fermentable substrates
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and starting materials for flavonoid biosynthesis. As more degradation pathways are elucidated, other cellulostic materials may also have the potential to act as substrates for production of flavonoids. One of the biggest challenges however for engineering the flavonoid (and many other phytochemical’s) biosynthetic pathway in E. coli is the functional expression of ER-associated plant cytochromic P450 monoxygenases. Due to requirements of P450 systems, functional expression of these enzymes in E. coli is a daunting task since E. coli lacks ER. Efforts to engineer a soluble P450 monooxygenase have generally resulted in enzymes with very low solubility and formation of inclusion bodies resulting in extensive cell lysis, especially in cases with high expression levels. Such was the case for the generation of an active flavonoid 3′5′-hydroxylase (F3′5′H) derived from Catharanthus roseus for the biosynthesis of hydroxylated flavonols such as quercetin and myricetin (Leonard et al. 2006). Both the F3′5′H and a P450-reductase from C. roseus (the enzyme responsible for transferring electrons from NADPH to the P450) were modified by either removing terminal codons and/or changing existing codons in the N- and C-terminus regions then fused together using a linker sequence with no preference for secondary structure formation. When the engineered chimera was expressed in E. coli together with a grafted flavanone-biosynthetic pathway, small amounts of quercetin could be recovered from the culture media (Leonard et al. 2006). Recently, a series of artificial isoflavone synthases were designed that allowed robust production by E. coli (Leonard and Koffas 2007). In this work, by assembling the overall architecture to mimic that of a self-sufficient bacterial P450s and at the same time including tailor-made membrane recognition signals, one identified chimera had up to 20-fold higher in vivo production than that achieved by the native enzyme expressed in a eukaryotic host. Nevertheless, cell lysis was evident, indicating that there are still several other parameters, including the use of weaker promoters and lower copy number plasmids, that need to be tuned for biocatalysis optimization. Other challenges have still yet to be fully investigated, including the selection of the right cluster of flavonoid genes to allow a high kinetic turnover (and possibly a broad substrate specificity); codon optimization of recombinant plant proteins; and identification of suitable promoters for achieving optimal, as opposed to maximal, expression levels of the recombinant proteins. Additionally, methods that modify the flavanone-biosynthetic genes in order to improve the catalytic activity or relieve product inhibition, are viable directions for achieving production-scalable titers. These issues provide golden opportunities for the development of new methodologies and technologies in
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metabolic engineering that can be utilized in several other metabolic engineering projects related to natural product biosynthesis.
References Ae Park S, Choi MS, Cho SY, Seo JS, Jung UJ, Kim MJ, Sung MK, Park YB, Lee MK (2006) Genistein and daidzein modulate hepatic glucose and lipid regulating enzyme activities in C57BL/ KsJ-db/db mice. Life Sci 79:1207–1213 Allister EM, Borradaile NM, Edwards JY, Huff MW (2005) Inhibition of microsomal triglyceride transfer protein expression and apolipoprotein B100 secretion by the citrus flavonoid naringenin and by insulin involves activation of the mitogenactivated protein kinase pathway in hepatocytes. Diabetes 54:1676–1683 Alper H, Jin YS, Moxley JF, Stephanopoulos G (2005a) Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metab Eng 7:155–164 Alper H, Miyaoku K, Stephanopoulos G (2005b) Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat Biotechnol 23:612–616 An JH, Kim YS (1998) A gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putative dicarboxylate carrier protein (MatC) in Rhizobium trifolii—cloning, sequencing, and expression of the enzymes in Escherichia coli. Eur J Biochem 257:395–402 Andersen OM, Markham KR (eds) (2006) Flavonoids: chemistry, biochemistry, and applications. CRC, Boca Raton Benoit I, Navarro D, Marnet N, Rakotomanomana N, Lesage-Meessen L, Sigoillot JC, Asther M (2006) Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial byproducts. Carbohydr Res 341:1820–1827 Bergbauer M, Eggert C (1994) Degradability of chlorine-free bleachery effluent lignins by two fungi: effects on lignin subunit type and on polymer molecular weight. Can J Microbiol 40:192–197 Birt DF, Hendrich S, Wang W (2001) Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol Ther 90:157–177 Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546 Caltagirone S, Rossi C, Poggi A, Ranelletti FO, Natali PG, Brunetti M, Aiello FB, Piantelli M (2000) Flavonoids apigenin and quercetin inhibit melanoma growth and metastatic potential. Int J Cancer 87:595–600 Chemler JA, Yan Y, Leonard E, Koffas MA (2007) Combinatorial mutasynthesis of flavonoid analogues from acrylic acids in microorganisms. Org Lett 9:1855–1858 Chemler JA, Fowler ZL, Koffas MA, Leonard E (2009) Trends in microbial synthesis of natural products and biofuels. Adv Enzymol Relat Areas Mol Biol 76:151–217 Davis MS, Solbiati J, Cronan JE Jr (2000) Overproduction of acetylCoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli. J Biol Chem 275:28593–28598 Fong SS, Burgard AP, Herring CD, Knight EM, Blattner FR, Maranas CD, Palsson BO (2005) In silico design and adaptive evolution of Escherichia coli for production of lactic acid. Biotechnol Bioeng 91:643–648 Forkmann G, Martens S (2001) Metabolic engineering and applications of flavonoids. Curr Opin Biotechnol 12:155–160
Appl Microbiol Biotechnol (2009) 83:799–808 Fowler ZL, Gikandi WW and Koffas M (2009) Increasing malonylCoA biosynthesis by tuning the Escherichia coli metabolic network and its application to flavanone production. Appl Environ Microbiol (in press). Furlong JJP, Nudelman NS (1985) Mechanism of cyclization of substituted 2'-hydroxychalcones to flavanones. J Chem Soc Perkin Trans 2:633–639 Greenwald P (2004) Clinical trials in cancer prevention: current results and perspectives for the future. J Nutr 134:3507S–3512S Guglielmetti S, De Noni I, Caracciolo F, Molinari F, Parini C, Mora D (2008) Bacterial cinnamoyl esterase activity screening for the production of a novel functional food product. Appl Environ Microbiol 74:1284–1288 Hall RD, Yeoman MM (1986) Temporal and spatial heterogeneity in the accumulation of anthocyanins in cell-cultures of Catharanthusroseus (L) Don, G. J Exp Bot 37:48–60 Hanagata N, Ito A, Uehara H, Asari F, Takeuchi T, Karube I (1993) Behavior of cell aggregate of Carthamus-tinctorius L culturedcells and correlation with red pigment formation. J Biotechnol 30:259–269 Harborne JB, Williams CA (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504 Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422 Hou DX, Fujii M, Terahara N, Yoshimoto M (2004) Molecular mechanisms behind the chemopreventive effects of anthocyanidins. J Biomed Biotechnol 5:321–325 Hsu CL, Yen GC (2006) Induction of cell apoptosis in 3T3–L1 preadipocytes by flavonoids is associated with their antioxidant activity. Mol Nutr Food Res 50:1072–1079 Hwang EI, Kaneko M, Ohnishi Y, Horinouchi S (2003) Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster. Appl Environ Microbiol 69:2699–2706 Jiang H, Wood KV, Morgan JA (2005) Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Appl Environ Microbiol 71:2962–2969 Kaneko M, Hwang EI, Ohnishi Y, Horinouchi S (2003a) Heterologous production of flavanones in Escherichia coli: potential for combinatorial biosynthesis of flavonoids in bacteria. J Ind Microbiol Biotechnol 30:456–461 Kaneko M, Ohnishi Y, Horinouchi S (2003b) Cinnamate:coenzyme A ligase from the filamentous bacterium streptomyces coelicolor A3(2). J Bacteriol 185:20–27 Katsuyama Y, Funa N, Horinouchi S (2007) Precursor-directed biosynthesis of stilbene methyl ethers in Escherichia coli. Biotechnol J 2:1286–1293 Kim JS, Kwon CS, Son KH (2000) Inhibition of alpha-glucosidase and amylase by luteolin, a flavonoid. Biosci Biotechnol Biochem 64:2458–2461 Kinghorn AD, Su BN, Jang DS, Chang LC, Lee D, Gu JQ, CarcacheBlanco EJ, Pawlus AD, Lee SK, Park EJ, Cuendet M, Gills JJ, Bhat K, Park HS, Mata-Greenwood E, Song LL, Jang M, Pezzuto JM (2004) Natural inhibitors of carcinogenesis. Planta Med 70:691–705 Kobayashi Y, Akita M, Sakamoto K, Liu HF, Shigeoka T, Koyano T, Kawamura M, Furuya T (1993) Large-scale production of anthocyanin by Aralia-cordata cell-suspension cultures. Appl Microbiol Biotechnol 40:215–218 Kontogiorgis C, Mantzanidou M, Hadjipavlou-Litina D (2008) Chalcones and their potential role in inflammation. Mini Rev Med Chem 8:1224–1242 Kumar A, Katiyar SB, Agarwal A, Chauhan PM (2003) Perspective in antimalarial chemotherapy. Curr Med Chem 10:1137–1150 Kumazawa T, Kimura T, Matsuba S, Sato S, Onodera J (2001) Synthesis of 8-C-glucosylflavones. Carbohydr Res 334:183–193
807 Kwon O, Eck P, Chen S, Corpe CP, Lee JH, Kruhlak M, Levine M (2007) Inhibition of the intestinal glucose transporter GLUT2 by flavonoids. FASEB J 21:366–377 Latha GM, Srinivas P, Muralikrishna G (2007) Purification and characterization of ferulic acid esterase from malted finger millet (Eleusine coracana, Indaf-15). J Agric Food Chem 55:9704–9712 Lee JS (2006) Effects of soy protein and genistein on blood glucose, antioxidant enzyme activities, and lipid profile in streptozotocininduced diabetic rats. Life Sci 79:1578–1584 Leonard E, Koffas MA (2007) Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli. Appl Environ Microbiol 73:7246–7251 Leonard E, Yan Y, Lim KH, Koffas MA (2005) Investigation of two distinct flavone synthases for plant-specific flavone biosynthesis in Saccharomyces cerevisiae. Appl Environ Microbiol 71:8241– 8248 Leonard E, Chemler J, Lim KH, Koffas MA (2006) Expression of a soluble flavone synthase allows the biosynthesis of phytoestrogen derivatives in Escherichia coli. Appl Microbiol Biotechnol 70:85–91 Leonard E, Lim KH, Saw PN, Koffas MA (2007) Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl Environ Microbiol 73:3877–3886 Leonard E, Yan Y, Fowler ZL, Li Z, Lim CG, Lim KH, Koffas MA (2008) Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids. Mol Pharm 5:257–265 Leonowicz A, Cho NS, Luterek J, Wilkolazka A, Wojtas-Wasilewska M, Matuszewska A, Hofrichter M, Wesenberg D, Rogalski J (2001) Fungal laccase: properties and activity on lignin. J Basic Microbiol 41:185–227 Li F, Jin Z, Qu W, Zhao D, Ma F (2006) Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco. Plant Physiol Biochem 44:455–461 Lim SS, Jung SH, Ji J, Shin KH, Keum SR (2001) Synthesis of flavonoids and their effects on aldose reductase and sorbitol accumulation in streptozotocin-induced diabetic rat tissues. J Pharm Pharmacol 53:653–668 Martone PT, Estevez JM, Lu F, Ruel K, Denny MW, Somerville C, Ralph J (2009) Discovery of lignin in seaweed reveals convergent evolution of cell-wall architecture. Curr Biol 19:169–175 McDougall GJ, Shpiro F, Dobson P, Smith P, Blake A, Stewart D (2005) Different polyphenolic components of soft fruits inhibit alpha-amylase and alpha-glucosidase. J Agric Food Chem 53:2760–2766 Meyer JE, Pepin MF, Smith MA (2002) Anthocyanin production from Vaccinium pahalae: limitations of the physical microenvironment. J Biotechnol 93:45–57 Miranda CL, Stevens JF, Ivanov V, McCall M, Frei B, Deinzer ML, Buhler DR (2000) Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro. J Agric Food Chem 48:3876–3884 Miyahisa I, Kaneko M, Funa N, Kawasaki H, Kojima H, Ohnishi Y, Horinouchi S (2005) Efficient production of (2S)-flavanones by Escherichia coli containing an artificial biosynthetic gene cluster. Appl Microbiol Biotechnol 68:498–504 Mojzis J, Varinska L, Mojzisova G, Kostova I, Mirossay L (2008) Antiangiogenic effects of flavonoids and chalcones. Pharmacol Res 57:259–265 Moore BS, Hopke JN (2001) Discovery of a new bacterial polyketide biosynthetic pathway. Chembiochem 2:35–38 Nakai M, Fukui Y, Asami S, Toyoda-Ono Y, Iwashita T, Shibata H, Mitsunaga T, Hashimoto F, Kiso Y (2005) Inhibitory effects of oolong tea polyphenols on pancreatic lipase in vitro. J Agric Food Chem 53:4593–4598 Park JB, Levine M (2000) Intracellular accumulation of ascorbic acid is inhibited by flavonoids via blocking of dehydroascorbic acid
808 and ascorbic acid uptakes in HL-60, U937 and Jurkat cells. J Nutr 130:1297–1302 Popiolkiewicz J, Polkowski K, Skierski JS, Mazurek AP (2005) In vitro toxicity evaluation in the development of new anticancer drugs—genistein glycosides. Cancer Lett 229:67–75 Potter SM, Baum JA, Teng HY, Stillman RJ, Shay NF, Erdman JW (1998) Soy protein and isoflavones: their effects on blood lipids and bone density in postmenopausal women. Am J Clin Nutr 68:1375S–1379S Pouget C, Lauthier F, Simon A, Fagnere C, Basly JP, Delage C, Chulia AJ (2001) Flavonoids: Structural requirements for antiproliferative activity on breast cancer cells. Bioorg Med Chem Lett 11:3095–3097 Ro DK, Douglas CJ (2004) Reconstitution of the entry point of plant phenylpropanoid metabolism in yeast (Saccharomyces cerevisiae): implications for control of metabolic flux into the phenylpropanoid pathway. J Biol Chem 279:2600–2607 Shi HC, Huang QY, Yamaji R, Inui H, Fujita T, Miyatake K, Nakano Y, Tada T, Nishimura K (1998) Suppression by water extracts of Sophora plants of sucrose-induced hyperglycemia in rats and inhibition of intestinal disaccharidases in vitro. Biosci Biotechnol Biochem 62:1225–1227 Smith MAL, Spomer LA (1995) Vessels, gels, liquid media, and support systems. In: Aitken-Christie J, Kozai T, Smith MAL (eds) Automation and environmental control in plant tissue culture. Kluwer Academic, Dordrecht, pp 371–404 Song J, Kwon O, Chen SL, Daruwala R, Eck P, Park JB, Levine M (2002) Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and glucose transporter isoform 2 (GLUT2), intestinal transporters for vitamin C and glucose. J Biol Chem 277:15252–15260 Sternberg Z, Chadha K, Lieberman A, Hojnacki D, Drake A, Zamboni P, Rocco P, Grazioli E, Weinstock-Guttman B, Munschauer F (2008) Quercetin and interferon-beta modulate immune response (s) in peripheral blood mononuclear cells isolated from multiple sclerosis patients. J Neuroimmunol 205:142–147 Tanaka H, Stohlmeyer MM, Wandless TJ, Taylor LP (2000) Synthesis of flavonol derivatives as probes of biological processes. Tetrahedron Lett 41:9735–9739
Appl Microbiol Biotechnol (2009) 83:799–808 Tanaka H, Miyoshi H, Chuang YC, Ando Y, Takahashi T (2007) Solid-phase synthesis of epigallocatechin gallate derivatives. Angew Chem Int Ed Engl 46:5934–5937 Tokimitsu I (2004) Effects of tea catechins on lipid metabolism and body fat accumulation. Biofactors 22:141–143 Tsuda T (2008) Regulation of adipocyte function by anthocyanins; possibility of preventing the metabolic syndrome. J Agric Food Chem 56:642–646 Ueda K, Kim KM, Beppu T, Horinouchi S (1995) Overexpression of a gene cluster encoding a chalcone synthase-like protein confers redbrown pigment production in Streptomyces griseus. J Antibiot (Tokyo) 48:638–646 Wan SB, Chan TH (2004) Enantioselective synthesis of afzelechin and epiafzelechin. Tetrahedron 60:8207–8211 Watts KT, Lee PC, Schmidt-Dannert C (2004) Exploring recombinant flavonoid biosynthesis in metabolically engineered Escherichia coli. Chembiochem 5:500–507 Wellmann E (1975) UV dose-dependent induction of enzymes related to flavonoid biosynthesis in cell suspension cultures of parsley. FEBS Lett 51:105–107 Wolfram S, Raederstorff D, Preller M, Wang Y, Teixeira SR, Riegger C, Weber P (2006) Epigallocatechin gallate supplementation alleviates diabetes in rodents. J Nutr 136:2512–2518 Yan YJ, Kohli A, Koffas MAG (2005) Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Appl Environ Microbiol 71:5610–5613 Yu O, Jung W, Shi J, Croes RA, Fader GM, McGonigle B, Odell JT (2000) Production of the isoflavones genistein and daidzein in nonlegume dicot and monocot tissues. Plant Physiol 124:781–794 Yu O, Shi J, Hession AO, Maxwell CA, McGonigle B, Odell JT (2003) Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63:753–763 Zava DT, Duwe G (1997) Estrogenic and antiproliferative properties of genistein and other flavonoids in human breast cancer cells in vitro. Nutr Cancer 27:31–40 Zhong JJ, Seki T, Kinoshita S, Yoshida T (1991) Effect of light irradiation on anthocyanin production by suspended culture of Perilla-frutescens. Biotechnol Bioeng 38:653–658
