Xinxiao Sun,Xianglai Li,Xiaolin Shen,Jia Wang,Qipeng Yuan*

State Key Laboratory of Chemical Resource Engineering,Beijing University of Chemical Technology,Beijing 100029,China

ABSTRACT Phenolic compounds(PCs)are a group of compounds with various applications in nutraceutical,pharmaceutical and cosmetic industries.Their supply by plant extraction and chemical synthesis is often limited by low yield and high cost.Microbial production represents as a promising alternative for efficient and sustainable production of PCs.In this review,we summarize recent advances in this field,which include enzyme mining and engineering to construct artificial pathways,balance of enzyme expression to improve pathway efficiency,coculture engineering to alleviate metabolic burden and side-reactions,and the use of genetic circuits for dynamic regulation and high throughput screening.Finally,current challenges and future perspectives for efficient production of PCs are also discussed.

Keywords:Phenolic compounds Pathway design Protein engineering Coculture engineering Modular pathway optimization Dynamic regulation

1.Introduction

Phenolic compounds (PCs) comprise a diverse group of plant secondary metabolites,and can be categorized into polyphenols,phenolic acids,phenylpropanoids,flavonoids,stilbenes,coumarins and their derivatives(e.g.esters,glycosides and polymers)[1].PCs possess the aromatic ring(s)bearing one or more hydroxyl groups,and exhibit multifaceted activities (antioxidation,antiinflammation,anti-tumor,antiviral,anti-bacteria and so on) [2,3].Due to these health-beneficial properties,PCs are widely used in nutraceutical,pharmaceutical and cosmetic industries.

Currently,PCs are mainly manufactured by either plant extraction or chemical synthesis.However,the plant extraction process often encounters bottlenecks such as low yield,fluctuation of active ingredients caused by seasonal/climatic variations,and over-exploitation of plant resources,and thus is difficult to fulfill the growing demands.Chemical synthesis also faces problems such as expensive precursors,inadequate regio-and stereoselectivity,use of toxic catalysts and harsh reaction conditions.Therefore,engineering microbial cell factories has become a promising alternative approach to produce these high-value PCs.

PCs like other aromatic compounds are generally biosynthesized through the shikimate pathway.This pathway consists of seven enzymatic steps that convert the precursors erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to chorismate.The involved enzymes and the underlying regulation mechanism are well elucidated,and numerous studies have been conducted to boost the shikimate pathway for the production of various aromatic compounds by metabolic strategies like increasing the supply of PEP and E4P,alleviating feedback inhibition of endproducts on upstream enzymes,and blocking/weakening the competing pathways.These strategies are well summarized already[4–6]and thus will not be the emphasis of this review.

The rapid expansion of available information on plant genomes and transcriptomes has accelerated enzyme mining and identification of the biosynthetic pathways from the native species.However,so far there are many PCs whose native pathways are still elusive.Fortunately,artificial pathways can be designed by mining genes from different species based on the bioinformatics databases.To meet the industrial application requirements,the titer,yield and productivity of the recombinant microorganisms need to be improved.To this end,the advancements in protein engineering,metabolic engineering and synthetic biology provide powerful tools to remove obstacles in the construction of microbial cell factories to produce PCs.In this review,we summarize the recent progress on the design of artificial pathways for the biosynthesis of PCs,and the strategies used to improve the production efficiency,which include enzyme mining and engineering,modular pathway assembly and optimization,coculture engineering,and dynamic regulation of carbon flux (Table 1).

2.Enzyme Mining to Construct Artificial Pathways

To achieve heterologous production of a PC,a straightforward way is to reconstruct the entire native pathway in a genetic tract-able microorganism.However,in many cases the native pathway may be partially characterized or even completely unknown.Thanks to the enormous enzyme information documented in the databases as BRENDA,one can dig up alternative enzymes from other organisms to fill the gap(s)in a pathway,or even design artificial pathways by connecting the target product with the native metabolismviaseveral enzymatic steps.

Table 1 Production of some representative phenolic compounds in metabolic engineered microbes

Poor expression of pathway enzymes in a heterologous host would generate metabolic bottlenecks limiting the flux to the product.For example,due to the lack of inner membrane structures and the post-translational modification mechanisms,combined with the bias in codon usage,prokaryotic microorganisms such asEscherichia coliare not competent to express some plant enzymes.Moreover,low enzyme activity toward non-natural substrates can also limit the holistic efficiency of a pathway.To tackle these obstacles,structure-based protein engineering or random mutagenesis followed by high-throughput screening has been applied for the microbial synthesis of a variety of PCs as described in the following.

Hydroxytyrosol(HT)is a PC with strong antioxidant activity and exhibits diverse health-beneficial effects.HT mostly exists in olives as a product of oleuropein degradation.To achieve HT biosynthesis,several pathways have been designed,which are extended from tyrosine or its precursor 4-hydroxyphenylpyruvate (4-HPP)(Fig.1).In one pathway,4-HPP is decarboxylated and reduced to tyrosol by the sequential catalysis of ketoacid decarboxylase(KDC) and alcohol dehydrogenase (ADH).Further,tyrosol is converted to HT by the promiscuous 4-hydroxyphenylacetate 3-hydroxylase (HpaBC).This pathway was constructed inE.coli,and by (1) selecting a superior KDC,(2) strain,pathway and medium optimization,and (3)in situproduct extraction using 1-dodecanol,the final HT titer reached 647 mg·L-1in shake flask culture [7].

In another pathway,tyrosine is converted to HTvia L-DOPA,dopamine and 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA),which are sequentially catalyzed by tyrosine hydroxylase (TH),LDOPA decarboxylase (DDC),tyramine oxidase (TYO),and alcohol dehydrogenase (ADH).This pathway was constructed inE.coliby using mouse TH,pig DDC,Micrococcus luteusTYO andE.colinative ADH,and the final strain produces 0.08 mmol·L-1HT from glucose;notably,TYO requires a special cofactor tetrahydrobiopterin(BH4)and its regeneration pathway,andE.colinative tetrahydromonapterin(MH4)was shown to be able to replace BH4 as an alternative cofactor for TYO [24].

HpaBC,a versatile hydroxylase with a broad substrate spectrum,was engineered to replace the rate-limiting TH.E.coliHpaBC shows weak activity on tyramine,tyrosol and tyrosine,and therefore semi-rational protein engineering was conducted to obtain mutations with improved activities and specificities toward these substrates.Specifically,the structure modeling determined S210,A211 and Q212 in HpaB as the target residues for simultaneous saturation mutagenesis,and a high throughput screening method was developed based on the colorimetric reaction between sodium periodate and the hydroxylated products.A HpaBC mutant H7(S210T/A211P/Q212Y) showed～271-and～17-fold higher activity on tyramine and tyrosol,and combing the mutant H7 with TDC,TYO,and ADH to construct the synthetic pathway resulted in the production of 1890 mg·L-1of HT from tyrosine [8].

Similarly,HpaBC mutant 23F9-M4 was obtained,and the strain expressing this enzyme produced 15-fold higher amount ofLDOPA from tyrosine than the control expressing the wild-type HpaBC.Further,the activity of TYO was also improved by using a biosensor-based screening method.Specifically,a vanillate responsive protein VanR was engineered to switch its specificity,and a variant HyT12 showed a good specific response toward HT.With this biosensor,in vivo-directed evolution was used to screen a TYO mutation library,and the catalytic efficiency (kcat/Km) of the mutant YM9-2 showed～3.3-fold improvement than that of the wild-type TYO.A strain containing the mutated HpaBC and TYO realized a 95% conversion rate of tyrosine [9].

Fig.1.Microbial synthesis of hydroxytyrosol.Multiple pathways have been designed as indicated by the arrow colors.4-HPP,4-hydroxyphenylpyruvate;4-HPAA,4-hydroxyphenylacetaldehyde;3,4-DHPAA,3,4-dihydroxyphenylacetaldehyde.Enzymes:ADH,alcohol dehydrogenase;DDC,L-DOPA decarboxylase;HpaBC,4-hydroxyphenylacetate 3-hydroxylase;KDC,ketoacid decarboxylase;TH,tyrosine hydroxylase;TYO,tyramine oxidase;TyrB,tyrosine aminotransferase.* and ** indicate mutated enzymes.

Gallic acid(GA)is a natural PC with strong anti-oxidative activity,and is widely used in food,pharmaceutical and chemical industries.Currently,GA is mainly manufactured by acid/alkaline hydrolysis of tannins.To achieve microbial production of GA,artificial pathways were designed by extending the shikimate pathwayvia3-dehydroshikimate (3-DHS) or chorismate (Fig.2).In one pathway,3-DHS is converted to 3,4-dihydroxybenzoate (3,4-DHBA) by 3-DHS dehydratase (AroZ),which is further hydroxylated to GA by a mutated 4-hydroxybenzoate hydroxylase (PobA Y385F).The engineered strain produced 20 g·L-1GA under fedbatch conditions [11].In another study,a double mutation Y385F/T294A was created based on structure analysis of PobA[12].Compared with the single mutant Y385F,the catalytic efficiency (kcat/Km) of Y385F/T294A toward 4-hydroxybenzoate (4-HBA)and PCA was improved by 1-and 5-fold,respectively.On this basis,a novel pathway was established,in which chorismate lyase(UbiC)converts chorismate to 4-HBA,and then PobA Y385F/T294A catalyzes the two-step sequential hydroxylation of 4-HBA to GA.By enhancing the shikimate pathway and modulating expression of the pathway genes,the final titer of GA reached 1266 mg·L-1in shake flask culture [11].

Pyrogallol (1,2,3-trihydroxybenzene) is a simple PC with broad applications in agricultural,food,dyeing,printing,cosmetic and pharmaceutical industries.Currently,pyrogallol is commercially produced by thermal decarboxylation of gallic acid under high temperature and pressure.In an attempt to pyrogallol biosynthesis,the GA biosynthetic pathway was extended by introducing a 3,4-DHBA decarboxylase,which shows promiscuous activity on GA.Unfortunately,the engineered strain only produced catechol instead of pyrogallol,indicating that 3,4-DHBA is decarboxylated to catechol prior to be hydroxylated [10].

Recently,pyrogallol biosynthesis was achievedviadecarboxylation of 2,3-dihydroxybenzoate(2,3-DHBA)[13](Fig.2).2,3-DHBA is a native metabolites inE.coli,and its biosynthesis had been achieved and extended for muconate production [25].Thus,the emphasis was put on the identification of efficient 2,3-DHBA decarboxylases.Five candidates including monooxygenases and dioxygenases that catalyze oxidative decarboxylation of substrates structurally similar to 2,3-DHBA,were tested.Salicylate 1-monooxygenase (NahG) was shown to be the most effective and was used for pathway assembly.By enhancement of the shikimate pathway,modular pathway optimization and alleviation of product autoxidation,the final strain produced gram-level pyrogallol in shake flask culture [12].

Fig.2.Microbial synthesis of several representative phenolic compounds.2,3-DHBA,2,3-dihydroxybenzoate;3,4-DHBA,3,4-dihydroxybenzoate;3-DHS,3-dehydroshikimate;4-HBA,4-hydroxybenzoate;4-HBAD,4-hydroxybenzaldehyde;4-HBAL,4-hydroxybenzyl alcohol;4-HPP,4-hydroxyphenylpyruvate;4-HPAA,4-hydroxyphenylacetaldehyde;HQ,hydroquinone.Enzymes:ADH,alcohol dehydrogenase;AroZ,3-DHS dehydratase;CAR-Sfp;carboxylate reductase;KDC,ketoacid decarboxylase;MNX1,4-hydroxybenzoate 1-hydroxylase;NahG,salicylate 1-monooxygenase;PobA,4-hydroxybenzoate hydroxylase;UGT,glycosyltransferase;UbiC,chorismate lyase.*indicates a mutated enzyme.

Arbutin is a hydroquinone (HQ) glycoside with an excellent skin-lightening property.It naturally exists in plants such as bearberry.Plant extraction is the major approach for its commercial supply.To fulfill its growing demand,efficient and sustainable routes need to be developed.Enzymatic and whole-cell conversion processes have been investigated for arbutin production.However,the low conversion rate and the high cost for biocatalyst preparation hinder their commercial application.For example,using the purified dextransucrase,only 544 mg·L-1arbutin was produced from 49.5 g·L-1HQ [26];an engineeredE.colistrain expressing surface-anchored transglucosidase produced 21 g·L-1arbutin from 200 g·L-1glucose under high-density fed-batch conditions [27].Thus,an artificial pathway was designed for arbutin biosynthesis[13].In this pathway,4-HBA is converted to arbutinviaHQ,which are sequentially catalyzed by a 4-hydroxybenzoate 1-hydroxylase(MNX1) fromCandida parapsilosis CBS604and arbutin synthase(AS) fromRauvolfia serpentine.The initial strain produced only 54.71 mg·L-1arbutin from glucose.Further efforts including enhancing the shikimate pathway and optimizing glucose concentration resulted in a 77-fold improvement in arbutin titer to 4.19 g·L-1in shake flasks culture,which shows scale-up potential[13].

Gastrodin,a phenolic glycoside,is an active ingredient of the herbal plantGastrodia elata.It exhibits sedative,hypnotic,anticonvulsive and neuroprotective properties and has been used clinically to treat various diseases such as headache,dizziness,and convulsion.Microbial production of gastrodin was hampered by the incomplete elucidation of its native biosynthetic pathway,and thus an artificial pathway was established [14].In this pathway,4-HBA is reduced to 4-hydroxybenzyl alcohol by the sequential catalysis of aNocardiacarboxylic acid reductase(CAR)and endogenous alcohol dehydrogenases,and 4-hydroxybenzyl alcohol is further converted to gastrodin by aRhodiolaglycosyltransferase (GT)UGT73B6(Fig.2).The low GT activity was demonstrated to be one of the rate-limiting steps.Because the crystal structure of UGT73B6 was undetermined,directed evolution was adopted to improve its activity and regioselectivity.To this end,a random mutant library was createdviaerror-prone polymerase chain reaction,and 4-methylumbelliferone was used as a mimic of 4-hydroxybenzl alcohol to facilitate high-throughput screening of the desired variants based on fluorescence quenching after glycosylation.Finally,by combining directed evolution,pathway engineering and metabolic flux enhancement,the final strain produced 545 mg·L-1gastrodin from glucose in shake flask culture [14].

Biosynthesis of other PCs such as phenol [28,29],vanillyl alcohol [30]has also been achieved by designing the corresponding artificial pathways.

3.Balancing Enzyme Expression to Improve Pathway Efficiency

Flavonoids comprise a highly diverse group of plant secondary metabolites,which can be further categorized into flavones,flavonols,flavonones,isoflavones,anthocyanins and catechins according to their structure variability.Due to their diverse biological properties,flavonoids have been widely used in human health and nutrition[31].To achieve economical and sustainable production of flavonoids,increasing efforts have been made to improve the efficiency of the biosynthetic pathways.

Flavonoids are synthesized from long and complex biosynthetic pathways (Fig.3),and balancing gene expression is essential to maximize production.Given the difficulty of modulating the expression level of each individual gene,the entire pathway is usually divided into several modules to allow more efficient pathway optimization.

p-Coumaric acid is a gateway precursor for the biosynthesis of stibenes and flavonoids.Recently,aSaccharomyces cerevisiaeplatform strain was constructed for efficient biosynthesis ofpcoumaric acid through (1) relieving bottlenecks in the aromatic amino acid biosynthesis pathway,(2) redirecting carbon distribution toward erythrose-4-phosphate (E4P) by implementing a heterologous phosphoketolase pathway,(3) dynamic control of the expression of heterologous genes by using the GALp expression system,and (4) balancing the availability of PEP and E4P.With these efforts,p-coumaric acid titer reached 12.5 g·L-1in fedbatch fermentation [15].

Pinocembrin is a backbone molecule to synthesize a variety of other flavonoids by the tailoring reactions as hydroxylation,reduction,oxidation,alkylation and glycosylation.Pinocembrin biosynthesis is extended from L-phenylalanine and involves the sequential catalysis of phenylalanine ammonia lyase (PAL),4-coumarate:CoA ligase(4CL),chalcone synthase(CHS)and chalcone isomerase (CHI) (Fig.3).In addition,3-deoxy-D-arabinoheptuloso nate-7-phosphate synthase (AroF) and chorismate mutase/prephenate dehydratase(PheA)catalyze the key steps in the shikimate pathway and the phenylalanine/tyrosine branch,respectively,whilst malonate synthetase (MatB) and malonate carrier protein (MatC) are required to enhance the supply of malonyl-CoA from malonate.These eight enzymes were divided into four modules (AroF/PheA,PAL/4CL,CHS/CHI and MatB/MatC),and cloned into four separate plasmids.These modules were balanced by varying the plasmid copy numbers,resulting in a strain that produced 40 mg·L-1pinocembrin from glucose in shake flask culture.In a following study,the expression levels of the last three modules were further tuned by adjusting both plasmid copy number and promoter strength.In addition,expression level of the rate-limiting PAL was customized by modifying the 5′region of mRNA secondary structure.These efforts resulting in a strain that produced 432.4 mg·L-1pinocembrin under fed-batch conditions[16].

Fine-tuning pathway gene expression often relies on the construction of a vast number of module combinations,which is laborious and time-consuming.To overcome these conundrums,high throughput assembly and screening strategies have been developed and applied for improve the biosynthesis of PCs such as(2S)-naringenin and catechins.

Naringenin is an early compound in flavonoid biosynthesis with broad nutritional and pharmaceutical properties.It is derived from tyrosine catalyzed by the same set of enzymes as for pinocembrin biosynthesis.An iterative high-throughput balancing (IHTB) strategy was established to enhance naringenin production inE.coli[32].Specifically,a series of gradient constitutive promoters were randomly cloned upstream of the pathway genes encoding PAL,4CL,CHS and CHI,and an ultraviolet spectrophotometryfluorescence spectrophotometry method based on the interaction between naringenin and AlCl3was established for high throughput screening of the combinatorial library.After several rounds of selection,the final titer reached 191.9 mg·L-1,representing a 1.1-fold increase than that of a previous modular optimized strain[32].The oleaginous yeastYarrowia lipolyticanaturally possesses high flux through acetyl and malonyl-CoA,which are precursors to stibenes and flavonoids.Recently,this yeast was engineered as a platform host for the production of naringenin,resveratrol and bisdemethoxycurcumin.The titer of naringenin reached 898 mg·L-1in defined media bioreactors,the highest value reported to date in any host organism [17].In another study,Y.lipolyticawas extensively engineered by increasing the activities of Aro4p and Aro7p,and integrating multiple resveratrol biosynthetic genes into the chromosome.The final strain produced (12.4±0.3)g·L-1resveratrol in controlled fed-batch bioreactor,the highest reported titer to date forde novoresveratrol production [18].

Fig.3.Microbial synthesis of flavonoids.PAL/TAL,phenylalanine/tyrosine ammonia lyase;4CL,4-coumarate:CoA ligase;CHS,chalcone synthase;CHI,chalcone isomerase;F3H,flavanone 3β-hydroxylase;DFR,dihydroflavonol 4-reductase;LAR,leucoanthocyanidin reductase.

(+)-Catechin is an active ingredient in green tea,and its biosynthesis from eriodictyol requires three enzymes,which are flavanone 3β-hydroxylase (F3H),dihydroflavonol 4-reductase (DFR)and leucoanthocyanidin reductase (LAR) (Fig.3).The ePathBrick system,which utilizes four isocaudamer pairs (AvrII,NheI,SpeI,and XbaI) and supports combinatorial generation of pathway diversities,was applied to optimize the catechin biosynthetic pathway [19].A total of 18 pathway variants were created by using three F3H,three DFR and two LAR genes from different plant species,and the best enzyme combination was demonstrated.The accumulation of the intermediate dihydroquercetin was reduced by introducing additional gene copies encoding DFR and LAR,resulting in a titer of 374.6 mg·L-1of(+)-catechin from eriodictyol.Further increasing the availability of NADPH and oxygen increased the titer to 910.9 mg·L-1,which is the highest titer reported [19].

4.Coculture Engineering to Alleviate Metabolic Burden and Unwanted Side-reactions

Modular optimization proved an effective strategy to balance the biosynthetic pathways.However,introducing long pathways in a single strain may cause severe metabolic burden.Thus,coculture engineering has been used to improve the biosynthesis of a variety of PCs.Compared with monocultures,cocultures shows several advantages,including division of labor to alleviate cell burden,compartmentalization of reactions to reduce metabolic interference [33].

Salidroside,a glucoside of tyrosol,is the major bioactive component ofRhodiola.The glucosyltransferase UGT73B6 isolated fromRhodiola sachalinensisewas found to be able to convert tyrosol into salidroside.Accordingly,a biosynthetic pathway was designed,in which tyrosine is converted to salidroside by three consecutive reactions catalyzed by KDC,ADH and UGT73B6.An engineeredE.colistrain produced 56.9 mg·L-1salidroside after pathway and strain optimization,and icariside D2 was accumulated as a byproduct due to the glycosylation of the phenolic hydroxyl group of tyrosol by UGT73B6 [34].

In order to improve the production efficiency,alternative enzymes(KDC fromPichia pastorisGS115 and UGT85A1 fromArabidopsis thaliana) were used for pathway construction.Further,a syntrophicE.colicoculture system was constructed,which consists of the aglycone (AG) strain and the glycoside (GD) strain.The AG strain,which is engineered to overproduce tyrosol,is deficient in phenylalanine and preferentially utilizes xylose while the GD strain,which is engineered to overexpress UGT85A1 and enhance UDP-glucose availability,is deficient in tyrosine and exclusively utilizes glucose.Thus,in the coculture system the two strains are mutualistic through the cross-feeding of tyrosine and phenylalanine.The syntrophic coculture was shown to be robust and stable,and produced 6.03 g·L-1of salidroside by fed-batch fermentation,which is 20 times higher than that of the monoculture [20].

Rosmarinic acid (RA) is a plant-derived PC with various activities(e.g.antioxidant,anti-inflammatory,neuroprotective and anticarcinogenic).Structurally,RA is an ester of caffeic acid (CA) and salvianic acid A (SAA),and thus the RA biosynthetic pathway involves biosynthesis of the two parallel precursors and the following condensation reaction(Fig.4).CA can be derived from tyrosine by two consecutive reactions catalyzed by tyrosine ammonia lyase(TAL) and HpaBC.SAA can be produced from 4-HPP by two-step enzymatic conversion using HpaBC and D-lactate dehydrogenase(D-LDH).CA is activated to caffeoyl-CoA by 4CL,which is condensed with SAA catalyzed by RA synthase(RAS).AnE.colimonoculture produced only 4.5 mg·L-1RA,indicating the challenge to balancing the whole pathway in a monoculture.Therefore,the pathway was divided into three modules (CA,SAA and RA modules),which were assigned to threeE.colistrains.By adjusting the strain ratios,the RA titer reached 172 mg·L-1,which is 38 times that of monoculture [21].

Coculture engineering has also been applied to prevent sidereactions caused by enzyme promiscuity as shown for monolignol biosynthesis.Monolignols includingp-coumaryl,caffeyl,coniferyl and sinapyl alcohols are direct precursors to lignin,and their natural derivatives such as silibin and pinoresinol diglucoside possess various physiological and pharmaceutical functions.p-Coumaryl alcohol biosynthesis is extended from tyrosine,which involves TAL,4CL,cinnamoyl-CoA reductase (CCR) and ADH.Efficient enzymes were selected for pathway construction,leading tode novoproduction of (501.8±41.4) mg·L-1p-coumaryl alcohol.The pathway was extended to produce caffeyl alcohol by introducing the hydroxylase HpaBC,but the monoculture produced only 30.9 mg·L-1caffeyl alcohol.A main reason is that HpaBC converts tyrosine to the instableL-DOPA,causing loss of the carbon sources.To solve this problem,the coculture strategy was used to minimize the accessibility of HpaBC to tyrosine.After optimization of the inoculation ratio,the titer reached 401 mg·L-1,which is nearly 13 times that of the monoculture [22].The coculture strategy has also proved effective to improve biosynthesis of naringenin [35],hydroxyphenyl-pyranoanthocyanins [36],acacetin [37],sakuranetin [38].

Fig.4.Coculture engineering for Rosmarinic acid biosynthesis.4-HPP,4-hydroxyphenylpyruvate;3,4-DHPP,3,4-dihydroxyphenylpyruvate;4-DHPL,4-hydroxyphenyllactate.Enzymes:4CL,4-coumarate:CoA ligase;HpaBC,4-hydroxyphenylacetate 3-hydroxylase;D-LDH,D-lactate dehydrogenase;RAS,rosmarinic acid synthase;TAL,tyrosine ammonia lyase;TyrB,tyrosine aminotransferase.

Besides coculture of strains of the same species,coculture of strains from different species has also been developed for the production of PCs.In one example,coculture ofE.coliandS.cerevisiaewas developed for naringenin production from xylose.E.coliwas engineered to overproduce tyrosine from xylose whileS.cerevisiaewas engineered to assimilate acetate produced byE.coliand converts tyrosine to naringenin.Naringenin titer reached(21.16±0.41) mg·L-1after optimization,which is nearly eightfold that of the yeast mono-culture [39].AnE.coli-Streptomycescoculture system was developed for the production of methylated PCs [40].Specifically,E.coliproduced the unmethylated stibenes and flavonoids,which were further modified byStreptomycesexpressing a versatile methyltransferase.

5.Genetic Circuit for Dynamic Regulation and High Throughput Screening

The objective of microbial production is to maximize the titer,yield and productivity of the engineered strains,which requires systematic remodeling of cell metabolism to coordinate the distribution of metabolic flux between growth and production.Oftentimes,too early expression of pathway enzymes and inhibition of competing pathways can impair cell growth.

Toggle switches have been used to shift the cell status by adding small molecule inducers.Malonyl-CoA is a versatile building block for the biosynthesis of fatty acids and a variety of secondary metabolites such as flavonoids and stilbenes.For example,biosynthesis of naringenin and resveratrol involves the condensation ofpcoumaroyl-CoA with three molecules of malonyl-CoA.Thus,the adequate supply of malonyl-CoA is essential for efficient biosynthesis of these PCs.However,the intracellular concentration of malonyl-CoA is usually tightly regulated and maintained at low levels.One way to enrich the intracellular pool is to increase malonyl-CoA synthesis by overexpressing the acetyl-CoA carboxylase(ACC)and enhance acetyl-CoA supply while another way is to reduce its consumption.However,inactivation of fatty acid biosynthesis by gene knockout is lethal to the host cells.Thus,an antisense RNA (asRNA)-based strategy was used for downregulating gene expression.The synthetic asRNA features a stem-loop structure,in which the loop is responsible to pair with the target mRNA while the stem enhances the asRNA stability.The factors that influence interference efficiency such as the loop length and the relative abundance of the asRNAs were evaluated and optimized.When tested,the strategy led to a remarkable increase in the production of 4-hydroxycoumarin,resveratrol and naringenin [41].However,this strategy relies on the addition of an exogenous signal molecule such as isopropyl-β-D-thiogalactopyranoside (IPTG) to manually switch the cell status.

Biosensors,which include the transcriptional factor-based and the riboswitch-based ones,are used to construct genetic circuits for dynamic regulation of gene expression in response to the intracellular or extracellular signals in an autonomous and continuous manner [42].These biosensors,in combination with anti-sense RNA/CRISPR technologies,can achieve dual(up and down) regulation of gene expression.

A quorum-sensing linked RNA interference strategy was used for dynamic control of 4-HBA biosynthesis inS.cerevisiae,which is an integration of the previously developed pheromone QS circuit and the Argonaute/Dicer based RNAi module,to enable both gene activation and repression,and the final titer of 4-HBA reached 148 mg·L-1,representing a 37 fold increase over the base strain[23].

Biosensors can also be used to improve the heterogeneity of the producing strains based on the social reward-punishment rules[43].Recently,taking naringenin biosynthesis in oleaginous yeast as an example,a strategy coupling metabolic addiction with negative autoregulation was developed to improve strain stability and pathway yield [44].The oleaginous yeast accumulates large amount of lipids,which compete with naringenin for the precursor malonyl-CoA.To achieve negative autoregulation of fatty acid synthesis,a fatty acid inducible promoter POX2 was engineered to adjust its dynamic output and range,and used to control transcription of the guide RNAs (gRNAs) that guide dCas9 to downregulate expression of the key genesFAS1,FAS2andFabD,either individually or in combination.To build the naringenin-inducible genetic circuits,the well-characterized transcriptional activator FdeR and its cognate DNA binding site FdeO were used.A panel of hybrid promoters was constructed by fusion of FdeO to several core promoters,and was used to controlLEU2expression in the leucine auxotrophic yeast.As a result,those productive cells will hold the growth advantage over those cheaters.With this design,the final engineered yeast retained 90.9% of the naringenin titer after cultivation for 324 generations [44].

In addition to dynamic regulation,genetic circuits can also be used for the high-throughput screening of high-producing strains[45,46].The FdeR/FdeO-based prokaryotic biosensor was transplanted intoS.cerevisiaeby a multiparametric engineering strategy;on the other hand,the Golden gate-directed combinatorial assembly of promoters and genes generated a naringeninproducing strain library.Using this biosensor with the mCherry fluorescence protein as the output signal,an improved recombinant strain was successfully identified [47].Similarly,a salicylate sensor-reporter system was constructed to screen a combinatorial library,and optimal gene expression patterns were rapidly identified,resulting in up to 123%increase in salicylate production[48].A resveratrol responsive biosensor was generated by engineering the TtgR regulatory protein fromPseudomonas putidaand used to screen ap-coumarate:CoA ligase (4CL) mutation library.A 4CL variant with improved activity was obtained,leading to increased production of not only resveratrol but also naringenin [49].

Unlike transcriptional factor-based biosensors,the riboswitchbased biosensors cause less metabolic burden.Riboswitches control gene expression by changing their conformation upon binding of specific molecules.However,natural riboswitch in response to flavonoids has not been identified yet [50].Thus,anin vitroselection method was used to screen RNA aptamers in response to naringenin.Briefly,naringenin was anchored to epoxy-activated sepharose 6B to construct the affinity matrix and used to enrich the RNA pool for 7 rounds.Further,in vivoselection was conducted by cloning the aptamer pool upstream of tetA-sGFP.The final selected riboswitches showed up to 2.91-fold increase in gene expression in response to naringenin,and were used for flowcytometry-based high-throughput screening of naringenin overproducingE.colistrains in a coculture system [51].

6.Challenges and Perspectives

Although significant progress has been made in microbial production of PCs,many challenges still need to be overcome on the way to their economical production.First,many PCs are derived from plants,and some types of plant enzymes such as cytochrome P450s are often poorly expressed in yeast or bacteria.Thus,the sequence,codon usage,expression level and subcellular localization usually need to be optimized.Emerging new tools such as machine learning [52]andde novocomputational protein design[53]could be applied to accelerate the engineering of the ratelimiting enzymes.Second,the native pathways of many PCs are still not fully elucidated.For this,computational tools have been developed for design and evaluation of artificial pathways based on the constantly growing enzyme data.Third,the native metabolism of the host cells needs to be remodeled to balance the redistribution of cell resources between growth and production.For this,in silicogenome-scale metabolic analysis [54]will continue to play important roles in identifying gene targets for knockout and overexpression.Fourth,some PCs such as coumarins exhibit inhibitory effect on cell growth.Adaptive laboratory evolution[55]can be used to improve the tolerance.The underlying mechanism can be elucidated by whole genome sequencing and comparison,and used to guide reverse metabolic engineering.Through systems metabolic engineering,combined with the process engineering,it is promising that increasing numbers of PCs will be economically produced using engineered microorganisms.

Acknowledgements

This work was supported by National Key Research and DevelopmentProgramofChina(2018YFA0901800and 2018YFA0901400) and National Natural Science Foundation of China (21978015,21636001,and 21776008).