Wenqiang Li,Wentao Sun,Chun Li*

Key Lab for Industrial Biocatalysis,Ministry of Education,Department of Chemical Engineering,Tsinghua University,Beijing 100084,China

ABSTRACT Plant natural products are a kind of active substance widely used in pharmaceuticals and foods.However,the current production mode based on plant culture and extraction suffer complex processes and severe concerns for environmental and ecological.With the increasing awareness of environmental sustainability,engineered microbial cell factories have been an alternative approach to produce natural products.Many engineering strategies have been utilized in microbial biosynthesis of complex phytochemicals such as dynamic control and substructure engineering.Meanwhile,Enzyme engineering including directed evolution and rational design has been implemented to improve enzyme catalysis efficiency and stability as well as change promiscuity to expand product spectra.In this review,we discussed recent advances in microbial biosynthesis of complex phytochemicals from the following aspects,including pathway construction,strain engineering to boost the production.

Keywords:Plant natural products Microorganisms Synthetic biology Enzyme engineering

1.Introduction

Plant natural products usually belong to plants secondary metabolites including flavonoids,alkaloids,and terpenoids,which always naturally act as defending molecules against diseases,insects,and xenobiotics[1,2].As a result,these compounds turned out to be the major bioactive constituents of some traditional herbal medicines,such as breviscapine fromErigeron breviscapus,and vinblastine fromCatharanthus roseus.Because of the versatile function,plant natural products have been widely used in the cosmetics and pharmaceutical industries [3].

However,due to the complex molecular structure of plant natural products,their total chemical synthesis was restricted.Thus,phytoextraction becomes the mainstream method in industrial manufacture,which is time-consuming,labor-intensive,and environment unfriendly[4].The phytoextraction generally suffers from low content and diverse analogs in plants,resulting in inefficient production and high cost.Thus,the replacement of phytoextraction by novel approaches is of great value[5].Compared with phytoextraction,a promising solution emerged as the fast-growing and easy culturing microorganism cell factories.In recent years,the complex plant biosynthetic pathways have been constructed in microbes includingE.coli,S.cerevisiae,andY.lipolyticafor the production of several medically important compounds,and many efforts have been made to boost the microbial biosynthesis efficiency.

During building heterogeneous pathways in microbes,a collection of enzymes catalyzing diverse reactions is recruited to assemble and decorate the final natural product [6].Although enzymes integrated into plant biosynthetic pathways are efficient and stable in plant cells,their applications are sometimes restricted in microbes due to the different intracellular conditions [7].Thus,many engineering strategies such as rational design,directed evolution also have been applied to transform plant natural enzymes in microbes.In this review,we discussed recent advances in the production of plant natural products in microbes and engineering natural enzymes for higher efficiency,selectivity,and stability.

2.Biosynthesis Pathways of Plant Natural Products

2.1.The biosynthesis pathways of terpenoids

The biosynthesis of terpenoids begins from the universal C-5 building blocks,isopentenyl pyrophosphate(IPP)and dimethylallyl pyrophosphate (DMAPP),synthesized from mevalonic acid (MVA)or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.The MVA pathway is found in plants [8],yeasts [9],animals [10],archaebacteria [11],and some gram-positive bacteria located in the cytoplasm and ER.In this pathway,acetyl-CoA is converted to MVA by the sequential action of Acetyl-CoA C-acetyltransferase(AACT),hydroxymethylglutaryl-CoA synthase (HMGS),and hydroxymethylglutaryl-CoA reductase (HMGR),which is subsequently converted to IPP by mevalonate kinase (MVK),phosphomevalonatekinase(PMVK)anddiphosphomevalonate decarboxylase (MVD).In contrast,the MEP pathway was plastidlocated in plants and distributed in cyanobacteria,green alga as well as gram-negative bacteria,starting from D-glyceraldehyde-3-phosphate.D-glyceralaldehyde-3-phosphate is converted to the landmark intermediate MEP by 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR),and finally converted to IPP [8].In higher plants and some alga,these two pathways could occur simultaneously[8,12]and isopentenyl pyrophosphate isomerase(IDI) can reversibly inter-isomerize IPP to DMAPP almost in all cases.IPP and DMAPP are condensed to the monoterpenoid precursor geranyl pyrophosphate (GPP),diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) which can also be further transformed to tretaterpenes,and triterpenoid precursor farnesyl pyrophosphate(FPP)(Fig.1).These skeletons will be further modified by tailoring enzymes such as P450s and UGTs to introduce the functional groups (hydroxyl,carbonyl,or carboxyl groups) and glycosyl expending the diversity of physiological activities.For example,glycyrrhizin is 170 folds sweeter than sucrose,and has improved solubility in water than the unsweetened glycyrrhetinic acid.

2.2.The biosynthesis pathways of flavonoids

Flavonoids begin with the aromatic amino acid L-Phenylalanine or L-tyrosine synthesized from shikimate pathway existing in prokaryotic,eukaryotic,and archaeal microorganisms,which are used to synthesizep-coumaric acid,an important intermediate and a common precursor for flavonoid compounds.p-coumaric acid is synthesized by tyrosine ammonia lyase (TAL) catalyzed deamination of tyrosine or by P450 cinnamic acid 4-hydroxylase(C4H) mediated oxidation of (E)-cinnamic acid derived from phenylalanine ammonia lyase (PAL) mediated deamination of phenylalanine.4-coumaroyl-CoA ligase (4CL) activatespcoumaric acid to form 4-coumaroyl-CoA,which is condensed to naringenin chalcone together with three molecular of malonyl-CoA by chalcone synthase(CHS),acting as the C6–C3–C6 backbone unit in all flavonoids.Naringenin chalcone could be tailored directly or be converted into naringenin as a precursor to form various sub-classes of flavonoids and isoflavonoidsby chalcone isomerase (CHI) catalyzed ring-closing reaction.Other than flavonoids,the biosynthesis of another phenylpropanoid class,stilbenoids starts from a single step condensation reaction catalyzed by stilbene synthase (STS) using 4-coumaroyl-CoA and malonyl-CoA to form a C6-C2-C6 backbone (Fig.2).All these skeleton molecules would be further modified by methyltransferases,glycosyltransferases,hydroxylases,acyltransferases,etc.,bring more molecular diversity [13].

2.3.The biosynthesis pathways of alkaloids

Alkaloid compounds,especially plant-derived monoterpene indole alkaloids (MIA) and benzylisoquinoline alkaloids (BIA) are considered valuable sources of pharmaceuticals for their anticancer,antiviral,and antimalarial activities,etc.MIA are widely used in the treatment of disease including cancer and heart disorders.Although it contains approximately 2000 structurally and pharmaceutically diverse compounds,all MIAs share the common precursor strictosidine,which was condensed from tryptamine and secologanin through a Pictet-Spengler reaction catalyzed by strictosidine synthases (STR),which reaction was first verified fromR.serpentine[14].Tryptamine derived from the decarboxylation reaction of tryptophan catalyzed by tryptophan decarboxylase (TDC),while secologanin is synthesized from the monoterpenoid geraniol[15].Geraniol was first transformed to 10-oxogernial through sequential oxidation reaction by geraniol 10-oxidase (G10O) and 10-hydroxygeraniol oxidoreductase (10HGO).By the tailoring function of iridoid synthase (IS) and iridoid oxidase (IO),10-oxogernial was further transformed to 7-deoxyloganetic acid,which was then converted to loganin by 7-deoxyloganetic acid glucosyltransferase (7-DLGT),7-deoxyloganic acid hydroxylase(7-DLH),and loganic acid O-methyltransferase (LAMT).Finally,loganin was transformed to secologanin by an oxidative C-C cleavage reaction catalyzed by a second P450 monooxygenase(CYP72A1)named SLS(secologanin synthase).After the deglycosylation of strictosidine β-glucosidase (SGS),strictosidine aglycone becomes very reactive and the rearrangement of the monoterpenoid moiety will lead to diversiform MIA ring systems,such as tabersonine polyneuridine aldehyde and catharanthine (Fig.3).

Benzylisoquinoline alkaloids(BIA)possess potent pharmacological properties,including the prominent narcotic analgesic morphine,the cough suppressant codeine,and the muscle relaxant papaverine.BIAs are produced from one molecular of dopamine forisoquinolinemoietyandonemolecularof4-hydroxyphenylacetaldehyde as the benzyl component,which is all derived from L-tyrosine.4-hydroxyphenylacetaldehyde was produced through the sequential catalysis reaction by L-tyrosine aminotransferase (TyrAT) and 4-hydroxyphenylpyruvate decarboxylase(4HPPDC).Dopamine could be synthesized by two routes.In one route,tyrosine decarboxylase (TYDC) catalyzes the decarboxylation of tyrosine to tyramine,which is then oxidized to dopamine by tyramine 3-hydroxylase (3OHase).In another route,tyrosine was firstly oxidized by a P450 monooxygenase CYP76AD1 to L-DOPA,which could be decarboxylated by DOPA decarboxylase(DODC) resulting in dopamine.Norcoclaurine synthase (NCS)brings the dopamine and 4-hydroxyphenylacetaldehyde into BIA biosynthesis through a Pictet-Spengler type condensation producing (S)-norcoclaurine.Followed byO-methylation,N-methylation,and hydroxylation reactions,(S)-norcoclaurine is further converted to(S)-reticuline,the branch point to different BIAs structural types.(Fig.3) For example,morphine starting from the formation of (R)-reticuline catalyzed by the fusion protein containing a cytochrome P450 monooxygenase and oxidoreductase (STORR) and noscapine starting from the formation of(S)-scoulerine catalyzed by the berberine bridge enzyme (BBE).As an exception,the dimeric bisbenzylisoquinoline alkaloids are not produced using (S)-reticuline,such as the bisbenzylisoquinoline alkaloid berbamunine [16].

2.4.Key tailoring enzymes involved in natural products

The synthesis of natural products involves various kinds of enzymes catalyzing diverse reaction types,for example,cyclization reactions mediated by the terpene synthase to form the terpenoid skeletons,and the condensation reactions mediated by NCS or CHS to form the core structure of BIA or flavonoid.Although the synthase enzymes integrate in different compounds are quite different,almost all natural plant products involves 3 kinds of tailoring enzymes to form active intermediates and specific compounds with functional groups.These enzymes are Cytochrome P450 monooxygenase (P450),O-methyltransferases (OMT),and UDP-glucosyltransferases (UGT) (Fig.4).

2.4.1.Cytochrome P450 monooxygenase (P450)

P450 was the most widely used tailoring enzyme in the biosynthesis of natural products,which intensively involved in the C-H bond activation,introducing active groups (including hydroxyl,carbonyl,or carboxyl) and modifying the skeletons in terpenoids,flavonoids,and alkaloids.

Fig.1.Biosynthesis pathway of terpenoids.AACT:Acetyl-CoA C-acetyltransferase,HMGS:hydroxymethylglutaryl-CoA synthase,HMGR:hydroxymethylglutaryl-CoA reductase,PMVK:phosphomevalonate kinase,MVD:diphosphomevalonate decarboxylase.DXS:1-deoxy-D-xylulose-5-phosphate synthase,DXR:1-deoxy-D-xylulose-5-phosphate reductoisomerase,GGPPS:geranylgeranyl pyrophosphate synthase,FPPS:farnesyl-diphosphate synthase,SQS:squalene synthase;SQE:squalene epoxidas.

In the biosynthesis of plant flavonoids,C4H hydroxylating the(E)-cinnamic acid top-coumaric acid is the first P450 that brings the phenolic hydroxyl group into core intermediate chalcones.When the chalcones are branched into specific products such as plant flavonoid or isoflavonoid,P450 is still an important enzyme activating the C-H bond and modifying the molecular skeleton.The total flavonoid,breviscapine fromE.breviscapushas been used for the clinical treatment of cardiovascular and cerebrovascular diseases for 30 years.In the biosynthesis of scutellarin,a compound of breviscapine derived from naringenin chalcones,the precursor apigenin or apigenin-7-O-glucuronide is finally hydroxylated at C-6 forming scutellarin by the F6H,a P450 whose coding gene was cloned fromE.breviscapus[17].Isoflavonoids have the B-ring attached to the C-ringviathe C-3 instead of the C-2 position in flavonoids.The oxidative aryl migration from C-2 to C-3 is exactly catalyzed by the P450 enzyme 2-hydroxy-iso-flavanone synthase (2HIS,also known as iso-flavonesynthase,IFS) [18].The isoflavonoid aglycones will undergo further modifications by P450s.For example,the hydroxylation of biochanin A and formononetin topratensein catalyzed by CYP81E9 as well as the hydroxylation of pseudobaptigenin catalyzed by P450 from CYP81E family [19].

Fig.2.Biosynthesis pathway of plant flavonoids.TAL:tyrosine ammonia lyase,C4H:cinnamic acid 4-hydroxylase,4CL:4-coumaroyl-CoA ligase,PAL:phenylalanine ammonia lyase,CHS:chalcone synthase,CHI:chalcone isomerase,STS:stilbene synthase.

In the biosynthesis of the known MIA and BIA biosynthetic pathways in plants,P450 is also a remarkable oxidation enzyme[20].The synthesis of MIA core intermediate secologain starts from geraniol C-10 hydroxylation by the P450 geraniol-10-hydroxylase(CYP76B6),and in the final step of this pathway,loganin was converted to secologanin through an oxidative C-C cleavage reaction catalyzed CYP72A1[21].In the biosynthesis of vindoline,the pathway begins with the hydroxylation of tabersonine at C-16 by tabersonine 16-hydroxylase (T16H),which also belongs to the P450 monooxygenase family named CYP71D12 [22].

Uncommonly,the intermediates used in the MIA pathway of Cornales were secologanic acid and strictosidinic acid,not the commonly used secologanin and strictosidine.The key reaction of the hydroxylation 7-deoxyloganic acid and the oxidative C-C cleavage to form loganic acid and secologanic acid was also catalyzed by P450s,CYP72A565,and CYP72A610 fromC.acuminate.These reactions are closely related to the reactions catalyzed by secologanin synthase (CYP72A1) and 7-deoxyloganic acid hydroxylase (CYP72A224) [23].

(S)-Reticuline is the central intermediate of most BIAs,whose synthesis involves two P450s.The oxidation of tyrosine to L-DOPA is the first committed reaction and catalyzed by CYP76AD1.In the later step,theN-methylcoclaurine would be hydroxylated by CYP80B3 to generate the 3-OH-N-methylcoclaurine which is furtherO-methylated to form (S)-Reticuline.In the derivation of(S)-Reticuline to morpine,(S)-Reticuline should firstly be converted to (R)-Reticuline.The corresponding enzyme reticuline epimerase (REPI,also known as STORR) catalyzing this epimerization reaction was identified to be a unique fused protein consist of an N-term P450 domain oxidizing (S)-reticuline to 1,2-dehydroreticuline and a C-term aldo–keto reductase domain reducing 1,2-dehydroreticuline to (R)-reticuline [24].Subsequently,(R)-reticuline is converted to salutaridine by salutaridine synthase (SalSyn),a P450 named CYP719B1 through a stereoand region-specific C-C phenol-coupling reaction [25].

The noscapine gene cluster encodes 10 enzymes,which includes four cytochrome P450 monooxygenases (CYP719A21,CYP82Y1,CYP82X1,and CYP82X2).Substrate-specific CYP719A21 is known as canadine synthase (CAS),catalyzes the formation of a methylenedioxy bridge to form (S)-canadine,which is further converted toN-methylcanadine by tetrahydroprotoberberine cis-N-methyltransferase.N-methylcanadine would be hydroxylated to 1-hydroxy-N-methylcanadine by CYP82Y1.CYP82X2 could further hydroxylate this compound introducing the active C-13 hydroxyl for the acylation to form 1-OH-13-O-acetyl-Nmethylcaadine,which is subsequently converted to 4′-Odesmethyl-3-O-acetypapaveroxine by CYP82X1 and a spontaneous reaction.

In the biosynthesis of terpenoids,the most involved skeletontailoring reactions are the oxidation reactions catalyzed by P450 to introduce active groups used for further modification mainly is glycosylation.In the biosynthesis of glycyrrhetinic acid,the triterpenoid skeleton β-amyrin is firstly oxidized at C-11 to form 11-oxo-β-amyrin[26],which is subsequently oxidized at C-30 to produce glycyrrhetinic acid [27].Siraitia grosvenoriiCYP87D18 is responsible for the production of 11-oxo-24,25-epoxy cucurbitadienol,11-oxo cucurbitadienol,and 11-hydroxy cucurbitadienol.Except for the monooxygenation,P450s,such as Csa3G903540 and Csa6G088160,from cucumber can catalyze the C-25 hydroxylation of 19-hydroxy cucurbitadienol and the double bond shifting between C-24,25 and C-23,24 [28].

2.4.2.O-methyltransferases (OMT)

Methylation is another common post modification of many plant secondary metabolites.Plant natural products,especially flavonoids and alkaloids,are always methylated byS-adenosyl-Lmethionine (SAM)-dependent caffeoyl-CoAO-methyltransferases(OMTs)[13],such as the methylation on the B-ring of anthocyanins catalyzed by anthocyanin-O-methyltransferases (AOMT).The biosynthesis of the key BIA intermediate (S)-reticuline from (S)-norcoclaurine undergoes three methylation reactions including twoO-methylation steps at position 6 of (S)-norcoclaurine and position 4 of 3-OH-N-methylcoclaurine.These reactions are catalyzed by norcoclaurine 6-O-methyltransferase (6OMT) and 3-hydroxy-N-methylcoclaurine 4-Omethyltransferase (4OMT)respectively,which belong to class IIO-methyltransferases always displaying strict regiospecificity.In the palmatine biosynthesis,palmatine was produced through methylation of columbamine by columbamineO-methyltransferase (CoOMT) [29].(S)-reticuline can also be consecutivelyO-methylated to form (S)-laudanosine.Firstly,(S)-reticulinewasmethylatedby(S)-reticuline 7-Omethyltransferase(7OMT)to(S)-laudanine.This intermediate was potentially further N-demethylated to(S)-tetrahydropapaverine by aN-demethylase,which will beO-methylated to (S)-laudanosine by an uncharacterized 30OMT [30].

Fig.3.Biosynthesis pathway of BIA and MIA.TDC:tryptophan decarboxylase,G10O:geraniol 10-oxidase,10HGO:10-hydroxygeraniol oxidoreductase,IS:iridoid synthase,IO:iridoid oxidase,7-DLGT:7-deoxyloganetic acid glucosyl transferase,7-DLH:7-deoxyloganic acid hydroxylase,LAMT:loganic acid O-methyltransferase,SLS:secologanin synthase,STR:strictosidine synthase,TYDC:tyrosine decarboxylase TyrAT:L-tyrosine aminotransferase,4HPPDC:4-hydroxyphenylpyruvate,NCS;norcoclaurine synthase decarboxylase,6OMT:norcoclaurine 6-O-methyltransferase;CNMT:coclaurine N-methyltransferase,4OMT,3-hydroxyl-N-methylcoclaurine 4-O-metyltransferase,NMCH CYP80B3, N-methylcoclaurine 30-hydroxylase;NMCH: N-methylcoclaurine 30-hydroxylase,BBE:berberine bridge enzyme,STORR:the fusion protein containing a cytochrome P450 monooxygenase and oxidoreductase.

Fig.4.An overview of reactions catalyzed by P450,OMT,and UGT.

2.4.3.UDP-glucosyltransferases (UGT)

Glycosylation is usually the final step in flavonoids and terpenoids especially triterpenoids biosynthesis,which further increased the diversity and modified the solubility,storage,and stabilization of aglycones.Plant flavonoids are always stored in their glycosylated forms,such as quercetin,apigenin,and luteolin.Glycosylation was catalyzed by glycosyltransferases(GTs)through attaching activated monosaccharide units to the skeleton.So far,111GTfamilieshavebeenidentifiedandUDPglucosyltransferases (UGTs)from GT superfamily 1 play vital roles in the glucosylation of flavonoids and triterpenoids.

In the flavonoid biosynthesis,some UGTs are multifunctional.For example,UGT73G1 can glucosylate various flavonoids forming flavonoids attached with either mono-or diglucosides.Medicago truncatula UGT78G1 can glycosylate various substrates,including kaempferol,myricetin,and cyanidin [31,32],and can catalyze the reverse reaction type such as deglycosylating quercetin 3-Oglucoside to quercetin.UGT71G1 fromM.truncatuladisplaying more promiscuity,which involvs in both saponin biosynthesis and flavonoids biosynthesis,can glycosylate quercetin and genistein [33].The Multifunctional UGT85H2 can decorate flavonols,isoflavones as well as chalcones [34].Conversely,UGT73J1 specifically tailored isoquercitrin and genistein on the C-7 site [35],UGT88F2 from Pyrus displayed full selective for phloretin and glucosylate leading to phloretin 2′-O-glucoside [36]and a black soybean UDP-flavonoid-3-O-glucosyltransferase was also found specifically tailoring anthocyanidins and flavonols on the C-3 site[37].

In plants,triterpenoids are always stored as glycosylation forms(saponins),such as glycyrrhizin in licorice.UGT73C11 could glycosylate oleanolic acid on C-3 OH forming 3-O-Glc-oleanolic andM.truncatulaUGT73F3 could glycosylate medicagenic acid on C-28 COOH producing medicagenic acid-28-O-glucoside [38].Based on the function of UGTPg100 and UGTPg1 from ginseng,protopanaxatriol(PPT)can be transformed to ginsenoside F1 and Rh1[39,40].Moreover,other important ginsenoside CK,Rh2,F2,Rd,and Rg3 also can be synthesized by the glycosylation of protopanaxadiol(PPD) catalyzed by UGTPgs,UGTPg45,UGTPg29,PgUGT74AE2,and PgUGT94Q2,respectively [41–43].

3.Microorganisms Engineering for Highly Efficient Production of Natural Products

3.1.Production of natural products using whole-cell catalyst

The functional modification of the skeleton is beneficial for the bioactivity of the natural products,for example,the hydroxylation on flavonoids would significantly enhance the bioactivity and facilitate further modification [44].As a result,naturally occurring fungi were screened for hydroxylation of flavones and isoflavones to produce 4′-hydroxyl and 3′,4′-hydroxyl derivatives as early as 1990,and aPenicillium chermesinumwas found to convert flavanone to 6-hydroxyflavanone and 4′-hydroxyflavanone [13].In these years,designed microbes with target function were used as whole-cell catalysts to produce natural products.Horinuchiet al.constructed a series ofE.colistrains for the biotransformation of various natural and unnatural flavonoids as well as stilbenoids[45].Using theE.colistrain harboring flavone biosynthesis genes,functionally hydroxylation of (E)-2-fluoro-cinnamic acid,(E)-4-fluoro-cinnamic acid,(E)-2-cinnamic acid,(E)-3-cinnamic acid,and(E)-4-amino cinnamic acid was achieved to form their hydroxylated derivatives [46].

Similar toE.coli,S.cerevisiaewas also engineered to a useful biotransformation platform.By exogenously supplementing natural or chemically synthesized phenylpropanoic acids,includingpcoumaric acid,p-fluorocinnamic acid,and fluorocinnamic acid,yeast strains harboring 4CL,CHS,and CHI produced various derivatives,such asp-aminocinnamic acid,(2S)-naringenin,(2S)-2′-fluo ro-5,7-dihydroxyflavanone,and (2S)-4′-amino-5,7-dihydroxyflava none.Based on theS.cerevisiaeharboring IFS,CPR,and 2-hydroxyisoflavanone dehydratase(HID),natural and unnatural isoflavonoids were also produced through the precursor directed biotransformation inS.cerevisiaeby the same group [47,48].By displaying β-glucuronidases (GUSs) fromAspergillus oryzaeusing the anchoring motif Pir1 or Agα1,an efficientS.cerevisiaebiotransformation platform was constructed to produce glycyrrhetinic acid from hydrolysis of glycyrrhizin.Based on the platform,the robust glycyrrhetinic acid biotransformation process can be conducted at an elevated temperature of 60°C making the process more suitable for the large-scale production of glycyrrhetinic acid [49].

3.2.De novo synthesis of natural products by engineered microorganisms

3.2.1.Pathway construction

The rapid progress in sequencing technology on plant genomes and transcriptomes enables the discovery of novel enzyme candidates involved in natural product biosynthesis.This progress facilities the reconstruction of complex plant pathways in microbes.With the determined pathway genes,we can redesign the synthesis pathways according to its native host using stepwise reconstitution.For example,(S)-reticuline synthesis pathway from dopamine was introduced intoE.colias the first proof of concept to produce plant BIAs in a microbial host by the supplemented dopamine.Endogenous dopamine synthesis genes were further introduced to realize thede novosynthesis of (S)-reticuline from glycerol [50].Liet al.expressed 16 plant enzymes related to the synthesis of noscapine from canadine inS.cerevisiae,reconstituting the biosynthetic pathway of noscapine as long as 14-steps from the alkaloid precursor norlaudanosoline achieving thede novoproduction of noscapine in yeast [51].For the purpose of producing morphine in microbes,codon-optimized T6ODM,COR1.3 and CODM were introduced intoS.cerevisiaefor the redesign of morphine biosynthetic pathway based on a single yeast artificial chromosome (YAC) vector (pYES1L).When feeding thebaine for 96 h,codeinone,codeine,and morphine were detected in the culture medium[5].The heterologous enzymes such as plant P450s always exhibit low activity in microbes,which is the primary bottleneck of the process.Mining orthologs from public databases for both efficient native and nonnative enzymes is the most direct way to overcome this bottleneck.Through miningGlycyrrhiza uralensistranscriptome database,the Unigene25647 with higher activity was mined to replace the ever used CYP88D6 in the glycyrrhetinic acid biosynthesis by yeast,leading to 1.94-fold increase of 11-oxoβ-amyrin production [52].Similarly,by BLASTX search against NCBI and some available leguminous transcriptome datasets,P.vulgarisCYP93E9 was identified to be more active thanM.truncatulaCYP93E2,and finally increased the β-amyrin oxidative activity by 61-fold in yeast [53].To improve the efficiency of α-amyrin biosynthesis inS.cerevisiae,Yuet al.identified MdOSC1 from Eriobotrya japonica through BLASTX criteria and phylogenetic analysis,which produced α-amyrin,β-amyrin,and lupeol at a ratio of 86:13:1,with higher specific activity than ever used EjAS.By introducing MdOSC1 intoS.cerevisiaetogether with the promotion of(3S)-2,3-oxidosqualene supplementation,the titer of α-amyrin increased by 5.8 folds[54].Another efficient way to facilitate pathway reconstruction was mining enzymes with booster actions.To promote the artemisinic acid production in yeast,anA.annuacytochrome b5 was introduced into the artemisinic acid-producing strain harboring CYP71AV1 to increase the activity of this P450,and by further expressing of ALDH1 and ADH1 to oxidize the hydroxylated products,artemisinic acid production was significantly improved.Aided by isopropyl myristate (a hydrophobic overlay),the addition of which would enable the extraction of artemisinic acid from the medium,finally 25 g·L-1of artemisinic acid was obtained [55].Moreover,the reconstruction can be optimized by regulating the expression ratio of pathway enzymes.The ratio of 2:1:3 was proved to be the most favorable for T6ODM:COR:CODM.Under this condition,5.2 mg·L-1morphine and 4.8 mg·L-1neomorphine were obtained [5].

3.2.2.Precursor and co-factor supplement

The integration of heterogeneous pathways in microbe cells would result in great consumption of common precursors and cofactors leading to disrupted cell growth and low final product production.Thus,increasing the precursor and cofactor availability displays key roles in the generation of efficient microbial cell factories.

The function of most plant P450s need electrons from NADPH competing co-factor with endogenous metabolism,which is the main reason limiting the cell growth rate and production,thus the co-factor regeneration becomes one of the main obstacles to overcome for high production.Through introducingmBDH1,a mutated 2,3-butanediol dehydrogenase gene,andmvhb,a vitreoscilla hemoglobin gene to regulate NADPH and oxygen supplementation,the production of triterpenoid betulinic acid was increased by 1.5 and 3.2 fold,respectively,in yeast[56].Because of the complexity of the metabolic network,regulating targets may be difficult to identify due to the interconnectivity.Except for simply introducing heterogeneous genes,Chemleret al.employed a stoichiometric-based model to identify combinations of gene knockouts for improving NADPH availability inE.coli.Compared to the control,the modified strain carrying a triple deletion ofDpgi,DppcandDpldAaccumulated up to 817 mg·L-1of leucocyanidin and 39 mg·L-1(+)-catechin,which increased by 4-fold and 2-fold,respectively [48].

Precursor enhancement is a straightforward way to boost the production efficiency,which can be achieved by overexpressing targeted genes on the synthesis pathways,for example,overexpressing of tHMG1 (the truncated version of HMG1 with higher intracellular activity) and other MVA related genes (e.g.ERG9,ERG1,IDI,and ERG20) was intensively studied for terpenoids[57–59].By introducing extra copies of IDI,farnesyl pyrophosphate synthase(FPPS),and squalene synthase(SQS)in the rDNA site ofS.cerevisiaegenome,the total oleanane-type triterpenoids production increased by 1.55-fold in our studies [52,60].In other studies,different precursor producing pathways were synergized to promote the production of s a promising anti-cancer agent perillyl alcohol (POH).All MVA pathway genes were introduced intoE.colionly carrying MEP pathways by a single plasmid to boost the C5 units supplement increasing limonene titers to 400 mg·L-1from glucose.Followed by hydroxylation of a cytochrome P450,100 mg·L-1of POH was obtained [61].

Except for targeted regulation,a global transcription factor in ergosterol synthesis of yeast,UPC2-1 was overexpressed to upregulate the transcription of MVA genes,further incorporated with the targeted regulation strategy,the ginsenoside CK production increased by 5-fold and reached up to 1 mg·L-1[41].In our study of producing oleanane-type triterpenoid by yeast,the promoters were reconstructed with the binding site of UPC2,which led to 65-fold increase of the β-amyrin titer [60,62].

The generation of chalcone skeleton consumes malonyl-CoA,a precursor also required in the biosynthesis of polyketides and fatty acids.As a result,the supplement of malonyl-CoA is considered one of the major bottlenecks for flavonoid biosynthesis.Thus,a strategy of overexpressing ACC complex genes together with the deletion ofackAand acetaldehyde dehydrogenase (adhE) was implemented in microbe cells to enlarge the malonyl-CoA pool[63].To overcome the similar difficulty as in co-factor regulating(The complex metabolic network hindered the recognition of regulating target),Reedet al.developed a Cipher of Evolutionary Design (CiED) method for strain design using genetic algorithm based on a previously publishedE.colimetabolic model (EciJR904 GSM/GPR).Based on the CiED algorithm,carbon flux towards malonyl-CoA and other cofactors was enhanced,and the production of flavanones was improved by the deletion of succinate dehydrogenase (sdhCDAB) and citrate lyase (citE) in the citrate cycle,acetaldehyde dehydrogenase (adhE) consuming pyruvate and the amino acid transporter brnQ,combined with overexpression of genes responsible for the biosynthesis of CoA and malonyl-CoA[47,64].

3.2.3.Redirection of metabolic flux

Redirecting the metabolic flux is also vital to improve the efficiency of microbial cell factories,which always implemented through synchronously enlarge the flux to target compound and prevent the flux from competing or branched pathways.To draw more malonyl-CoA to the flavonoid,a fatty acid biosynthesis pathway and a native malonyl-CoA utilized pathway were inhibited by adding inhibitor cerulenin in the medium leading to more than 9-fold enhancement of flavonoid production including pinocembrin,naringenin,and eriodictyol compared withE.colistrain only expressing genes from the flavonoid biosynthetic pathway [47].For efficiently producing p-coumaric acid inS.cerevisiae,the metabolic flux between p-coumaric acid and aromatic amino acid synthesiswasrebalancedthroughintroducinga phosphoketalose-based pathway to pull glycolytic flux toward erythrose 4-phosphate formation,and optimizing the carbon distribution between glycolysis and the aromatic amino acid biosynthesis pathway by promoters engineering of several genes at key nodes of these two pathways,the highest 12.5 mg·L-1ofp-coumaric acid was finally obtained [65].

During terpenoid biosynthesis,the MVA pathway produces C5 terpenoid units using cytosolic acetyl-CoA as a precursor in yeast.To refine acetyl-CoA consumption a combinational acetyl-CoA supply route was developed with balanced redox cofactors,lower energy consumption,and more efficient glucose utilization.Combined with further disrupting acetyl-CoA competing pathway,(279.0±13.0)mg·L-1β-amyrin production,which was the highest titer,was obtained using glucose fed-batch fermentation[66].Due to the linkage between terpenoid biosynthesis and glucose metabolism,these pathways suffer unbalanced metabolic flux.To overcome these limitations,an isopentenol utilization pathway (IUP)was developed to synthesize IPP or DMAPP by mining enzymes that can sequentially phosphorylate isopentenol isomers isoprenol or prenol,which displayed competitive results with the highest isoprenoid fluxes reported [67].

As the main competing pathway,ergosterol biosynthesis consumes 2,3-oxidosqualene,but its gateway geneERG7coding lanosterol synthase is essential for yeast.Directly knocking out ofERG7will cause lethality.In order to down-regulate ERG7,the methionine repressible promoter PMET3p was used to substitute its native promoter,which improved the β-amyrin production by inhibiting the competitive pathway in the engineered yeast strain [68].The CRISPRi strategy was also used to redirect the metabolic flux.In one study,we focused on two competing pathways consuming precursors 2,3-oxidosqualene and cytosolic acetyl-CoA for triterpenoid synthesis and developed a CRISPRi strategy to downregulate the related genes,including ERG7,ADH1,ADH4,ADH5,ADH6,CIT2,and MLS2,leading to a 44.3%increase in β-amyrin production [69].

3.2.4.Substructure engineering

Substructure engineering refers to the organelle engineering to change the morphological and biochemical properties of specific organelles as well as compartmentalization of biosynthetic pathways based on different organelles.Besides the commonly used method in strain engineering such as redirecting metabolism flux to target pathway,compartmentalization to relocate the pathway enzymes at different subcellular site also turns out to be an efficient strategy for boosting the final production [5].During morphine biosynthesis,the intervening spontaneous step between the reactions catalyzed by T6ODM and COR is the key factor leading to byproduct neomorphine.To make enough time for the spontaneous rearrangement of neopinone to codeinone reducing the byproduct,an endoplasmic reticulum routing tag (ER1) was fused to the C-term of COR1.3,and the ER-localized COR1.3-ER1 separated from T6ODM leading to increased specificity and titer for morphine [5].

Organelle engineering was also used to strengthen the triterpenoid synthesis.Knocking outPAH1(phosphatidic acid phosphatase gene) to extend the endoplasmic reticulum significantly boosted the accumulation of several triterpenoids and triterpenoid saponins [38].The high plasticity of peroxisome makes it an ideal structure for compound synthesis and storage.By engineering the inflated peroxisome for dynamic storage of squalene inS.cerevisiae,the squalene titer was improved from by 138-fold improvement up to 1312.82 mg·L-1[70].Due to the inner lipid body,Y.lipolyticaturned to be a promising host for hydrophobic natural products.To create such a microenvironment inS.cerevisiae,DGA1coding for diacylglycerol acyltransferase was overexpressed to promote the formation of lipid droplets along with pathway engineering,as a result,lycopene content increased to 70.5 mg·g-1DCW compared with that of the starting strain(56.2 mg·g-1DCW).Using a similar strategy,a-amyrin production in yeast was increased to gram level in our recent research [71,72].

3.2.5.Dynamic control

In the biosynthesis of natural products by microbes,dynamic control is used to overcome the toxic effect that resulted from some intermediates or final products,which will disrupt cell growth[73].Employing different ergosterol-responsive promoters,Yuanet al.developed a dynamic control strategy regulating the expression ofERG9to improve the production of plant isoprenoids in yeast.Using this method,the production of amorpha-4,11-diene could increase by 2 to 5-fold compared to the control,while the using of PERG1to controlERG9demonstrated the best amorpha-4,11-diene production of 350 mg·L-1[74].

Besides the regulation of the transcriptional level,protein degradation is also used for dynamic control of natural product biosynthesis.The endoplasmic reticulum-associated protein degradation (ERAD) mechanism was introduced to ERG9 protein to decrease cellular levels of ERG9 using PEST sequence for the regulation of the flux between production and growth.Without any inducers,repressors,or specific repressing conditions,nerolidol titer was improved by 86% to～100 mg·L-1with no negative effect on cell growth [75].

3.2.6.Co-culture

Microbial co-cultures is efficient method for removing pathway bottlenecks,which can reduce metabolic burden,gene expression burden,side reactions and improve metabolic robustness.The most widely used form of co-culture is dividing the complete biosynthetic pathway into the individual module in different strains.For example,the alkaloid norlaudanosoline synthesis undergoes side enzymatic oxidation by TYR reducing the conversing efficiency of dopamine to reticuline.To avoid this side reaction,norlaudanosoline synthesis was divided into two parts,which were glycerol-to-dopamine and dopamine-to-norlaudanosoline,and were introduced into twoE.colistrains,respectively.When cultured sequentially,norlaudanosoline accumulation improved by 300-fold[50].For improving the biosynthesis efficiency of complex flavonoids such as naringenin,co-culture also turned out to be a promising method.Through introducing naringenin biosynthesis into twoE.colirespectively,naringenin production was improved significantly due to reduced metabolic burden,optimized catalytic reaction,and simplified regulation of the related long biosynthetic pathways [76].

4.Enzyme Engineering for Highly Efficient Production of Natural Products

Heterologous expression of natural enzymes for plant natural products has met great success in recent years.Knowledges from enzyme discovery and pathway assembly have accelerated production of important products.The biosynthesis of taxol,glycyrrhetinic acid,and naringenin have all proved this method effective.However,industrial production requires more than natural enzymes in terms of efficiency and stability.When expressed in microbes,plant enzymes always suffer low catalytic efficiency.To solve this problem,high-throughput sequencing and genomics technology have been used for mining new enzymes from plants.However,this approach remains time-consuming and difficult to find proper enzymes.

Many engineering strategies are approved for improving the natural enzymes including directed evolution and rational design.Directed evolution emulates the natural evolution process to construct a mutant library and further select the beneficial mutant.Although most mutant is fruitless or unfavorable,beneficial mutations can be accumulated with proper evolution and screening strategy.In the end,mutants with better efficiency,specificity,and stability will reserve.Rational design is based on an understanding of the molecular basis of the enzyme and can be achieved by a single mutation,exchange of elements,or fusion of enzymes[77].An increasing number of enzyme crystal structures provide a better understanding of their catalytic mechanisms,which greatly facilitates rational design.Computational methods such as homology modeling,docking,molecular dynamics simulation,quantum mechanics also provide instructional tools to guide rational design.

4.1.Enzyme engineering to improve the efficiency and selectivity of plant enzymes

Enzymes offer incredible catalytic efficiency with fine selectivity toward substrates.However,plant natural enzymes in microbe cells are hard to be as same efficient and selective as themselves in source cells,which is one of the main bottlenecks in the biosynthesis of natural products[7].Increasing the efficiency and selectivity of a rate-limiting pathway enzyme toward the synthesis of the desired product can be achieved by both random directed evolution and more rational protein design [78,79].Although directed evolution method can bear the missing of crystal structure,the labour and time costs cannot be estimated.Wanget al.constructed a library with error-prone PCR and screened in 96-well plates,the enzyme variant UGTPg45-HV which has two missense mutations showed 70% increase of ginsenoside Rh2 yield compared with UGTPg45.TheVmaxis nearly doubled and the catalytic efficiency(kcat/Km) of UGTPg45-HV increased by more than 40%.During the process,more than 1500 transformants were screened which means over 125 h were cost on HPLC [80].Directed evolution is still generally approved when less is known about enzyme catalysis mechanism and high-throughput screening method can accelerate directed evolution [81].After developing a color-indicated high-throughput screening method,linalool synthase t67OMcLIS was evolved and expression of the t67OMcLIS variant in the engineeredS.cerevisiaeenhanced precursor supply yielded 53.14 mg·L-1of linalool [82].

On the basis of enzyme structure,rational or semi-rational design is most developed to improve efficiency and selectivity.Structure-guided alanine scanning and saturation mutations hlep chang the regioselectivity of a UGT that synthesizes ginsenoside F12 and Rh2 at a ratio of 7:3 leading to a mutant which can efficiently synthesize Rh2 (～99%) [83],and obtain two P450LaMO mutants with not only improved (S)-enantioselectivity (er 98:2)but also excellent product selectivity (ak 99:1)[84].In the study of pentacyclic triterpenoids glucosylation UGTs,Liuet al.mined UGT73C11 fromBarbarea vulgarisproducing glycyrrhetinic acid-3 O monoglucose,which displayed low activity in yeast.To promote the glycyrrhetinic acid-3 O monoglucose production by the yeast cell factory,UGT73F24 was mined inG.uralensiswith fine regioselectivity.After rationally designing site-directed mutagenesis aided by computation,the use of mutant UGT73F24-I23G/L84N led to a highest 26.31 mg·L-1glycyrrhetinic acid-3 O monoglucose in engineered yeast [85,86].

Besides,the combination of random and rational protein design also displayed fine outcomes.Carloset al.exploited derivational information from mutability landscapes and molecular dynamics simulations to rationally design iterative saturation mutagenesis.P450BM3 mutants evolved with this combined approach achieved unusually high activity for regio-and diastereoselective hydroxylation of five steroids specifically at the C16-position[87].By successive use of random mutagenesis and sequence consensus analysis,one triterpene glycosyltransferase UGT74AC1 variant exhibited up to 4.17 × 104-fold increase in catalytic efficiency toward mogrol and 1.53 × 104-fold increase to UDP-glucose [88].As these examples demonstrated,engineered enzymes with enhanced activity and selectivity have resulted in new breakthroughs in boosting the production of plant natural products.Random protein design can also be considered as the foreshadowing of the rational protein design.

4.2.Enzyme engineering for strengthened stability of plant enzymes

Another problem that hinders plant natural product production is enzyme stability.In realistic engineering production,enzymes must keep high catalytic efficiency in an environment of improper pH,elevated temperature,or organic solvent.Increased stability can extend the lifespan of each enzyme molecule.

Thermostability is of the highest priority in improving enzyme stability.Increasing enzyme tolerance in higher temperatures can allow enzymes to meet the productivity targets required for commercial viability [89].Directed evolution is a common strategy which can improve enzyme thermostability without complicated operation on protein structure corroborated by some examples.Lauchliet al.developed a high-throughput screen using a modified substrate to detect the cyclization byproduct methanol and the thermostability of terpene synthase BcBOT2 increased by 12 °C was obtained using this screening method [90].

For rational approaches,pieces of evidence have shown that the accumulation of minor improvements in weak interactions can enhance the inherent stability of enzymes[91].The major obstacle remains the prediction of mutation sites and optimal amino acid substitutions which have little side effect on activity[92].Previous studies with the comparison of enzymes and their thermophilic homologs can guide us to recognize the proper mutation site[92,93].Using a thermostable sesquiterpene synthase as a model for thermostability,11 residues on the C terminus were truncated which improvedTm1.9 °C in the buffer [94].

Such mutation sites can also be gained by free energy calculation and molecular dynamics simulation.Juanet al.reported the computational design of tobacco 5-epi-aristolochene synthase(TEAS) at elevated temperatures and the thermostable terpene synthase variant denatures above 80 °C,approximately twice the temperature of the wild-type enzyme[95].In the field of biotransforming natural molecule for modified functions,β-glucuronidase has been widely studied for the breaking down of natural glucuronides,which was also limited by the poor thermostability.By the rational protein design,various β-glucuronidase was engineered for increased stability and thermostability.Site-directed mutagenesis was designed to replace five critical aspartic acid and glutamic acid residues with arginine on the surface based on PGUS structure analysis and molecular dynamics simulation.Mutant 5Rs was obtained with optimal pH shifting from 4.5 to 6.5 and thekcat/Kmat pH 6.5 was 10.7-fold higher than that of the wild type [96].The thermostability of PGUS-E was improved by rationally mutating key residues within the catalytic domain based on in-depth structure analysis and sequence alignment.Three mutants F292L/T293K,S35P,R304L were obtained that showed significantly improved thermostability [97].

Besides site-directed mutagenesis,the engineering of structural region was also approved.To increase the catalytic efficiency and thermal stability during the conversion of glycyrrhizin into glycyrrhetinic acid 3-O-mono-β-D-glucuronide and glycyrrhetinic acid by a β-glucuronidase named AtGUS fromAspergillus terreusLi-20,Xuet al.developed a rational sequence-editing strategy through partial truncation or splicing of non-conserved sequence at the C-terminal.The truncated mutant AtGUS-t3 harboring a deletion of the C-terminal hydrophilic coil peptide increased the catalytic efficiency by 3.8-fold with expanded optimal pH range,while a splicing mutant AtGUS(NP) showed increased thermostability [98].Aided by computation based on structural analysis,a highly dynamic C-terminal region was recognized to regulate the expression level,stability,and activity of a GH2 fungal glucuronidase,PGUS fromAspergillus oryzaeLi-3.By rationally truncating the C-terminal region in different lengths,mutant D591-604 improved the half-life at 65 °C by 3.8-fold and Gibbs free energy by 6.8 kJ·mol-1,resulting in obviously improved kinetic and thermodynamic stability compared to the wild type [99].In another study,they developed a loop transplant strategy to refine the thermostability of PGUS-E.Based on a common residue skeleton of DXXTX(X)R,three chimera loops of RSQTSND,RSSTQRD,and DDQTSR were rationally designed by homology structure modeling to replace the unstable loops of PGUS-E.Mutants M1,M3,and M8 showed 1.8,3.3,and 9.4 times higher half-life at 70°C than that of wild-type(8.5 min)respectively[100].These results provided new insights into the interaction between structure and stability of the β-glucuronidase.

Moreover,combinedrandommutagenesis,rational computation-aided design,and the introduction of “clamp”to a TIM-barrel domain led to a mutant TpGUS79A-P4 which increased the thermostability at 55 °C by 2.9-fold [101,102].Recently,protein modifications were also explored to improve the catalytic properties of enzymes.Protein stability was influenced by Nglycosylation,so we designed and placed glyco-linkers in the unusual form of glycobridge and glyco-hairpin at interfaces between domains and monomers with higher structural level,respectively,which conferred dramatically higher kinetic stability and thermodynamic stability of PGUS than the inherent N-glycans [103,104].

4.3.Enzyme engineering to improve the promiscuity of plant enzymes

Tailoring enzymes including P450,OMT,and UGT can add various functional groups to natural products to obtain natural product derivatives.Some derived natural products have played a significant role in medicine and cosmetic areas such as glycyrrhetinic acid 3-O-mono-β-D-glucuronide and dihydroarteannuin.Enzyme promiscuity enables the combination of diverse substrates and functional groups to generate different derivatives.From this point of view,enzymes are expected to have the ability to catalyze linkages between a variety of donors and acceptors[105].Brandenberget al.evolved a cytochrome P450 for highly efficient carbene transfer to pyrroles,indoles,and cyclic alkenes for heterocycle carbene functionalization [106].Malbertet al.generated libraries of GH70 α-transglucosylases mutants targeting pair-wise mutations inferred by molecular docking simulations and found mutants showing remarkable ability for luteolin,morin,and naringenin glucosylation with conversion ranging from 30%to 90% [107].

Substrate entrance and creation,stabilization and repurposing of reaction intermediates are key for unlocking new activities in an existing or designed active site[108].The structure of substrate entrance and binding pocket can help the substrate enter,orient,and bind as well as optimizing catalytic efficiency.Based on this point,efforts to expand substrate entrance and pocket by identifying essential residues are facilitative to ameliorate the promiscuity.With structures ofO-methyltransferase,Valenticet al.identified important residues for enzyme regiospecificity and then expanded substrate scopes toward various 1-benzylisoquinoline substrates[109].In other studies,completely altered product spectra of the 2-methylenebornane synthase was obtained by a semi-rational replacement of active site amino acids[110]and the the simultaneous production of 2-and 3-hydroxy-β-ionone was achieved by CYP109Q5 mutants based on it crystal structure[111].

5.Perspective

Although significant progress has been made in the biosynthesis of plant natural products using microbes,and various complex compounds including morphine and noscapine have been produced by microbes,many challenges remained to meet the commercialization.Diverse metabolic strategies have been developed to boost production,but the final promotion effect is still limited,for the final production of most natural products produced by microbes was at the level of mg·L-1or even μg·L-1.This may hint that the low efficiency of related synthase is the key constraint factors that need to solve firstly.To this end,enzyme engineering by both directed evolution and rational design strategies have been implemented and greatly fascinated plant natural product biosynthesis.

Directed evolution requires inestimable time and effort unless high-effective screen exits.Proper biosensors can convert intracellular information to detectable signals and are regarded as an ideal screen method which can easily identify enzyme mutant with desired properties.However,biosensor mining or design is still a challenge.The universal or migratable biosensor will deepen the application of directed evolution in plant natural product biosynthesis.Rational design can reduce the mutant library but relying more on enzyme structure and calculation method [112,113].Whereas,complex protein structures such as membrane protein are still hard to get,and the precision and speed of homology modeling,docking,molecular dynamics modeling,and quantum mechanics cannot fully meet the demand for engineering methods.Thus,new algorithms and tools need to be developed to rational engineer enzymes.

On the other hand,the difference between the cell internal environment of microbes and plants may also be the reason leading to the low efficiency of plant enzymes in microbes.As a result,the catalytic mechanism of plant enzymes in different cells needs to be illustrated,which would promote the rational desgin of target enzymes.Plant natural products refer to complex synthesis pathways and muti-step enzyme reactions.The construction of the complex pathway,such as noscapine,is still challenging.To solve such problem,efficient assembly methods should be developed to realize the easy construction of complex biosyntheis pathways.

Compared withS.cerevisiae,E.colialways displayed higher final production especially when the synthesis pathway uninvolved membrane proteins,for example,the highest published yields of aromatic AAs or aromatic AA-derived compounds are currently an order of magnitude higher inE.colithan in yeast,both in shake-flask (300 mg·L-1vs 2 g·L-1) and fermentation (2 g·L-1vs 55 g·L-1)conditions.However,in the complex pathways especially involving membrane proteins such as P450,the reconstruction would be significantly limited.It is known that the total synthesis of reticuline from tyrosine has been demonstrated inE.coli.However,the downstream modification of reticuline to form further functionalized molecules,including scoulerine,canadine,salutaridine,magnofluorine and corytuberine,has only been achieved in a yeast host.Yeast is better suited than bacteria to functionally express plant tailoring enzymes such as the endomembranelocalized cytochrome P450s [5].Even using the same species as hosts,different strains lead to different titers of product,one probable reason may be that different strains bear different protein overexpressing burdens.Two representativeS.cerevisiaestains,the diploid INVSc.1 and haploid CEN.PK 2-1C was used in triterpenoid production and INVSc.1 obtained higher concentrations of glycyrrhetinic acid and intermediates,which highlighted the importance of host and suggested the necessity of host screening fornatual products synthesis.

Moreover,the industrial production of natural products still faces the obstacle that the fermentation parameters and the extraction method of a specific compound are unclear.Developing intelligent control strategy with self-response circuits sensing the change of temperature,DO,and pHet al.,based on the illumination of the coupling mechanism between metabolic network and fermentation control would be helpful to facilitate the final production.Furthermore,combined with the exploit of novel materials and techniques for the exclusive separation of target compounds,the progress of the green manufacturing of natural plant products would be sharply improved.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2018YFA0901800),and the National Natural Science Foundation of China (No.21736002).