Yang Zhang,Jing Yu,Yilu Wu,Mingda Li,Yuxuan Zhao,Haowen Zhu,Changjing Chen,Meng Wang,Biqiang Chen,Tianwei Tan*

National Energy R&D Center for Biorefinery,Beijing University of Chemical Technology,Beijing 100029,China

ABSTRACT The use of traditional chemical catalysis to produce chemicals has a series of drawbacks,such as high dependence on fossil resources,high energy consumption,and environmental pollution.With the development of synthetic biology and metabolic engineering,the use of renewable biomass raw materials for chemicals synthesis by constructing efficient microbial cell factories is a green way to replace traditional chemical catalysis and traditional microbial fermentation.This review mainly summarizes several types of bulk chemicals and high value-added chemicals using metabolic engineering and synthetic biology strategies to achieve efficient microbial production.In addition,this review also summarizes several strategies for effectively regulating microbial cell metabolism.These strategies can achieve the coupling balance of material and energy by regulating intracellular material metabolism or energy metabolism,and promote the efficient production of target chemicals by microorganisms.

Keywords:Chemicals Synthetic biology Metabolic regulation Microbial cell factory

1.Introduction

In recent years,the depletion of fossil resources and global warming have received great attention from the international community.Synthesizing chemicals based on renewable resources is one of the promising solutions to achieve sustainable production[1–3].The use of microbial cell factories to construct important chemicals synthesis pathways has attracted more and more attention from scientists,and promotes the green biological manufacturing of chemicals.

Synthetic biology and metabolic engineering technologies are the key to the construction of microbial cell factories.Synthetic biology combines engineering and biology to design and construct novel biomolecular components,networks and pathways,and use these constructs to rearrange and reprogram organisms [4].With the development of synthetic biology and metabolic engineering,microorganisms have made great progress in the efficient synthesis of chemicals.Keaslinget al.used synthetic biology tools inEscherichia coliandSaccharomyces cerevisiaeto construct the synthetic pathway of artemisinin precursor amorphadiene.Through engineering transformation,the titer of amorphadiene can reach 25 g·L-1and 40 g·L-1respectively,realizing semi-synthetic artemisinin and industrial production [5].For small molecular organic alcohols and organic acids,they are widely used as material monomers.DuPont integrated the glycerol production pathway from yeast and the 1,3-propanediol (1,3-PDO) pathway fromKlebsiella pneumoniaeinEscherichia coli,and modified more than 70 genes,which successfully realized the production of 1,3-PDO from glucose as raw material.The output reached 135 g·L-1and the production intensity reached 3.5 g·L-1·h-1.The systematic fermentation and separation purification system was established,which was put into production in 2006 [6].These examples all prove the feasibility of microorganisms to synthesize chemicals efficiently.

Microbial cell metabolism is very complicated.In order to achieve the efficient synthesis of the target compound,it is necessary to maintain the coupling balance of material and energy in the microbial cell factory,and allow more carbon to flow to the target product without affecting cell growth.There are many strategies to regulate cell metabolism,such as cofactor engineering,dynamic regulation,multivariate modular metabolic engineering,CRISPRbased gene knocking-out or weakening,etc.Studies have shown that high-efficiency carbon metabolism pathways in microorganisms generally require two driving forces:kinetics and thermodynamics.Cofactors (NAD(H),NADP(H) and ATP) provide energy for intracellular metabolism [7].Therefore,cofactor engineering has become an important research direction of metabolic engineering and synthetic biology.Cofactor engineering mainly regulates the metabolic flux of specific metabolites or metabolic networks by changing the form and level of intracellular cofactors,which has become an important strategy to improve the production efficiency of bio-based chemicals and bioenergy [8,9].Tanet al.established the NADH regeneration system inKlebsiella pneumoniaeto effectively increase the yield and production intensity of 1,3-PDO[10].Dynamic regulation is a strategy widely used in organisms to help organisms adapt to real-time changes in intracellular or environmental conditions.From the perspective of metabolic engineering and synthetic biology,by maintaining cells in an optimal production state during the entire culture stage,dynamic control makes the host biologically stable,thereby obtaining higher productivity and yield.Multivariate modular metabolic engineering is to divide the entire metabolic pathway into different modules,and then use metabolic engineering technology and synthetic biology regulatory elements to adjust the strength of each module and coordinate the expression of different modules to finally optimize the entire metabolic pathway.CRISPR is the genome editing technology that has attracted much attention in recent years.Two scientists Emmanuelle Charpentier and Jennifer A.Doudna won the 2020 Nobel Prize in Chemistry for their development of CRISPRbased genome editing technology.Scientists can efficiently and accurately change,edit or replace genes in microorganisms,plants,animals and even human through CRISPR technology.The modified CRISPR technology is widely used in the fields of agriculture and biomedicine.

This review summarizes the microbial synthesis pathways of several important chemicals,including small molecular organic alcohols/acids,medical chemicals and aromatic compounds.In addition,we also summarized several commonly used metabolic regulation strategies,which can make the production of chemicals more efficient.

2.Synthesis of Several Important Chemicals by Microorganism

2.1.Synthesis of small molecule organic alcohol by microorganism

1,3-Propanediol (1,3-PDO) is an important chemical substance,which has been widely used in textiles,resins and pharmaceuticals,especially as a synthetic monomer of polymer polytrimethyl terephthalate (PTT) [11].The production methods of 1,3-propanediol mainly include chemical synthesis and biosynthesis.The representative processes of chemical synthesis are acrolein and ethylene oxide,but the pollution is more serious and there are many by-products [12],the biosynthetic method produced by microbial fermentation produces less pollution.The biosynthesis method is introduced in detail below.

In nature,some strains such asKlebsiella[13],Citrobacter[14],Lactobacillus[15]can produce 1,3-PDO through two-step reduction(Fig.1).Various strategies have been reported to increase the production of 1,3-propanediol.In order to strengthen the synthesis pathway,Zhaoet al.separately overexpressed two endogenous enzymes fromKlesiella pneumoniae.The molar yield is increased from 50.6% to 64.0%,and the concentration of by-products is reduced.Overexpression of glycerol dehydratase (GDH) reduces the concentration of ethanol and 2,3-butanediol (2,3-BDO) [16].Wuet al.blocked the 2,3-BDO pathway and introduced an FDHdependent NADH regeneration system into the host.The final concentration of 1,3-PDO reached 72.2 g·L-1[17].

Carbohydrates cannot be used as natural 1,3-propanediol production substrates,but carbohydrates can be converted to glycerol and then glycerol is converted to 1,3-PDO through two-step reduction.Currently,the highest titer is 135 g·L-1by DuPont,which is synthesized by expressing glycerol synthesis pathway fromyeastand 1,3-PDO synthesis pathway fromKlebsiella pneumoniae,and a large number of genes inE.colihave been modified [6].The 1,3-propanediol oxidoreductase (yqhD) and glycerol dehydratase(dhaB) are integrated into the chromosome through the transformation method mediated byAgrobacterium tumefaciens.Saccharomyces cerevisiaecan produce 0.4 g·L-11,3-PDO with glucose as raw material [18].Zenget al.synthesized 1,3-PDO inE.coliusing glucose as a substrate and homoserine as a precursor.Application of protein engineering to modify glutamate dehydrogenase to increase its affinity for homoserine,and the final yield reaches 51.5 mg·L-1[19].The Chen’s group of Tsinghua University optimized the pathway to synthesize 1,3-PDO using homoserine as the precursor.The key enzymes in this pathway were screened for glutamate dehydrogenase and pyruvate dehydrogenase and the point mutation technology was used.A fusion protein was constructed for these two key enzymes.At the same time,overexpression of alcohol dehydrogenase (yqhD) and deletion of by-product pathways can eventually produce 0.63 g·L-11,3-PDO inE.coli[20].Fraz?oet al.systematically described the construction of an anabolic pathway for the direct biosynthesis of 1,3-PDO from glucose through the Krebs cycle of malate.Using protein engineering and other methods to screen and modify DHB dehydrogenase and OHB dehydrogenase while expressing 1,3-PDO oxidoreductase to produce 0.1 mmol·L-11,3-PDO,but by co-cultivatingE.coliwith DHB-PDO pathway and malate-DHB pathway,1,3-PDO increased by 40 times[21].Recently,Zenget al.proposed the use of methanol as a substrate to produce 1,3-PDO and selected a pyruvatedependent aldolase to convert the formaldehyde (HCHO) channel into 2-keto-4-hydroxybutyrate as a biological An important intermediate for synthesis.By combining this reaction with three other enzymes,it is proved that the C1 metabolic pathway based on pyruvate can be used for the biosynthesis of 1,3-propanediol.The titer of 1,3-PDO is (508.3 ± 9.1) mg·L-1and (32.7 ± 0.8) mg·L-1when HCHO or methanol are used as co-substrates for glucose fermentation,respectively (Fig.2) [22].

1,4-Butanediol (1,4-BDO) is an important chemical raw material.The global annual market volume exceeds 2.5 million tons and the market value exceeds 4 billion US dollars.1,4-BDO is widely used in medicine.In the fields of chemical,textile,paper,automobile and daily-use chemicals,the traditional production method is to hydrogenate fossil raw materials.However,as fossil energy is withering day by day,biosynthetic 1,4-BDO will occupy more and more proportion.However,no strain has been reported to produce 1,4-BDO naturally.Researchers have made some progress in the biosynthesis of 1,4-BDO by microbial cell factories(Table 1).

Boldtet al.used their internally developed SimPheny Biopathway Predictor software to predict the potential pathways for the biosynthesis of 1,4-BDO inEscherichia coli.The algorithm identified 10,000 common metabolites (such as acetyl-CoA,α-ketoglutarate and Succinyl-CoA) to synthesize 1,4-BDO (four to six steps).Through the above screening,they identified two effective pathways,one with α-ketoglutarate as the precursor and the other with succinyl-CoA as the precursor.Succinyl semialdehyde is synthesized by α-keto acid decarboxylase or succinyl-CoA dehydrogenase (SucD).Succinyl semialdehyde can be converted to 4-hydroxybutyryl-CoA by 4-hydroxybutyrate dehydrogenase,and then 4-hydroxybutyryl-CoA is converted to 4-hydroxybutyraldehyde by 4-hydroxybutyryl-CoA transferase,which finally is converted to 1,4-BDO byE.coliendogenous alcohol dehydrogenase (yqhd) [24,28].Genomatica successfully transformed the industrial strain for producing 1,4-BDO,with a titer greater than 120 g·L-1,a productivity of 3.5 g·L-1·h-1,and a yield of 0.4 g·(g glucose)-1,which reached 80% of the theoretical yield[23].Hwanget al.obtained a dehydrogenase with a catalytic activity of about 2.5 times higher than before by performing directed evolution on 4-hydroxybutyryl-CoA dehydrogenase,and optimized the production pathway of 1,4-BDO to a certain extent [29].Taiet al.used the non-phosphorylated lignocellulose utilization pathway to construct a biosynthetic pathway of 1,4-BDO from xylose(the green pathway in Fig.3),with the highest titer of 3.8 g·L-1,which reached the theoretical yield of 63% [25].Wanget al.used the above approach combined with protein engineering to first increase the titer of 1,2,4-butanetriol to 1.5 g·L-1to provide the corresponding substrate for glycol dehydratase,and finally obtained the titer of 209 mg·L-1.1,4-BDO (purple pathway in Fig.3) [27].

Fig.1.Glycerol metabolism in Klebsiella.glycerol dehydratase (dhaB),1,3-propanediol oxidoreductase (dhaT),glycerol dehydrogenase (dhaD),Dihydroxyacetone kinase(dhaK),lactate dehydrogenase(ldhA),acetolactate synthase(als),acetolactate decarboxylase(adc),acetoin reductase(ard),aldehyde-alcohol dehydrogenase(adhE),phosphate acetyltransferase (pta),acetate kinase (ackA).

Fig.2.1,3-PDO is produced from glucose and methanol as substrates.glycerol 3-phosphate dehydrogenase (gpd1),glycerol 3-phosphate phosphatase (gpp2),pyruvate carboxylase (pycP458S),aspartokinase/homoserine dehydrogenase variant G433R (thrAG433R),aspartate transaminase (aspC),pyruvate decarboxylase (pdc),alcohol dehydrogenase (yqhD),malate kinase (mk),malate-b-semialdehyde dehydrogenase (msd),malate semialdehyde reductase (msr),putative DHB dehydrogenase V108C variant (LlDdV108C),pyruvate decarboxylase variant W392Q (pdcW392Q),NAD-dependent methanol dehydrogenase (mdh2),2-keto-4-hydroxybutyrate aldolase (khb),branched-chain alpha-keto acid decarboxylase (kdc).

Table 1 Summary of 1,4-BDO by microorganism

Fig.3.Bioproduction pathways of 1,4-BDO.

2.2.Synthesis of small molecule organic acid by microorganism

In addition to small molecular organic alcohols,microorganisms could also produce small molecular organic acids through cell metabolism,such as succinic acid,muconic acid,glutaric acid,and itaconic acid.The following is an example of microbial synthesis of glutaric acid and itaconic acid.

Glutarate is an important C5 dicarboxylic acid,which is the monomer for producing nylon 4,5 or nylon-5,5 [30,31].Glutarate can be synthesized through the aminovalerate (AMV) pathway in the lysine catabolism pathway by bacteria such asPseudomonas putida(Fig.4) [32,33].In AMV pathway,Lysine is converted into glutarate by lysine 2-monooxygenase,delta-aminovaleramidase,5-aminovalerate transaminase and glutarate semialdehyde dehydrogenase [34].Over the years,The AMV pathway was introduced intoE.coliorCorynebacterium glutamicumfor the production of glutarate (Table 2).Corynebacterium glutamicumis used as an excellent host strain for lysine production.Christoph Wittmannet al.selected the mutantC.glutamicumAVA-2 served as a starting strain and overexpressed 5-aminovalerate transaminase (gabT)and glutarate semialdehyde dehydrogenase (gabD).,a new 5-aminovalerate importer NCgl0464 was overexpressed to promote the convertion of 5-aminovalerate.This recombinant strain can accumulate glutarate to 90 g·L-1with a yield of 0.7 mol·mol-1in the production phase,which is the highest reported titer [37].In recent years,many other pathways have been proposed for glutarate production (Fig.4 and Table 2),including 1) Cadaverine pathway;Yuanet al.achieved efficient synthesis of glutaric acid by strengthening the endogenous cadaverine pathway ofE.coli,relieving lysine inhibition,enhancing the supply of oxaloacetate and overexpressing native transporters.The titer of glutarate reached 54.5 g·L-1with high yield (0.54 mol·(mol glucose)-1)[34]2) 2-ketoglutarate pathway divided into 2-hydroxyglutarate branch and homoisocitrate branch;Qianet al.constructed a 2-hydroxyglutarate branch pathway in Escherichia coli to achieve the production of glutarate.2-ketoglutarate is firstly converted to 2-hydroxyglutarate by 2-hydroxyglutarate dehydrogenase(HgdH),and then 2-hydroxyglutarate is converted to glutarate through 5 step enzymatic reactions [30].In addition,Yanet al.constructed a homoisocitrate branch pathway inE.coli,which involves homocitrate synthase (HCS),homoaconitase (HA),homoisocitrate dehydrogenase (HICDH),α-keto acid decarboxylase and glutarate semialdehyde dehydrogenase.The final strain can produce 0.42 g·L-1glutarate [31].3) reverse β-oxidation pathway from acetyl-CoA and malonyl-CoA.Denget al.introduced five-step reverse adipate degradation pathway (RADP) identified inThermobifida fuscainE.coli,and synthesized glutarate through acetyl-CoA and malonyl-CoA.The optimized strain produced 36.5 mmol·L-1glutarate by fed-batch fermentation [39].

Fig.4.Bioproduction pathways of glutarate.

Table 2 Summary of glutarate by microorganism

Itaconic acid (IA) is a white crystalline unsaturated dicarbonic acid.It is an important bulk chemical and is widely used in the industrial production of resins,acrylic plastics,acrylic latex and superabsorbents.It has a long and successful production history through the fermentation offilamentous fungi,most of which are produced by the fermentation ofAspergillus terreus.So far,the maximum productivity of theAspergillus terreus-based system is 1.2 g·L-1·h-1,and the final titer can reach 86.2 g·L-1[40].

Fig.5.Biosynthetic pathways of itaconic acid and its subcellular location in Aspergillus terreus (a) and corn smut (b).

Aspergillus terreusandCorn Smut Fungushave been used as model systems to study the biosynthetic pathway of itaconic acid,as shown in Fig.5a and b,respectively.Glucose or other carbon sources from the extracellular environment convert into citric acid through glycolysis and tricarboxylic acid cycle (TCA cycle).Citric acid is dehydrated to cis-aconitic acid,which is transported into the cytoplasm by the putative mitochondrial tricarboxylic acid transporter (MTTA) encoded bymttA[41],and converted into cisaconitic acid by cis-aconitic acid decarboxylase (CAD) The MFSA transporter encoded by themfsAgene is then used to transport itaconic acid into the extracellular environment[42](Fig.5a).The key enzyme for the formation of itaconic acid is CAD,which is encoded by the cadA gene.InCorn Smut Fungus,the isomer of cis-aconitic acid,trans-aconitic acid is the precursor of itaconic acid [43].The cis-aconitic acid is transported into the cytoplasm by the mitochondrial TCA transporter(MTT1),converted to trans-aconitic acid,and then decarboxylated to form itaconic acid (Fig.5b).The key enzymes in this process are cytosolic aconitic acid dimerase(ADI) and trans acid decarboxylase (TAD).Finally,the main promoter ITP1 mediates the transport of itaconic acid to the outside of the cell.

In industry,Aspergillus terreusproduces up to 85 g·L-1of itaconic acid from glucose[40,44].Genetic engineering is an effective strategy to increase the titer of itaconic acid.The overexpression of related key genes cadA,mttA and mfsA affects the accumulation of itaconic acid to varying degrees [42].In particular,the cooverexpression of cadA and mfsA genes resulted in an 8.7%increase in itaconic acid production [45,46].In addition,the overexpression of specific transcription factors (RIA1) or MTT1 inCorn Smut Fungusmay two times the titer of itaconic acid [43].By knocking out the Alg3 gene encoding GlcNAc-PP-Disulfide mannose transferase,or by overexpressing LaeA,the titer of itaconic acid was increased by about 27%.In addition,the LaeA gene was overexpressed in the Alg3 mutant,which increased the titer of itaconic acid by more than 1.5 times [47].

In addition toAspergillus terreusandCorn Smut Fungus,other host organisms were designed,includingAspergillus nigerstrains that produced 200 g·L-1of citric acid,which can accumulate itaconic acid [48].Yarrowia lipolyticathat produced 200 g·L-1citric acid may be a potential platform for itaconic acid production[49].In addition,Saccharomyces cerevisiae[50],Corynebacterium glutamicum[51]andEscherichia coli[52]were modified to produce itaconic acid.Due to the low level of endogenous multifunctional CAD,it is necessary to overexpress the cadA (cis-aconitic acid decarboxylase) gene fromAspergillus terreus.However,these engineered strains still show lower titers of itaconic acid.

2.3.Synthesis of medical chemicals by microorganism

Fig.6.Bioproduction pathways of N-acetylglucosamine and heparosan.

Table 3 Overview of GlcN and GlcNAc production by microorganisms

N-acetylglucosamine(GlcNAc)is a monosaccharide obtained by acetylation of GlcN.GlcN plays an important role in the protection of human joints [53].GlcNAc and GlcN are also important precursors for the synthesis of glycosaminoglycans and bifidus factors in humans.GlcNAc has a high content in the exoskeletons of crustaceans and is one of the important components of the exoskeletons of crustaceans.GlcNAc and GlcN are present in the cell walls of most bacteria,yeasts and filamentous fungi.GlcNAc and GlcN are also important precursors in the glucosamine sugar metabolism pathway of these microorganisms,and they play a vital role in the growth and metabolism of microorganisms[54–58].In view of the role and function of GlcNAc and GlcN in the growth and metabolism of organisms,GlcNAc and GlcN are mainly used in health care products,medicines,and cosmetics[59,60].In the past ten years,researchers have modified the GlcNAc biosynthesis pathway by engineering techniques inEscherichia coli,Saccharomyces cerevisiae,Bacillus subtilisandCorynebacterium glutamicum(Fig.6).The comparison of GlcNAc synthesized by various strains is shown in Table 3.Denget al.used a series of metabolic engineering strategies to construct high-performance recombinantE.colistrains for the production of GlcN and GlcNAc,and the titer of GlcNAc reached 110 g·L-1[67].The modification strategy includes modification of the key enzyme (glmS) inE.colito reduce GlcN inhibition,and heterologous expression of GlcN-6-P acetyltransferase (GNA1) fromSaccharomyces cerevisiae,knocking out the depletion genesnagEof GlcN and GlcNAc,as well as the operons manXYZ,nagBACD.Bacillus subtilisis a gram-positive bacteria that is widely used in the industrial production of enzymes,amino acids and vitamins[68].Compared withEscherichia coli,Bacillus subtilisis a recognized safe strain.Liuet al.built a key overexpression module and blocked GlcNAc and GlcN consumption modules inBacillus subtilis.The titer of GlcNAc in genetically modifiedBacillus subtilisreached 7.5 g·L-1[69].Liet al.optimized the fermentation process and the GlcNAc titer reached 35.77 g·L-1[70].

Heparin is an anticoagulant drug expressed by mast cells of mammalian connective tissue,which is an acid mucopolysaccharide composed of uronic acid and glucosamine.E.coliK5 capsular polysaccharide contains heparosan component,and its skeleton structure is similar to heparin,except that it has not undergone unsulfated modification.Therefore,heparosan can be modified to obtain heparin and its analogues.Heparosan is a disacchariderepeating unit formed by alternately linking glucuronic acid and glucosamine.So far,heparosan has been found inE.coliK5,Avian bacillus paragallinarumandPasteurella multocidatype D.But onlyE.coliK5 is used to produce heparosan by fermentation,and the titer reached 15 g·L-1[71,72].However,E.coliK5 is a pathogenic bacterium.In order to solve this problem,scholars have conducted a lot of research,including the construction of safe heparosanproducing recombinant strains and enzymatic preparation of heparosan in vitro and other methods,as shown in Fig.6 and Table 4.KfiAandKfiCfromE.coliK5,respectively encoded acetylglucosamine transferase and glucuronyl transferase,are responsible for the polymerization of heparosan sugar chains.PmHS1 and PmHS2 are heparosan synthetases from D-type Pasteurs The amino acid sequences of PmHS1 and PmHS2 are about 70% similar.Currently,Duet al.constructed a synthetic heparosan strain,with the titer of 7.25 g·L-1[79],which is the highest titer.

2.4.Synthesis of aromatic chemicals by microorganism

Aromatic compounds refer to the general term for a class of compounds containing benzene ring structure,which have a wide range of applications in the fields of food,chemical industry,medicine and feed[80].The shikimic acid pathway is the main pathway for microorganisms to synthesize aromatic compounds and their derivatives (Fig.7).Through metabolic engineering methods,the shikimic acid pathway is optimized,and synthetic biology is used as a guide to design and construct natural and non-natural aromatic compounds synthesis pathways [81–83].

Fig.7.Bioproduction pathways of several important aromatic compounds.

Styrene is a bulk petrochemical product,McKennaet al.[84]let the L-phenylalanine aminolyase genePAL2ofArabidopsis thalianaand the ferulic acid decarboxylase geneFDC1ofS.cerevisiaeoverexpressed in the L-phenylalanine high-producing strain.Finally,the engineered strain produced styrene by shaking flask fermentation,with the titer of styrene reaching 260 mg·L-1.Subsequent research will explore the modification of host strains to increase the production of precursor L-phenylalanine [85].

As an important industrial chemical,terephthalic acid is the main raw material for the manufacture of polyester fiber,film and polyester for bottles.In a patent,E.coliwas used to heterologously express four genes ofComamonas testosterone:tsaB,tsaM,tsaC,tsaD,and successfully achieved the de novo synthesis of terephthalic acid in microorganisms through the shikimic acid pathway using glucose as the raw material.Heet al.[86]found that a nitrilase from thegenus Rhodococcus-CCZU10-1can catalyze the hydrolysis of dinitrile to acid,and finally obtain 776 g·(g DCW)-1terephthalic acid.

As an important aromatic compound,vanillin is widely used in various flavored foods that need to increase the flavor of milk.Hansenet al.[87]first reported to useSaccharomyces cerevisiaeto make use of 3-dehydroshikimic acid as an intermediate,and heterologously expressed three enzymes (3DSD,OMT,ACAR) to convert 3-dehydroshikimate to vanillin,achieving the de novo synthesis of vanillin in microorganisms via the shikimic acid pathway using glucose as the raw material.Finally,the titer of vanillin is 65 mg·L-1.

The research on the microbial synthesis of aromatic compounds has attracted more and more attention,which not only provides a new green production method for industrialized high-yield aromatic compounds,but also provides a theoretical basis and technical methods for the research of microbial high-yield and high-value aromatic compounds.

3.Metabolic regulation promotes efficient synthesis of chemicals by microorganisms

3.1.Cofactor engineering for more efficient production of chemicals

To improve the efficiency of producing target products through microbial fermentation,it is necessary to increase the metabolic flux of its biosynthetic pathway.In addition to carbon flow,the effective production of target products also requires oxidation–reduction balance[88].Cofactors such as NAD(H),NADP(H)and ATP can provide energy for carbon metabolism.Cofactor coupling in redox reactions can promote the biocatalytic process and even push forward the development of unfavorable thermodynamic reactions.Cofactor regulation strategies mainly include:1)Regulation of endogenous cofactor system;Maintaining cell redox balance is the basic requirement for obtaining high product yield,and this balance requires proper consumption of cofactors and regeneration[89].There are three ways to regulate the endogenous cofactor system:(i)a competitive approach to reduce cofactor consumption,(ii)an endogenous pathway to enhance cofactor production,and (iii) modifying the overall regulator.2) Replenishing heterologous cofactor regeneration system.It is possible to regulate cofactors by introducing a heterologous cofactor regeneration system.The enzyme cascade is widely used for cofactor regeneration[90]and the most common NADH regeneration is through the expression of heterologous enzymes [91-93].On the other hand,the ATP regeneration system mainly relies on kinases [94].These enzyme-dependent cofactor regeneration systems can change the ratio of NADH/NAD+or ATP/ADP in the cell.The other two strategies to adjust the redox balance are the combination of substrates or the introduction of rationally designed approaches.3) Modification of cofactor preferences;Enzymes engineered by site-directed mutagenesis can change the priority of cofactors from NADH to NADPH,although changes in coenzymes usually impair the activity of mutant enzymes compared to the original cofactors.Compared with site-directed mutagenesis,random mutations with large libraries have limited application in specific changes in enzyme cofactor preferences due to cumbersome screening [95].However,high-throughput screening using computers can discover new enzymes for further rational design.A tool is based only on protein structure without any sequence/structure information about homologous proteins.This tool can systematically screen the mutation sites in the computer [96].Another tool,Cofactor Specific Reversal:Structural Analysis and Library Design(CSR-SALAD),is available online for free.CSR-SALAD can be used in synthetic biology,metabolic engineering and biocatalysis [97].These achievements will make cofactor preference engineering a routine task and will continue to expand the current strategies for adjusting the cofactor system.

Cofactor engineering has an important impact on cell metabolism.In cell growth and metabolism,cofactors participate in the transportation and metabolism of intracellular substances.In aerobic metabolism,cofactors are continuously consumed and produced,thereby promoting cell metabolism [98].The intracellular redox reaction directly affects the rate of cell growth and metabolic reactions [99].The fluctuation of the concentration of various cofactors will also affect the distribution of metabolic flux [100].Many studies have shown that cofactor engineering can affect gene transcription and expression.For example,NADPH indirectly affects the expression of genes related to methionine,lysine and other amino acid synthesis pathways [95].Through the combination of carbon metabolism and cofactor engineering,we could achieve efficient production of chemical substances and biofuels.Currently,researchers have introduced a variety of cofactor regulation strategies into host microorganisms to increase the microbial yield of target products.The coupling of carbon metabolism and cofactor engineering effectively coordinate the demand for substance and energy,and can greatly increase the output of target products.

3.2.Dynamic regulation for more efficient production of chemicals

With the development of synthetic biology technology,more and more high-value products can be produced by constructing cell factories,such as vanillin[101],p-hydroxybenzoic acid(PHBA)[102].Traditional static control methods have been successfully used to regulate metabolic pathways,such as gene knockout,promoter engineering [103],RBS library [104].These methods can produce high-yield chemicals,but due to their irreversibility,and the inability to monitor the concentration of intracellular substances and control the level of gene expression in real time,there are certain limitations.On this basis,dynamic control came into being.

Dynamic regulation is through the design and construction of gene pathway units,so that recombinant cells can dynamically sense and process specific signals,so as to perform adaptive regulation according to the changes in the signal to achieve the optimal state of product synthesis or target system[105].Dynamic control generally consists of three parts:input signal,biosensor,and actuator.According to the difference of input signals,it can be divided into dynamic regulation system induced by environmental signals and dynamic regulation system induced by cell endogenous signals.The dynamic regulation system induced by cell endogenous signals includes intracellular metabolite response system and quorum sensing system.

The dynamic regulation induced by environmental signals mainly includes light,temperature and chemical induction.Optogenetics tools are ideal for precise control,because their pulse and duration can be precisely controlled and are an instantaneously accessible signal.A light-controlled switch called OptoEXP was constructed inSaccharomyces cerevisiae.Use this light-controlled switch to control the mitochondrial isobutanol pathway,switching the metabolic state of the cell between the light-induced growth phase and the dark-induced production phase canproduce (8.49 ± 0.31) g·L-1isobutanol and (2.38 ± 0.06)g·L-12-methyl-1-butanol from glucose[106].Because of its versatility,sensitivity and uniformity,temperature is another common signal in dynamic path engineering applications.There are mainly two systems,temperature-sensitive promoters and temperaturesensitive molecules.PR/PL is a temperature-sensitive promoter.In the recently constructed itaconic acid producingE.coli,the promoter of isocitrate dehydrogenase coding gene was replaced by the Lambda promoter (pR).The growth phase is carried out at 37 °C,and it is converted to itaconic acid production at 28 °C.Through dynamic production,the titer of itaconic acid is increased to 47 g·L-1[107].Chemical induction is also a commonly used method of regulation.IPTG is the most commonly used chemical inducer.Excessive activity of the TCA cycle inE.coliwill lead to insufficient flux into the production pathway.By adjusting gltA to control the TCA cycle,the titer and yield of isopropanol increased by 3.7 and 3.1 times,respectively [108].

Dynamic regulation of intracellular metabolites mainly includes promoters,transcription factors and riboswitches that respond to intracellular metabolites (Table 5).Ergosterol is a competitive product for the production of amorpha-4,11-diene.The use of a promoter that responds to ergosterol to replace the endogenous PERG9 ofSaccharomyces cerevisiaereduces the FPP metabolic flux synthesized by the competitive pathway,making The production of amorpha-4,11-diene has been increased by 2 to 5 times [109].FapR is a transcription inhibitor derived fromBacillus subtilis.It can specifically sense the concentration of the intracellular malonyl-CoA.Useing this switch to dynamically regulate fatty acid biosynthesis inE.colican achieve a balanced metabolism between cell growth and product formation.Compared with wild-type strains,the titer of fatty acid increased by 15.7 times [110].The LysR family of transcriptional regulators represents the mostabundant transcriptional regulator type in the prokaryotic kingdom [111].CatR is a member of the LysR family,which can bind to muconic acid,allowing CatR to be released from the bound promoter and starting transcription of downstream genes.In a study,the authors proposed a sensor regulator based on RNAi and a dualfunction dynamic control network,which can simultaneously provide engineered biosynthesis,achieving up or down regulation of cell metabolism.This dynamic control network was used to regulate the phosphoenolpyruvate metabolism node inEscherichia coli,which can realize the autonomous distribution of carbon flow between its natural metabolism and the engineered muconic acid biosynthesis pathway,so that the titer of muconic acid reached 1.8 g·L-1[112].Riboswitch is a ligand-dependent RNA cis-acting element,which can specifically bind or release metabolite ligands according to changes in intracellular metabolite concentration,and regulate the expression of downstream coding sequences through conformational changes.A strategy for evolving and screening Nacetylneuraminic acid (NeuAc) riboswitches in vivo was established.The base saturation mutation on the binding ring changes the affinity of the riboswitch and affects the threshold of the riboswitch.A modified riboswitch with a high threshold and a large dynamic range is obtained.Using NeuAc riboswitch with highthreshold for Nacetylneuraminic acid production,glucose was used as the sole carbon source,and the highest NeuAc titer was 14.32 g·L-1[113].

Table 5 Research progress of dynamic regulation

Microorganisms can synthesize special signal molecules by themselves to achieve intra-and inter-species communication to initiate the expression of specific genes,thereby controlling the state of the entire community.At present,this phenomenon is generally called quorum sensing (quorum sensing,QS).QS includes gram-negative bacteria system such as the typical Lux system and gram-positive bacteria system such as Enterococcus faecalis is the Fsr system [114].LuxI-LuxR is a QS system fromVibrio fischeri.Under the control of the promoter PluxI,the expression of downstream genes is activated.For synthesizing 1,4-butanediol inEscherichia coli,LuxI/LuxR system was used to regulate the expression of D-xylose dehydrogenase (xdh),2-dehydro-3-deoxyd-xylonic acid dehydratase (xylX) and ketoacid decarboxylase(mdIC),which realized the expression of xylose to 1,4-butanediol pathway after mass growth,and the titer and yield reached 0.44 g·L-1and 0.07 mol·(mol xylose)-1[26].EsaI-EsaR system is a QS system fromPantoea stewartia.According to the EsaI–EsaR system,a gene circuit was designed to fine-tune the expression level of EsaI to control the expression of phosphofructokinase-1(Pfk-1) and oxalate kinase (AroK),thereby down-regulating the flux of glycolysis,allowing more carbon flux to flow to the glucaric acid synthesis pathway.The concentration of glucaric acid is increasedfrom0to>0.8g·L-1[115].

3.3.Multivariate modular metabolic engineering for more efficient production of chemicals

Inspired by the way,higher organisms divide their metabolic pathways into modules through organelle compartmentalization,scientists have proposed the concept of modularized modified biosynthesis pathways.In modular metabolic engineering,enzymes in metabolic pathways are grouped into a series of interacting modules to relieve metabolic pressure and rebuild metabolic balance,so as to improve the yield of target products [116].

In order to avoid the high-throughput screening challenges,the modular metabolic Engineering (MMME) and the modular coculture Engineering (MCE) have promoted the construction of modular strains and the development of co-culture engineering for high-efficiency biochemical production.Initially,MMME has been shown to be an effective strategy for modularization of biosynthetic pathways in a single host,reconstructing metabolic balance and improving metabolite production [117].At present,the concept of modularity has been further extended,and the use of the MCE strategy enables the construction of biosynthesis paths into two or more hosts,and the co-production of multiple hosts is more advantageous than that of a single host,thus further improving the production efficiency of the target product [118].

With the development of more sophisticated gene-editing techniques(CRISPR-Cas9,CRISPRi,etc.),candidate hosts for MCE strategies have been extended to non-model organisms.For example,clostridium-mediated ABE fermentation production pathways that have previously been industrialized inE.coli[119],are now being developed by using MCE strategies in combination with CRISPRi and optimized electroporation techniques.The mixed-fungal fermentation increased theN-butanol titer to 11.5 g·L-1[120].

In addition to the latest advances in experimental methods,the rapid development of gene scale modle(GSM)makes it possible to realize efficient modularization in metabolic engineering.Recent advances in GSM construction and optimization have improved our ability to simulate and predict biological phenotypes based on genomic information [121].GSM also provides a solid foundation for new engineering strategies in the modular unit design[122].In recent years,the GSM has expanded to the field of molecular biology,for example through gene knock-out and biological coupling insaccharomyces cerevisiaeto increase production of succinic acid,also through enhancing the activity and richness of enzyme to improve the production of olefin [123].In another recently developed method,transcriptional repressor prediction was combined with the STRAIN optimization algorithm of GSM to regulate the metabolic module and control the production of target compounds such as shikimic acid and muconic acid [124]

The modularization of metabolic engineering embodies the idea of block regulation and overall optimization.At present,researchers have introduced a variety of modularized regulation strategies in host microorganisms to improve the microbial target product yield.Multiple modular methods are used to realize the carbon balance of each host and the mutual promotion effect of the coculture system.After a lot of computer calculation and analysis,reasonable optimal design and improvement,it is expected to explore a more efficient and stable co-culture system,which can greatly increase the yield of the target product.

4.Application of CRISPR Technology in Synthesis of Chemicals by Microorganism

The industrial production of bulk chemicals usually requires robust strains,which can tolerate low pH,temperature fluctuations and the presence of various inhibitors [125].In order to maximize the fermentation yield and productivity of the strains in the industrial production of bulk chemicals,it is necessary to modify microorganisms by metabolic engineering(such as overexpression,knock-out or knock-down of multiple target genes) to design the best microbial cell factories.In the past several years,CRISPRCas9 system have been widely used in the construction of various genetically engineered strains to produce bulk chemicals [126].Nonetheless,its multiple applications are still limited by guide RNA (gRNA) processing efficiency and throughput [127].The CRISPR-Cas9 system was modified to further optimization to improve the microbial cell factory to produce bulk chemicals(Table 6).Here,we mainly summarized the application of CRISPR technology inSaccharomyces cerevisiaeandBacillus subtilis.

4.1.The application of CRISPR technology in Saccharomyces cerevisiae

The CRISPR-Cas9 system can be effectively applied to the production of bulk chemicals inSaccharomyces cerevisiae.The toolsfor CRISPR-Cas9-mediated genome editing was appiled in industrial strains,with the plasmid carrying Cas9 gene,the other performing gRNA cassette and natMX marker (Fig.8A).Pyruvate decarboxylasePDC1andPDC5was knocked out in the industrialS.cerevisiae,then the heterologous lactate dehydrogenase geneldhLwas knocked in,which facilitated the production of lactate.(5.1±0.65)g·L-1glucose was consumed and(2.5±0.3)g·L-1lactic acid was produced (the yield of lactic acid was (0.49 ± 0.03)g·(g glucose)-1within 100 hours [128].The industrialS.cerevisiaewas used for the production of second-generation lignocellulosic bioethanol.The obstacle is the high cost of enzymes that saccharify lignocellulosic biomass into fermentable-sugars and the utilization of xylose,which limited the production of bioethanol[129].Simultaneous saccharification and fermentation address the problem,using CRISPR/Cas9 protocol combined with two guide RNA to integrate 7 lignocellulolytic enzymes gene(endoglucanase geneAocelB,β-glucosidase geneTrbgl1,cellobiohydrolase I and II geneTecbh1andClcbh2b,xylanase geneAnxlnB,β-xylosidase geneAnxlnDand acetylxylan esterases geneCcxynA),obtaining 94.5 FPU per g CDW with a combined activity [128].For improving gene editing efficiency when expressing multiple gRNAs,the gRNA-tRNA array technology was used for CRISPR-Cas9 (GTR-CRISPR).The GTRCRISPR plasmid is assembled by Golden Gate.The fragment contains the initial Cas9 plasmid,and segments containing the gRNA-tRNA array cassette and the URA3 genefor selection(Fig.8C).Transforming into yeast,it is cleaved by the endogenous tRNA processing enzymes RNase P and RNase Z,leaving the different guide RNA to the genome editing operations.Then selected eight identified genes (FAA1,FAA4,POX1,ARE1,ARE2,PAH1,LPP1 and DPP1) for FFA production,the production (intracellular and extracellular) increased from 19.93 mg·L-1of wild-type yeast to 559.52 mg·L-1of engineered yeast,a 30-fold increase over wild-type yeast [130].In order to make the CRISPR system multifunctional,the CRISPR-AID system was developed,which requires at least three functional CRISPR proteins:CRISPRa,CRISPRi and CRISPRd.The production of β-carotene increased 2.8 fold with the synergistic effect of the three gRNA combinations including overexpression of a rate-limiting enzyme of the the mevalonate pathway HMG1,down-regulation of ERG9 and deletion of ROX1 encoding the stress responsive transcription regulaton [131].

Table 6 Application of the optimized CRISPR-Cas system in the production of bulk chemicals in microorganisms

4.2.The application of CRISPR technology in Bacillus subtilis

Bacillus subtilisis a safe Galans-positive bacterium,which is mainly used in the production of medical chemicals.There are three competing pathways for the production of GlcNAc from glucose byBacillus subtilis,namely glycolysis (EMP),pentose phosphate (HMP) and peptidoglycan synthesis (PSP).It is usually necessary to enhance the synthetic module at the transcription or translation level and try to knock out or down-regulation the competitive module in a dynamic manner [132].The autonomous dual-control (ADC) system was increased by combining synthetic genetic circuit based on GlcN6P biosensor and CRISPRi based NOT gates for regulating the production of GlcNAc inBacillus subtilis(Fig.8B).The GlcNcP biosensor up-regulated the synthesis of GlcNAc,while the inhibitory cascade in response to GlcN6P down-regulated the competition pathwaysencoded by zwf,pfkA,andglmMgene,which was related to cell growth and metabolism,and thenalsSD,a byproduct of the pyruvate pathway,wasknocked out,eliminating the formation of acetaldehyde.Finally,in the 15L batch fermentation bioreactor,the titer of GlcNAc increased from 81.7 g·L-1to 131.6 g·L-1without producing acetaldehyde [133],which also successfully demonstrated the stability and effectiveness of the combination of NOT gate based CRISPRi with the new biosensor in the synthesis of large bioreactor system.

5.Summary and Perspectives

Fig.8.Optimization and application of CRISPR-Cas9 system.(A) The tools for CRISPR-Cas9-mediated genome editing in industrial strains.The left replicative plasmid carry Cas9 gene and kanMX marker.The right plasmid perform gRNA cassette(including a specific 20-bp target sequence)and natMX marker.(B)Synthetic genetic circuit based on GlcN6P biosensor and CRISPRi based NOT gates for regulating the biological production of N-acetylglucosamine (GlcNAc) in Bacillus subtilis.(C) A gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering.

As the demand for sustainable and environmentally friendly biological manufacturing continues to increase,the use of synthetic biology technology to synthesize important chemicals has received more and more attention.Microbial cell factories can assemble a variety of metabolic engineering components to promote efficient and green synthesis of target compounds.Many important chemicals have been efficiently synthesized by microorganisms,such as terpenes,organic alcohols/acids,aromatic compounds,and medical chemicals.Metabolic regulation strategies provide an important guarantee for maintaining the balance of substances and energy in microbial cells,which is conducive to improving the output and yield of target chemicals.

In recent years,global climate change caused by greenhouse gas emissions has inspired scientists to use microbial cell factories to convert carbon dioxide (CO2) into chemicals and fuels,which will become the third generation of biorefinery.At present,some progress has been made in the application of microorganisms using CO2as a raw material.Microbial CO2utilization provides an opportunity to solve ecological and social problems through closed-loop recycling of resources and reduction of CO2emissions.However,the utilization efficiency of CO2by microorganisms is still low,which limits its large-scale application.The future research directions of biological manufacturing using CO2as raw materials may include:a.Designing and simulating calculation effective CO2immobilized enzymes and pathways.b.Improving metabolic engineering tools,constructing a synthetic biology automation platform for the integration of recombinant pathways for synthesizing various products and the recombinant expression of carbon fixation pathways in wild microbes.c.Accumulation of genome,transcriptome,proteome and metabolome data of organisms currently using carbon dioxide and optimization of CO2capture technology.We believe the use of microbial CO2may make a significant contribution to the establishment of a sustainable society with the advancement of synthetic biology technology[134].

In addition,combinatorial optimization provides a more efficient metabolic regulation method for microbial synthesis of chemicals,which has become the focus of research by scientists.Combinatorial optimization allows the rapid generation of a large number of diverse genetic constructs in a short period of time.It generates combinatorial libraries by assembling libraries of pathway elements such as promoters,RBS,coding sequences,and terminator,which can be obtained from high-throughput technologies.The optimized production strains were selected from the library.Combinatorial optimization will become an efficient strategy for screening high-yield chemical strains in the future[135].

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 financially supported by the National Natural Science Foundation of China (Grant Nos.21811530003,21861132017,U1663227,21706006)