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Critical Reviews in Biotechnology

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NADPH metabolism: a survey of its theoreticalcharacteristics and manipulation strategies inamino acid biosynthesis

Jian-Zhong Xu, Han-Kun Yang & Wei-Guo Zhang

To cite this article: Jian-Zhong Xu, Han-Kun Yang & Wei-Guo Zhang (2018): NADPH metabolism:a survey of its theoretical characteristics and manipulation strategies in amino acid biosynthesis,Critical Reviews in Biotechnology, DOI: 10.1080/07388551.2018.1437387

To link to this article: https://doi.org/10.1080/07388551.2018.1437387

Published online: 25 Feb 2018.

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REVIEW ARTICLE

NADPH metabolism: a survey of its theoretical characteristics andmanipulation strategies in amino acid biosynthesis

Jian-Zhong Xua,b , Han-Kun Yanga and Wei-Guo Zhanga

aThe Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, WuXi, PR China;bThe Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, JiangnanUniversity, WuXi, PR China

ABSTRACTReduced nicotinamide adenine nucleotide phosphate (NADPH), which is one of the key cofactorsin the metabolic network, plays an important role in the biochemical reactions, and physiologicalfunction of amino acid-producing strains. The manipulation of NADPH availability and form is anefficient and easy method of redirecting the carbon flux to the amino acid biosynthesis in indus-trial strains. In this review, we survey the metabolic mode of NADPH. Furthermore, we summarizethe research developments in the understanding of the relationship between NADPH metabolismand amino acid biosynthesis. Detailed strategies to manipulate NADPH availability are addressedbased on this knowledge. Finally, the uses of NADPH manipulation strategies to enhance themetabolic function of amino acid-producing strains are discussed.

ARTICLE HISTORYReceived 6 June 2017Revised 17 January 2018Accepted 20 January 2018

KEYWORDSNADPH; amino acid;metabolic modes;manipulation strategies;biosynthetic pathway

Introduction

Amino acids are types of organic compounds with carb-oxyl and amine groups that play important roles in reg-ulating the physiology of all life-forms. More than 300amino acids exist in nature, but only 20 of them are thebasic structural elements of proteins, and only 10 areconsidered to be essential amino acids for humans andanimals [1]. In addition to the synthesis of proteins andother compounds present in nature, amino acids alsoparticipate in a wide variety of biochemical reactions,and are vital for energy transfer and energy cycles [2].The improvement of amino acid yield and efficiency hasacquired significant interest because of the importanceof amino acids in food, fodder, medical, cosmetic, andother industrial applications and the growing marketdemand in the amino acid industry. At present, micro-bial fermentation via Corynebacterium glutamicum orEscherichia coli plays a leading role in the amino acidindustry [3]. Appropriate strains and fermentation tech-nologies are crucial for amino acid production becausethey promote increased carbon flux into the biosyn-thetic pathways of amino acids. However, the regulationmechanism of these pathways is very complex becauseseveral metabolic reactions require the involvement ofcofactors, such as adenosine tri-/di-phosphate (ATP/

ADP), reduced-/nicotinamide adenine dinucleotide(NADH/NADþ), and reduced-/nicotinamide adeninedinucleotide phosphate (NADPH/NADPþ) except formetabolic enzymes [4]. ATP and ADP are known asenergy cofactors, while NADH/NADþ and NADPH/NADPþ are referred to as redox cofactors [5]. Figure 1shows that these cofactors participate in the biosyn-thesis of several amino acids. Table S1 lists and numbersthe enzymes involved in the three cofactor metabolismfor the metabolic pathways of E. coli and C. glutamicum.

As one of the key cofactors in the metabolic network,NADPH plays an important role in the biochemical reac-tions and physiological function of amino acid-produc-ing strains. Similar to other redox cofactors, NADPH isalso known as a co-enzyme in the cellular electrontransfer that drives the biosynthetic pathways of DNA,amino acids, fatty acids, phospholipids, and steroids.Equally important is the universal reducing power ofNADPH to fuel the activities of enzymes, such as cata-lase, superoxide dismutase, and glutathione peroxidase,which play important roles in allowing microorganismsto thrive in aerobic environments [6]. Moreover, NADH/NADþ generated from NADPþ can be used to formnicotinamide and other products, including ADP-ribose(Figure 2) [6]. These metabolites can be employed as

CONTACT Jian-Zhong Xu xujianzhong@jiangnan.edu.cn The Key Laboratory of Industrial Biotechnology and The Key Laboratory of CarbohydrateChemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, WuXi, PR China

Supplemental data for this article can be accessed here.

� 2018 Informa UK Limited, trading as Taylor & Francis Group

CRITICAL REVIEWS IN BIOTECHNOLOGY, 2018https://doi.org/10.1080/07388551.2018.1437387

regulating factors to maintain cellular functions. Thus,NADPH plays an important part in cell growth andmetabolite production. However, unlike other cofactors,NADPH drives anabolic reactions [7]. Although high-producing amino acid strains were developed bymanipulating the NADPH availability and state, giventhe existence of several reviews on the subject topic,

their physiological functions are not the major emphasisof this study [6,8]. The review aims to survey the meta-bolic modes of NADPH and their relationship withamino acid biosynthesis, and provide an overview of itsapplications. This review also covers strategies formanipulating the NADPH availability and generatingamino acid-producing strains with a focus on two

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Figure 1. Metabolic pathways of NADPH and amino acids in E. coli and C. glutamicum. The abbreviations and the enzymesinvolved in the cofactor metabolism are listed in the Supplemental file and Table S1. The red lines (i.e. 19th–28th enzymatic reac-tions) indicate the biosynthetic pathway of GFAAs; the dark green lines (i.e. 29th–39th enzymatic reactions) indicate the biosyn-thetic pathway of AFAAs; the blue lines (i.e. 40th–41th enzymatic reactions) indicate the biosynthetic pathway of PFAAs; the pinklines (i.e. 42th–43th enzymatic reactions) indicate the biosynthetic pathway of SFAAs; and the spring-green lines (i.e. 44th–50thenzymatic reactions) indicate the biosynthetic pathway of AAAs.

2 J.-Z. XU ET AL.

dominant organisms for industrial production of aminoacids (i.e. C. glutamicum and E. coli) and the physio-logical consequences.

NADPH metabolism

The ratios of NADPH/NADPþ affects numerous enzym-atic activities and endogenous concentrations of regula-tors (e.g. reactive oxygen species and nicotinic acidadenine dinucleotide phosphate) that play importantroles in cellular functions and cell survival under severalconditions. Therefore, balancing the catabolic formationof NADPH with the anabolic demand is crucial to aminoacid biosynthesis via microbial fermentation.

NADPH anabolism

The following are three major methods by whichNADPH can be formed (Figure 3): I) NADPH is regener-ated from NADPþ by diversified NADPþ-dependentenzymes; II) NADPH is regenerated from NADþ usingNADþ kinases, or from NADH using NADH kinases; andIII) NADPH is regenerated from NADH and NADPþ usingtranshydrogenases. Method I is the major method forNADPH-regeneration in glucose-grown microorganismcells. However, the intracellular concentrations ofNADPH and NADPþ are low compared with those ofNADH and NADþ [9]. Therefore, using Method II forNADPH regeneration recently had a prominent role.NAD kinases (NADKs) are crucial enzymes in Method IIthat regulate the NAD(H/þ) and NADP(H/þ) levelsthrough NAD(H/þ) phosphorylation using nucleosidetriphosphates (NTP) or inorganic polyphosphates[Poly(P)] as phosphoryl donors to form NADP(H/þ)(Figure 3). NADKs exist abundantly in living organisms[10,11] and do not affect net catabolic fluxes [12].Therefore, Method II is widely used for NADPH regener-ation and regulation of intracellular NADPH levels.Similar to Method II, Method III has no effect onnet catabolic fluxes [13]. In Method III, hydride is

reversibly transferred by transhydrogenases betweenNADH/NADþ and NADPH/NADPþ in several microorgan-isms. The Supplemental file presents in detail theenzymes involved in the NADPH anabolism and theircharacteristics. Table S2 summarizes the commonenzymes involved in the NADPH regeneration and theirproperties.

NADPH catabolism

NADPH is an essential anabolic reducing cofactor in allliving organisms, which is involved in numerous ana-bolic reactions to form NADPþ. Wittmann and de Graaf[14] identified that the formation of 1 g of biomassrequires 16.4mmol of NADPH to form NADPþ. Thus,NADPH catabolism is closely related to the biomass for-mation rate, which will significantly vary with the envir-onmental conditions [7]. Most of the NADPH isconsumed by an NADPH-dependent reductase to formNADPþ as the hydride transfer is finally completed.However, the intracellular level of NADPþ is lower thanits theoretical value because NADPþ can be catabolizedby multiple families of enzymes to form differentnucleotide derivatives and other products includingADP-ribose (Figure 2) [15]. The enzymes directlyinvolved in NADPþ catabolism include ADP-ribosyl-cyclases, NADPþ nucleosidase, and NADPþ phosphatase(Figure 2). The Supplemental file describes in detail thecharacteristics and functions of these enzymes.

Relationships between NADPH and amino acidproduction

C. glutamicum and E. coli are two of the most importantbacteria used for the industrial production of variousamino acids, including: glutamate-family amino acids(GFAA), aspartate-family amino acids (AFAA), pyruvate-family amino acids (PFAA), serine-family amino acids(SFAA), and aromatic amino acids (AAA). Amino acidhigh-producing strains have been obtained in the past

NADP+NAADPcADPRP NAD+

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Figure 2. NADPH conversion and degradation. The abbreviations are listed in the Supplemental file.

CRITICAL REVIEWS IN BIOTECHNOLOGY 3

using classical mutagenesis and overexpression (orderegulation) of biosynthetic enzyme-coding genes aswell as deletion of some enzymes that are irrelevant forthe biosynthesis of the target amino acid. However,aside from modifying biosynthetic pathways andincreasing precursor availability, the supply of NADPH isa critical factor for amino acid production [16], becausesome enzymatic reactions in the biosynthetic pathwayrequire the NADPH participation (Figure 1). Tables 1and S3 summarize the participating enzymes and thedemand for NADPH in the amino acid biosynthetic pro-cess. The Supplemental file describes the relationshipsbetween NADPH and the production of PFAAs, SFAAs,and AAAs in detail.

NADPH is closely related to GFAA production

According to descriptions by Jensen et al. [17], GFAAcontains L-glutamate, L-glutamine, L-proline, and L-arginine, which derive part or all of their carbon froma-ketoglutarate. They also contain several non-proteinamino acids, such as L-ornithine [18] and L-citrulline[19]. Figure 1 (red lines indicating section) presents themetabolic pathways of the GFAA and cofactor systems.

L-glutamate can be biosynthesized through eitherthe glutamate dehydrogenase (GDH) pathway or theglutamine synthetase (GS)/glutamate synthase (GOGAT)pathway. However, the GDH pathway is the main route[20] leading to the reductive amination of a-ketogluta-rate by GDH (No.19; [21,22]). Nevertheless, GDH can besubdivided into the three following subfamilies accord-ing to the cofactor specificity: (a) NADþ-dependentGDH, (b) NADPþ-dependent GDH, and (c) NADþ/NADPþ

dual-specific GDH [23]. NADPþ-dependent GDH is oneof the major enzymes for the L-glutamate biosynthesisin C. glutamicum and E. coli because it is required for

ammonium assimilation [22]. Therefore, 1mol ofNADPH must be supplied for 1mol of L-glutamate bio-synthesis. L-glutamine, which is an amide of L-glutam-ate, biosynthesized from L-glutamate, and is catalyzedby GS [24]. GS can be subdivided into three subfamiliesbased on the diverse bacterial groups, namely, GSI, GSII,and GSIII (for a review, see Ref. [25]). However, GSI isthe main catalyzing enzyme [26]. According to previ-ously reported studies, GSI is an ATP-dependent L-glu-tamate:ammonia ligase, which requires ATP instead ofNADPH to catalyze the L-glutamine biosynthesis [24].Therefore, 1mol NADPH is required for the biosynthesisof 1mol L-glutamine in C. glutamicum and E. coli. Notethat GDH is associated with isocitrate dehydrogenase toform a conjugated complex in the redox reaction; thusNADPH can become self-sufficient (Figure 1). NADPH isnot important for the L-glutamate and L-glutaminebiosynthesis, and consequently, studies on breedingL-glutamate or L-glutamine high-yielding strains aremainly focused on optimizing culture conditions [27],enhancing precursor supply [28], regulating energycofactors level [29], and modifying export systems [30].

In a widespread biosynthetic route for L-proline pro-duction, L-glutamate is converted into L-proline viathree enzymatic reactions and one spontaneous reac-tion (Figure 1; [31,32]). The redox and dephosphoryla-tion reaction is the second enzymatic reaction catalyzedby c-glutamyl phosphate reductase (No. 27) andrequires NADPH as a cofactor [31]. The third enzymaticreaction type is a redox reaction, and is catalyzed bypyrroline-5-carboxylate reductase (No. 28). This reactionalso requires NADPH as cofactor [31]. L-proline can alsobe biosynthesized from L-ornithine catalyzed by orni-thine cyclodeaminase (OCD). However, the overexpres-sion of the putative OCD-coding gene does not result in

I.NADP+ NADPH

NADP+-dependent dehydrogenases mainly in PP andacetate pathway, eg., G6PDH, 6PGDH and ALDH

II.

NAD+

Poly(P)n/NTP

NADP+

NADH NADPH

Poly(P)n-1/NDP

NAD+ Kinase

NADH Kinase

NADP+-dependentoxido-reductases

NAD+-dependentoxido-reductases

III. NAD+ NADPH+NADH NADP++Membrane-bound transhydrogenase (mTH), eg., PntAB

Soluble transhydrogenase (sTH), eg., UdhA and Sth

Figure 3. Three general approaches for NADPH regeneration. The abbreviations are listed in the Supplemental file.

4 J.-Z. XU ET AL.

L-proline accumulation in the medium despite the gen-ome of C. glutamicum or E. coli containing a putativeOCD-coding gene [31]. Based on these findings, 2molNADPH must be supplied for the biosynthesis of 1molL-proline from L-glutamate in C. glutamicum and E. coli(Figure 1, where the red lines indicate the section).Despite these findings (to the best of our knowledge)reports regarding the generation of L-proline high-yielding strains have not been presented in the recentyears.

L-arginine in both C. glutamicum and E. coli is biosyn-thesized from L-glutamate through eight enzymaticsteps (Table S3). The third enzyme in the biosyntheticpathway is N-acetyl-c-glutamyl-phosphate reductase(No. 21), which is an NADPH-dependent reductase thatrequires NADPH to catalyze the biosynthesis of N-acetylglutamyl-c-semialdehyde [33]. The fourth enzymaticreaction is catalyzed by acetyl-ornithine aminotransfer-ase (No. 22), in which the amino group from L-glutam-ate is transferred to N-a-acetylornithine. The fifthenzymatic reaction is catalyzed by bifunctionalornithine acetyltransferase (No. 23), in which acetylfrom N-a-acetylornithine is transferred to L-glutamate[33]. Meanwhile, the seventh reaction is catalyzed byargininosuccinate synthetase (No. 24) and requires L-aspartate and ATP intervention [33]. However, the L-glu-tamate and L-aspartate biosynthesises need NADPHparticipation [22,34]. Therefore, 3mol NADPH isrequired to biosynthesize 1mol of L-arginine fromL-glutamate in C. glutamicum and E. coli (Figure 1).Engineering of the NADPH metabolism has beengradually regarded by researchers as one of the keymethods for breeding an L-arginine high-yielding strainbecause of the importance of NADPH for improvingL-arginine production [35–37], with the exception of thepathways modification of carbon metabolism [38,39],and the export system to breed L-arginine high-produc-ing strains [40].

NADPH is closely related to AFAA production

AFAA contains L-aspartate, L-asparagine, L-threonine, L-lysine, L-isoleucine, and L-methionine. L-aspartate isderived from oxaloacetate (OAA; [41]), while the othersderive part or all of their carbon from L-aspartate [42].Figure 1 (dark green lines indicating the section)presents the metabolic pathways of the AFAA andcofactor systems.

The L-aspartate biosynthesis is the amination of OAAby aspartate aminotransferase (No. 29) that requires1mol NADPH to biosynthesize 1mol L-aspartate (Figure1, the dark green lines indicate the section). An add-itional biosynthetic pathway is needed for L-aspartate

synthesis in E. coli, but it will not be discussed herebecause it is unimportant [34]. The L-asparagine biosyn-thesis normally begins with the catalysis of L-aspartateby asparagine synthetase (No. 30). This enzymatic steprequires ATP instead of NADPH as a cofactor [43]; thusthe biosynthesis of 1mol L-asparagine requires only1mol NADPH.

L-threonine is synthesized from L-aspartate using fiveenzymatic steps (Table S3; [44,45]). Figure 1 shows thattwo key enzymes, namely, aspartate-semialdehydedehydrogenase (No. 32) and homoserine dehydrogen-ase (No. 33), require NADPH to catalyze the biosynthesisof aspartate semialdehyde and homoserine, respect-ively. Combined with the NADPH requirement in the L-aspartate biosynthesis, 3mol NADPH must be suppliedfor the biosynthesis of 1mol L-threonine from OAA inC. glutamicum and E. coli. However, the strategies forgenerating L-threonine high-yielding strains mainlyfocus on the following six aspects: (1) increasing theefficiency of the biosynthetic pathway by overexpress-ing the key enzyme-coding genes [46], (2) weakening orblocking the competitive pathways to increase the pre-cursor availability and reduce the by-products formation[45], (3) reducing the L-threonine degradation pathways[47], (4) increasing the L-threonine secretion [48], (5)expanding the useful range of available sugars [49], and(6) integrating (1) to (4) via system-level metabolicengineering [50,51]. Until now, only Xie et al. [52]reported that blocking the Embden–Meyerhof pathway(EMP) in E. coli leads to an increase in the intracellularNADPH level that would enhance L-threonineaccumulation.

The sulfur-containing amino acid L-methionine issynthesized via seven enzymatic steps from L-aspartateand derived from the precursor L-homoserine (Table S3;[53]). Interestingly, the L-methionine biosynthesis sharesa partial biosynthetic pathway with L-threonine(Figure 1, the dark green lines indicate the section).Similar to L-threonine biosynthesis, the biosynthesis of1mol L-homoserine requires 3mol NADPH. The sulfateassimilation also requires NADPH for sulfur reduction[53,54]. Therefore, approximately 8mol NADPH and 8.5mol NADPH are required for the biosynthesis of 1molL-methionine in C. glutamicum and E. coli, respectively[53]. Although NADPH is important for improving the L-methionine production, to our knowledge, very fewstudies focused on NADPH metabolism engineering forbreeding L-methionine high-yielding strains. The major-ity of these studies have focused on the enhancementof the precursor supply [28,55], deregulation of thesynthetic pathway [56], and modification of the exportsystem [57]. Therefore, engineering the NADPH

CRITICAL REVIEWS IN BIOTECHNOLOGY 5

metabolism will be a tendency for breeding L-methio-nine high-yielding strains.

Starting with L-aspartate, L-isoleucine biosynthesisinvolves 10 enzymatic steps with L-threonine serving asan indirect precursor for its biosynthesis (Table S3).Therefore, the L-isoleucine biosynthesis proceeds withfive enzymatic steps starting from L-threonine (TableS3). The third enzymatic reaction is catalyzed by aceto-hydroxy acid isomeroreductase (No. 35), and NADPH isused as a cofactor in this reaction [58,59]. The lastenzymatic reaction is catalyzed by branched-chainamino acid aminotransferase (No. 36), in which theamino group from L-glutamate is transferred to L-iso-leucine [60,61]. However, the L-glutamate biosynthesisneeds NADPH participation [21,22]. Therefore, 5molNADPH must be supplied for the biosynthesis of 1molL-isoleucine from OAA. Apart from modification of themetabolic pathways and export systems [58,61–63], sev-eral studies have proven that increasing the NADPHsupply is beneficial for the increase of L-isoleucine pro-duction by either overexpressing the NAD [60,64] orNADH kinases [21], or strengthening the carbon fluxinto the oxidative pentose phosphate (OPP) pathway[11].

C. glutamicum comprises two L-lysine biosyntheticpathways from L-aspartate (i.e. acetylase pathway anddehydrogenase pathway), whereas E. coli comprisesonly one pathway (i.e. acetylase pathway) (Figure 1, thedark green lines indicate the section; [65]). The L-lysinebiosynthesis can be improved by overexpressing thekey enzyme genes in the biosynthetic pathway [66–69],weakening or blocking the competitive metabolic path-ways [70,71], increasing the sugar availability [16,69,72],enhancing export systems [48], and expanding the use-ful range of available sugars [16,73,74]. However,NADPH-regenerating systems are also important forL-lysine biosynthesis because 4mol NADPH must besupplied for the biosynthesis of 1mol L-lysine from L-aspartate (Table 1). Numerous studies have focused onNADPH metabolism engineering in order to preciselyimprove L-lysine high-producing strains because of theimportance of NADPH for L-lysine production[66–68,70,75–79].

Manipulation strategy for NADPH regenerationduring amino acid biosynthesis

NADPH regeneration in micro-organisms is in a dynamicbalance. It is strictly dependent on the microbial growthstate and the genetic background [67,68,80,81], or onthe applied carbon source [16,82,83]. NADPH supply isin excess in most cases. However, the NADPH availabil-ity is a major limitation in the efficient production of

several value-added products, particularly amino acids[84]. Although NADPH regeneration can be achieved byexternal regulation, such as the addition of the electronacceptor or adjustment of the dissolved oxygen con-centration [85,86], an endogenous regulation based onthe metabolic pathway of NADPH in vivo is one of themajor methods for the NADPH regeneration. Thisdepends on the regulation of the enzyme activityinvolved in NADPH regeneration using geneticengineering.

Increasing the activity of enzymes involved inNADPH regeneration

Numerous enzymes are involved in the NADPH-regener-ating reactions in C. glutamicum and E. coli (Table S2)that can be subdivided into three groups based on thecofactor specificity (see above; Figure 3). AlthoughNADPþ-dependent dehydrogenases originate from theOPP pathway, the tricarboxylic acid (TCA) cycle (i.e. iso-citrate dehydrogenases, ICD, No. 15), and anapleroticreactions (i.e. malic enzyme, MalE, No. 11), several stud-ies have identified that NADPþ-dependent dehydrogen-ases from the OPP pathway are the major enzymes forNADPH generation in C. glutamicum and E. coli (forreview, see Refs. [46,84]).

Figure 1 shows the first and third enzymatic steps inthe OPP pathway involved in NADPH generation thatare catalyzed by glucose-6-phosphate dehydrogenase(G6PDH, No. 2; encoded by zwf gene in C. glutamicumand E. coli) and 6-gluconate phosphate dehydrogenase(6PGDH, No. 3; encoded by gnd gene in C. glutamicumand E. coli), respectively. The key enzyme for controllingthe flux of the OPP pathway is G6PDH [87]. Therefore,one of the most efficient methods of increasing theNADPH availability is to increase the G6PDH and 6PGDHactivities. Several studies reported that the overexpres-sion of G6PDH or/and 6PGDH is effective in improvingthe target amino acid production because of theincrease of the NADPH availability [21,66,88,89].However, the G6PDH and 6PGDH activities are regu-lated by ATP and fructose-1,6-diphosphate (Fru-1,6-P2),whereas the introduction of the A243T mutation intothe zwf gene or the S361F mutation into the gnd generelieves feedback regulation by ATP and Fru-1,6-P2,thereby increasing the L-lysine production [82,90]. Eventhough ICD and MalE are also the NADPH sources in C.glutamicum and E. coli, the overexpression of ICD andMalE was not beneficial in improving the amino acidproduction [82,84]. Conversely, decreasing ICD activitycan promote L-lysine production in C. glutamicum[66,71], showing that compared to G6PDH and 6PGDH,

6 J.-Z. XU ET AL.

Table 1. Demand for NADPH and the participating enzymes in the amino acids biosynthetic process during cultivation onglucosea.

Encoding gene

Group Amino acidNADPH

(mol [mol AA]�1) Enzymes E. coli C. glutamicum Substrates Enzymatic reaction

GFAAs L-glutamate 1 GDH gdhA gdh a-KG Reductive amination reactionL-glutamine 1 GDH gdhA gdh a-KG Reductive amination reactionL-proline 3 GDH gdhA gdh a-KG Reductive amination reaction

GPR proA proA Glu-P Reductive dephosphorylationreaction

P5CR proC proC P5C Redox reactionL-arginine 4 GDH gdhA gdh a-KG Reductive amination reaction

AGPR argC argC AcGlu-P Reductive dephosphorylationreaction

ACO-OT argD argD AcGlu-c-Saldþ L-glutamate

Amino group transfer reaction

ASS argG argG L-citrulineþ L-aspartate

Condensation reaction

AFAAs L-asparagine 1 AAT aspB aspB OAAþ L-glutamate Amino group transfer reactionL-asparagine 1 AAT aspB aspB OAAþ L-glutamate Amino group transfer reactionL-threonine 3 AAT aspB aspB OAAþ L-glutamate Amino group transfer reaction

ASADH asd asd Asp-P Reductive dephosphorylationreaction

HDH thrA, metL hom Asp-SAld Redox reactionL-methionine 8 for C. glutamicum

and 8.5 for E. coliAAT aspB aspB OAAþ L-glutamate Amino group transfer reaction

ASADH asd asd Asp-P Reductive dephosphorylationreaction

HDH thrA, metL hom Asp-SAld Redox reaction–b – – – Sulfate assimilation reaction

L-isoleucine 5 AAT aspB aspB OAAþ L-glutamate Amino group transfer reactionASADH asd asd Asp-P Reductive dephosphorylation

reactionHDH thrA, metL hom Asp-SAld Redox reactionAHAIR ilvC ilvC AcHB Redox and acetoin rearrange-

ment reactionBCAT ilvE ilvE KMVAþ L-

glutamateAmino group transfer reaction

L-lysine 4 AAT aspB aspB OAAþ L-glutamate Amino group transfer reactionASADH asd asd Asp-P Reductive dephosphorylation

reactionDHDPR dapB dapB DHDP Redox reactionDapC dapC dapC AKVþ L-glutamate Amino group transfer reactionDDHc – ddh THDP Reductive amination reaction

PFAAs L-alanine 1/2d AlaT/AvtA alaT/avtA alaT/avtA Pyrþ L-glutamate/L-valine

Amino group transfer reaction

L-valine 2 AHAIR ilvC ilvC AcHB Redox and acetoin rearrange-ment reaction

BCAT/AvtA ilvE/avtA ilvE/avtA KIValþ L-glutam-ate/L-alanine

Amino group transfer reaction

L-leucine 2 AHAIR ilvC ilvC AcHB Redox and acetoin rearrange-ment reaction

BCTA ilvE ilvE KICapþ L-glutamate

Amino group transfer reaction

SFAAs L-serine 1 PSAT serC serC PHPþ L-glutamate Amino group transfer reactionL-glycine 1 PSAT serC serC PHPþ L-glutamate Amino group transfer reactionL-cysteine 6 for C. glutamicum

and 6.5 for E. coliPSAT serC serC PHPþ L-glutamate Amino group transfer reaction

–a – – – Sulfate assimilation reactionAAAs L-phenylalanine 2 SHKH aroE aroE DHS Hydro-oxidation reaction

AAATm tyrB pat PKUþ L-glutamate Amino group transfer reactionL-tyrosine 2 SHKH aroE aroE DHS Hydro-oxidation reaction

AAATm tyrB pat HPKUþ L-glutamate

Amino group transfer reaction

L-tryptophan 3 SHKH aroE aroE DHS Hydro-oxidation reactionAS trpDE trpE, NCgl2928 CHAþ L-glutamine TAmide group transfer

reactionTS trpAB trpA, NCgl2931 Indoleþ L-serine Condensation reaction

aThe abbreviations and the enzymes involved in the cofactor metabolism are listed in the Supplemental file.bMany enzymes participate in sulfate assimilation and 5–6mol per mol sulfate assimilation is needed [53].cNo meso-diaminopimelate dehydrogenase (DDH) can be found in E. coli.dThe NADPH consumption depends on the substrates.

CRITICAL REVIEWS IN BIOTECHNOLOGY 7

neither ICD nor MalE are better choices for NADPHregeneration in amino acid biosynthesis.

The second types of NADPH-regenerating enzymesare NADKs, which are not coupled to the central carbonmetabolism [12,84]. NADKs are divided into ATP-NADK(e.g. YfjB from E. coli), and Poly(P)/ATP-NADK (e.g. PpnKfrom C. glutamicum) types [10,11]. YfjB and PpnK arethe only NADKs in E. coli and C. glutamicum [91,92].PpnK was first isolated from C. glutamicum ssp. flavusby Kawai et al. [93], which completely and specificallyphosphorylated NADþ by utilizing both Poly(P) and NTPas phosphoryl donors. The phosphorylation reactioncatalyzed by this enzyme required the participation ofbivalent metal ions, but the effects of different ionswere different [93,94]. Moreover, PpnK from differentsources, or the introduction of a novel mutation intothe PpnK-coding gene PpnK exhibits different enzymeproperties, which provides different NADPH concentra-tions for the target product production [60,93,95].Dissolved oxygen levels will also affect the PpnK activityand function [35]. PpnK does not affect the net cata-bolic fluxes; thus, it is now widely used to regulate theintracellular NADPH level and improve amino acid bio-synthesis. The overexpression of the PpnK gene from C.glutamicum in the corresponding amino acid-producercan contribute to the enhancement of NADPH, therebyincreasing the production of: L-lysine [10], L-isoleucine[21,63,64], L-arginine [92], and L-ornithine [96]. YfjBobtained from E. coli was first purified and identified byZerez et al. [97]. Unlike PpnK from C. glutamicum, YfjBfrom E. coli phosphorylates NADþ to form NADPþ withNTP as the only phosphoryl donor [98]. However, similarto PpnK from C. glutamicum, it requires the participa-tion of bivalent metal ions, and Mn2þ is one of its mostefficient activators [93,98]. Moreover, this enzyme actsat an optimal reaction temperature of 60 �C and at mildalkali conditions (pH� 7.5) [98]. The overexpression ofthe YfjB-coding gene yfjB in E. coli to improve variousvalue-added materials (e.g. poly-3-hydroxybutyrate[99,100], thymidine [49], isobutanol [91], and shikimicacid [101]) has been reported. However, no relevantreport on the modification of the yjfB gene for improv-ing the amino acid production in E. coli has yet beenpresented.

The third NADPH-regenerating enzymes are transhy-drogenases coupled to the translocation of hydridebetween NAD(H/þ) and NADP(H/þ) rather than to themetabolism of the central carbon [9,77]. PntAB andUdhA, which are two transhydrogenases coexisting in E.coli, maintain the cellular NADPH/NADH balance [102],but they have different requirements for cofactors anddiffer in their physiological roles. PntAB only requirescofactors NAD(H/þ) and NADP(H/þ) for activity, whereas

UdhA additionally requires flavin adenine dinucleotide[84,103]. Moreover, several prior reports suggested thatPntAB catalyzes the transfer of hydride from NADH toNADPþ to increase the NADPH supply, whereas UdhAcatalyzes the transfer of hydride from NADPH to NADþ

to prevent the excess production of NADPH (for areview, see Ref. [84]). Interestingly, the overexpressionof the UdhA-coding gene UdhA was beneficial inimproving various NADPH-dependent products in E.coli, such as thymidine [49], (S)-2-chloropropionate[104], and fatty acids (C12 – C18) [105]. UdhA also partici-pates in NADPH regeneration in E. coli. The overexpres-sion of the PntAB-coding gene PntAB is expected toplay an important role in enhancing the yields ofseveral NADPH-dependent products in E. coli, such as 3-hydroxypropionic acid [106], and isobutanol [91].However, until now, no relevant report has outlined themodification of PntAB or/and UdhA in E. coli to improveamino acid production. Although C. glutamicum lackstranshydrogenases, it functions through a transhydroge-nase-like shunt [107,108]. Blombach et al. [107] indi-cated that deactivating MalE would interrupt isobutanolsynthesis in C. glutamicum because of an insufficientNADPH supply. Cocaign-Bousquet and Lindley [109]also indicated that increasing the activity of MalEenhanced carbon conversion efficiency in C. glutamicumbecause of improvement of the NADPH supply. Theysuggested that this approach can be proposed as astrategy for breeding amino acid high-yielding strains.However, overexpression of the MalE-coding gene MalEin C. glutamicum strains producing L-lysine did notimprove L-lysine production using glucose, fructose orsucrose [82].

Reducing or blocking-up the competitivebypass of NADPH regeneration

Weakening the carbon fluxes in the competitive bypassof NADPH regeneration is also a good strategy forimproving the NADPH supply. In the previous section,the OPP pathway was mentioned to be a major routefor NADPH generation in C. glutamicum and E. coli.Thus, redirecting the carbon flux in the EMP and OPPpathways is an obvious choice to increase the intracel-lular NADPH level.

Phosphoglucose isomerase (PGI) is an importantenzyme in EMP that catalyzes the reversible reactionbetween glucose-6-phosphate and fructose-6-phos-phate. Disrupting the PGI-coding gene pgi can disor-ganize the competitive EMP, thereby forcing the carbonflux completely through the OPP pathway. The PGIinactivation is beneficial in increasing several NADPH-dependent products in C. glutamicum and E. coli,

8 J.-Z. XU ET AL.

including amino acids [110–113]. However, a PGI-knock-out strain exhibits a low specific growth rate and is onlyone-third as fast as the parental strain during growthon glucose [114]. Interestingly, Park et al. [36] reportedthat downregulating the expression level of the pgigene, by replacing the start codon ATG with GTG, willincrease the carbon flux entering the OPP pathway witha minor effect on the cell growth (more than two-thirdsof the parental strain), thereby resulting in an increaseL-arginine production in C. glutamicum. However, thisstrategy is only available for glucose-based processesbecause sucrose- or fructose-based processes requirean active PGI for recycling carbon into the OPP pathway[83].

Another method is to redirect the carbon flux in theEMP and OPP pathways by decreasing the intracellularFru-1,6-P2 concentration. This method has attracted theattention of researchers because it inhibits the activitiesof G6PDH [115], 6PGDH [90], and fructose-1,6-bisphos-phatase (FBPase) [116]. FBPase catalyzes the hydrolysisof Fru-1,6-P2 to fructose-6-phosphate (Fru-6-P) and Pi,which is a key enzyme in the gluconeogenic pathway.A metabolic flux analysis indicated that overexpressionof the FBPase-coding gene fbp resulted in enhancedOPP pathway flux, thus increasing the supply of NADPHand L-lysine production [82,83]. Similarly, the inactiva-tion of phosphofructokinase (PFK, No. 4, encoded bygenes pfkA and pfkB in C. glutamicum and E. coli), thatcatalyzes the phosphorylation of Fru-6-P to form Fru-1,6-P2, will decrease the intracellular Fru-1,6-P2 concen-tration, thus forcing the carbon flux through the OPPpathway. Wang et al. [117] and Siedler et al. [118]reported that PFK knockouts will direct Fru-6-P into theOPP pathway and increase NADPH generation, thusincreasing the production of NADPH-dependent prod-ucts in E. coli. However, the inactivation of PFK seems toact against L-threonine production in E. coli accordingto the report by Xie et al. [52]. They found that deletingthe pfkA gene decreased cell growth and L-threonineproduction because NADPH synthesis is blocked. In con-trast, deleting the pfkB gene had no effect on cellgrowth and L-threonine production because theincrease in the NADPH supply was not large [52].Moreover, several reports demonstrated that overex-pressing the pfkAB gene increased the carbon flux inEMP, resulting in an increase in the production of prod-ucts, such as L-alanine [119] and L-serine [120].

C. glutamicum has two routes for the formation of 6-phosphogluconate, namely, the OPP pathway, and thegluconate bypass. The gluconate bypass consisted oftwo key enzymes [i.e. glucose dehydrogenase (GlcDH)and gluconate kinase (GntK)] that decrease the carbonflux through the OPP pathway [121]. An alternative

method to maximize the carbon flux through the OPPpathway is to block the gluconate bypass by inactivat-ing GlcDH and/or GntK. Hwang and Cho [38,122]reported that blocking the gluconate bypass by a dis-ruption of the GlcDH- or GntK-coding gene significantlyforced carbon flux from glucose toward the OPP path-way with a concomitant increase in L-ornithine produc-tion. However, the effect that stimulated the OPPpathway was different for GlcDH and GntK inactivation.In the case of the GlcDH-deficient strain, the carbon fluxfrom glucose- 6-phosphate was entirely in the OPPpathway, thereby resulting in an increase of key enzymeactivities in the OPP pathway. However, the GntK-knockout leads to an increase of key enzyme activitiesin the OPP pathway because of accumulation of gluco-nate that hindered binding of the GntR transcriptionalrepressor types (i.e. GntR1 and GntR2) to their targetgene promoters (e.g. the gnd promoter) [121].

Introduction of an extrinsic pathway forNADPH regeneration

Introducing an extrinsic pathway is another appropriatestrategy for intracellular NADPH regeneration, and ithas been gaining interest amongst researchers. Thereplacement of the native NADþ-dependent enzymewith a non-native NADPþ-dependent enzyme or theintroduction of a new and extrinsic pathway in the hoststrain has been extensively used for intracellular NADPHregeneration.

Glyceraldehyde-3-phosphate dehydrogenase (GADPH,No. 5) catalyzes an essential step in the central metabol-ism. Therefore, the GADPH modification is one of theimportant research areas in enzyme replacement forNADPH regeneration. GADPHs are classified into twotypes according to differences in the specificities of thecofactors: NADþ- and NADPþ-dependent GADPHs. In E.coli, GADPH is an NADþ-dependent enzyme that cata-lyzes the reversible oxidation of glyceraldehyde-3-phos-phate into 1,3-bisphosphoglycerate (1,3-BPG) usingNADþ as the cofactor [123]. In contrast, C. glutamicumhas two GAPDHs, namely GapA (NADþ-dependentenzyme) and GapB (NADPþ-dependent enzyme, No. 8)[124]. Although GapB catalyzes NADPH synthesis, theup-regulation of GapB activity was not advantageousfor cell growth and L-lysine production because GapBwas only involved in gluconeogenesis [75]. These resultsforced the replacement of native NADþ-dependentGAPDH with a non-native NADPþ-dependent GADPH.The NADPþ-dependent GADPH can be classified intotwo types: the phosphorylating type catalyzing the syn-thesis of 1,3-BPG and the non-phosphorylating type(GapN) catalyzing the synthesis of 3-PG (for a review,

CRITICAL REVIEWS IN BIOTECHNOLOGY 9

see Ref. [84]). However, the heterologous expression ofany type of NADPþ-dependent GADPH-coding gene isconducive to the production of NADPH-dependentproducts in C. glutamicum and E. coli. For examplereplacing the native GAPDH-coding the gapA gene withthe non-native gene gapC (encoding phosphorylatedNADPþ-dependent GADPH) from Clostridium acetobuty-licum significantly increased the production of L-lysine[68] and L-ornithine [125] in C. glutamicum. Similarly,the introduction of the gapN gene (encoding GapN)from Streptococcus mutans in the gene locus of gapAfrom C. glutamicum can also significantly increase theproduction of L-lysine with different industrial sugars,such as glucose (increased by �70%), fructose(increased by �120%), and sucrose (increased by�100%), as compared to the parental strain [75].Meanwhile, altering the cofactor specificity by site-directed modification of GADPH binding sites can real-ize the regeneration of NADPH rather than NADH in theGADPH-catalyzed step. Bommareddy et al. [78] shownthat introducing the D35G, L36T/L36R, T37K, and/orP192S mutations into the gapA gene of C. glutamicumwill alter the cofactor specificity of GapA to NADPþ,thereby significantly increasing L-lysine production.However, the effect of directing the carbon flux was dif-ferent from introducing the non-native GADPH and amutation in the native GADPH. The metabolic flux ana-lysis indicated that the heterologous expression of theNADPþ-dependent GADPH-coding gene significantlydecreased the carbon flux to the OPP pathway(decreased by 80%), whereas introducing a mutation inthe native GADPH did not dramatically change the car-bon flux to the OPP pathway [76,78].

Another strategy for regenerating NADPH in the cellis to strengthen the hydride transfer from NADH toNADPH by enhancing the activity of transhydrogenases.As was previously mentioned, C. glutamicum does notcontain any transhydrogenase. In contrast, E. coli hastwo transhydrogenases, namely PntAB and UdhA. As aresult, some researchers attempted to regenerateNADPH from NADH by introducing an exogenous trans-hydrogenase in C. glutamicum. They overexpressed thePntAB-coding gene PntAB from E. coli, which resulted inrelieving the GADPH inhibition by NADH and increasingthe NADPH supply. Numerous reports indicated thatthe expression of the PntAB gene from E. coli to C. gluta-micum improves the production of L-lysine [67,80] andL-valine [126]. Bartek et al. [126] also suggested that theintroduction of heterologous PntAB redirects the carbonflux from the OPP pathway to the EMP (decreased by�12%), suggesting that the introduction of transhydro-genase is feasible for increasing the conversion ratios of

glucose to the target products because of the reductionin CO2 formation via the OPP pathway.

However, the intracellular NADP(H/þ) concentrationwas lower than that of NAD(H/þ) and NADH can be eas-ily regenerated through several pathways (e.g. EMP andTCA cycle) [9,127]. Therefore, how to direct the phos-phorylation of NAD(H/þ) to NADP(H/þ) has become animportant issue in NADPH regeneration for researchers.E. coli and C. glutamicum only contain YfjB and PpnK;hence, they have been used to increase the NADPHsupply by overexpression of corresponding nativegenes [11,63,92,100]. Several attempts have been madeto supply more efficiently NADKs for NADPH regener-ation. For example the introduction of Pos5, which is anNADH kinase, from Saccharomyces cerevisiae to E. colicould increase the production yield of poly-3-hydroxy-butyrate (increased by 5.6%), GDP-L-fructose (increasedby 51%), and e-caprolactone (increased by 96%),because Pos5 directly catalyzes NADH phosphorylationto form NADPH [46,128]. Shi et al. [11,21] pointed thatthe heterologous expression of the Pos5-coding genepos5 from S. cerevisiae into C. glutamicum will signifi-cantly increase L-isoleucine production (increased by26%), and it is better than that of the PpnK-expressionstrain. These results indicated that direct phosphoryl-ation of NADH via the heterologous expression of theNADH kinase can be considered to be a more efficientmethod for NADPH regeneration.

Alternative methods for NADPH regeneration

In addition to increasing the NADPH supply by overex-pressing the key enzyme-coding genes involved inNADPH regeneration, reducing the competitive bypassor introducing an extrinsic pathway, a rational designfor the cofactor specificity of enzymes in the biosyn-thetic pathway of metabolites can be considered asanother method for indirectly enhancing NADPH regen-eration. We know that the native acetohydroxy acid iso-meroreductase (AHAIR, No. 41; encoded by ilvC gene)from C. glutamicum is an NADPH-dependent enzyme inthe biosynthetic pathway of branched-chain aminoacids (BCAAs). However, Hasegawa et al. [129,130] intro-duced three mutations (i.e. Ser34Gly, Leu48Glu, andArg49Phe) into the ilvC gene to replace the NADPH-dependent AHAIR with the NADH-dependent AHAIR,thereby resulting in an increase in the NADPH/NADHratio and L-valine production under oxygen-deprivedconditions. They also found that replacing C. glutami-cum transaminase B (No. 36, an NADPH-dependentenzyme) with leucine dehydrogenase (an NADH-dependent enzyme) from Lysinibacillus sphaericusimproved the redox balance (i.e. regulating the

10 J.-Z. XU ET AL.

NADH/NADPH supply), and increased L-valine produc-tion in C. glutamicum. However, occasionally, alteringthe cofactor specificity of enzymes is not good forNADPH regeneration. Marx et al. [131] demonstratedthat replacing C. glutamicum NADPH-dependent GDHwith NADH-dependent GDH from Peptostreptococcusasaccharolyticus decreased the NADPH biosynthesis(decreased from 210 to 139%) because of rerouting thecentral carbon metabolism. These results indicated thatthe central carbon metabolism had an extraordinaryflexibility, and we should reasonably select the strategyfor NADPH regeneration.

Conclusions and future prospects

Amino acids play an important role regulating thephysiology of all life-forms because they participate inthe synthesis of proteins and other compounds presentin nature, regulate diverse biochemical reactions, andare vital during energy transfer and energy cycles. [2].Over the past 60 years, numerous research efforts werespent on increasing amino acid production via engin-eering cellular central carbon metabolic pathways andoptimizing the fermentation process. The NADPH/NADPþ ratios affect various enzymatic activities in thecarbon metabolism and intracellular environment.Therefore, NADPH should be supplied in sufficientquantities to sustain cellular and enzyme activities.Several metabolic engineering studies have alsofocused on improving the intracellular NADPH supply inamino acid-producing genetically engineered bacteria.

This review has provided an overview of the majormetabolic mode of NADPH and the key enzymes relatedto NADPH metabolism (Figures 2 and 3; Table S2). Italso summarizes research developments in the under-standing of the relationship between NADPH, theamino acid biosynthesis, and the manipulation strat-egies for NADPH regeneration in order to increaseamino acid production in C. glutamicum and E. coli.Although diverse NADPH-regenerating enzymes weresuccessfully overexpressed and had proven their validityfor NADPH regeneration, other NADPH-regeneratingenzymes (e.g. NADPþ-dependent dehydrogenase, phos-phate dehydrogenase and ferredoxin:NADPþ oxidore-ductase) are not used in genetically modifying aminoacid-producing strains despite clear evidence of theirbeneficial property for increasing the NADPH supply(for reviews, see Refs. [8,46,84]). Future research effortsare required for engineering amino acid high-yieldingstrains through NADPH-regenerating systems.Therefore, we will expend our efforts to understand thevarious types of NADPH-regenerating enzymes andtheir effects on the production of target amino acids,

thereby predicting strategies that will effectivelyincrease NADPH availability for the amino acid produc-tion processes.

Sebastiaan K. Spaans from Wageningen Universityhas also argued that a microbial cell is a complex sys-tem that usually contains various integrated units [84],leading to unexpected physiological and metabolic per-turbations during modification of NADPH-regeneratingpathways. However, a few studies on the optimizationof cellular physiological and metabolic functions maxi-mized carbon flux through the biosynthetic route foramino acids based on regulation of the intracellularNADPH level. We can predict the three following criticalscientific issues in developing high-yielding tamino acidstrains via regulation of the intracellular NADPH levelsbased on completed research programs and the futuredevelopments:

i. How to quantitatively study the effects of NADPHlevels based on microbial metabolome, proteome,and transcriptome with applied systems biologyand high-throughput analysis: systems biologyand high-throughput analysis are extensivelyapplied in biology. Also, these systems can beused to study changes in metabolic fingerprinting,metabolic fluxes, protein expression levels, tran-scription levels, and signal transduction.Correspondingly, they are able to lay the founda-tion for revealing the physiological mechanism ofthe microbial functional regulation based on intra-cellular NADPH levels [132,133].

ii. How to reveal the role of intracellular NADPH lev-els to regulate the flow and direction of the meta-bolic fluxes based on the relations betweenchanges in the carbon metabolic fluxes and theflow of cofactors, on clarifying the relationshipbetween regulatory and metabolic networks.

iii. How to correctly choose the right control strategyto maximize the carbon metabolic fluxes and rap-idly guide them to a target amino acid via theregulation of intracellular NADPH levels.

Disclosure statement

The authors report no declarations of interest.

Funding

This work was financially supported by the National NaturalScience Foundation of China [No. 31601459], the NaturalScience Foundation of Jiangsu Province [No. BK20150149],China Postdoctoral Science Foundation Grant [No.2016M590410], and Fundamental Research Funds for theCentral Universities [No. JUSRP115A19].

CRITICAL REVIEWS IN BIOTECHNOLOGY 11

ORCID

Jian-Zhong Xu http://orcid.org/0000-0003-0750-6875

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