NADPH Metabolism: a Survey of Its Theoretical Characteristics and Manipulation Strategies in Amino Acid Biosynthesis

Critical Reviews in Biotechnology

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NADPH metabolism: a survey of its theoretical characteristics and manipulation strategies in amino 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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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ibty20 CRITICAL REVIEWS IN BIOTECHNOLOGY, 2018 https://doi.org/10.1080/07388551.2018.1437387

REVIEW ARTICLE NADPH metabolism: a survey of its theoretical characteristics and manipulation 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, Jiangnan University, WuXi, PR China

ABSTRACT ARTICLE HISTORY Reduced nicotinamide adenine nucleotide phosphate (NADPH), which is one of the key cofactors Received 6 June 2017 in the metabolic network, plays an important role in the biochemical reactions, and physiological Revised 17 January 2018 function of amino acid-producing strains. The manipulation of NADPH availability and form is an Accepted 20 January 2018 efficient and easy method of redirecting the carbon flux to the amino acid biosynthesis in indus- KEYWORDS trial strains. In this review, we survey the metabolic mode of NADPH. Furthermore, we summarize NADPH; amino acid; the research developments in the understanding of the relationship between NADPH metabolism metabolic modes; and amino acid biosynthesis. Detailed strategies to manipulate NADPH availability are addressed manipulation strategies; based on this knowledge. Finally, the uses of NADPH manipulation strategies to enhance the biosynthetic pathway metabolic function of amino acid-producing strains are discussed.

Introduction ADP), reduced-/nicotinamide adenine dinucleotide (NADH/NADþ), and reduced-/nicotinamide adenine Amino acids are types of organic compounds with carb- dinucleotide phosphate (NADPH/NADPþ) except for oxyl and amine groups that play important roles in reg- metabolic enzymes [4]. ATP and ADP are known as ulating the physiology of all life-forms. More than 300 energy cofactors, while NADH/NADþ and NADPH/ amino acids exist in nature, but only 20 of them are the þ basic structural elements of proteins, and only 10 are NADP are referred to as redox cofactors [5]. Figure 1 considered to be essential amino acids for humans and shows that these cofactors participate in the biosyn- animals [1]. In addition to the synthesis of proteins and thesis of several amino acids. Table S1 lists and numbers other compounds present in nature, amino acids also the enzymes involved in the three cofactor metabolism participate in a wide variety of biochemical reactions, for the metabolic pathways of E. coli and C. glutamicum. and are vital for energy transfer and energy cycles [2]. As one of the key cofactors in the metabolic network, The improvement of amino acid yield and efficiency has NADPH plays an important role in the biochemical reac- acquired significant interest because of the importance tions and physiological function of amino acid-produc- of amino acids in food, fodder, medical, cosmetic, and ing strains. Similar to other redox cofactors, NADPH is other industrial applications and the growing market also known as a co-enzyme in the cellular electron demand in the amino acid industry. At present, micro- transfer that drives the biosynthetic pathways of DNA, bial fermentation via Corynebacterium glutamicum or amino acids, fatty acids, phospholipids, and steroids. Escherichia coli plays a leading role in the amino acid Equally important is the universal reducing power of industry [3]. Appropriate strains and fermentation tech- NADPH to fuel the activities of enzymes, such as cata- nologies are crucial for amino acid production because lase, superoxide dismutase, and glutathione peroxidase, they promote increased carbon flux into the biosyn- which play important roles in allowing microorganisms thetic pathways of amino acids. However, the regulation to thrive in aerobic environments [6]. Moreover, NADH/ þ þ mechanism of these pathways is very complex because NAD generated from NADP can be used to form several metabolic reactions require the involvement of nicotinamide and other products, including ADP-ribose cofactors, such as adenosine tri-/di-phosphate (ATP/ (Figure 2)[6]. These metabolites can be employed as

CONTACT Jian-Zhong Xu [email protected] The Key Laboratory of Industrial Biotechnology and The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, WuXi, PR China Supplemental data for this article can be accessed here. ß 2018 Informa UK Limited, trading as Taylor & Francis Group 2 J.-Z. XU ET AL.

Glc 1 2 G6P 6PGlac 6PGluc 3 RL5P NADPH NADH ATP

NADP NAD ADP F6P X5P R5P 51 PRPP His 52/53/54 4

S7P G3P F1,6BP

DHAP GA3P E4P F6P

8 5

1,3BPG Tyr Cys 47/48 6

CHA 48 Phe 44/45/46 42 Ser 43 PHP 3PG Trp 49/50 Val Gly PEP 36 Ala 40

Asp 30 Asn 7

KIVal DHIV 35 Alac Pyr 31/32 41 9

Lac 12 13 14 29 33 Eth 13 AspSAld HSer KICap 34 HSerP AcCoA 10 36 DHDP Leu Cit OAA 37 Met Thr ICit 18 35 15 11 THDP Mal DHMV αKG

7 Fum SucAKP 36 1

Succ 1 9 1 38 Ile 39 Glu SucDAP 2 0 7 2 /2 / 1 6 2 Glu-γ-SAld AGlu-γ-SAld 25 LL-DAP

22 Spontaneous meso-DAP Arg α-AOrn Gln P5C Pro 28 23/24

Lys Figure 1. Metabolic pathways of NADPH and amino acids in E. coli and C. glutamicum. The abbreviations and the enzymes involved in the cofactor metabolism are listed in the Supplemental file and Table S1. The red lines (i.e. 19th–28th enzymatic reactions) indicate the biosynthetic pathway of GFAAs; the dark green lines (i.e. 29th–39th enzymatic reactions) indicate the biosynthetic pathway of AFAAs; the blue lines (i.e. 40th–41th enzymatic reactions) indicate the biosynthetic pathway of PFAAs; the pink lines (i.e. 42th–43th enzymatic reactions) indicate the biosynthetic pathway of SFAAs; and the spring-green lines (i.e. 44th–50th enzymatic reactions) indicate the biosynthetic pathway of AAAs. regulating factors to maintain cellular functions. Thus, their physiological functions are not the major emphasis NADPH plays an important part in cell growth and of this study [6,8]. The review aims to survey the meta- metabolite production. However, unlike other cofactors, bolic modes of NADPH and their relationship with NADPH drives anabolic reactions [7]. Although high- amino acid biosynthesis, and provide an overview of its producing amino acid strains were developed by applications. This review also covers strategies for manipulating the NADPH availability and state, given manipulating the NADPH availability and generating the existence of several reviews on the subject topic, amino acid-producing strains with a focus on two CRITICAL REVIEWS IN BIOTECHNOLOGY 3

NOX NADHK Electron transport chain ROS NADPH NADH ATP

NADP+-dependent NAD+-dependent oxido-reductase oxido-reductase NAADP ARCs NADPase NADP+ NAD+ cADPRP NADK A + RCs, cARHs, LT ) P ase d AD( N ARTs PARPs dependent , e nucleosi Cs tc. ADPR(P) NAD- HD Poly(ADP-ribosyl- Deacetylation of Mono(ADP-ribosyl- ADP-ribose Regulating the ation) of protein (non-)histones protein ation) of protein cell function

Figure 2. NADPH conversion and degradation. The abbreviations are listed in the Supplemental file. dominant organisms for industrial production of amino reversibly transferred by transhydrogenases between acids (i.e. C. glutamicum and E. coli) and the physio- NADH/NADþ and NADPH/NADPþ in several microorgan- logical consequences. isms. The Supplemental file presents in detail the enzymes involved in the NADPH anabolism and their characteristics. Table S2 summarizes the common NADPH metabolism enzymes involved in the NADPH regeneration and their þ The ratios of NADPH/NADP affects numerous enzym- properties. atic activities and endogenous concentrations of regulators (e.g. reactive oxygen species and nicotinic acid NADPH catabolism adenine dinucleotide phosphate) that play important roles in cellular functions and cell survival under several NADPH is an essential anabolic reducing cofactor in all conditions. Therefore, balancing the catabolic formation living organisms, which is involved in numerous ana- of NADPH with the anabolic demand is crucial to amino bolic reactions to form NADPþ. Wittmann and de Graaf acid biosynthesis via microbial fermentation. [14] identified that the formation of 1 g of biomass requires 16.4 mmol of NADPH to form NADPþ. Thus, NADPH catabolism is closely related to the biomass for- NADPH anabolism mation rate, which will significantly vary with the envir- The following are three major methods by which onmental conditions [7]. Most of the NADPH is NADPH can be formed (Figure 3): I) NADPH is regener- consumed by an NADPH-dependent reductase to form þ þ ated from NADP by diversified NADP -dependent NADPþ as the hydride transfer is finally completed. þ enzymes; II) NADPH is regenerated from NAD using However, the intracellular level of NADPþ is lower than þ NAD kinases, or from NADH using NADH kinases; and its theoretical value because NADPþ can be catabolized þ III) NADPH is regenerated from NADH and NADP using by multiple families of enzymes to form different transhydrogenases. Method I is the major method for nucleotide derivatives and other products including NADPH-regeneration in glucose-grown microorganism ADP-ribose (Figure 2)[15]. The enzymes directly cells. However, the intracellular concentrations of involved in NADPþ catabolism include ADP-ribosyl- þ NADPH and NADP are low compared with those of cyclases, NADPþ nucleosidase, and NADPþ phosphatase þ NADH and NAD [9]. Therefore, using Method II for (Figure 2). The Supplemental file describes in detail the NADPH regeneration recently had a prominent role. characteristics and functions of these enzymes. NAD kinases (NADKs) are crucial enzymes in Method II that regulate the NAD(H/þ) and NADP(H/þ) levels þ Relationships between NADPH and amino acid through NAD(H/ ) phosphorylation using nucleoside production triphosphates (NTP) or inorganic polyphosphates [Poly(P)] as phosphoryl donors to form NADP(H/þ) C. glutamicum and E. coli are two of the most important (Figure 3). NADKs exist abundantly in living organisms bacteria used for the industrial production of various [10,11] and do not affect net catabolic fluxes [12]. amino acids, including: glutamate-family amino acids Therefore, Method II is widely used for NADPH regener- (GFAA), aspartate-family amino acids (AFAA), pyruvate- ation and regulation of intracellular NADPH levels. family amino acids (PFAA), serine-family amino acids Similar to Method II, Method III has no effect on (SFAA), and aromatic amino acids (AAA). Amino acid net catabolic fluxes [13]. In Method III, hydride is high-producing strains have been obtained in the past 4 J.-Z. XU ET AL.

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

NAD+ Kinase NAD+ NADP+

NAD+-dependent NADP+-dependent Poly(P)n/NTP Poly(P)n-1/NDP II. oxido-reductases oxido-reductases

NADH NADPH NADH Kinase

Membrane-bound transhydrogenase (mTH), eg., PntAB III. NADH+ NADP+ NAD+ + NADPH Soluble transhydrogenase (sTH), eg., UdhA and Sth

Figure 3. Three general approaches for NADPH regeneration. The abbreviations are listed in the Supplemental file. using classical mutagenesis and overexpression (or ammonium assimilation [22]. Therefore, 1 mol of deregulation) of biosynthetic enzyme-coding genes as NADPH must be supplied for 1 mol of L-glutamate bio- well as deletion of some enzymes that are irrelevant for synthesis. L-glutamine, which is an amide of L-glutam- the biosynthesis of the target amino acid. However, ate, biosynthesized from L-glutamate, and is catalyzed aside from modifying biosynthetic pathways and by GS [24]. GS can be subdivided into three subfamilies increasing precursor availability, the supply of NADPH is based on the diverse bacterial groups, namely, GSI, GSII, a critical factor for amino acid production [16], because and GSIII (for a review, see Ref. [25]). However, GSI is some enzymatic reactions in the biosynthetic pathway the main catalyzing enzyme [26]. According to previ- require the NADPH participation (Figure 1). Tables 1 ously reported studies, GSI is an ATP-dependent L-glu- and S3 summarize the participating enzymes and the tamate:ammonia ligase, which requires ATP instead of demand for NADPH in the amino acid biosynthetic pro- NADPH to catalyze the L-glutamine biosynthesis [24]. cess. The Supplemental file describes the relationships Therefore, 1 mol NADPH is required for the biosynthesis between NADPH and the production of PFAAs, SFAAs, of 1 mol L-glutamine in C. glutamicum and E. coli. Note and AAAs in detail. that GDH is associated with isocitrate dehydrogenase to form a conjugated complex in the redox reaction; thus NADPH is closely related to GFAA production NADPH can become self-sufficient (Figure 1). NADPH is not important for the L-glutamate and L-glutamine According to descriptions by Jensen et al. [17], GFAA biosynthesis, and consequently, studies on breeding contains L-glutamate, L-glutamine, L-proline, and L- L-glutamate or L-glutamine high-yielding strains are arginine, which derive part or all of their carbon from mainly focused on optimizing culture conditions [27], a-ketoglutarate. They also contain several non-protein amino acids, such as L-ornithine [18] and L-citrulline enhancing precursor supply [28], regulating energy [19]. Figure 1 (red lines indicating section) presents the cofactors level [29], and modifying export systems [30]. metabolic pathways of the GFAA and cofactor systems. In a widespread biosynthetic route for L-proline pro- L-glutamate can be biosynthesized through either duction, L-glutamate is converted into L-proline via the glutamate dehydrogenase (GDH) pathway or the three enzymatic reactions and one spontaneous reac- glutamine synthetase (GS)/glutamate synthase (GOGAT) tion (Figure 1;[31,32]). The redox and dephosphoryla- pathway. However, the GDH pathway is the main route tion reaction is the second enzymatic reaction catalyzed [20] leading to the reductive amination of a-ketogluta- by c-glutamyl phosphate reductase (No. 27) and rate by GDH (No.19; [21,22]). Nevertheless, GDH can be requires NADPH as a cofactor [31]. The third enzymatic subdivided into the three following subfamilies accord- reaction type is a redox reaction, and is catalyzed by ing to the cofactor specificity: (a) NADþ-dependent pyrroline-5-carboxylate reductase (No. 28). This reaction GDH, (b) NADPþ-dependent GDH, and (c) NADþ/NADPþ also requires NADPH as cofactor [31]. L-proline can also dual-specific GDH [23]. NADPþ-dependent GDH is one be biosynthesized from L-ornithine catalyzed by orni- of the major enzymes for the L-glutamate biosynthesis thine cyclodeaminase (OCD). However, the overexpres- in C. glutamicum and E. coli because it is required for sion of the putative OCD-coding gene does not result in CRITICAL REVIEWS IN BIOTECHNOLOGY 5

L-proline accumulation in the medium despite the gen- synthesis in E. coli, but it will not be discussed here ome of C. glutamicum or E. coli containing a putative because it is unimportant [34]. The L-asparagine biosyn- OCD-coding gene [31]. Based on these findings, 2 mol thesis normally begins with the catalysis of L-aspartate NADPH must be supplied for the biosynthesis of 1 mol by asparagine synthetase (No. 30). This enzymatic step L-proline from L-glutamate in C. glutamicum and E. coli requires ATP instead of NADPH as a cofactor [43]; thus (Figure 1, where the red lines indicate the section). the biosynthesis of 1 mol L-asparagine requires only Despite these findings (to the best of our knowledge) 1 mol NADPH. reports regarding the generation of L-proline high- L-threonine is synthesized from L-aspartate using five yielding strains have not been presented in the recent enzymatic steps (Table S3;[44,45]). Figure 1 shows that years. two key enzymes, namely, aspartate-semialdehyde L-arginine in both C. glutamicum and E. coli is biosyn- dehydrogenase (No. 32) and homoserine dehydrogen- thesized from L-glutamate through eight enzymatic ase (No. 33), require NADPH to catalyze the biosynthesis steps (Table S3). The third enzyme in the biosynthetic of aspartate semialdehyde and homoserine, respect- c pathway is N-acetyl- -glutamyl-phosphate reductase ively. Combined with the NADPH requirement in the L- (No. 21), which is an NADPH-dependent reductase that aspartate biosynthesis, 3 mol NADPH must be supplied requires NADPH to catalyze the biosynthesis of N-acetyl for the biosynthesis of 1 mol L-threonine from OAA in c glutamyl- -semialdehyde [33]. The fourth enzymatic C. glutamicum and E. coli. However, the strategies for reaction is catalyzed by acetyl-ornithine aminotransfer- generating L-threonine high-yielding strains mainly ase (No. 22), in which the amino group from L-glutam- focus on the following six aspects: (1) increasing the a ate is transferred to N- -acetylornithine. The fifth efficiency of the biosynthetic pathway by overexpress- enzymatic reaction is catalyzed by bifunctional ing the key enzyme-coding genes [46], (2) weakening or ornithine acetyltransferase (No. 23), in which acetyl blocking the competitive pathways to increase the pre- from N-a-acetylornithine is transferred to L-glutamate cursor availability and reduce the by-products formation [33]. Meanwhile, the seventh reaction is catalyzed by [45], (3) reducing the L-threonine degradation pathways argininosuccinate synthetase (No. 24) and requires L- [47], (4) increasing the L-threonine secretion [48], (5) aspartate and ATP intervention [33]. However, the L-glu- expanding the useful range of available sugars [49], and tamate and L-aspartate biosynthesises need NADPH (6) integrating (1) to (4) via system-level metabolic participation [22,34]. Therefore, 3 mol NADPH is engineering [50,51]. Until now, only Xie et al. [52] required to biosynthesize 1 mol of L-arginine from reported that blocking the Embden–Meyerhof pathway L-glutamate in C. glutamicum and E. coli (Figure 1). (EMP) in E. coli leads to an increase in the intracellular Engineering of the NADPH metabolism has been NADPH level that would enhance L-threonine gradually regarded by researchers as one of the key accumulation. methods for breeding an L-arginine high-yielding strain The sulfur-containing amino acid L-methionine is because of the importance of NADPH for improving synthesized via seven enzymatic steps from L-aspartate L-arginine production [35–37], with the exception of the pathways modification of carbon metabolism [38,39], and derived from the precursor L-homoserine (Table S3; and the export system to breed L-arginine high-produc- [53]). Interestingly, the L-methionine biosynthesis shares ing strains [40]. a partial biosynthetic pathway with L-threonine (Figure 1, the dark green lines indicate the section). Similar to L-threonine biosynthesis, the biosynthesis of NADPH is closely related to AFAA production 1 mol L-homoserine requires 3 mol NADPH. The sulfate AFAA contains L-aspartate, L-asparagine, L-threonine, L- assimilation also requires NADPH for sulfur reduction lysine, L-isoleucine, and L-methionine. L-aspartate is [53,54]. Therefore, approximately 8 mol NADPH and 8. derived from oxaloacetate (OAA; [41]), while the others 5 mol NADPH are required for the biosynthesis of 1 mol derive part or all of their carbon from L-aspartate [42]. L-methionine in C. glutamicum and E. coli, respectively Figure 1 (dark green lines indicating the section) [53]. Although NADPH is important for improving the L- presents the metabolic pathways of the AFAA and methionine production, to our knowledge, very few cofactor systems. studies focused on NADPH metabolism engineering for The L-aspartate biosynthesis is the amination of OAA breeding L-methionine high-yielding strains. The major- by aspartate aminotransferase (No. 29) that requires ity of these studies have focused on the enhancement 1 mol NADPH to biosynthesize 1 mol L-aspartate (Figure of the precursor supply [28,55], deregulation of the 1, the dark green lines indicate the section). An add- synthetic pathway [56], and modification of the export itional biosynthetic pathway is needed for L-aspartate system [57]. Therefore, engineering the NADPH 6 J.-Z. XU ET AL. metabolism will be a tendency for breeding L-methio- several value-added products, particularly amino acids nine high-yielding strains. [84]. Although NADPH regeneration can be achieved by Starting with L-aspartate, L-isoleucine biosynthesis external regulation, such as the addition of the electron involves 10 enzymatic steps with L-threonine serving as acceptor or adjustment of the dissolved oxygen con- an indirect precursor for its biosynthesis (Table S3). centration [85,86], an endogenous regulation based on Therefore, the L-isoleucine biosynthesis proceeds with the metabolic pathway of NADPH in vivo is one of the five enzymatic steps starting from L-threonine (Table major methods for the NADPH regeneration. This S3). The third enzymatic reaction is catalyzed by aceto- depends on the regulation of the enzyme activity hydroxy acid isomeroreductase (No. 35), and NADPH is involved in NADPH regeneration using genetic used as a cofactor in this reaction [58,59]. The last engineering. enzymatic reaction is catalyzed by branched-chain amino acid aminotransferase (No. 36), in which the amino group from L-glutamate is transferred to L-iso- Increasing the activity of enzymes involved in leucine [60,61]. However, the L-glutamate biosynthesis NADPH regeneration needs NADPH participation [21,22]. Therefore, 5 mol Numerous enzymes are involved in the NADPH-regener- NADPH must be supplied for the biosynthesis of 1 mol ating reactions in C. glutamicum and E. coli (Table S2) L-isoleucine from OAA. Apart from modification of the that can be subdivided into three groups based on the – metabolic pathways and export systems [58,61 63], sev- cofactor specificity (see above; Figure 3). Although eral studies have proven that increasing the NADPH NADPþ-dependent dehydrogenases originate from the supply is beneficial for the increase of L-isoleucine pro- OPP pathway, the tricarboxylic acid (TCA) cycle (i.e. iso- duction by either overexpressing the NAD [60,64]or citrate dehydrogenases, ICD, No. 15), and anaplerotic NADH kinases [21], or strengthening the carbon flux reactions (i.e. malic enzyme, MalE, No. 11), several stud- into the oxidative pentose phosphate (OPP) pathway ies have identified that NADPþ-dependent dehydrogen- [11]. ases from the OPP pathway are the major enzymes for C. glutamicum comprises two L-lysine biosynthetic NADPH generation in C. glutamicum and E. coli (for pathways from L-aspartate (i.e. acetylase pathway and review, see Refs. [46,84]). dehydrogenase pathway), whereas E. coli comprises Figure 1 shows the first and third enzymatic steps in only one pathway (i.e. acetylase pathway) (Figure 1, the the OPP pathway involved in NADPH generation that dark green lines indicate the section; [65]). The L-lysine are catalyzed by glucose-6-phosphate dehydrogenase biosynthesis can be improved by overexpressing the (G6PDH, No. 2; encoded by zwf gene in C. glutamicum key enzyme genes in the biosynthetic pathway [66–69], and E. coli) and 6-gluconate phosphate dehydrogenase weakening or blocking the competitive metabolic path- (6PGDH, No. 3; encoded by gnd gene in C. glutamicum ways [70,71], increasing the sugar availability [16,69,72], and E. coli), respectively. The key enzyme for controlling enhancing export systems [48], and expanding the use- the flux of the OPP pathway is G6PDH [87]. Therefore, ful range of available sugars [16,73,74]. However, one of the most efficient methods of increasing the NADPH-regenerating systems are also important for NADPH availability is to increase the G6PDH and 6PGDH L-lysine biosynthesis because 4 mol NADPH must be supplied for the biosynthesis of 1 mol L-lysine from L- activities. Several studies reported that the overexpres- aspartate (Table 1). Numerous studies have focused on sion of G6PDH or/and 6PGDH is effective in improving NADPH metabolism engineering in order to precisely the target amino acid production because of the improve L-lysine high-producing strains because of the increase of the NADPH availability [21,66,88,89]. importance of NADPH for L-lysine production However, the G6PDH and 6PGDH activities are regu- [66–68,70,75–79]. lated by ATP and fructose-1,6-diphosphate (Fru-1,6-P2), whereas the introduction of the A243T mutation into the zwf gene or the S361F mutation into the gnd gene Manipulation strategy for NADPH regeneration relieves feedback regulation by ATP and Fru-1,6-P2, during amino acid biosynthesis thereby increasing the L-lysine production [82,90]. Even NADPH regeneration in micro-organisms is in a dynamic though ICD and MalE are also the NADPH sources in C. balance. It is strictly dependent on the microbial growth glutamicum and E. coli, the overexpression of ICD and state and the genetic background [67,68,80,81], or on MalE was not beneficial in improving the amino acid the applied carbon source [16,82,83]. NADPH supply is production [82,84]. Conversely, decreasing ICD activity in excess in most cases. However, the NADPH availabil- can promote L-lysine production in C. glutamicum ity is a major limitation in the efficient production of [66,71], showing that compared to G6PDH and 6PGDH, CRITICAL REVIEWS IN BIOTECHNOLOGY 7

Table 1. Demand for NADPH and the participating enzymes in the amino acids biosynthetic process during cultivation on glucosea. Encoding gene NADPH Group Amino acid (mol [mol AA]1) Enzymes E. coli C. glutamicum Substrates Enzymatic reaction GFAAs L-glutamate 1 GDH gdhA gdh a-KG Reductive amination reaction L-glutamine 1 GDH gdhA gdh a-KG Reductive amination reaction L-proline 3 GDH gdhA gdh a-KG Reductive amination reaction GPR proA proA Glu-P Reductive dephosphorylation reaction P5CR proC proC P5C Redox reaction L-arginine 4 GDH gdhA gdh a-KG Reductive amination reaction AGPR argC argC AcGlu-P Reductive dephosphorylation reaction ACO-OT argD argD AcGlu-c-Sald þ L- Amino group transfer reaction glutamate ASS argG argG L-citruline þ L- Condensation reaction aspartate AFAAs L-asparagine 1 AAT aspB aspB OAA þ L-glutamate Amino group transfer reaction L-asparagine 1 AAT aspB aspB OAA þ L-glutamate Amino group transfer reaction L-threonine 3 AAT aspB aspB OAA þ L-glutamate Amino group transfer reaction ASADH asd asd Asp-P Reductive dephosphorylation reaction HDH thrA, metL hom Asp-SAld Redox reaction L-methionine 8 for C. glutamicum AAT aspB aspB OAA þ L-glutamate Amino group transfer reaction and 8.5 for E. coli ASADH asd asd Asp-P Reductive dephosphorylation reaction HDH thrA, metL hom Asp-SAld Redox reaction –b ––– Sulfate assimilation reaction L-isoleucine 5 AAT aspB aspB OAA þ L-glutamate Amino group transfer reaction ASADH asd asd Asp-P Reductive dephosphorylation reaction HDH thrA, metL hom Asp-SAld Redox reaction AHAIR ilvC ilvC AcHB Redox and acetoin rearrange- ment reaction BCAT ilvE ilvE KMVA þ L- Amino group transfer reaction glutamate L-lysine 4 AAT aspB aspB OAA þ L-glutamate Amino group transfer reaction ASADH asd asd Asp-P Reductive dephosphorylation reaction DHDPR dapB dapB DHDP Redox reaction DapC dapC dapC AKV þ L-glutamate Amino group transfer reaction DDHc – ddh THDP Reductive amination reaction PFAAs L-alanine 1/2d AlaT/AvtA alaT/avtA alaT/avtA Pyr þ L-glutamate/ Amino group transfer reaction L-valine L-valine 2 AHAIR ilvC ilvC AcHB Redox and acetoin rearrange- ment reaction BCAT/AvtA ilvE/avtA ilvE/avtA KIVal þ L-glutam- Amino group transfer reaction ate/L-alanine L-leucine 2 AHAIR ilvC ilvC AcHB Redox and acetoin rearrange- ment reaction BCTA ilvE ilvE KICap þ L- Amino group transfer reaction glutamate SFAAs L-serine 1 PSAT serC serC PHP þ L-glutamate Amino group transfer reaction L-glycine 1 PSAT serC serC PHP þ L-glutamate Amino group transfer reaction L-cysteine 6 for C. glutamicum PSAT serC serC PHP þ L-glutamate Amino group transfer reaction and 6.5 for E. coli –a ––– Sulfate assimilation reaction AAAs L-phenylalanine 2 SHKH aroE aroE DHS Hydro-oxidation reaction AAATm tyrB pat PKU þ L-glutamate Amino group transfer reaction L-tyrosine 2 SHKH aroE aroE DHS Hydro-oxidation reaction AAATm tyrB pat HPKU þ L- Amino group transfer reaction glutamate L-tryptophan 3 SHKH aroE aroE DHS Hydro-oxidation reaction AS trpDE trpE, NCgl2928 CHA þ L-glutamine TAmide group transfer reaction TS trpAB trpA, NCgl2931 Indole þ 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 5–6 mol 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. 8 J.-Z. XU ET AL. neither ICD nor MalE are better choices for NADPH UdhA additionally requires flavin adenine dinucleotide regeneration in amino acid biosynthesis. [84,103]. Moreover, several prior reports suggested that The second types of NADPH-regenerating enzymes PntAB catalyzes the transfer of hydride from NADH to are NADKs, which are not coupled to the central carbon NADPþ to increase the NADPH supply, whereas UdhA metabolism [12,84]. NADKs are divided into ATP-NADK catalyzes the transfer of hydride from NADPH to NADþ (e.g. YfjB from E. coli), and Poly(P)/ATP-NADK (e.g. PpnK to prevent the excess production of NADPH (for a from C. glutamicum) types [10,11]. YfjB and PpnK are review, see Ref. [84]). Interestingly, the overexpression the only NADKs in E. coli and C. glutamicum [91,92]. of the UdhA-coding gene UdhA was beneficial in PpnK was first isolated from C. glutamicum ssp. flavus improving various NADPH-dependent products in E. by Kawai et al. [93], which completely and specifically coli, such as thymidine [49], (S)-2-chloropropionate þ phosphorylated NAD by utilizing both Poly(P) and NTP [104], and fatty acids (C12 – C18)[105]. UdhA also partici- as phosphoryl donors. The phosphorylation reaction pates in NADPH regeneration in E. coli. The overexpres- catalyzed by this enzyme required the participation of sion of the PntAB-coding gene PntAB is expected to bivalent metal ions, but the effects of different ions play an important role in enhancing the yields of were different [93,94]. Moreover, PpnK from different several NADPH-dependent products in E. coli, such as 3- sources, or the introduction of a novel mutation into hydroxypropionic acid [106], and isobutanol [91]. the PpnK-coding gene PpnK exhibits different enzyme However, until now, no relevant report has outlined the properties, which provides different NADPH concentra- modification of PntAB or/and UdhA in E. coli to improve tions for the target product production [60,93,95]. amino acid production. Although C. glutamicum lacks Dissolved oxygen levels will also affect the PpnK activity transhydrogenases, it functions through a transhydroge- and function [35]. PpnK does not affect the net cata- nase-like shunt [107,108]. Blombach et al. [107] indi- bolic fluxes; thus, it is now widely used to regulate the cated that deactivating MalE would interrupt isobutanol intracellular NADPH level and improve amino acid biosynthesis in C. glutamicum because of an insufficient synthesis. The overexpression of the PpnK gene from C. NADPH supply. Cocaign-Bousquet and Lindley [109] glutamicum in the corresponding amino acid-producer also indicated that increasing the activity of MalE can contribute to the enhancement of NADPH, thereby enhanced carbon conversion efficiency in C. glutamicum increasing the production of: L-lysine [10], L-isoleucine because of improvement of the NADPH supply. They [21,63,64], L-arginine [92], and L-ornithine [96]. YfjB suggested that this approach can be proposed as a obtained from E. coli was first purified and identified by strategy for breeding amino acid high-yielding strains. Zerez et al. [97]. Unlike PpnK from C. glutamicum, YfjB However, overexpression of the MalE-coding gene MalE þ þ from E. coli phosphorylates NAD to form NADP with in C. glutamicum strains producing L-lysine did not NTP as the only phosphoryl donor [98]. However, similar improve L-lysine production using glucose, fructose or to PpnK from C. glutamicum, it requires the participa- sucrose [82]. tion of bivalent metal ions, and Mn2þ is one of its most efficient activators [93,98]. Moreover, this enzyme acts Reducing or blocking-up the competitive at an optimal reaction temperature of 60 C and at mild bypass of NADPH regeneration alkali conditions (pH 7.5) [98]. The overexpression of the YfjB-coding gene yfjB in E. coli to improve various Weakening the carbon fluxes in the competitive bypass value-added materials (e.g. poly-3-hydroxybutyrate of NADPH regeneration is also a good strategy for [99,100], thymidine [49], isobutanol [91], and shikimic improving the NADPH supply. In the previous section, acid [101]) has been reported. However, no relevant the OPP pathway was mentioned to be a major route report on the modification of the yjfB gene for improv- for NADPH generation in C. glutamicum and E. coli. ing the amino acid production in E. coli has yet been Thus, redirecting the carbon flux in the EMP and OPP presented. pathways is an obvious choice to increase the intracel- The third NADPH-regenerating enzymes are transhy- lular NADPH level. drogenases coupled to the translocation of hydride Phosphoglucose isomerase (PGI) is an important between NAD(H/þ) and NADP(H/þ) rather than to the enzyme in EMP that catalyzes the reversible reaction metabolism of the central carbon [9,77]. PntAB and between glucose-6-phosphate and fructose-6-phos- UdhA, which are two transhydrogenases coexisting in E. phate. Disrupting the PGI-coding gene pgi can disor- coli, maintain the cellular NADPH/NADH balance [102], ganize the competitive EMP, thereby forcing the carbon but they have different requirements for cofactors and flux completely through the OPP pathway. The PGI differ in their physiological roles. PntAB only requires inactivation is beneficial in increasing several NADPH- cofactors NAD(H/þ) and NADP(H/þ) for activity, whereas dependent products in C. glutamicum and E. coli, CRITICAL REVIEWS IN BIOTECHNOLOGY 9 including amino acids [110–113]. However, a PGI-knock- method to maximize the carbon flux through the OPP out strain exhibits a low specific growth rate and is only pathway is to block the gluconate bypass by inactivat- one-third as fast as the parental strain during growth ing GlcDH and/or GntK. Hwang and Cho [38,122] on glucose [114]. Interestingly, Park et al. [36] reported reported that blocking the gluconate bypass by a dis- that downregulating the expression level of the pgi ruption of the GlcDH-orGntK-coding gene significantly gene, by replacing the start codon ATG with GTG, will forced carbon flux from glucose toward the OPP path- increase the carbon flux entering the OPP pathway with way with a concomitant increase in L-ornithine produc- a minor effect on the cell growth (more than two-thirds tion. However, the effect that stimulated the OPP of the parental strain), thereby resulting in an increase pathway was different for GlcDH and GntK inactivation. L-arginine production in C. glutamicum. However, this In the case of the GlcDH-deficient strain, the carbon flux strategy is only available for glucose-based processes from glucose- 6-phosphate was entirely in the OPP because sucrose- or fructose-based processes require pathway, thereby resulting in an increase of key enzyme an active PGI for recycling carbon into the OPP pathway activities in the OPP pathway. However, the GntK- [83]. knockout leads to an increase of key enzyme activities Another method is to redirect the carbon flux in the in the OPP pathway because of accumulation of gluco- EMP and OPP pathways by decreasing the intracellular nate that hindered binding of the GntR transcriptional

Fru-1,6-P2 concentration. This method has attracted the repressor types (i.e. GntR1 and GntR2) to their target attention of researchers because it inhibits the activities gene promoters (e.g. the gnd promoter) [121]. of G6PDH [115], 6PGDH [90], and fructose-1,6-bisphosphatase (FBPase) [116]. FBPase catalyzes the hydrolysis Introduction of an extrinsic pathway for of Fru-1,6-P to fructose-6-phosphate (Fru-6-P) and P , 2 i NADPH regeneration which is a key enzyme in the gluconeogenic pathway. A metabolic flux analysis indicated that overexpression Introducing an extrinsic pathway is another appropriate of the FBPase-coding gene fbp resulted in enhanced strategy for intracellular NADPH regeneration, and it OPP pathway flux, thus increasing the supply of NADPH has been gaining interest amongst researchers. The þ and L-lysine production [82,83]. Similarly, the inactiva- replacement of the native NAD -dependent enzyme þ tion of phosphofructokinase (PFK, No. 4, encoded by with a non-native NADP -dependent enzyme or the genes pfkA and pfkB in C. glutamicum and E. coli), that introduction of a new and extrinsic pathway in the host catalyzes the phosphorylation of Fru-6-P to form Fru- strain has been extensively used for intracellular NADPH

1,6-P2, will decrease the intracellular Fru-1,6-P2 concen- regeneration. tration, thus forcing the carbon flux through the OPP Glyceraldehyde-3-phosphate dehydrogenase (GADPH, pathway. Wang et al. [117] and Siedler et al. [118] No. 5) catalyzes an essential step in the central metabol- reported that PFK knockouts will direct Fru-6-P into the ism. Therefore, the GADPH modification is one of the OPP pathway and increase NADPH generation, thus important research areas in enzyme replacement for increasing the production of NADPH-dependent prod- NADPH regeneration. GADPHs are classified into two ucts in E. coli. However, the inactivation of PFK seems to types according to differences in the specificities of the þ þ act against L-threonine production in E. coli according cofactors: NAD - and NADP -dependent GADPHs. In E. þ to the report by Xie et al. [52]. They found that deleting coli, GADPH is an NAD -dependent enzyme that cata- the pfkA gene decreased cell growth and L-threonine lyzes the reversible oxidation of glyceraldehyde-3-phos- production because NADPH synthesis is blocked. In con- phate into 1,3-bisphosphoglycerate (1,3-BPG) using trast, deleting the pfkB gene had no effect on cell NADþ as the cofactor [123]. In contrast, C. glutamicum þ growth and L-threonine production because the has two GAPDHs, namely GapA (NAD -dependent increase in the NADPH supply was not large [52]. enzyme) and GapB (NADPþ-dependent enzyme, No. 8) Moreover, several reports demonstrated that overex- [124]. Although GapB catalyzes NADPH synthesis, the pressing the pfkAB gene increased the carbon flux in up-regulation of GapB activity was not advantageous EMP, resulting in an increase in the production of prod- for cell growth and L-lysine production because GapB ucts, such as L-alanine [119] and L-serine [120]. was only involved in gluconeogenesis [75]. These results C. glutamicum has two routes for the formation of 6- forced the replacement of native NADþ-dependent phosphogluconate, namely, the OPP pathway, and the GAPDH with a non-native NADPþ-dependent GADPH. gluconate bypass. The gluconate bypass consisted of The NADPþ-dependent GADPH can be classified into two key enzymes [i.e. glucose dehydrogenase (GlcDH) two types: the phosphorylating type catalyzing the syn- and gluconate kinase (GntK)] that decrease the carbon thesis of 1,3-BPG and the non-phosphorylating type flux through the OPP pathway [121]. An alternative (GapN) catalyzing the synthesis of 3-PG (for a review, 10 J.-Z. XU ET AL. see Ref. [84]). However, the heterologous expression of glucose to the target products because of the reduction þ any type of NADP -dependent GADPH-coding gene is in CO2 formation via the OPP pathway. þ conducive to the production of NADPH-dependent However, the intracellular NADP(H/ ) concentration þ products in C. glutamicum and E. coli. For example was lower than that of NAD(H/ ) and NADH can be eas- replacing the native GAPDH-coding the gapA gene with ily regenerated through several pathways (e.g. EMP and the non-native gene gapC (encoding phosphorylated TCA cycle) [9,127]. Therefore, how to direct the phos- þ þ NADPþ-dependent GADPH) from Clostridium acetobuty- phorylation of NAD(H/ ) to NADP(H/ ) has become an licum significantly increased the production of L-lysine important issue in NADPH regeneration for researchers. [68] and L-ornithine [125]inC. glutamicum. Similarly, E. coli and C. glutamicum only contain YfjB and PpnK; the introduction of the gapN gene (encoding GapN) hence, they have been used to increase the NADPH from Streptococcus mutans in the gene locus of gapA supply by overexpression of corresponding native from C. glutamicum can also significantly increase the genes [11,63,92,100]. Several attempts have been made production of L-lysine with different industrial sugars, to supply more efficiently NADKs for NADPH regener- such as glucose (increased by 70%), fructose ation. For example the introduction of Pos5, which is an (increased by 120%), and sucrose (increased by NADH kinase, from Saccharomyces cerevisiae to E. coli 100%), as compared to the parental strain [75]. could increase the production yield of poly-3-hydroxy- Meanwhile, altering the cofactor specificity by site- butyrate (increased by 5.6%), GDP-L-fructose (increased e directed modification of GADPH binding sites can real- by 51%), and -caprolactone (increased by 96%), ize the regeneration of NADPH rather than NADH in the because Pos5 directly catalyzes NADH phosphorylation GADPH-catalyzed step. Bommareddy et al. [78] shown to form NADPH [46,128]. Shi et al. [11,21] pointed that the heterologous expression of the Pos5-coding gene that introducing the D35G, L36T/L36R, T37K, and/or pos5 from S. cerevisiae into C. glutamicum will signifi- P192S mutations into the gapA gene of C. glutamicum þ cantly increase L-isoleucine production (increased by will alter the cofactor specificity of GapA to NADP , 26%), and it is better than that of the PpnK-expression thereby significantly increasing L-lysine production. strain. These results indicated that direct phosphoryl- However, the effect of directing the carbon flux was dif- ation of NADH via the heterologous expression of the ferent from introducing the non-native GADPH and a NADH kinase can be considered to be a more efficient mutation in the native GADPH. The metabolic flux ana- method for NADPH regeneration. lysis indicated that the heterologous expression of the NADPþ-dependent GADPH-coding gene significantly decreased the carbon flux to the OPP pathway Alternative methods for NADPH regeneration (decreased by 80%), whereas introducing a mutation in In addition to increasing the NADPH supply by overex- the native GADPH did not dramatically change the car- pressing the key enzyme-coding genes involved in bon flux to the OPP pathway [76,78]. NADPH regeneration, reducing the competitive bypass Another strategy for regenerating NADPH in the cell or introducing an extrinsic pathway, a rational design is to strengthen the hydride transfer from NADH to for the cofactor specificity of enzymes in the biosyn- NADPH by enhancing the activity of transhydrogenases. thetic pathway of metabolites can be considered as As was previously mentioned, C. glutamicum does not another method for indirectly enhancing NADPH regen- contain any transhydrogenase. In contrast, E. coli has eration. We know that the native acetohydroxy acid iso- two transhydrogenases, namely PntAB and UdhA. As a meroreductase (AHAIR, No. 41; encoded by ilvC gene) result, some researchers attempted to regenerate from C. glutamicum is an NADPH-dependent enzyme in NADPH from NADH by introducing an exogenous trans- the biosynthetic pathway of branched-chain amino hydrogenase in C. glutamicum. They overexpressed the acids (BCAAs). However, Hasegawa et al. [129,130] intro- PntAB-coding gene PntAB from E. coli, which resulted in duced three mutations (i.e. Ser34Gly, Leu48Glu, and relieving the GADPH inhibition by NADH and increasing Arg49Phe) into the ilvC gene to replace the NADPH- the NADPH supply. Numerous reports indicated that dependent AHAIR with the NADH-dependent AHAIR, the expression of the PntAB gene from E. coli to C. gluta- thereby resulting in an increase in the NADPH/NADH micum improves the production of L-lysine [67,80] and ratio and L-valine production under oxygen-deprived L-valine [126]. Bartek et al. [126] also suggested that the conditions. They also found that replacing C. glutami- introduction of heterologous PntAB redirects the carbon cum transaminase B (No. 36, an NADPH-dependent flux from the OPP pathway to the EMP (decreased by enzyme) with leucine dehydrogenase (an NADH- 12%), suggesting that the introduction of transhydro- dependent enzyme) from Lysinibacillus sphaericus genase is feasible for increasing the conversion ratios of improved the redox balance (i.e. regulating the CRITICAL REVIEWS IN BIOTECHNOLOGY 11

NADH/NADPH supply), and increased L-valine produc- thereby predicting strategies that will effectively tion in C. glutamicum. However, occasionally, altering increase NADPH availability for the amino acid produc- the cofactor specificity of enzymes is not good for tion processes. NADPH regeneration. Marx et al. [131] demonstrated Sebastiaan K. Spaans from Wageningen University that replacing C. glutamicum NADPH-dependent GDH has also argued that a microbial cell is a complex sys- with NADH-dependent GDH from Peptostreptococcus tem that usually contains various integrated units [84], asaccharolyticus decreased the NADPH biosynthesis leading to unexpected physiological and metabolic per- (decreased from 210 to 139%) because of rerouting the turbations during modification of NADPH-regenerating central carbon metabolism. These results indicated that pathways. However, a few studies on the optimization the central carbon metabolism had an extraordinary of cellular physiological and metabolic functions maxi- flexibility, and we should reasonably select the strategy mized carbon flux through the biosynthetic route for for NADPH regeneration. amino acids based on regulation of the intracellular NADPH level. We can predict the three following critical scientific issues in developing high-yielding tamino acid Conclusions and future prospects strains via regulation of the intracellular NADPH levels Amino acids play an important role regulating the based on completed research programs and the future physiology of all life-forms because they participate in developments: the synthesis of proteins and other compounds present in nature, regulate diverse biochemical reactions, and i. How to quantitatively study the effects of NADPH are vital during energy transfer and energy cycles. [2]. levels based on microbial metabolome, proteome, Over the past 60 years, numerous research efforts were and transcriptome with applied systems biology spent on increasing amino acid production via engin- and high-throughput analysis: systems biology eering cellular central carbon metabolic pathways and and high-throughput analysis are extensively optimizing the fermentation process. The NADPH/ applied in biology. Also, these systems can be þ NADP ratios affect various enzymatic activities in the used to study changes in metabolic fingerprinting, carbon metabolism and intracellular environment. metabolic fluxes, protein expression levels, tran- Therefore, NADPH should be supplied in sufficient scription levels, and signal transduction. quantities to sustain cellular and enzyme activities. Correspondingly, they are able to lay the founda- Several metabolic engineering studies have also tion for revealing the physiological mechanism of focused on improving the intracellular NADPH supply in the microbial functional regulation based on intra- amino acid-producing genetically engineered bacteria. cellular NADPH levels [132,133]. This review has provided an overview of the major ii. How to reveal the role of intracellular NADPH lev- metabolic mode of NADPH and the key enzymes related els to regulate the flow and direction of the meta- to NADPH metabolism (Figures 2 and 3; Table S2). It bolic fluxes based on the relations between also summarizes research developments in the under- changes in the carbon metabolic fluxes and the standing of the relationship between NADPH, the flow of cofactors, on clarifying the relationship amino acid biosynthesis, and the manipulation strat- between regulatory and metabolic networks. egies for NADPH regeneration in order to increase iii. How to correctly choose the right control strategy amino acid production in C. glutamicum and E. coli. to maximize the carbon metabolic fluxes and rap- Although diverse NADPH-regenerating enzymes were idly guide them to a target amino acid via the successfully overexpressed and had proven their validity regulation of intracellular NADPH levels. for NADPH regeneration, other NADPH-regenerating enzymes (e.g. NADPþ-dependent dehydrogenase, phosphate dehydrogenase and ferredoxin:NADPþ oxidore- Disclosure statement ductase) are not used in genetically modifying amino The authors report no declarations of interest. acid-producing strains despite clear evidence of their beneficial property for increasing the NADPH supply Funding (for reviews, see Refs. [8,46,84]). Future research efforts are required for engineering amino acid high-yielding This work was financially supported by the National Natural strains through NADPH-regenerating systems. Science Foundation of China [No. 31601459], the Natural Science Foundation of Jiangsu Province [No. BK20150149], Therefore, we will expend our efforts to understand the China Postdoctoral Science Foundation Grant [No. various types of NADPH-regenerating enzymes and 2016M590410], and Fundamental Research Funds for the their effects on the production of target amino acids, Central Universities [No. JUSRP115A19]. 12 J.-Z. XU ET AL.

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