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Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 538790 12 pageshttpdxdoiorg1011552013538790

Review ArticleImproved Succinate Production by Metabolic Engineering

Ke-Ke Cheng1 Gen-Yu Wang1 Jing Zeng2 and Jian-An Zhang1

1 Institute of Nuclear and New Energy Technology Tsinghua University Beijing 100084 China2 Institute of Applied Chemistry Department of Chemical Engineering Tsinghua University Beijing 100084 China

Correspondence should be addressed to Jian-An Zhang zhangjatsinghuaeducn

Received 27 November 2012 Revised 12 March 2013 Accepted 17 March 2013

Academic Editor Eugenio Ferreira

Copyright copy 2013 Ke-Ke Cheng et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Succinate is a promising chemical which has wide applications and can be produced by biological route The history of thebiosuccinate production shows that the joint effort of different metabolic engineering approaches brings successful results In orderto enhance the succinate production multiple metabolical strategies have been sought In this review different overproducersfor succinate production including natural succinate overproducers and metabolic engineered overproducers are examined andthe metabolic engineering strategies and performances are discussed Modification of the mechanism of substrate transportationknocking-out genes responsible for by-products accumulation overexpression of the genes directly involved in the pathway andimprovement of internal NADH and ATP formation are some of the strategies applied Combination of the appropriate genesfrom homologous and heterologous hosts extension of substrate integrated production of succinate and other high-value-addedproducts are expected to bring a desired objective of producing succinate from renewable resources economically and efficiently

1 Introduction

Succinate and its desirable properties have been knownfor a long time Succinate can be used as an importantC4 building-block chemical and its demand is sharplyincreasing since new applications of this chemical compoundare reported in many publications Of particular interestis that it can be used for 14-butanediol synthesis andfurther as a monomer in a polycondensation reaction yield-ing biodegradable poly(butylene succinate) 14-Butanediol-based polymers have better properties and greater stabilityin comparison to polymers produced from 12-propanediolor ethylene glycol Further CO

2is assimilated during the

succinate biosynthesis which can be considered as an envi-ronmental advantage The commercialization of the polymerproduced from biobased succinate by some multinationalcorporations such as BioAmber BASF Myriant Mitsubishiand DuPont showed that the aim of biobased bulk chemicalsis feasible [1ndash4]

Biotechnological processes are particularly attractivesincemicroorganisms usually utilize renewable feedstock andonly produce few toxic by-products However there are limi-tations ofmicrobial production like limited yields concentra-tions and productivities difficulties in the product recovery

from the broth or the need of pretreatment ofmost of the rawsubstrates These limitations can be significantly improvedthrough application of metabolic engineering New strate-gies for succinate production improvement by metabolicengineering are frequently reported Metabolic engineer-ing of the microbial succinate production covers naturalsuccinate overproducers (like Actinobacillus succinogenesAnaerobiospirillum succiniciproducens andMannheimia suc-ciniciproducens) as well as metabolic engineered overproduc-ers like Escherichia coli Corynebacterium glutamicum andSaccharomyces cerevisiae Genes from homologous and het-erologous hosts were often used in combination to completethe pathway [1 2] In this review different overproducers forsuccinate production are examined and the metabolic engi-neering strategies and performances are discussed Finallystrategies for successful commercialization of succinate pro-duction by improvement of metabolic engineered are pro-posed

2 Succinate Formation Pathway

Besides as an intermediate of the tricarboxylic acid (TCA)cycle succinate can also be a fermentation end product when

2 BioMed Research International

Succinate

Fumarate

Malate

Oxaloacetate

Carbon resource

Phosphoenolpyruvate

NADH

NADH

CO2

NAD+

NAD+

(a)

Citrate

Isocitrate

SuccinateGlyoxylate

Malate

Oxaloacetate

NADH

Acetyl-CoA

Acetyl-CoA

Carbon resource

NAD+

(b)

Citrate

Isocitrate

Succinate

Fumarate

Malate

Oxaloacetate

NADH

Acetyl-CoA

Carbon resource

Pyruvate

NADH

FAD

NAD+

NAD+

CO2

2NAD+

2NADH2CO2

FADH2

(c)

Figure 1 Succinate production pathway from (a) the reductive branch of the TCA cycle Succinate accumulates derived fromphosphoenolpyruvate via some intermediate including oxaloacetate malate and fumarate (b) The glyoxylate pathway The glyoxylatepathway operates as a cycle to convert 2mol acetyl CoA to 1mol succinate (c) The oxidative TCA cycle This pathway converts acetyl-CoA to citrate isocitrate and succinate and subsequently converted to fumarate by succinate dehydrogenase Under aerobic conditions theproduction of succinate is not naturally possible and to realize succinate accumulation under aerobic condition inactivation of sdhA gene toblock the conversion of succinate to fumarate in TCA cycle is necessary

sugar or glycerol is used as a carbon source There are threepathways for succinate formation including the reductivebranch of the TCA cycle the glyoxylate pathway and theoxidative TCA cycle [5ndash10]

Under anaerobic conditions succinate is the H-acceptorinstead of oxygen and therefore the reductive branch of theTCA cycle is used Succinate accumulates derived from phos-phoenolpyruvate (PEP) via some intermediate compoundsof TCA reductive branch including oxaloacetate (OAA)malate and fumarate (Figure 1(a)) The pathway convertsoxaloacetate to malate fumarate and then succinate whichrequires 2 moles of NADH per mole of succinate produced[5ndash8] The equation of anaerobic pathway is

PEP + CO2+ 2NADH 997888rarr succinate + 2NAD+ (1)

The maximum possible succinate yield based solely on acarbon balance is 2molmolminus1 glucose when all the succinateis formed via the anaerobic pathway One major obstacle toobtain high succinate yield through the anaerobic pathwayis NADH limitation This is because 1mole glucose canprovide only 2 moles of NADH through the glycolyticpathway Therefore the molar yield of succinate is limited to1molmolminus1 glucose assuming that all the carbon flux will goonly through the anaerobic fermentative pathway

Another potential biosynthetic route for succinate isthrough the glyoxylate pathway which is an anaplerotic reac-tion to fill up the molecule pool of the TCA The glyoxylatecycle is essentially active under aerobic conditions uponadaptation to growth on acetate (Figure 1(b)) The glyoxylatepathway operates as a cycle to convert 2mol acetyl CoA to1mol succinate [9] The equation of glyoxylate pathway is

2Acetyl CoA + 2H2O +NAD+

997888rarr Succinate + 2CoASH + NADH + 2H+(2)

Since the conversion of glucose to succinate via glyoxylatepathway generates NADH this route alone is not sufficientto balance the electrons However during anaerobic cultureand in the absence of an additional electron donor activatedglyoxylate pathway will provide extra NADH to anaerobicfermentative pathway and benefit to achieve higher succinateyield

Succinate can also be formed from acetyl-CoA gener-ated from pyruvate via oxidative TCA cycle under aerobicconditions [10] This pathway converts acetyl-CoA to citrateisocitrate and succinate which is subsequently converted tofumarate by succinate dehydrogenase Under aerobic condi-tions the production of succinate is not naturally possiblesince it is only an intermediate of the TCA cycle To realize

BioMed Research International 3

succinate accumulation under aerobic condition inactivationof sdhA gene to block the conversion of succinate to fumaratein TCA cycle is necessary (Figure 1(c)) The equation ofaerobic pathway is

2Pyrurate + 2H2O + 3NAD+

997888rarr Succinate + 3NADH + 2CO2

(3)

3 Debottlenecking of the Succinate Pathway

Theyield of succinate in anaerobic culture from sugar or otherfeedstock is strongly decided by available NADHproduced inthe glycolysis route which results in by-productsrsquo accumula-tions [11ndash13]Theby-product formation is caused by the redoxbalance from substrate to product By-product accumulationsresult in a substrate loss as well as usual product inhibitionsuch as formate acetate and lactate accumulation whoseundissociated form will be harmful for biomass formationand substrate consumption Also succinate itself harms themicroorganisms in the same way like other weak organicacids Since the dissociation constants of these weak organicacids and the resistance of microorganisms to these acidsinhibition are different genetic engineering tools can alsobe used to manipulate the metabolic pathways so thattarget product pathway can be strengthened or by-productpathways can be selectively eliminated [14]

Deletion of the one of undesired metabolitesrsquo pathwayswill divert carbon resource to other metabolites Howeverin some case the desired product is not improved due tounreasonable metabolic flux redistribution and new unde-sired metabolitersquos accumulation Furthermore eliminationof some by-products will break original intracellular redoxbalance So a reasonable design of metabolic net is neededbefore genetic manipulations so that the redox or ATP is keptat good balance and the production of desired product isenhanced

4 Metabolic Engineering ofthe Succinate Producer

A succinogenes A succiniciproducens M succiniciproducensare well-known natural overproducers and there were someefforts to improve the yield E coli C glutamicum and Scerevisiae are not natural overproducers and therefore com-pletely genetic engineered pathway was sought for acquiredability to form succinate Succinate production using differentbacteria species in terms of performances and engineeringstrategies is compared in Table 1

A succinogenes mutant strain FZ-6 with pyruvate for-mate lyase and formate dehydrogenase deletion did notshow improved succinate production Only when electricallyreduced neutral red or hydrogen was fed as the electrondonor the mutant can use fumarate alone for succinate pro-duction [34] Major metabolic pathways in M succinicipro-ducens resulting in acetate formate and lactate accumulationwere successfully deleted by disrupting the ldhA pflB ptaand ackA genes A modified strain LPK7 was developed toexcrete 134 g Lminus1 succinate using 20 g Lminus1 glucose with little

or no by-product accumulation In fed-batch fermentationby occasional glucose feeding M succiniciproducens LPK7produced 524 g Lminus1 succinate giving a yield of 076 g gminus1glucose and a productivity of 18 g Lminus1 hminus1 [15]

41 Escherichia coli Due to plentiful of genetic tools avail-able fast cell growth and simple culture medium E colihas turned to one of the most wholly studied systems forsuccinate production Strategies in metabolic engineering ofE coli can be classified as four main methods improvementof substrate or product transportation enhancement of path-ways directly involved in the succinate production deletionof pathways involved in by-product accumulation and theircombinations (Figure 2) These methods have been studiedin many reports and some high efficient succinate producershave been constructed [35ndash38]

5 Improvement of Substrate orProduct Transportation

A fundamental change imposed on E coli is the elimina-tion of glucose transport by the phosphotransferase system(PTS)Thismodification addresses the limitation that glucosephosphorylation in E coli is largely PEP dependent IfPEP is generated solely via the Embden-Meyerhof-Parnaspathway this constraint imposes an artificial ceiling yieldof 1molmolminus1 glucose in succinate production Althoughalternate routes for the generation of PEP exist glucosephosphorylation is energetically more efficient if the PEP-dependent system is replaced with ATP-dependent phos-phorylation Further more PEP are reserved and used forthe succinate formation route Some successful constructedE coli strains for succinate production such as AFP111 andKJ060 mainly rely on glucokinase for glucose uptake whichhas been confirmed bymetabolic flux and enzymatic analysis[35 39]

Succinate export in E coli is normally active underanaerobic conditions and import only under aerobic con-ditions The dicarboxylic acid transport system of E coliwas modified by Beauprez et al to enhance productionof succinate The engineering comprised the elimination ofsuccinate uptake and the enhancement of succinate outputThe gene responsible for succinate import dctA was knockedout and the gene coding for succinate export dcuC wasoverexpressed with a constitutive artificial promoter Thecombination of altered import (ΔdctA) and export (ΔFNR-pro37-dcuC) increased the specific production rate by about55 and the yield by approximately 53 [40]

6 Enhancement of Pathways Directly Involvedin the Succinate Production

Overexpression of genes directly involved in the succinateproduction pathway including PEP carboxylase PEP car-boxykinase pyruvate carboxylase and malic enzyme wasreported in a lot of publications In a study by Millard et alsuccinate production using E coli JCL 1208 increased from


Table 1 Comparison of succinate production using different bacteria species in terms of performance and engineering strategies

StrainsSuccinate production

Engineeringstrategies

Culturemethods Concentration

(g Lminus1)Productivity(g Lminus1 hminus1)

Yield(g gminus1)

Biomass By-product References

Mannheimia succiniciproducens

LPK7Deletion of theLDH PFL PTA

ACK

Anaerobicbatch 134 118 067 15 g Lminus1 Pyruvate

malateAnaerobicfed-batch 524 175 076 25 g Lminus1 Pyruvate

malate

[15]

Escherichia coli

JCL1208 (pCP201) OverexpressedPEPC

Anaerobicbatch 107 059 029 mdash

Ethanolacetatelactateformate

[16]

MG1655-pyc Overexpressed PYC Anaerobicbatch 177 0177 0177 09 g Lminus1 Lactate

formate[17]

NZN111Deletion of PFL and

LDHOverexpressed MAE

Anaerobicbatch 128 029 064 mdash Acetate [18]

AFP111

Glucose transport byATP-dependentphosphorylation

Deletion of PFL andLDH

Anaerobicbatch 128 mdash 07 016 g Lminus1 Acetate

ethanol [19]

Dual phaseaerationfed-batch

mdash 121 096 10 (OD600) Acetate [20]

AFP111-pyc AFP111 withoverexpressed PYC

Dual-phaseaerationfed-batch

992 131 11 mdash Pyruvateformate [21]

SBS110MGDeletion of LDH

PFLOverexpressed PYC

Anaerobicbatch 156 033 085 mdash Acetate

formate[22]

SBS550MG (pHL314)

Deletion of ADHLDH ICLR and

ACK-PTAOverexpressed PYC

Anaerobicfed-batch 40 042 106 17

(OD600)Formateacetate

[23]

SBS990MG (pHL314)Deletion of ADHELDHA ACK-PTAOverexpressed PYC

Anaerobicbatch 159 064 107 9

(OD600) Formate [24]

GJT (pHL333 pHL413)

Coexpression ofPEPC and

pantothenate kinase(PANK)

Anaerobicbatch 205 0085 01 mdash Lactate [25]

YBS132 (pHL333pHL413)

Coexpression ofPEPC and PYCDeletion of LDHand ACK-PKA


ethanol[26]

HL51276k

Mutation in thetricarboxylic acid

cycle (SDHAB ICDICLR) and acetatepathways (POXBACKA-PTA)

Aerobicbatch 461 006 043 mdash Pyruvate

acetate[27]

HL51276k- pepc HL51276k withoverexpressed PEPC


acetate [27]


Table 1 Continued





Yield(g gminus1)


KJ060

Deletion of LDHADHE ACKAFOCA PFLB

Glucose transport byATP-dependentphosphorylationPEPC was replaced

by thegluconeogenic PEPC

Anaerobicfed-batch 866 09 092 22 g Lminus1 Malate

acetate[8]

KJ134

Deletion of LDHADHE

FOCA-PFLBMGSA POXBTDCDE CITFASPC SFCAPTA-ACKA



Anaerobicfed-batch 716 075 1 23 g Lminus1

Acetatepyruvatemalate

[28]

Corynebacterium glutamicum

ΔldhA-pCRA717 Deletion of LDHaOverexpressed PYC

Micro-aerobicfed-batchwith

membranefor cellrecycling

146 317 092 60 g Lminus1 Acetate [29]

Saccharomyces cerevisiae

Suc-200

Deletion of ADH1ADH2 and GPD1Overexpressed

PCKa FUMR FUMMDH and MAE1

Aerobicfed-batch 345 024 mdash mdash mdash [30]

Suc-297


PEPCNADH-dependentFUMR FUM MDH

and malic acidtransporter protein

PYC


Kura Deletion of SDH1and FUM1

Aerobicbatch 232 0005 0015 mdash

Ethanolmalatelactate

[31]

8D (pRS426T-ICL1-C)Deletion of SDHand SER3SER33Overexpression of

ICL1

Aerobicbatch 09 005 gg mdash mdash mdash [32]

AH22ura3Δsdh2Δsdh1Δidh1Δidp1

Deletion of SDHand IDH

Aerobicbatch 362 0022 0072 70 g Lminus1

Ethanolglycerolacetate

[33]


Citrate

IsocitrateFumarate

Malate

Acetyl-CoA

Acetyl-CoA

Glucose

Pyruvate

Phosphoenolpyruvate

1

Lactate

2

Ethanol

Acetate5 6

39

8 10

Formate4

7

Oxaloacetate

H2 + CO2

Acetyl-phosphate

Succinate

Glyoxylate

Figure 2 Fermentation of glucose to succinate by genetically engineered central anaerobic metabolic pathway Bold vertical bar meansthat the relative gene was inactivated Bold arrows show overexpression (1) The PEP-dependent glucose uptake system is replaced withATP-dependent phosphorylation (2) the activation of the glyoxylate pathway (3) the knockouts of lactate (lactate dehydrogenase) (4)the knockouts of formate pathway (pyruvate formate-lyase) (5) and (6) the knockouts of acetate pathway (acetate kinase phosphateacetyltransferase) (7) the knockouts of ethanol pathway (alcohol dehydrogenase) (8) overexpressed phosphoenolpyruvate carboxylase (9)overexpressed malic enzyme (10) overexpressed pyruvate carboxylase

3 g Lminus1 to 107 g Lminus1 by overexpressing native PEP carboxylase[16] However overexpression of PEP carboxykinase did notaffect succinate production Due to the intimate role thatPEP is needed as substrate for glucose transport by the phos-photransferase system in wild-type E coli the consequencesof overexpressing PEP carboxylase are decreasing rate ofglucose uptake and organic acid excretion [41] Anothermethod is to improve pyruvate to the succinate synthesisroute through the expression of pyruvate carboxylase anenzyme which can convert pyruvate to oxaloacetate butnot contained in E coli A wild-type E coli strain MG1655transformed with vector pUC18-pyc which contained thegene encoding for Rhizobium etli pyruvate carboxylase led toa succinate formation of 177 g Lminus1 corresponding to a 50increase in succinate concentration using the parent strainThe increased succinate was due to decreased lactate for-mation whose final concentration decreased from 233 g Lminus1to 188 g Lminus1 The expression of pyruvate carboxylase had noeffect on the glucose uptake but decreased the rate of cellgrowth [17]

7 Deletion of Pathways Involved inBy-Product Accumulation

Only shift of carbon flux to succinate route is not enoughto control the formation of other undesired metabolitesAs a consequence genetically modified strains without lac-tate and formate forming routes are developed to improvesuccinate fermentation Mutant of E coli LS1 which only

lacked lactate dehydrogenase had no effect on anaerobicbiomass formation However E coli NZN111 deficient inboth the pyruvate-formate lyase and lactate dehydrogenasegenes respectively gave few biomass formations on glu-cose NZN111 accumulated 018ndash026 g Lminus1 pyruvate beforemetabolism ceased even when supplied with acetate forbiosynthetic needs However when transformed with themdh gene encoding NAD+-dependent malic enzyme andcultured from aerobic environment converting to anaerobicenvironment gradually (by metabolically depleting oxygeninitially present in a sealed culture tube) E coli NZN111 wasallowed to consume all the glucose 128 g Lminus1succinate wasproduced as one of the major metabolites [18] Analogouslywhen the gene encoding malic enzyme from Ascaris suumwas transformed into NZN111 succinate yield was 039 g gminus1and productivity was 029 g Lminus1 hminus1 [18]

8 Optimization of the Succinate Yield bya Combination of Gene Operations

Donnelly et al screened a spontaneous chromosomal muta-tion in NZN111 and this mutation was named AFP111 whichcan grow on glucose in anaerobic environment A succinateyield of 07 g gminus1 was obtained using AFP111 in anaerobic fer-mentations under 5 H

2-95 CO

2flow and the molar ratio

between succinate and acetate is 197 [19] Further if AFP111first grew under aerobic conditions for biomass formationand then was shifted to anaerobic fermentation with CO

2

aeration (dual-phase fermentation) higher succinate yield


Table 2 Comparison of maximum theoretical yield of each pathway in E coli

Means of culture Means of glucose intake Succinate production pathway Maximum theoretical yield(molmolminus1)

PTS The reductive branch of the TCA cycle 1

Anaerobic cultureGlucokinase The reductive branch of the TCA cycle 133

PTS The reductive branch of the TCA cycle and activated glyoxylatepathway 12

Glucokinase The reductive branch of the TCA cycle and activated glyoxylatepathway 171

PTS The oxidative TCA cycle with inactivation of sdhA gene 1

Aerobic cultureGlucokinase The oxidative TCA cycle with inactivation of sdhA gene and

activated glyoxylate pathway 1


Glucokinase The oxidative TCA cycle with inactivation of sdhA gene andactivated glyoxylate pathway 1

These theoretical yields in anaerobic culture were calculated assuming that NADH and NAD are balanced as a result of central carbon metabolismThese theoretical yields in aerobic culture were calculated assuming that oxygen is the H-acceptor

(096 g gminus1) with a productivity of 121 g Lminus1 hminus1 was obtained[20 21] In Chatterjee et alrsquos report the difference betweenNZN111 and AFP111 was found to be the PTS Because ofthe PTS mutation AFP111 mainly relied on glucokinase forglucose uptake No matter anaerobic or aerobic environmentfor cell growth AFP111 exhibited obviously higher glucoki-nase activity than that of NZN111 Compared with the wild-type parent strainW1458 AFP111 also showed a lower glucoseuptake [39] According to Vemuri et al the routes for PEPconversion to succinate also varied between NZN111 andAFP111 The key glyoxylate shunt enzyme isocitrate lyasewas not present with both NZN111 and AFP111 grown underanaerobic environment but was detected after 8 h of aerobicculture and NZN111 exhibited 4-fold higher isocitrate lyaseactivity than AFP111 [20] Because the two strains havedifferent modes of glucose uptake and different isocitratelyase activity level the distribution of end products in thetwo strains is different Further they concluded that themaximum theoretical succinate yield based on the necessaryredox balance is 112 g gminus1 glucose (Table 2) For obtaining themaximal succinate yield the molar ratio of the carbon fluxfrom fumarate to succinate to the carbon flux from isocitrateto succinate must be 50 Insufficient carbon flux throughthe PEP-to-fumarate branch or elevated carbon flux throughthe glyoxylate shunt lowers the observed yield Achieving theoptimal ratio of fluxes in the two pathways involved in anaer-obic succinate accumulation requires a concomitant balancein the activities of the participating enzymes which have beenexpressed during aerobic growth The global regulation ofaerobic and anaerobic pathways is further complicated by thepresence of different isozymes Based on carbon flux analysisthis group transformed AFP111 with the pyc gene (encodeRhizobium etli pyruvate carboxylase) to provide metabolicflexibility at the pyruvate node A two-stage aeration strat-egy (firstly aerobic fermentations with 119870

119871119886 52 hminus1 when

dissolved oxygen concentration decreases to 90 of initialconcentration shift to anaerobic fermentation with oxygen-free CO

2sparged) was applied in the fermentation for higher

succinate yield and productivity Using this strategy a final992 g Lminus1 succinate concentration with a yield of 11 g gminus1 anda productivity of 13 g Lminus1 hminus1 was obtained [21]

Serial publications on modification of E coli MG1655strain for improved succinate production were reportedby Sanrsquos group Firstly SBS110MG (pHL413) was createdfrom an adhE ldhA mutant strain of E coli SBS110MGharboring plasmid pHL413 which encoded the Lactococcuslactis pyruvate carboxylase After 48 h fermentation usingthis strain 156 g succinate Lminus1 was produced with a yieldof 085 g gminus1 [22] E coli SBS550MG (pHL413) was furtherdeveloped by deleting adhE ldhA and ack-pta routes and byactivating the glyoxylate routes by deactivation of iclR Bya repeated glucose feeding SBS550MG (pHL413) produced40 g Lminus1 succinate with a yield of 105 g gminus1 glucose Theyfound that the best distribution ratio for the highest succinateyield was the fractional partition of OAA to glyoxylate of032 and 068 to the malate [24] In addition this groupstudied the effect of overexpressing a NADH insensitivecitrate synthase from B subtilis on succinate fermentationThey found that this change had no effect on succinate yieldbut affected formate and acetate distribution FurthermoreSanchez et al tried to place an arcAmutation within the hostSBS550MG leading to a strain designated as SBS660MGThe phosphorylated arcA is a dual transcriptional regulatorof aerobic respiration control which also suppresses tran-scription of the aceBAK Deactivation of arcA could furtherimprove the transcription of aceBAK and hence led to amore efficient glyoxylate pathway Unfortunately SBS660MGdid not improve the succinate yield and it reduced glucoseconsumption by 80 It should be pointed out that succinateproduction experiments in the previous description werecarried out in a two-stage aeration culture in bioreactorswhere the first stage was aerobic for cell growth followed byan anaerobic stage for succinate accumulation and the initialinoculum was very large (initial dry cell weight 56 g Lminus1)Aeration condition during the cell growth stage has greatimpact on anaerobic succinate accumulation using E coli


SBS550MG (pHL413) [23] Martınez et al found that amicroaerobic environment was more suitable for succinateproduction Compared with microaerobic environment thehigh aeration experiment led tomore pyruvate accumulationwhich correlated with a lower pflAB expression during thetransition time and a lower flux towards acetyl-CoA duringthe anaerobic stage The improvement in glyoxylate shunt-related genes expression (aceA aceB acnA acnB) during thetransition time anaerobic stage or both increased succinateyield in microaerobic environment [42]

Because intracellular acetyl-CoA and CoA concentra-tions can be increased by overexpression of E coli pantothen-ate kinase (PANK) and acetyl-CoA is a promising activatorfor PEP carboxylase (PEPC) and pyruvate carboxylase (PYC)Lin et al constructed E coliGJT (pHL333 pRV380) and GJT(pTrc99A pDHK29) by coexpressing of PANK and PEPCand PANK and PYC respectively They found that coexpres-sion of PANK and PEPC or PANK and PYC did enhancesuccinate accumulation but lactate production decreasedsignificantly [25] In a subsequent report GJT (pHL333pHL413) coexpression of PEPC and PYC contributed to205 g Lminus1 succinate accumulation When both the acetate(ackA-pta) and lactate pathways (ldhA) were deactivatedin YBS132 (pHL333 pHL413) succinate concentration andyield increased by 67 and 76 respectively comparedwith the control strain GJT (pHL333 pHL413) (34 g Lminus1versus 21 g Lminus1 02 g gminus1 versus 011 g gminus1) No lactate wasdetected and acetate accumulation reduced by 76 [27]For higher cell growth faster substrate consumption andproduct accumulation mutation in the tricarboxylic acidcycle (sdhAB icd iclR) and acetate pathways (poxB ackA-pta) of E coli HL51276k was developed to construct the gly-oxylate cycle for producing succinate aerobically After 80 hfermentation succinate production reached 461 g Lminus1 witha yield of 043 g gminus1 glucose The substantial accumulationsof pyruvate and TCA cycle C

6intermediates were observed

during aerobic fermentation which hindered achieving themaximum succinate theoretical yield PEPC from Sorghumwas overexpressed in the strain HL51276k [26] At approxi-mately 58 h HL51276k (pKK313) produced 8 g Lminus1 succinatewith a succinate yield of 072 g gminus1 glucose Overexpression ofPEPC was also effective in decreasing pyruvate accumulation(30mM in HL51276k (pKK313) versus 48mM in HL51276k)Further they found that an overexpression of PEPC anddeactivation of ptsG combined strategy was the most efficientin decreasing pyruvate accumulation

By a combination of gene deletions on E coli ATCC8739 and multiple generations of growth-based selection ahigh succinate producing strain KJ060 (ldhA adhE ackAfocA pflB) was obtained producing 866 g Lminus1 of succi-nate with a yields of 092 g gminus1 glucose and a productivityof 09 g Lminus1 hminus1 [28] After selection PEP carboxylase wasreplaced by the gluconeogenic PEP carboxykinase throughspontaneous mutation to the major carboxylation pathwayfor succinate formation The PEP-dependent phosphotrans-ferase system was deactivated by a spontaneous point muta-tion and functionally replaced by the GalP permease andglucokinase By these improvements the net ATP molar

yield during succinate formation was increased to 20 ATPper glucose This improved E coli pathway is similar tothe pathway of native succinate producing rumen bacteriaFor higher yield and lower by-product formation newgeneration KJ134 (ΔldhA ΔadhE ΔfocA-pflB ΔmgsA ΔpoxBΔtdcDE ΔcitF ΔaspC ΔsfcA Δpta-ackA) was constructedproducing 716 g Lminus1 succinate with a yield of 1 g gminus1 glucoseand a productivity of 075 g Lminus1 hminus1 in batch fermentationsusing mineral salts medium under anaerobic environmentCompared with KJ060 by-product acetate of KJ134 decreases85 (44 g Lminus1 versus 295 g Lminus1) which is very useful inproduct recovery for the commercial production of succinate

81 Corynebacterium glutamicum C glutamicum is a fast-growing nonmotile gram-positive microorganism with along history in the microbial fermentation industry foramino acids and nucleic acids C glutamicum under oxygendeprivation without growth produces organic acids such aslactic acid succinate and acetic acid from glucose Thesefeatures allow for the use of high-density cells leading toa high volumetric productivity Some studies have beencarried out on C glutamicum with respect to the microbialproduction of succinate [29 43]

In order to eliminate lactic acid production deletion ofldhA gene coding for L-lactate dehydrogenase was targeted inC glutamicum R A lactate-dehydrogenase-(LDH-) deficientmutant was not able to produce lactate suggesting that thisenzyme has no other isozyme Moreover overexpression ofgenes coding for anaplerotic enzymes in the mutant demon-strated that the rate of succinate production of the resul-tant strain C glutamicum ΔldhA-pCRA717 with enhancedpyruvate carboxylase activity was 15-fold higher than thatof parental mutant strain Using this strain at a dry cellweight of 50 g Lminus1 under oxygen deprivation succinate wasproduced efficiently with intermittent addition of sodiumbicarbonate and glucose The succinate production rate andyield depended onmedium bicarbonate concentration ratherthan glucose concentration Succinate concentration reached146 g Lminus1 with a yield of 092 g gminus1 and a productivity of32 g Lminus1 hminus1 [29]

82 Saccharomyces cerevisiae S cerevisiae is genomesequenced genetically andphysiologicallywell characterizedand can produce organic acids even at the low pH thatfacilitates downstream Many tools for genetic improvementare established These features make S cerevisiae suitablefor the biotechnological production of succinate Due tothis fact attempts to engineer S cerevisiae for the succinateproduction were made

Using 13C flux analysis Camarasa et al found that duringanaerobic glucose fermentation by S cerevisiae the reductivebranch generating succinate via fumarate reductase operatesindependently of the nitrogen source This pathway is themain source of succinate during fermentation unless gluta-mate is the sole nitrogen source in which case the oxidativedecarboxylation of 2-oxoglutarate generates additional succi-nate [44]


S cerevisiae is a well-known glycerol and ethanol pro-ducer Therefore with respect to this microorganism syn-thesis of succinate must limit the formation of glycerol andethanol In a patent issued by Verwaal et al S cerevisiaeRWB064 with a deletion of the genes alcohol dehydro-genase 1 and 2 and the gene glycerol-3-phosphate dehy-drogenase 1 was used for the parental strain The genesPEP carboxykinase from A succinogenes NADH-dependentfumarate reductase fromTrypanosoma brucei fumarase fromRhizopus oryzae malate dehydrogenase from S cerevisiaeandmalic acid transporter protein from Schizosaccharomycespombe were overexpressed The recombinant SUC-200 pro-duces 345 g Lminus1succinate and the main by-products include45 g Lminus1 ethanol 77 g Lminus1 glycerol and 78 g Lminus1 malateFurther improvement of the recombinant SUC-297 includedoverexpression of pyruvate carboxylase from S cerevisiaeSuccinate formation increases to 43 g Lminus1 and no malateaccumulates Since succinate glycerol and ethanol havedifferent volatility they can be easily purified [30]

Arikawa et al reported an improved succinate productionusing sake yeast strains with some TCA cycle genes deletions[45] Compared with the wild-type strain succinate produc-tionwas increased up to 27-fold in a strainwith simultaneousdisruption of a subunit of succinate dehydrogenase (SDH1)and fumarase (FUM1) under aerobic conditions The singledeletion of gene SDH1 led to a 16-fold increase of succinateThese enhancements were not observed under strictly anaer-obic or sake brewing conditions Absence or limitation ofoxygen resulted in decreased succinate production in sdh1andor fum1 deletion strains [31] In another study on sakeyeast strains the deletion of genes encoding for succinatedehydrogenase subunits (SDH1 SDH2 SDH3 and SDH4)also resulted in increased succinate production only underaerobic conditions [46]

In order to redirect the carbon flux into the glyoxylatecycle and to improve succinate accumulation the disrup-tion of isocitrate dehydrogenase activity is also part of ametabolic strategy for succinate production in the oxidativebranch Succinate production reduced to approximate half incomparison with the parental strain was observed for yeaststrains with disruptions of isocitrate dehydrogenase subunits(IDH1 or IDH2) under sake brewing conditions [47] Theconstructed yeast strains with disruptions in the TCA cycleafter the intermediates isocitrate and succinate by four genedeletions (Δsdh1 Δsdh2 Δidh1 Δidp1) produce 362 g Lminus1succinate at a yield of 0072 g gminus1 glucose and do not exhibitserious growth constraints on glucoseThemain by-productsare 14 g Lminus1 ethanol 38 g Lminus1 glycerol and 08 g Lminus1 acetate[33] With the aim to reduce fermentation by-products andto promote respiratory metabolism by shifting the fermen-tativeoxidative balance the constitutive overexpressions ofthe SAK1 and HAP4 genes in S cerevisiae were carried outalso by Raab et al Sak1p is one of three kinases responsiblefor the phosphorylation and thereby the activation of theSnf1p complex which plays a major role in the glucosederepression cascade Hap4p is the activator subunit of theHap2345 transcriptional complex Hap4p overexpressionresulted in increased growth rates and biomass formation

while levels of ethanol and glycerol were decreased Thesdh2 deletion strain with SAK1 and HAP4 overexpressionproduced 85 g Lminus1 succinate with a yield of 026molmolminus1glucose No glycerol formation was found and the ethanolproduced after 24 h fermentation was consumed for acetateformation [48]

A multigene deletion S cerevisiae 8D was constructed byOtero et al and directed evolution was used to select a suc-cinate producingmutantThemetabolic engineering strategyincluded deletion of the primary succinate consuming reac-tion (succinate to fumarate) and interruption of glycolysisderived serine by deletion of 3-phosphoglycerate dehydroge-naseThe remodeling of central carbon flux towards succinateminimized the conversion of succinate to fumarate andforced the biomass-required amino acids L-glycine and L-serine to be produced from glyoxylate pools The mutantstrain 8D with isocitrate lyase overexpression represented a30-fold improvement in succinate concentration and a 43-fold improvement in succinate yield on biomass with only a28-fold decrease in the specific growth rate compared to thereference strain [32]

9 Final Remarks

At present some succinate productions usingA succinogenesand A succiniciproducens are studied under environmentsabsolutely free of oxygen for cultivation Anaerobic fermen-tation is preferred because of its lower capital and operationalcosts when compared to aerobic fermentations Howevertheir applicability in the industrial process is limited tosome extent because of their maximum possible succinateyield (1mol molminus1 glucose) Also few genetic tools mayseriously limit these strainsrsquo reconstruction of metabolicengineering E coli is the ideal host of choice in futuredue to in-depth knowledge on this species gained over thepast decades high possible succinate yield (172molmolminus1glucose) and general acceptance of their usage in industrialprocesses Other metabolic engineered overproducers like Cglutamicum ΔldhA-pCRA717 which can produce 146 g lminus1succinate in a cell recycling fed-batch culture deserve moreattention in the future

In order to improve the succinate production bymetabolic engineering approaches various strategies havebeen investigated and applied in different hosts with acquiredability to produce succinate Multiple studies have beendedicated to overcome current limitations of the processlike introduction of an ATP-dependent glucose transportsystem knockout of LDH ADHE or ACKA encoding genesto eliminate by-products formation to favorably change theATP formation to restrict accumulation of pyruvate andso forth To seek economical and robust biotechnologicalproduction of succinate considerable progress has beenmade However much work needs to be done in order toachieve the desired process efficiency Better understandingof metabolic background of both native succinate producersand heterologous hosts is essential for further efforts ofdirected metabolic engineering Improvements with internalredox balance efficient overexpression of required enzyme


Table 3 Coproduction of succinate and other high-value-added products using different bacteria species

Strains Strategies Products Substrates Reference

E coli KJ071 Deletion of LDH ADHEFOCA-PFLB MGSA PTA-ACKA 331 g Lminus1 succinate 692 g Lminus1 malate Glucose [8]

E coli KJ073Deletion of LDH ADHEFOCA-PFLB MGSA POXBPTA-ACKA

789 g Lminus1 succinate 158 g Lminus1 malate Glucose [8]

E coli SBS990MG Deletion of LDH ADHE PTA-ACKAOverexpression of PYC and AAT

122 g Lminus1 isoamyl acetate 537 g Lminus1succinate

Glucose andisoamyl alcohol [9]

E coli QZ1112Deletion of SDHA POXBPTA-ACKAOverexpression of PHBCAB

246 g Lminus1 succinate 495 g Lminus1polyhydroxybutyrate Glucose [10]

E coli KNSP1Deletion of FADR PTAS ATOCFADA SDHA PTA-ACKAOverexpression of PHAC1

2107 g Lminus1 succinate 054 g Lminus13-hydroxyoctanoate +3-hydroxydecanoate

Glycerol and fattyacid [49]

K pneumoniae LDH526 Deletion of LDH 1021 g Lminus1 13-propanediol138 g Lminus1 succinate Glycerol [50]

K pneumoniae CICC 10011 Enhanced CO2 level in the medium 771 g Lminus1 23-butanediol287 g Lminus1 succinate Glucose [51]

A succinogenes ATCC 55618 Fractional treatment and separateutilization for succinate production 64 g Lminus1 succinate glucoamylase Wheat [52]

activities and modification of by-products formation areexpected to approach higher product concentration andyield

After the great success of succinate production fromglucose it is the time to develop an efficient process fromraw glycerol and biomass (like lignocellulosic substrates oragroindustrial waste products) Being more reduced thanglucose each glycerol could be converted to succinate andcould maintain redox balance Thus the use of glycerol ismore favorable for succinate yield Hydrolysate from biomassis a mixture of sugars containing mainly glucose and xyloseWhen glucose and xylose are present at the same timexylose consumption generally does not start until glucose isdepleted Microbial preference for glucose is caused by theregulatory mechanism named carbon catabolite repressionMetabolic engineering approach to eliminate glucose repres-sion of xylose utilization is very important so that glucoseand xylose could be consumed simultaneously to producesuccinate [53ndash60] Furthermore to increase the competi-tiveness of biological succinate strategies can be efficientlyutilized for the coproduction of succinate and other high-value-added products such as propanediol and succinateisoamyl acetate and succinate and polyhydroxybutyrate andsuccinate (Table 3) This type of fermentation producingtwo commercial interests at the same fermentation processmight be considered for a promising biological productionprocess which will decrease the production cost by sharingthe recovery cost and operation cost

10 Conclusion

Metabolic engineering focuses on the improvement ofmicro-bial metabolic capabilities via improving existing pathwaysandor introducing new pathways The metabolic engineer-ing approach has been widely applied to improve biological

succinate production As a consequence there has beensignificant progress in optimization of succinate producingmachineries elimination of biochemical reactions competingwith succinate production and the incorporation of non-native metabolic pathways leading to succinate productionHowever the performance of most succinate producers interms of concentration productivity yield and industrialrobustness is still not satisfactoryMore studies like extensionof substrate combination of the appropriate genes fromhomologous and heterologous hosts and integrated produc-tion of succinate with other high-value-added products arein progress

Acknowledgments

This study was supported by the National Natural ScienceFoundation of China (21176139) and the National BasicResearch Program of China (2011CB707406)

References

[1] J J Beauprez M de Mey and W K Soetaert ldquoMicrobialsuccinic acid production natural versus metabolic engineeredproducersrdquo Process Biochemistry vol 45 no 7 pp 1103ndash11142010

[2] K K Cheng X B Zhao J Zeng and J A Zhang ldquoBiotechno-logical production of succinate-current state and perspectivesrdquoBiofuels Bioproducts and Biorefining vol 6 pp 302ndash318 2012

[3] A Cukalovic and C V Stevens ldquoFeasibility of productionmethods for succinic acid derivatives a marriage of renewableresources and chemical technologyrdquo Biofuels Bioproducts andBiorefining vol 2 no 6 pp 505ndash529 2008

[4] J Xu and B H Guo ldquoPoly(butylene succinate) and its copoly-mers Research development and industrializationrdquoBiotechnol-ogy Journal vol 5 no 11 pp 1149ndash1163 2010


[5] J B McKinlay J G Zeikus and C Vieille ldquoInsights intoActinobacillus succinogenes fermentativemetabolism in a chem-ically defined growth mediumrdquo Applied and EnvironmentalMicrobiology vol 71 no 11 pp 6651ndash6656 2005

[6] J B McKinlay Y Shachar-Hill J G Zeikus and C VieilleldquoDetermining Actinobacillus succinogenes metabolic pathwaysand fluxes by NMR and GC-MS analyses of 13C-labeledmetabolic product isotopomersrdquo Metabolic Engineering vol 9no 2 pp 177ndash192 2007

[7] M J van der Werf M V Guettler M K Jain and J GZeikus ldquoEnvironmental and physiological factors affecting thesuccinate product ratio during carbohydrate fermentation byActinobacillus sp 130Zrdquo Archives of Microbiology vol 167 no6 pp 332ndash342 1997

[8] K Jantama M J Haupt S A Svoronos et al ldquoCombiningmetabolic engineering and metabolic evolution to developnonrecombinant strains of Escherichia coli C that producesuccinate andmalaterdquoBiotechnology andBioengineering vol 99no 5 pp 1140ndash1153 2008

[9] C R Dittrich G N Bennett and K Y San ldquoMetabolicengineering of the anaerobic central metabolic pathway inEscherichia coli for the simultaneous anaerobic production ofisoamyl acetate and succinic acidrdquo Biotechnology Progress vol25 no 5 pp 1304ndash1309 2009

[10] Z Kang C J Gao Q Wang H M Liu and Q S Qi ldquoA novelstrategy for succinate and polyhydroxybutyrate co-productionin Escherichia colirdquo Bioresource Technology vol 101 no 19 pp7675ndash7678 2010

[11] A Singh K C Soh V Hatzimanikatis and R T Gill ldquoManip-ulating redox and ATP balancing for improved production ofsuccinate in E colirdquoMetabolic Engineering vol 13 no 1 pp 76ndash81 2011

[12] R M Liu L Y Liang K Q Chen et al ldquoFermentationof xylose to succinate by enhancement of ATP supply inmetabolically engineeredEscherichia colirdquoAppliedMicrobiologyand Biotechnology vol 94 pp 959ndash968 2012

[13] S Y Lee S H Hong and S Y Moon ldquoIn silico metabolic path-way analysis and design succinic acid production by metabol-ically engineered Escherichia coli as an examplerdquo GenomeInformatics vol 13 pp 214ndash223 2002

[14] Q Li D Wang Y Wu et al ldquoKinetic evaluation of productsinhibition to succinic acid producers Escherichia coli NZN111AFP111 BL21 andActinobacillus succinogenes 130ZTrdquo Journal ofMicrobiology vol 48 no 3 pp 290ndash296 2010

[15] S J Lee H Song and S Y Lee ldquoGenome-based metabolicengineering of Mannheimia succiniproducens for succinic acidproductionrdquo Applied and Environmental Microbiology vol 72no 3 pp 1939ndash1948 2006

[16] C S Millard Y P Chao J C Liao and M I DonnellyldquoEnhanced production of succinic acid by overexpression ofphosphoenolpyruvate carboxylase in Escherichia colirdquo Appliedand Environmental Microbiology vol 62 no 5 pp 1808ndash18101996

[17] R R Gokarn M A Eiteman and E Altman ldquoExpressionof pyruvate carboxylase enhances succinate production inEscherichia coliwithout affecting glucose uptakerdquo BiotechnologyLetters vol 20 no 8 pp 795ndash798 1998

[18] L Stols G Kulkarni B G Harris and M I Donnelly ldquoExpres-sion of Ascaris suum malic enzyme in a mutant Escherichiacoli allows production of succinic acid from glucoserdquo AppliedBiochemistry and Biotechnology A vol 63ndash65 no 1ndash3 pp 153ndash158 1997

[19] M I Donnelly C S Millard D P Clark M J Chen and JW Rathke ldquoA novel fermentation pathway in an Escherichiacoli mutant producing succinic acid acetic acid and ethanolrdquoApplied Biochemistry and Biotechnology A vol 70ndash72 pp 187ndash198 1998

[20] G N Vemuri M A Eiteman and E Altman ldquoEffects of growthmode and pyruvate carboxylase on succinic acid productionby metabolically engineered strains of Escherichia colirdquo Appliedand Environmental Microbiology vol 68 no 4 pp 1715ndash17272002

[21] G N Vemuri M A Eiteman and E Altman ldquoSuccinate pro-duction in dual-phase Escherichia coli fermentations dependson the time of transition from aerobic to anaerobic conditionsrdquoJournal of IndustrialMicrobiology and Biotechnology vol 28 no6 pp 325ndash332 2002

[22] A M Sanchez G N Bennett and K Y San ldquoEfficient succinicacid production from glucose through overexpression of pyru-vate carboxylase in an Escherichia coli alcohol dehydrogenaseand lactate dehydrogenase mutantrdquo Biotechnology Progress vol21 no 2 pp 358ndash365 2005

[23] A M Sanchez G N Bennett and K Y San ldquoNovel pathwayengineering design of the anaerobic central metabolic pathwayin Escherichia coli to increase succinate yield and productivityrdquoMetabolic Engineering vol 7 no 3 pp 229ndash239 2005

[24] AM SanchezGN Bennett andKY San ldquoBatch culture char-acterization and metabolic flux analysis of succinate-producingEscherichia coli strainsrdquoMetabolic Engineering vol 8 no 3 pp209ndash226 2006

[25] H Lin R V Vadali G N Bennett and K Y San ldquoIncreas-ing the acetyl-CoA pool in the presence of overexpressedphosphoenolpyruvate carboxylase or pyruvate carboxylaseenhances succinate production in Escherichia colirdquo Biotechnol-ogy Progress vol 20 no 5 pp 1599ndash1604 2004

[26] H Lin K Y San and G N Bennett ldquoEffect of Sorghum vul-gare phosphoenolpyruvate carboxylase and Lactococcus lactispyruvate carboxylase coexpression on succinate production inmutant strains of Escherichia colirdquo Applied Microbiology andBiotechnology vol 67 no 4 pp 515ndash523 2005

[27] H Lin G N Bennett and K Y San ldquoMetabolic engineeringof aerobic succinate production systems in Escherichia colito improve process productivity and achieve the maximumtheoretical succinate yieldrdquoMetabolic Engineering vol 7 no 2pp 116ndash127 2005

[28] K Jantama X Zhang J C Moore K T Shanmugam S ASvoronos and L O Ingram ldquoEliminating side products andincreasing succinate yields in engineered strains of EscherichiacoliCrdquo Biotechnology and Bioengineering vol 101 no 5 pp 881ndash893 2008

[29] S Okino R Noburyu M Suda T Jojima M Inui and HYukawa ldquoAn efficient succinic acid production process in ametabolically engineered Corynebacterium glutamicum strainrdquoApplied Microbiology and Biotechnology vol 81 no 3 pp 459ndash464 2008

[30] R Verwaal L Wu R A Damveld and C M J Sagt ldquoSuccinateproduction in a eukaryotic cellrdquo WO patent 2009065778A1

[31] Y Arikawa M Kobayashi R Kodaira et al ldquoIsolation of sakeyeast strains possessing various levels of succinate- andormalate-producing abilities by gene disruption or mutationrdquoJournal of Bioscience and Bioengineering vol 87 no 3 pp 333ndash339 1999

[32] JMOteroD Cimini K R Patil S G Poulsen LOlsson and JNielsen ldquoIndustrial systems biology of Saccharomyces cerevisiae


enables novel succinic acid cell factoryrdquo Plos One vol 1 ArticleID e54144 2013

[33] A M Raab G Gebhardt N Bolotina D Weuster-Botz andC Lang ldquoMetabolic engineering of Saccharomyces cerevisiaefor the biotechnological production of succinic acidrdquoMetabolicEngineering vol 12 no 6 pp 518ndash525 2010

[34] D H Park M Laivenieks M V Guettler M K Jain and JG Zeikus ldquoMicrobial utilization of electrically reduced neutralred as the sole electron donor for growth and metaboliteproductionrdquo Applied and Environmental Microbiology vol 65no 7 pp 2912ndash2917 1999

[35] X Zhang K Jantama J C Moore L R Jarboe K T Shan-mugam and L O Ingram ldquoMetabolic evolution of energy-conserving pathways for succinate production in Escherichiacolirdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 106 no 48 pp 20180ndash20185 2009

[36] D Wang Q Li Z Y Song W Zhou Z G Su and J M XingldquoHigh cell density fermentation via a metabolically engineeredEscherichia coli for the enhanced production of succinic acidrdquoJournal of Chemical Technology and Biotechnology vol 86 no4 pp 512ndash518 2011

[37] Y J Cao Y G Cao and X Z Lin ldquoMetabolically engineeredEscherichia coli for biotechnological production of four-carbon14-dicarboxylic acidsrdquo Journal of Industrial Microbiology andBiotechnology vol 38 pp 649ndash656 2011

[38] J Du Z Y Shao and H M Zhao ldquoEngineering microbialfactories for synthesis of value-added productsrdquo Journal ofIndustrialMicrobiology and Biotechnologyl vol 38 pp 873ndash8902011

[39] R Chatterjee C S Millard K Champion D P Clark andM IDonnelly ldquoMutation of the ptsG gene results in increased pro-duction of succinate in fermentation of glucose by Escherichiacolirdquo Applied and Environmental Microbiology vol 67 no 1 pp148ndash154 2001

[40] J J Beauprez M R Foulquie-Moreno J Maertens et alldquoInfluence of C

4-dicarboxylic acid transporters on succinate

productionrdquo Green Chemistry vol 13 pp 2179ndash2186 2011[41] Y P Chao and J C Liao ldquoAlteration of growth yield by

overexpression of phosphoenolpyruvate carboxylase and phos-phoenolpyruvate carboxykinase in Escherichia colirdquo Appliedand Environmental Microbiology vol 59 no 12 pp 4261ndash42651993

[42] I Martınez G N Bennett and K Y San ldquoMetabolic impact ofthe level of aeration during cell growth on anaerobic succinateproduction by an engineered Escherichia coli strainrdquo MetabolicEngineering vol 12 no 6 pp 499ndash509 2010

[43] B Litsanov M Brocker and M Bott ldquoToward homosuccinatefermentation metabolic engineering of Corynebacterium glu-tamicum for anaerobic production of succinate from glucoseand formaterdquo Applied and Environmental Microbiology vol 78pp 3325ndash3337 2012

[44] C Camarasa J P Grivet and S Dequin ldquoInvestigation by 13C-NMR and tricarboxylic acid (TCA) deletion mutant analysisof pathways of succinate formation in Saccharomyces cerevisiaeduring anaerobic fermentationrdquoMicrobiology vol 149 no 9 pp2669ndash2678 2003

[45] Y Arikawa T Kuroyanagi M Shimosaka et al ldquoEffect ofgene disruptions of the TCA cycle on production of succinicacid in Saccharomyces cerevisiaerdquo Journal of Bioscience andBioengineering vol 87 no 1 pp 28ndash36 1999

[46] Y Kubo H Takagi and S Nakamori ldquoEffect of gene disruptionof succinate dehydrogenase on succinate production in a sake

yeast strainrdquo Journal of Bioscience and Bioengineering vol 90no 6 pp 619ndash624 2000

[47] T Asano N Kurose N Hiraoka and S Kawakita ldquoEffect ofNAD+-dependent isocitrate dehydrogenase gene (IDH1 IDH2)disruption of sake yeast on organic acid composition in sakemashrdquo Journal of Bioscience and Bioengineering vol 88 no 3pp 258ndash263 1999

[48] A M Raab V Hlavacek N Bolotina and C Lang ldquoShiftingthe fermentativeoxidative balance in Saccharomyces cerevisiaeby transcriptional deregulation of Snf1 via overexpression of theupstream activating kinase Sak1prdquo Applied and EnvironmentalMicrobiology vol 77 no 6 pp 1981ndash1989 2011

[49] Z Kang L Du J Kang et al ldquoProduction of succinate andpolyhydroxyalkanoate from substrate mixture by metabolicallyengineeredEscherichia colirdquoBioresource Technology vol 102 no11 pp 6600ndash6604 2011

[50] Y Z Xu N N Guo Z M Zheng X J Ou H J Liu and DH Liu ldquoMetabolism in 13-propanediol fed-batch fermentationby a D-lactate deficient mutant of Klebsiella pneumoniaerdquoBiotechnology and Bioengineering vol 104 no 5 pp 965ndash9722009

[51] K K Cheng J Wu G Y Wang W Y Li J Feng and J AZhang ldquoEffects of pH and dissolved CO

2level on simultaneous

production of 23-butanediol and succinate using Klebsiellapneumoniaerdquo Bioresource Technology vol 135 pp 500ndash5032013

[52] C Y Du S K C Lin A Koutinas R Wang P Doradoand C Webb ldquoA wheat biorefining strategy based on solid-state fermentation for fermentative production of succinic acidrdquoBioresource Technology vol 99 no 17 pp 8310ndash8315 2008

[53] G P da Silva M Mack and J Contiero ldquoGlycerol a promis-ing and abundant carbon source for industrial microbiologyrdquoBiotechnology Advances vol 27 no 1 pp 30ndash39 2009

[54] M G Adsul M S Singhvi S A Gaikaiwari and D V GokhaleldquoDevelopment of biocatalysts for production of commoditychemicals from lignocellulosic biomassrdquo Bioresource Technol-ogy vol 102 no 6 pp 4304ndash4312 2011

[55] P Zheng L Fang Y Xu J J Dong Y Ni and Z H SunldquoSuccinic acid production from corn stover by simultaneoussaccharification and fermentation using Actinobacillus succino-genesrdquo Bioresource Technology vol 101 no 20 pp 7889ndash78942010

[56] X J Li Z Zheng Z J Wei S T Jiang L J Pan and SB Weng ldquoScreening breeding and metabolic modulating ofa strain producing succinic acid with corn straw hydrolyterdquoWorld Journal of Microbiology and Biotechnology vol 25 no 4pp 667ndash677 2009

[57] R B Elcio and P J Nei ldquoSuccinate production from sugarcanebagasse hemicellulose hydrolysate by Actinobacillus succino-genesrdquo Journal of IndustrialMicrobiology and Biotechnology vol38 pp 1001ndash1011 2011

[58] D Wang Q A Li M H Yang Y J Zhang Z G Su andJ M Xing ldquoEfficient production of succinic acid from cornstalk hydrolysates by a recombinant Escherichia coli with ptsGmutationrdquo Process Biochemistry vol 46 no 1 pp 365ndash371 2011

[59] J Wang J Zhu G N Bennett and K Y San ldquoSuccinateproduction from different carbon sources under anaerobicconditions by metabolic engineered Escherichia coli strainsrdquoMetabolic Engineering vol 13 no 3 pp 328ndash335 2011

[60] Z B Zheng T Chen M N Zhao Z W Wang and X MZhao ldquoEngineering Escherichia coli for succinate productionfrom hemicellulose via consolidated bioprocessingrdquo MicrobialCell Factories vol 11 article 37 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Succinate

Fumarate

Malate

Oxaloacetate

Carbon resource

Phosphoenolpyruvate

NADH

NADH

CO2

NAD+

NAD+

(a)

Citrate

Isocitrate

SuccinateGlyoxylate

Malate

Oxaloacetate

NADH

Acetyl-CoA

Acetyl-CoA

Carbon resource

NAD+

(b)

Citrate

Isocitrate

Succinate

Fumarate

Malate

Oxaloacetate

NADH

Acetyl-CoA

Carbon resource

Pyruvate

NADH

FAD

NAD+

NAD+

CO2

2NAD+

2NADH2CO2

FADH2

(c)

Figure 1 Succinate production pathway from (a) the reductive branch of the TCA cycle Succinate accumulates derived fromphosphoenolpyruvate via some intermediate including oxaloacetate malate and fumarate (b) The glyoxylate pathway The glyoxylatepathway operates as a cycle to convert 2mol acetyl CoA to 1mol succinate (c) The oxidative TCA cycle This pathway converts acetyl-CoA to citrate isocitrate and succinate and subsequently converted to fumarate by succinate dehydrogenase Under aerobic conditions theproduction of succinate is not naturally possible and to realize succinate accumulation under aerobic condition inactivation of sdhA gene toblock the conversion of succinate to fumarate in TCA cycle is necessary

sugar or glycerol is used as a carbon source There are threepathways for succinate formation including the reductivebranch of the TCA cycle the glyoxylate pathway and theoxidative TCA cycle [5ndash10]

Under anaerobic conditions succinate is the H-acceptorinstead of oxygen and therefore the reductive branch of theTCA cycle is used Succinate accumulates derived from phos-phoenolpyruvate (PEP) via some intermediate compoundsof TCA reductive branch including oxaloacetate (OAA)malate and fumarate (Figure 1(a)) The pathway convertsoxaloacetate to malate fumarate and then succinate whichrequires 2 moles of NADH per mole of succinate produced[5ndash8] The equation of anaerobic pathway is

PEP + CO2+ 2NADH 997888rarr succinate + 2NAD+ (1)

The maximum possible succinate yield based solely on acarbon balance is 2molmolminus1 glucose when all the succinateis formed via the anaerobic pathway One major obstacle toobtain high succinate yield through the anaerobic pathwayis NADH limitation This is because 1mole glucose canprovide only 2 moles of NADH through the glycolyticpathway Therefore the molar yield of succinate is limited to1molmolminus1 glucose assuming that all the carbon flux will goonly through the anaerobic fermentative pathway

Another potential biosynthetic route for succinate isthrough the glyoxylate pathway which is an anaplerotic reac-tion to fill up the molecule pool of the TCA The glyoxylatecycle is essentially active under aerobic conditions uponadaptation to growth on acetate (Figure 1(b)) The glyoxylatepathway operates as a cycle to convert 2mol acetyl CoA to1mol succinate [9] The equation of glyoxylate pathway is

2Acetyl CoA + 2H2O +NAD+

997888rarr Succinate + 2CoASH + NADH + 2H+(2)

Since the conversion of glucose to succinate via glyoxylatepathway generates NADH this route alone is not sufficientto balance the electrons However during anaerobic cultureand in the absence of an additional electron donor activatedglyoxylate pathway will provide extra NADH to anaerobicfermentative pathway and benefit to achieve higher succinateyield

Succinate can also be formed from acetyl-CoA gener-ated from pyruvate via oxidative TCA cycle under aerobicconditions [10] This pathway converts acetyl-CoA to citrateisocitrate and succinate which is subsequently converted tofumarate by succinate dehydrogenase Under aerobic condi-tions the production of succinate is not naturally possiblesince it is only an intermediate of the TCA cycle To realize





(3)




















Yield(g gminus1)




ACK



malate

[15]

Escherichia coli




[16]


formate[17]




AFP111




ethanol [19]


mdash 121 096 10 (OD600) Acetate [20]







formate[22]

SBS550MG (pHL314)





[23]




GJT (pHL333 pHL413)







ethanol[26]

HL51276k




acetate[27]



acetate [27]


Table 1 Continued





Yield(g gminus1)


KJ060





acetate[8]

KJ134

Deletion of LDHADHE






[28]





146 317 092 60 g Lminus1 Acetate [29]


Suc-200




Suc-297




PYC





[31]


ICL1






[33]


Citrate

IsocitrateFumarate

Malate

Acetyl-CoA

Acetyl-CoA

Glucose

Pyruvate

Phosphoenolpyruvate

1

Lactate

2

Ethanol

Acetate5 6

39

8 10

Formate4

7

Oxaloacetate

H2 + CO2

Acetyl-phosphate

Succinate

Glyoxylate








2-95 CO



2
















119871119886 52 hminus1 when






















9 Final Remarks






















10 Conclusion



Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





(3)




















Yield(g gminus1)




ACK



malate

[15]

Escherichia coli




[16]


formate[17]




AFP111




ethanol [19]


mdash 121 096 10 (OD600) Acetate [20]







formate[22]

SBS550MG (pHL314)





[23]




GJT (pHL333 pHL413)







ethanol[26]

HL51276k




acetate[27]



acetate [27]


Table 1 Continued





Yield(g gminus1)


KJ060





acetate[8]

KJ134

Deletion of LDHADHE






[28]





146 317 092 60 g Lminus1 Acetate [29]


Suc-200




Suc-297




PYC





[31]


ICL1






[33]


Citrate

IsocitrateFumarate

Malate

Acetyl-CoA

Acetyl-CoA

Glucose

Pyruvate

Phosphoenolpyruvate

1

Lactate

2

Ethanol

Acetate5 6

39

8 10

Formate4

7

Oxaloacetate

H2 + CO2

Acetyl-phosphate

Succinate

Glyoxylate








2-95 CO



2
















119871119886 52 hminus1 when






















9 Final Remarks






















10 Conclusion



Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







Yield(g gminus1)




ACK



malate

[15]

Escherichia coli




[16]


formate[17]




AFP111




ethanol [19]


mdash 121 096 10 (OD600) Acetate [20]







formate[22]

SBS550MG (pHL314)





[23]




GJT (pHL333 pHL413)







ethanol[26]

HL51276k




acetate[27]



acetate [27]


Table 1 Continued





Yield(g gminus1)


KJ060





acetate[8]

KJ134

Deletion of LDHADHE






[28]





146 317 092 60 g Lminus1 Acetate [29]


Suc-200




Suc-297




PYC





[31]


ICL1






[33]


Citrate

IsocitrateFumarate

Malate

Acetyl-CoA

Acetyl-CoA

Glucose

Pyruvate

Phosphoenolpyruvate

1

Lactate

2

Ethanol

Acetate5 6

39

8 10

Formate4

7

Oxaloacetate

H2 + CO2

Acetyl-phosphate

Succinate

Glyoxylate








2-95 CO



2
















119871119886 52 hminus1 when






















9 Final Remarks






















10 Conclusion



Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued





Yield(g gminus1)


KJ060





acetate[8]

KJ134

Deletion of LDHADHE






[28]





146 317 092 60 g Lminus1 Acetate [29]


Suc-200




Suc-297




PYC





[31]


ICL1






[33]


Citrate

IsocitrateFumarate

Malate

Acetyl-CoA

Acetyl-CoA

Glucose

Pyruvate

Phosphoenolpyruvate

1

Lactate

2

Ethanol

Acetate5 6

39

8 10

Formate4

7

Oxaloacetate

H2 + CO2

Acetyl-phosphate

Succinate

Glyoxylate








2-95 CO



2
















119871119886 52 hminus1 when






















9 Final Remarks






















10 Conclusion



Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Citrate

IsocitrateFumarate

Malate

Acetyl-CoA

Acetyl-CoA

Glucose

Pyruvate

Phosphoenolpyruvate

1

Lactate

2

Ethanol

Acetate5 6

39

8 10

Formate4

7

Oxaloacetate

H2 + CO2

Acetyl-phosphate

Succinate

Glyoxylate








2-95 CO



2
















119871119886 52 hminus1 when






















9 Final Remarks






















10 Conclusion



Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















119871119886 52 hminus1 when






















9 Final Remarks






















10 Conclusion



Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















9 Final Remarks






















10 Conclusion



Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















10 Conclusion



Acknowledgments


References












































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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