bioproduction of fumaric acid: an insight into microbial
TRANSCRIPT
REVIEW ARTICLE
Bioproduction of fumaric acid: an insight into microbial strainimprovement strategies
Joseph Sebastiana, Krishnamoorthy Hegdea, Pratik Kumara, Tarek Rouissia and Satinder Kaur Brara,b
aINRS-ETE, Universit�e du Qu�ebec, Qu�ebec, Canada; bDepartment of Civil Engineering, Lassonde School of Engineering, York University,Toronto, Ontario, Canada
ABSTRACTFumaric acid (FA), a metabolic intermediate, has been identified as an important carbohydratederived platform chemical. Currently, it is commercially sourced from petrochemicals by chemicalconversion. The shift to biochemical synthesis has become essential for sustainable developmentand for the transition to a biobased economy from a petroleum-based economy. The main limi-tation is that the concentrations of FA achieved during bioproduction are lower than that from achemical process. Moreover, the high cost associated with bioproduction necessitates a higheryield to improve the feasibility of the process. To this effect, genetic modification of microorgan-ism can be considered as an important tool to improve FA yield. This review discusses variousgenetic modifications strategies that have been studied in order to improve FA production.These strategies include the development of recombinant strains of Rhizopus oryzae, Escherichiacoli, Saccharomyces cerevisiae, and Torulopsis glabrata as well as their mutants. The transformedstrains were able to accumulate fumaric acid at a higher concentration than the correspondingwild strains but the fumaric acid titers obtained were lower than that reported with nativefumaric acid producing R. oryzae strains. Moreover, one plausible adoption of gene editing tools,such as Agrobacterium-mediated transformation (AMT), CRISPR CAS-9 and RNA interference(RNAi) mediated knockout and silencing, have been proposed in order to improve fumaric acidyield. Additionally, the introduction of the glyoxylate pathway in R. oryzae to improve fumaricacid yield as well as the biosynthesis of fumarate esters have been proposed to improve the eco-nomic feasibility of the bioprocess. The adoption of some of these genetic engineering strategiesmay be essential to enable the development of a feasible bioproduction process.
ARTICLE HISTORYReceived 23 July 2018Revised 18 April 2019Accepted 30 April 2019
KEYWORDSFumaric acid;bioproduction; geneticmodification; metabolicengineering; transformation;R. oryzae; fumaric acid ester
Introduction
Microorganisms can produce a diverse range of chemi-cals and materials. These products of microbial metab-olism, such as organic acids, have a wide range ofapplications and can be used in our daily life. Humanshave a long history of reliance on microorganisms toproduce diverse products ranging from chemicals,such as ethanol and organic acids, to consumables,such as bread, yogurt, and cheese. Fumaric acid (FA),also referred to as (E)-2-butenedioic acid or trans-1,2-ethylene dicarboxylic acid, is one such microbial meta-bolic products that have been identified as a platformchemical by the US Department of Energy (DOE) in2004 [1,2]. It is as an important carbohydrate derivedplatform chemical, a key intermediate of microbialmetabolism, with several applications in the synthesisof bio-based chemicals and polymer production. Thediverse applications are rendered due to the presence
of the dicarboxylic groups. This makes FA amenable tochemical modifications and being suitable for use asa monomer for the synthesis of biodegradable poly-mers [3].
FA is mainly used as a food additive and foodacidulant and in the production of paper resins, unsat-urated polyester resins, and plasticizers [4–6]. Theapplication of fumaric acid, in general, has beenreviewed extensively by Das et al. [7,8] and the diverseindustrial applications of this acid as a resin, plasti-cizer, and component for polymer synthesis has beenreviewed by Doscher et al. [9]. More recently, deriva-tives of FA, especially fumaric acid esters (FAEs) havefound its application as an important chemical with awide array of biomedical applications, such as psoriasisand sclerosis treatment and a support material for tis-sue engineering. Clinical level studies on the pharma-cological effects of FA and its ester derivatives (FAEs)
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CONTACT Satinder Kaur Brar [email protected], [email protected] Institut national de la recherche scientifique, Centre Eau,Terre & Environnement, 490 de la Couronne, Qu�ebec (Qc) G1K 9A9, Canada� 2019 Informa UK Limited, trading as Taylor & Francis Group
CRITICAL REVIEWS IN BIOTECHNOLOGYhttps://doi.org/10.1080/07388551.2019.1620677
have confirmed their efficacy. Methyl esters of fumaricacid, mono-methyl fumarate (MMF) and dimethylfumarate (DMF) esters, has been even licensed for usein Europe for the treatment of severe psoriasis vulgaris[10,11]. The recent approval of FDA of DMF (Tecfidera)for use in the treatment of relapsing forms of multiplesclerosis, proves the importance of FA and FAEs. Inaddition, FAEs, specifically their ethyl esters, has foundits application in tissue engineering as a scaffold aswell as for drug delivery [12,13]. The review by Daset al. [8] provides comprehensive information aboutthe diverse medical applications of fumaric acid andits derivatives.
The diverse applications of FA have fueled thedemand for dicarboxylic acid, which is fulfilled by themeans of chemical synthesis from petroleum prod-ucts. Emphasis on the transition towards the bio-based economy has led to the recognition of interestin the development of bioproduction strategies forFA production. FA is a key intermediate of the meta-bolic pathway of a diverse group of microorganismsand has led to the investigation of these microorgan-isms as a potential means of FA production. Fungalstrains such as Rhizopus oryzae, has been identified asa FA overproducer and a potential source for FA. Thisreview discusses various strain improvement strat-egies to improve R. oryzae mediated FA productionas well as metabolic and genetic engineering strat-egies investigated to achieve microbial productionusing non-FA producers. These investigations havenot led to commercial bioproduction of FA andthe plausible reasons for this have been provided.Moreover, the advancement in metabolic engineeringtechniques have provided us with novel tools andpossible avenues for the use of these strategies forthe bioproduction of FA and its derivatives. Previousreviews have emphasized primarily on the control ofprocess parameters in order to improve and controlFA production. These reviews have provided little dis-cussion on genetic engineering strategies that can beadopted to improve FA production. This lack of com-piled information on strain improvement strategies,especially genetic engineering, is attempted to beconsidered in this review.
Current status of the commercial synthesis offumaric acid
At present, the global demand for FA is solely fulfilledby petrochemical methods of synthesis as representedin Figure 1. It is derived by the isomerization of maleicacid obtained from hydrolysis of maleic anhydride.
The maleic anhydride is obtained by the oxidation ofbutane or benzene in the presence of the catalyst,vanadyl pyrophosphate [3,4]. The catalyst mediatedconversion is the only feasible technique for theselective conversion of butane into maleic acid [14].This chemical catalyst mediated process provides ahigh process yield of FA (112% w/w) from maleicanhydride [3,14]. However, this process also leads tothe formation of the toxic gas, carbon monoxide, andgreenhouse gas, carbon dioxide, as byproducts, whichin turn leads to environmental pollution and contrib-utes to global warming [7]. The global demand for FAis expected to expand to over 300 Kilotons by theyear 2020 from 225.2 Kilotons (2012). This increase indemand is mainly fueled by a rise in the demand forthe acid used in the food and beverage industry,which accounts for over 35% of global consump-tion [15–17].
Moreover, FA is an important intermediate in thesynthesis of edible products, such as L-malic acid andL-aspartic acid. The increase in demand for artificialsweeteners, such as aspartame, in beverages andfoods, will further increase the worldwide demandfor FA and its derivatives [18]. Furthermore, therecent identification of its pharmaceutical propertiescan lead to increased usage of FA and its derivativesin the pharmaceutical industry, as discussed earlier.This will further enhance the global demand for thisacid. Additionally, the high price of crude oil, itsdepletion and the recent emphasis on green chemis-try for sustainable development, has necessitatedthe adoption of alternate means for increased FAproduction [15,19].
Bioproduction of fumaric acid
Microbial bioproduction of this carbohydrate derivedbiochemical provides an attractive alternative whichensures sustainable development by reducing thedependence on fossil fuels. Moreover, the use ofrenewable feedstocks helps the recovery of valuablecarbon. FA is one such compound synthesized bymany microorganisms, especially aerobic microbes, asan intermediate compound during the TCA cycle[7,20]. As early as 1911 the fungal strain, Rhizopus nig-ricans, was identified for its ability to synthesize FA asearly as 1911. Later studies, involving multiple fungalspecies, concluded that Rhizopus species was the bestproducer of fumaric acid. Xu et al. [5] provided a briefsummary of the history of the bioproduction of FA.Additionally, the adoption of a bioproduction strategy
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2 J. SEBASTIAN ET AL.
allows for carbon dioxide fixation and recovery, espe-cially when organic industrial residues are employed.
However, the advent of less expensive petrochem-ical means of fumaric acid synthesis led to the discon-tinuation of the bioproduction of FA. Higher yields ofFA are obtained during the chemical process (112%w/w against 85% w/w via fermentation using glucoseas carbon source). Also, the lower production cost andbetter control over production conditions led to thewidespread use of the chemical process for FA pro-duction [3]. The recent increase in the price of petrol-eum products, increasing the emphasis on low carbonfootprint production strategies and green chemistryapproaches for sustainable development. This hasresulted in the resurgence of interest in fumaric acidproduction by fermentation [5,15]. Moreover, theadoption of bioproduction strategy allows for the util-ization of renewable biomass as well as industrialresidues for FA production. This in turn significantlyreduces the production costs thereby improvingits feasibility. Additionally, the low solubility of FA,5–7 g/L (at room temperature), ensures its easy
recovery by simple precipitation following acidification.Hence, no specialized strategies are required for therecovery of FA [21,22]. The FA crystals so recoveredhave been observed to be of high purity, with inor-ganic components in the fermentation medium beingthe primary contaminant, and can be used thereafterfor low purity applications such as the manufacture ofresins or polymers as well as cattle feed supplements[3]. To further improve FA purity, the use of activatedcarbon has been investigated and found to be effect-ive [23,24]. The recovered FA (of purity >99%) can beused for biomedical applications such as psoriasisand multiple sclerosis treatment.
Metabolic pathway of fumaric acid synthesis
Fumaric acid is an important intermediate of the tri-carboxylic acid (TCA) cycle (Krebs cycle). Two differentmetabolic pathways are mainly involved in the synthe-sis of FA viz. oxidative TCA cycle and reductive TCApathway. Fixation of CO2 and conversion of pyruvateto oxaloacetate occurs during the TCA cycle. Reductive
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Figure 1. Chemical fumaric acid synthesis and its derivatives.
CRITICAL REVIEWS IN BIOTECHNOLOGY 3
CO2 fixation catalyzed by the pyruvate carboxylaseenzyme is the main reason for the accumulation offumaric acid during fermentation as it was observed inthe case of the Rhizopus species [5]. The reductive cyclehas a maximal theoretical yield of 2 moles of FA permole of glucose consumed and 2 moles of CO2 fixed.Reliance on reductive pyruvate carboxylation, as thesole pathway, will lead to no ATP formation. This ATP isrequired for cell maintenance and will affect overall cellgrowth. Hence, the TCA cycle is active during FA pro-duction and complete theoretical fumaric yield willnot be achieved [3,7]. Adoption of strategies to limitfungal growth has the potential to ensure optimumfumaric acid production. The metabolic pathway andenzymes involved in the key steps of this pathway arerepresented in Figure 2 and a brief summary of themetabolic pathways involved in the microbial fumaricacid production is provided in reviews by Engel et al.[3] and Xu et al. [5]. Additionally, it was identifiedthat amino acid and fatty acid metabolism, as wellas activation of the glyoxylate pathway, can have apotential impact on FA accumulation [25–27].
Rhizopus sp. mediated fumaric acid production
Fungal species have been employed to produce a vastarray of compounds and consumables. Multicellular
filamentous fungi or molds are extensively used toproduce fermented foods, secondary metabolites, andindustrial enzymes. Rhizopus sp. is an example of fila-mentous fungi, belonging to the “Mucoraceae” family,that is used to produce a wide array compoundsranging from fermented food products to platformchemicals such as fumaric acid [20,28,29].
Rhizopus oryzae, generally regarded as safe (GRAS),has usually been employed for FA production via fer-mentation at neutral pH values to produce fumaratesalts [22]. This fungal species is subdivided into twogroups (Type I and Type II) according to their geneticand phenotypic differences. Type I accumulates lacticacid whereas Type II mostly synthesize FA in the pres-ence of fermentable sugars. The main differencebetween these two types is that Type I contains twolactate dehydrogenase (ldh) genes, ldhA, and ldhB,whereas Type II only has one of the genes ldhB. Thelactate dehydrogenase enzyme, LdhA, encoded by theldhA gene enables the conversion of pyruvic acid tolactic acid whereas the enzyme encoded by ldhB genepossesses insufficient reductive capacity. This reducedcapacity allows for the conversion of excess pyruvateto fumaric acid and/or ethanol [28,30]. Table 1 pro-vides a summary of FA production studies conductedusing R. oryzae.
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Figure 2. Overview of metabolic pathway and enzymes involved in the fumaric acid synthesis in R. oryzae. EMP stands forEmbden–Meyerhof–Parnas pathway or glycolysis. The enzymes involved in the various steps of the pathway are PDC—pyruvatedecarboxylase (EC 4.1.1.1); ADH—alcohol dehydrogenase (EC 1.1.1.1); LDH—lactate dehydrogenase (EC 1.1.1.27); PDH—PYRUVATEdehydrogenase (EC 1.2.4.1); PYC—pyruvate carboxylase (EC 6.4.1.1); MDH—malate dehydrogenase (EC 1.1.1.37) and FUM—fumarase (EC 4.2.1.2). For color version of the figure refer e-print.
4 J. SEBASTIAN ET AL.
Limitations of R. oryzae based production
R. oryzae mediated FA production is usually per-formed at neutral pH values. The production of theacid during fermentation leads to a significantdecrease in pH from 6 to 4 within the first 48 h offermentation. This reduction in pH causes the rate ofproduction to slow down and eventually cease com-pletely. To prevent this drop in pH, the fumaric acidis converted to fumarate salts by using neutralizers,such as calcium carbonate and sodium carbonate.Fumaric acid is then recovered from the fermentationbroth by precipitation, due to its low solubility, fol-lowing acidification. The requirement of neutralizersand acids during the recovery step leads to highconsumption of bases and acids as along with theproduction of large quantities of salts. Calcium car-bonate is commonly used as the neutralizer butthe insolubility of calcium salts causes considerablepower consumption and process difficulties, such asincreased viscosity and associated power consump-tion [22,39,48]. Hence, sodium carbonate is mainlyproposed as a neutralizing agent because the sodiumsalt of fumarate is soluble. Gangl et al. [48] have per-formed a comprehensive comparison of the advan-tages and disadvantages of using calcium carbonateand sodium carbonate as the neutralizing agent.
Some of the above-mentioned limitations of thefungal strain mediated fumaric acid fermentation canbe overcome by performing the fermentation at alower pH. The move to a lower pH fermentationreduces the quantity of neutralizing agent required inorder to maintain pH close to neutral as well as thequantity of salt generated during the recovery offumaric acid from the fermented broth. Moreover, the
undissociated fumaric acid so formed can be recov-ered by the crystallization from the broth due to thelow solubility of FA. Additionally, the direct recoveryof fumaric acid from the broth using adsorbents, suchas polyvinyl pyridine (PVP) resin and Amberlite IRA-900, would be possible [5]. This strategy of FA recov-ery using adsorbents was successfully used by Caoet al. [47] to recover high purity FA following desorp-tion and acidification. To this effect, the shortenedduration of pH control and fermentation at pH 5 wasproposed [22]. It was observed that eliminating off pHcontrol at 90 h instead of 120 h did not lead toimportant effects on FA production. The drawback ofthis strategy is that pH control is required during theinitial stages of fermentation even though reducedconsumption of the neutralizing agent is achieved.
Moreover, it has been observed that the morph-ology of the fungus during fermentation has a pro-found impact on FA production and has beenextensively studied by Engel et al. [22] and Zhou et al[49]. It was also observed by them that the fungalpellet diameter played a key role in FA productivity.Zhou et al. [49] concluded that lower pellet diameterenhanced for mass transfer as well as better contactwith more of the actively growing fungi presenton the surface of the culture. Furthermore, it wasobserved by Engel et al. [22] that an anaerobic envir-onment was present at the core of the fungal pellet.Therefore, fungal pellets of smaller diameter are pre-ferred which requires the optimization of conditions,such as agitation rate, pH conditions, temperature,and working volume, to achieve the optimum pelletdiameter. This dependence on fungal morphologyfurther adds to the complexities associated with
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Table 1. Summary of fumaric acid production studies carried out using R. oryzae.
Carbon source FermenterFumaric acidconcentration
Productivity(g/L/h) Reference
Glucose Stirred tank 56.2 g/L 0.7 [31]Glucose Stirred tank 41.1 g/L 0.37 [32]Glucose Stirred tank 32.1 g/L 0.32 [33]Glucose Stirred tank 30.2 g/L 0.19 [22]Xylose Shake flask 28.4 g/L – [34]Xylose Shake flask (immobilization) 45.3 g/L – [35]Glucose/xylose Shake flask 46.7 g/L – [36]Corn straw Shake Flask 27.8 g/L 0.33 [37]Cornstarch Shake flask (Simultaneous saccharification fermentation) 44.1 g/L 0.53 [38]Cornstarch Shake Flask 45.0 g/L 0.55 [32]Brewery wastewater Shake Flask 31.3 g/L – [39]Apple juice extraction waste Shake Flask 25.2 g/L 0.35 [40]Diary manure Stirred tank 31.0 g/L 0.32 [41]Apple pomace Solid state-Rotating drum fermenter 138 g/kg dry weight – [40]Glucose/crude glycerol Shake flask 22.81 g/L 0.34 [42]Synthetic medium Immobilized fungi 32.03 g/L 1.33 [43]Synthetic medium Immobilized fungi 40.13 g/L 0.32 [44]Synthetic medium Immobilized fungi 30.3 g/L 0.21 [45]Brewery wastewater Immobilized fungi 43.67 g/L 1.21 [46]Synthetic medium Immobilized fungi- fed-batch 85 g/L 4.25 [47]
CRITICAL REVIEWS IN BIOTECHNOLOGY 5
fungal bioproduction of fumaric acid. To this effect,immobilization of fungi on the support have beenproposed to reduce the effects of pellet morphologyand has been found effective in increasing FA prod-uctivity as well as its yield [43,46,47]. Reduction in theduration of fermentation, leading to increased prod-uctivity, from 144 h to 24 h was observed by Gu et al.[43]. The fumaric acid yield of 32.03 g/L was obtainedpost immobilization of the fungi R. arrhizus duringthis study. In addition, a glucose to nitrogen ratio ofthe production media has been observed to have aprofound impact on FA production. To enable max-imum FA yield, nitrogen limitation is recommended tolimit fungal growth and achieve optimal diversion ofsugars towards fumarate production. It has also beenobserved that optimal activity of the cytosolic fumar-ase enzyme was obtained under nitrogen limitingconditions and this activity increased 300% uponreduction of the urea concentration from 2.0 to0.1 g l–1 [3,19,50].
To overcome the above-mentioned complexitiesof R. oryzae mediated fumaric acid production, thegenetic transformation of microorganisms, such asS. cerevisiae (capable of growing at low pH), to accu-mulate fumarate has been considered. These strat-egies of genetic engineering microorganisms toproduce fumaric acid are discussed in the follow-ing sections.
Improved fumaric acid production throughstrain improvement and genetic modification
The lower yield of FA, during bioproduction, com-pared to chemical synthesis has motivated researchersto investigate strategies for improving the FA yieldof the filamentous fungus R. oryzae. These strainimprovement strategies primarily rely on increasingproduct yields thereby enhancing the feasibility ofcommercial bioproduction. These techniques of strainimprovement have been extensively reviewed byMeussen et al. [51] and Li et al. [52] but strainimprovement techniques targeted towards FA
production have so far not been discussed and thisdiscussion occurs in later sections of this review.Moreover, under low pH conditions (3–5), due toreduced fungal growth as well as the complexities ofthis fermentative fungal strain mediated fumaric acidproduction has led researchers to investigate the pos-sibility of using alternate microorganisms for the pro-duction of FA. Developments in genetic engineeringtechniques, such as genetic transformation and over-expression of homologous genes, such as fum, pycand mdh, and metabolic engineering, have providedthe tools for improving microbial production oforganic acids, such as: malic acid, succinic acid, andfumaric acid [29,51].
Strain improvement strategies for R. oryzae
Random mutagenesis
Random mutagenesis involves the use of chemicalmutagens, such as diethyl sulfate, nitrosoguanidineand diethyl sulfate, or radiation, such as UV light andgamma radiation, to induce changes in the geneticmakeup of an organism. This technique has for centu-ries been employed for the development of novelstrains. The main drawback of this technique is thatfollowing induction of the mutation, multiple mutantstrains are generated. These strains generate the needto be screened and a suitable strain selected which istime-consuming and laborious [19,51]. The mutantstrains selected were able to produce higher yieldsof fumaric acid than the parent strain but these titerswere significantly lower than the maximum titersreported for strains obtained from culture collections(represented in Table 2) but comparable to other titersthat have been reported [31–33]. Hence, randommutagenesis can be used as a viable tool for strainimprovement. The adoption of novel strategies, dis-cussed in a later section, in combination with randommutagenesis can be useful for the selection of appro-priate high yielding strains. Q1Moreover, the use ofthis technique generates multiple mutations in the
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Table 2. Random mutagenesis of R. oryzae and fumaric acid concentrations obtained (R. oryzae reassigned as R.arrhizus, [53]).Fungal strain Mutagen Fumaric acid titer (g/L) Yield (g/g) Reference
Pure cultureR. arrhizus NRRL 2582 – 103 0.79 [54]R. arrhizus NRRL1526 – 98 0.82 [55]
MutantR. oryzae RUR709 UV and c-ray mutagenesis 32.1 0.45 [33]R. oryzae ZD-35 UV irradiation 57.4 0.67 [32]R. oryzae ME-F01 UV and nitrosoguanidine 52.7 – [56]R. oryzae ME-F12 Nitrogen ion implantation 44.1 0.44 [38]R. oryzae FM19 Femtosecond laser irradiation 49.4 0.56 [25]
6 J. SEBASTIAN ET AL.
microorganism and the other metabolic activities maybe hampered in addition to the targeted metaboliteproduction. Also, the damage to the DNA by the useof high energy radiations may prevent DNA replicationand hamper the overall transfer of the desired trait.
Homologous and heterologous gene expression inR. oryzae for fumaric acid production
Fumaric acid is an intermediate of the TCA cycle, asmentioned in the earlier section, but the fumaricacid generated during the oxidative pathway is notaccumulated as it is consumed for cell growth andmaintenance. The accumulation of fumaric acidoccurs via the reductive TCA pathway, as observedin the case of R. oryzae. Multiple enzymes areinvolved in the synthesis of fumaric acid from pyru-vate. Pyruvate carboxylase (PYC) catalyzes the ATP-dependent condensation of pyruvate and CO2 toform oxaloacetic acid (OAA). The OAA, so formed,is converted to malate by malate dehydrogenase(MDH), followed by conversion of malate to fumarateby the enzyme fumarase. [57] The overexpression ofthe enzymes involved should be able to increasefumarate production.
Zhang et al. [57] observed a 26% increase infumaric acid production in their study by transformingR. oryzae to express homologous and heterologousenzymes. They introduced the exogenous phosphoe-nolpyruvate carboxylase enzyme (PEPC) in addition toPYC using the plasmid (pPyrF2.1A) mediated trans-formation of fungi R. oryzae M16. The PEPC enzyme,not present in R. oryzae, is involved in the conversionof phosphoenolpyruvate to OAA with CO2 fixation.Increased PYC and PEPC activity of 3–6mU/mg wereobserved. The redirection of this metabolic pathwayresulted in the introduction of these genes that leadto an increase in fumarate production [57]. In anotherstudy, the impact of the overexpression of the fumRgene that encodes for the fumarase enzyme wasinvestigated. It was observed that the over-expressionof fumarase in R. oryzae lead to malic acid productioninstead of FA. The over-expressed fumarase is alsoable to catalyze the hydration of FA to malic acid.Hence, it can be concluded that the presence offumarase alone may not be responsible for FA accu-mulation during R. oryzae mediated fermentation [58].To this effect, the observation by Peleg et al. has aparticular significance [59]. They identified two isoen-zymes of fumarase with a different localization withinthe cell. One isoenzyme was solely present in thecytosol whereas the other was found in both thecytosol and in the mitochondria. It was also observed
that the inhibition of fumarase activity by cyclohexi-mide only affected fumaric acid production anddecreased the activity of the cytosolic fumarase isoen-zyme [59]. Therefore, the transformation of R. oryzaewith the appropriate isoenzyme has the potential toimprove FA production as the reductive TCA cycleoccurs in the cytosol.
In addition, a dicarboxylic acid transporter withhigh selectivity for fumaric acid can also play animportant role in its production with R. oryzae. Thisconclusion is supported by the observation that malicacid production by Aspergillus oryzae NRRL 3488 wasimproved twofold by the overexpression of a C4-dicar-boxylate transporter. The overexpression of malatedehydrogenase and pyruvate carboxylase in conjunc-tion with the transporter led to an additional increaseof 27% and achieved a malic acid concentration of154 g/L in 164 h [60]. The high concentration of malicacid achieved in this study points to the possibility ofachieving a similar titer for fumaric acid by providingan efficient system for the conversion of the malicacid into fumaric acid.
Fumaric acid production in recombinantmicroorganisms
The limitations of R. oryzae mediated synthesis offumaric acid, discussed previously in Limitations of R.oryzae based production section, has motivatedresearchers to investigate the possibility of usingother microorganisms, particularly Escherichia coli andSaccharomyces cerevisiae, to produce fumaric acid. Tothis effect, the metabolic pathways that influence FAproduction have to be investigated prior to the gen-eration of recombinant strains. Cellular metabolismis highly regulated and made up of a plethora of vari-ous components, such as transcripts, proteins, andmetabolites, and the production of the compound ofinterest is directly linked to the cellular activities.Hence, an accurate understanding of the metabolicstates and regulatory mechanisms are important.
The most effective way to evaluate the metabolicstate is to quantify various metabolites. Therefore, theadvances in analytical techniques, such as NMR, gaschromatography-mass spectrometry (GC-MS), gaschromatography time-of-flight mass spectrometry (GC-TOF) and liquid chromatography-mass spectrometry(LC-MS), has enabled high-throughput analysis of themetabolites. This has allowed for quick comparativeanalysis of metabolite profiles post-genetic modifica-tion and provides an understanding of the physio-logical state of the cell, which in turn provides us
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CRITICAL REVIEWS IN BIOTECHNOLOGY 7
with an understanding of their metabolic status. Tofurther improve an understanding of the metabolicstate of the cell, isotopomer experiments can be per-formed. These studies provide us with additionalinformation on intracellular fluxes and metabolic rates.13C-labeled glucose is the most frequently used iso-topomer. The monitoring of the distribution of theisotopomer, as the labeled glucose is metabolized,can be used to estimate metabolic reaction rates.Hence, provide quantitative information regarding car-bon flow through the metabolic pathways [60,61]. Anunderstanding of this would allow for the selection ofthe metabolic pathway of interest that would enablemaximum conversion of glucose to fumaric acid withreduced byproduct formation.
Improvement of the cellular systems by metabolicengineering strategies is vital to enhance the overallproductivity and yield of the target compound. Thedevelopment of stoichiometric metabolic modelswhich includes all enzymatic reactions of organisms,such as E. coli and S. cerevisiae, has enabled the pre-diction of metabolic states using in silico simulations.Metabolic flux analysis (MFA), flux balance analysis(FBA), and metabolic pathway analysis (MPA) are themost popular tools in stoichiometric metabolic net-work analysis. Additionally, database resources, suchas UniProt, BRENDA, BioCyc, KEGG, and the EnzymeCommission database, can be used for the reconstruc-tion of a metabolic pathway [29,62]. The above-men-tioned tools for metabolic engineering have beenextensively used for transformation of E. coli and S.cerevisiae strains but have not been readily used forthe transformation of R. oryzae. There are only a fewreports the transformation of R. oryzae that adoptedmetabolic flux analysis for metabolic engineering ofconsidering this fungal strain.
One such early study used TELUX, a specific radio-activity curve-matching program, to investigate glu-cose metabolism by R. oryzae. In the study, 14Cisoprotomer labeled glucose was used to understandglucose metabolism and thereby increase lactate yield.This was followed by UV-mediated random mutagen-esis and the selection of a lactate overproducingstrain. The prediction of metabolic pathway modifica-tion that led to lactate overproduction, provided byglucose flux analysis, helped in the identification of asuitable strain. This selected strain showed reducedethanol production and chitin synthesis, as predicted,and a lactic acid yield of 30 g/L was obtained [63]. Inanother study, the results of the carbon flux analysisof a mutant lactic acid overproducing strain, R. oryzaeR1021, and the parent strain, R. oryzae R3017, showed
that pyruvate was the key metabolic intermediate. Themutant strain showed reduced loss of carbon to etha-nol and acetyl-CoA and provided a lactic acid yield of79.4 g/L, which was 52% higher than that of the par-ent strain [64]. These studies focused on the synthesisof lactic acid but the understandings can possibly beadopted for FA production. Such a study on metabolicprofiling, post femtosecond laser-mediated randommutagenesis, identified that modifications to aminoacid and fatty acid metabolism led to a 1.59-foldincrease in FA production [25]. These studies usedMFA or metabolic profiling for understanding carbonmetabolism either before or after random mutagenesisbut not for designing metabolic pathways and trans-forming the fungi. The importance of a better under-standing of metabolic activities using metaboliteprofiling is further signified by the observation thatthe FA production was enhanced by 39.7% as a resultof supplementation with 1% linoleic acid during R.oryzae mediated fermentation at low pH (pH 3) condi-tion. The strategy of supplementation with linoleicacid was adopted based on the understanding of thechanges to the metabolic activity at low pH conditionsprovided by GC-MS assisted metabolite profiling [65].The use of in silico metabolic modeling was found tobe effective in designing a metabolic engineeringstrategy to achieve a higher FA titer in T. glabrata[66]. Hence, an in silico model of fumarate synthesisof R. oryzae is essential to achieve effective conversionof glucose to fumarate.
The successful adoption of MFA assisted strainselection, in combination with strain improvementstrategies, can also be adopted to improve FA yield inR. oryzae. MFA can act as an important tool for theselection of an appropriate high FA producing strainfollowing random mutagenesis. Moreover, it would bepossible to obtain far higher titers for FA by usingMFA mediated design and then transform the fungalstrain using targeted genetic modification approaches.These strategies would enable reduced byproductsynthesis, such as ethanol and malic acid, as wellas effective redirection of a metabolic pathway thatallows for maximum productivity by optimal conver-sion of glucose to FA, as observed in case of both E.coli and S. cerevisiae discussed below.
Escherichia coli
High concentrations of fumaric acid are not normallyproduced in E. coli and 20 g/L has been shown tohave an impact on cell growth [26]. The adoption ofgenetic engineering strategies has allowed the useof E. coli for the production of a diverse range of
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8 J. SEBASTIAN ET AL.
compounds and possibly fumarate. This possibility hasbeen investigated. The de-repression of the glyoxylatepathway in combination with overexpression anddeletion of specific genes was exploited to induce FAaccumulation [26,27]. Song et al. [26] used rationalmetabolic engineering strategies coupled with in silicoflux response simulation to develop a novel E. colistrain. The in silico flux modeling simulation (E. coligenome-scale metabolic model EcoMBEL979) wasused to investigate the influence of PEPC flux onfumarate production and helps to understand theresponse to a metabolic shift. It was observed theoverexpression of PEPC was able to increase the FAproduction and further genetic modifications selectedbased on their effect on FA production. The final con-centration of FA was achieved by transforming E. colito overproduce FA via the noncyclic glyoxylate route.The isocitrate lyase repressor gene iclR was deletedto enhance the carbon flow into the glyoxylate shuntin addition to the silencing of fumarase isozymes.Moreover, endogenous PEPC was also overexpressedto increase the reductive TCA cycle flux to achieve FAproduction. Additionally, genes involved in the furtherconversion of FA and byproduct syntheses, such asarcA, aspA, lacL, and ptsG, were deleted. FA produc-tion with the final strain obtained was 28.2 g/L, and aproductivity of 0.448 g/L/h under aerobic fed-batchculture conditions [26].
The glyoxylate pathway flux was enhanced simi-larly to obtain a genetically modified strain of E. colicapable of utilizing glycerol as a feedstock for FAproduction. The conversion of fumaric acid to malicacid was prevented by the deletion of fumA, fumB,and fumC genes that code for three fumarases. Itwas observed that the overexpression of glyoxylateshunt and PEPC enzyme led to enhanced fumaricacid production as well as reduction of acetate for-mation (a byproduct). Additionally, the gene codingfor aspartase, which converts fumarate to aspartate,was deleted to prevent the reverse conversion. AnFA concentration of 41.5 g/L was achieved with theoverall productivity of 0.51 g/L/h following fermenta-tion under fed-batch conditions. This correspondedto 70% of the maximum theoretical yield [27]. Theconcentration of FA achieved during these investiga-tions is still lower than the titers obtained by cultur-ing R. oryzae. These studies focused primarily on theredirection of the TCA cycle towards the glyoxylatepathway for FA accumulation. However, the reduc-tive TCA pathway has the potential to produce 2moles of FA for every mole of glucose. This yield isdouble than that of the theoretical maximum of the
oxidative glyoxylate pathway (1mol/mol glucose)[67]. Hence, it may be possible to obtain even ahigher titer for FA with the enhancement of thereductive TCA pathway along with the genetic modi-fication strategies adopted during these studies.
Another metabolic pathway that has been investi-gated for fumarate bioproduction in E. coli was theintroduction of the mammalian urea cycle [68].Fumaric acid is produced as a byproduct in this meta-bolic pathway during the conversion of L-arginosucci-nate to arginine. The recombinant E. coli (ABCDIA-RAC) was produced by blocking the Krebs cycle andintroducing the urea cycle. To improve FA production,several genes, such as fumA, fumB, fumC, frdABCD,iclR, and arcA, were deleted. Additionally, the ureacycle was introduced by overexpressing the homolo-gous carAB, argI, and heterologous rocF genes whichencoded for carbamoyl phosphate synthase (CPS),ornithine carbamoyl transferase (OTC), and arginase,respectively. The strain isolated was able to yield afumarate titer of 11.38mmol/L (1.32 g/L) and more-over, it was concluded that cell growth and the dere-pression of the Krebs cycle were required for high-titer FA production in aerobic conditions [68]. Inanother study by the same research group, the FAyield of the strain was further improved to 9.42 g/L bythe overexpression of the homologous C-4 dicarboxy-late carrier gene dcuB [69]. These studies have failedto achieve the FA yields reported for R. oryzae butshows that targeted multilevel genetic transformationsare essential to improve fumarate yield.
Saccharomyces cerevisiae
The yeast, S. cerevisiae is a well-established industrialproduction organism and it possesses exceptional cul-tivation characteristics. They require only simplemedia for growth and are tolerant to the presence ofinhibitors. Moreover, there is a high degree of toler-ance to (high) sugar concentrations and their abilityto grow under acidic conditions. The inability to growunder low pH conditions is one of the main limita-tions to R. oryzae mediated FA production. S. cerevi-siae is not naturally capable of accumulating FA andthe feasibility of the induction of FA accumulation byengineering S. cerevisiae has been investigated [5].
The introduction of heterologous cytosolic R. oryzaemalate dehydrogenase, high-level expression of the R.oryzae fumarase and overexpression of the nativecytosolic pyruvate carboxylase was observed to onlyyield an FA concentration of 3.2 g/L [70]. In a similarstudy, simultaneous co-expression of both reductiveand oxidative routes of FA was investigated. This
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CRITICAL REVIEWS IN BIOTECHNOLOGY 9
co-expression of heterologous RoMDH and RoFUM1failed to accumulate FA in the engineered strain. Itwas also observed that the fumarase deleted straincould produce FA via the oxidative route. Moreover, itwas observed that the introduction of the heterol-ogous reductive route of FA led to lower productionof FA but the introduction of a heterologous gene forpyruvate carboxylase (RoPYC) led to a higher FA titer(2.48 g/L). The maximum FA titer obtained, under opti-mal conditions of nitrogen limitation and the presenceof biotin (co-factor of PYC), was 5.6 g/L [5].
Another strategy investigated improved FA yield,investigated by the same group, was to subject the S.cerevisiae CEN.PK2-1C pdc1adh1fum1 strain to site-directed mutagenesis for improving the catalytic activ-ity of RoPYC as well as overexpression of the samestrain. The site-directed mutagenesis was able toimprove the catalytic activity of it by reducing theconformational instability afforded by the presence ofthe amino acid proline at 474 positions. Substitutionof the amino acid with asparagine led to improved FAproduction than the wild type strains. The best FAyield of 448.4mg/L was obtained following biotinsupplementation of 56 mg/L [71]. This yield is consid-erably lower than their previous study which yielded5.6 g/L fumarate [5]. Hence, a better understanding ofthe underlying mechanism of the metabolic pathwaysinfluencing fumarate production is essential for suc-cessful genetic modification strategies to improveits yield.
Torulopsis glabrata
Recently, multivitamin auxotrophic mutant strains ofT. glabrata have been investigated for fumaric acid pro-duction following genetic modifications. The auxo-trophic strains have been known to produce high titersof pyruvic acid and reported to have a higher glucoseand acid tolerance than S. cerevisiae [66]. Hence, isan ideal strain for use in the industrial production oforganic acids, mainly pyruvate, but has been subjectedto genetic modification to enable the production ofmalic acid as well as FA [66,72].
The genetic modification strategy adopted involvedidentification of the metabolic pathways that contrib-uted to the accumulation of FA in the cytosol fol-lowed by the introduction of enzymes involved in themetabolic pathways. To this effect, key enzymes affect-ing fumarate accumulation, involved in both carbonand nitrogen metabolism such as argininosuccinatelyase (ASL), adenylosuccinate lyase (ADSL), fumarylace-toacetase (FAA), and fumarase (FUM1), were identifiedusing in the silico metabolic model (iNX804). The
overexpression of the enzymes individually led to theaccumulation of FA in the modified strains comparedto the parent auxotrophic T. glabrata strain but ASLand ADSL were identified as the key enzymes leadingto higher FA accumulation. Optimization of overex-pression levels of enzymes ASL and ADSL gave a FAtiter of 5.62 g/L. This was further improved by intro-ducing a heterogeneous gene, SpMAE1, that encodesfor a C4-dicarboxylic acids transporter in S. pombe.The final titer obtained was 8.83 g/L [66].
Genetic modification of T. glabrata was performed inanother study which involved the introduction of heter-ogenous R. oryzae genes that code for the fumaraseenzyme (RoFUM), pyruvate carboxylase (RoPYC) andmalate dehydrogenase (RoMDH) as well as the C4-dicarboxylic transporter, SpMAE1, from S. pombe. Thebinding site B of RoFUM was subject to site-directedmutagenesis to enhance the catalytic efficiency of theenzyme. The resulting modified strain yielded a fumar-ate titer of 5.1 g/L. Further increases in FA yield wereachieved by subjecting the strain to additional modifi-cation. This additional modification involved deletionof the gene, ade12, that codes for adenylosuccinatesynthetase that is involved in purine metabolism. Theobtained strain resulted in a significantly higher FAyield of 9.2 g/L as well as increased acid tolerance [72].
Limitations of current genetic engineering strategies
The concentrations of the fumaric acid obtained usinggenetically engineered strains of E. coli, S. cerevisiae,and T. glabrata are significantly lower than thatreported for R. oryzae, this is summarized in Table 3.Despite the lower yields, genetic modification techni-ques are an important tool to improve to furtherimprove fumarate yield. These genetic and metabolicengineering strategies exhibit a special significance todecrease the cost of production as well as efficientutilization of biomass and industrial residues as feed-stocks for fumaric acid production. Moreover, thesemodifications allow for the alleviation of the problemsassociated with R. oryzae mediated FA bioproductiondiscussed in Limitations of R. oryzae based productionsection. Advanced genetic and metabolic engineeringstrategies that may be adopted for strain improve-ment are discussed in the following sections.
Advanced genetic and metabolic engineeringstrategies for fumaric acid production and itsderivatives
Advances in genetic and metabolic engineering havepermitted for modifications to inherent metabolic
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10 J. SEBASTIAN ET AL.
pathways to be carried out as well as the expressionof relevant heterologous pathways in desired micro-organisms. Few of such strategies that can be con-sidered for the production of fumaric acid as wellas its derivative esters are discussed in the follow-ing sections.
Genetic and metabolic engineering tools for thebioproduction of fumaric acid
Genetic transformation systems have allowed for theintroduction of foreign DNA in filamentous fungi, suchas R. oryzae, to produce strains with desired character-istics. Most of these transformation techniques, suchas plasmid vector-mediated transformation, producestrains with the transforming DNA remaining extrac-hromosomally leading to low mitotic stability [75].Moreover, alternate transformation techniques, such asprotoplast (spheroplast) fusion, are complicated aswell as inconsistent due to inherent variability in thecomposition of fungal cell walls of different strains[52]. To this effect, alternate transformation techniquesthat provide for stable chromosomal integration of theintroduced DNA are required.
Agrobacterium-mediated transformation (AMT)
For successful genetic transformation, an efficienttransformation technique is required to insert genesof interest. Agrobacterium tumefaciens-mediated trans-formation (ATMT) is a such technique and has beenused for the transformation of filamentous fungi. Themain advantage of this method is that it generatestransformants that have single genomic integration ofthe desired gene [76]. The soil bacterium A. tumefa-ciens has the ability to transfer a part of its DNA calledtransfer DNA (T-DNA) and this ability is exploited forheterologous protein expression in the host organism.For this purpose, the T-DNA is altered to contain the
gene of interest. After the introduction of the modi-fied T-DNA into the host by A. tumefaciens, the T-DNAintegrates into the chromosome of the host, therebyachieving the transformation of the host organism. Abrief summary of the technique and factors affectingAMT efficiency has been provided by Li et al. [52].
The filamentous fungus R. oryzae has been sub-jected to transformation using the AMT technique[75]. It was concluded that the technique was not suit-able for the expression of the gene of interest inR. oryzae due to the presence of a possible defensemechanism. However, more recent researches haveproved that heterologous gene expression is possible.The ectomycorrhizal fungus Laccaria laccata was suc-cessfully transformed through the ATM technique toexpress hygromycin B phosphotransferase (hph) [76].In another study, the filamentous fungus Trichodermareesei QM9414 was transformed using the AMT tech-nique which resulted in the formation of a hygromycinB-resistant fungi [77]. On the other hand, a decreasein resistance to hygromycin B was observed duringseveral cultivation cycles of the fungus Backusellalamprospora post successful integration of the heterol-ogous gene [78].
The AMT technique has been used for the geneticengineering of a diverse group of organisms but thereare no reports of using this technique for the trans-formation of fungi for organic acid production, espe-cially fumaric acid. The requirement of binary vectorsfor this method may be one of the probable reasons.These binary vector systems are tedious to prepare[79]. Also, there are numerous factors that play a keyrole in determining the transformation efficiencywhich needs to be optimized. This genetic modifica-tion and successful integration is a time-consumingprocess. Moreover, the presence of a possible defensemechanism against the presence of heterologousgenes may have contributed to the non-adoption ofthe AMT technique for the fungal transformation [75].
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Table 3. Comparison of fumaric acid titer and yield of natural strains and genetically engineered strains.Strains Fumaric acid titer (g/L) Yield (g/g) Reference
Natural strainsR. arrhizus 103 0.79 [54]
98 0.82 [55]R. oryzae 56.2 0.54 [31]
56.5 0.94 [73]40.5 0.51 [74]
Engineered strainsE. coli CWF812 28.2 0.39 [26]E. coli EF02(pSCppc) 41.5 0.44 [27]R. oryzae ppc 25 0.78 [57]S. cerevisiae FMME 001 PYC2 þ RoMDH 3.18 0.05 [70]S. cerevisiae FMME 006 DFUM1 þ RoPYCþ RoMDHþ RoFUM1 5.64 0.11 [5]T. glabrata ASL(H) – ADSL(L) – SpMAE1 8.83 0.15 [66]T. glabrata (Dade12) – PMS – P160A 9.2 0.15 [72]
CRITICAL REVIEWS IN BIOTECHNOLOGY 11
The loss of introduced heterologous characteristicswith time further contributes to the unsuitability ofthe technique [78].
crispr cas-9
The advances in genetic engineering technologies havecreated a number of opportunities for basic biologicalresearch with filamentous fungi. Clustered regularlyinterspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) has recently emerged as apotent genetic engineering technique. It is a potentialcandidate to resolve the issue of low edit gene fre-quency observed in the case of filamentous fungi. Acomprehensive review of using the system for editingthe fungal genome is provided by Deng et al. [80] andShi et al. [81]. The efficient, simple, and fast techniqueof genetic modification allows for systemic researchon filamentous fungi. This technique is in its infancyand researchers are continuously learning about thepotential applications of this potent genome-edit-ing tool.
This genetic engineering technique has been mostlyused to perform gene deletion or creating a knockouttransformants. The versatility of the Cas9-multipleSgRNA allows for insertion, deletion, and replacementof target genes, thereby generating positive multiplegene modifications. From the fumaric acid productionpoint of view, the system can be used to efficiently pro-duce transformants with the deletion of genes thatencode enzymes, such as lactate dehydrogenase andalcohol dehydrogenase, that causes the production ofbyproducts. This enables the redirection of metabolicflux towards fumarate production [82].
Moreover, this system was used in the develop-ment of a genetic engineering toolbox for A. niger.The toolbox allows for the generation of strains car-rying heterologous expression cassettes at a definedgenetic locus. The proof of this concept was shownby the transformation of A. niger to produce aconiticacid. The transformed cells produced aconitate onlyin the presence of the inducer 10 mg/mL doxycycline[83]. Similar expression systems may be used for theproduction of fumaric acid whereas R. oryzae can betransformed to overexpress the enzymes involved inthe reductive pathway. Additionally, the enzymesinvolved in the glyoxylate pathway may be overex-pressed leading to the accumulation of succinateand malate which can then be converted to fumaricacid by succinate dehydrogenase and fumaraserespectively.
Gene knockout and silencing
Gene knockout and gene silencing have becomeimportant tools for genetic engineering. During geneknockout, the DNA is modified deliberately to removethe entire or part of the gene whereas gene silencing isa natural process where the expression is reduced. Theinterlinked nature of metabolic pathways, as repre-sented in Figure 3(a), can lead to the production ofmultiple byproducts in addition to the compound ofinterest. For example, during the synthesis of fumaratefrom the glucose, byproducts, such as ethanol, lacticacid, and glycerol, are also synthesized. This leads tothe loss or redirection of some of the glucose in theculture towards the synthesis of these byproducts. Theinactivation of the genes responsible for the synthesisof the enzymes, or the prevention of their synthesis,involved in the production of these byproducts canlead to the redirection of the metabolic pathwaytowards the synthesis of only the desired product, asdepicted in Figure 3(b).
For example, the inactivation of enzymes pyruvatedecarboxylase and lactate dehydrogenase can preventthe formation of the byproducts ethanol and lactate,respectively. This redirects the metabolic pathwaytowards fumarate synthesis. The inactivation of theenzymes involved can be achieved by the use oftechniques such as gene knockout or RNAi (RNA inter-ference) [84]. Gheinani et al. [85] were able to suc-cessfully silence the ldhA and ldhB genes of R. oryzaewith siRNA technology, the most commonly used RNAinterference (RNAi) tool and redirects metabolic activ-ity towards ethanol synthesis. Similar deactivationof the lactate dehydrogenase gene of an anaerobicgut fungus, Pecoramyces ruminantium strain C1Awas performed and reduced lactate production wasobserved [86].
Possible metabolic pathway modifications inR. oryzae
The modification of the metabolic pathway of E. coliby activation of the glyoxylate pathway has proved tobe successful in enabling the transformed strain toaccumulate FA [26,27]. Hence, this re-direction of themetabolic pathway in conjunction with the inherentreductive TCA cycle can be introduced in R. oryzae toenhance the production of FA. This possibility oftransformation has not been reported and the redir-ected metabolic pathway is represented in Figure 4.This redirection via the glyoxylate pathway hasthe potential to enhance FA accumulation in this fun-gal strain. It has been reported that the glyoxylate
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pathway is required for fungal virulence [87]. Hence, acomprehensive understanding of the underlying riskof enhanced fungal virulence, if any, needs to bestudied extensively before any commercial applicationof the transformed strain is considered.
Additionally, metabolic modifications similar to thatdescribed by Chen et al. [66,72] can be investigatedwith R. oryzae. These modifications have been able to
increase FA titers in T. glabrata. Hence, the introduc-tion of metabolic enzymes involved in nitrogen andpurine metabolism are that carbon metabolism canbe considered to improve fumarate yield. Moreover,the introduction of an effective C4-dicarboxylicacid transporter, such as SpMAE1 and DcuB, that hasbeen shown to significantly improve fumarate produc-tion can be considered [66]. The acid tolerance of
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Figure 3. (a) Metabolic pathways involved in the production of fumaric acid and byproducts and (b) reduction of byproduct for-mation by gene inactivation (represented in red) to obtain a high fumaric acid titer. gdp—Glyceraldehyde-3-phosphate dehydro-genase; ldh—lactate dehydrogenase, and pdc—pyruvate decarboxylase. For color version of the figure refer e-print.
CRITICAL REVIEWS IN BIOTECHNOLOGY 13
T. glabrata, as well as fumarate production, wasimproved by the deletion of the gene that codes foradenylosuccinate synthase (ade12). Similarly, themodification can be studied to improve the acid toler-ance of R. oryzae. This modification will have a dra-matic impact on the feasibility of commercial FAbioproduction strategies as the cost associated withthe use of the neutralizing agents as well as therecovery of FA from the fumarate complex formed bythe use of a neutralizing agent.
In situ biosynthesis of fumarate esters
The derivative of the fumaric acid, fumaric acid ester(FAEs), has recently been identified as an importantderivative due to its diverse medical applications.Hence, in situ bioconversion of fumaric acid to its esterforms provided an attractive alternative for the greensynthesis of the esters. The current methods for indus-trial synthesis of esters are nonselective, tedious, andconsume considerable energy. Most often these com-mercial processes rely on direct chemical esterification.
This high-temperature inorganic catalyst mediatedesterification reactions convert organic acids to estersin the presence of alcohol [88]. Due to the drawbacksof chemical processes, such as non-selectivity and highenergy consumption, biosynthesis has been proposedas a suitable alternative.
Lipases are the most used enzymes for the biosyn-thesis of a diverse group of pharmaceutical com-pounds. These enzymes have excellent stability evenin the presence of organic solvents, hence, their appli-cation in the production of diverse biosynthetic com-pounds. This application of enzymes in the synthesisof a diverse array of compounds has been reviewedextensively by Carvalho et al. [89]. R. oryzae is aknown producer of lipases and widely used for indus-trial applications [90,91]. Hence, it would be possibleto bioconvert FA produced to FAEs by inducing lipasesynthesis either via genetic modification or inductionof their synthesis. The alcohol needed for conversionof FA to FAEs may be provided externally. Ethanolproduction during FA production fermentation hasbeen reported [40]. The induction of this ethanol
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Figure 4. Possible fumaric acid production via the introduction of the glyoxylate pathway (highlighted in blue) in conjunctionwith reductive TCA pathway (highlighted in blue) in R. oryzae. The various enzymes involved in the metabolic pathway havebeen represented in numbers. 1. Isocitrate lyase (EC 4.1.3.1); 2. malate synthase (EC 2.3.3.9); 3. pyruvate carboxylase (EC 6.4.1.1);4. malate dehydrogenase (EC 1.1.1.37); 5. fumarase (EC 4.2.1.2); 6. pyruvate dehydrogenase (EC 1.2.4.1); 7. citrate synthase (EC2.3.3.1); 8. aconitase (EC 4.2.1.3); 9. isocitrate dehydrogenase (EC 1.1.1.42); 10. 2-oxoglutarate dehydrogenase (EC 1.2.4.2); 11. suc-cinyl CoA synthase (EC 6.2.1.5); and 12. succinate dehydrogenase (complex III) (EC 1.3.99.1). For color version of the figure refere-print.
14 J. SEBASTIAN ET AL.
synthesis would not be recommended as the diversionof sugars to ethanol synthesis can lead to reducedFA production.
Conclusion
The depletion of the fossil fuels, their impact on theenvironment and the emphasis on green chemistryhas necessitated the production of microorganismmediated synthesis of platform chemicals. This shiftallows for the use of the renewable substrate as wellas industrial and domestic residues as feedstocksfor the manufacture of biochemicals. Due to the lowprice of commodity chemicals, such as fumaric acid,the main factors that determine economic viability isthe costs of substrate and downstream processing.These issues can be overcome by the use of biomassor organic residues as feedstocks for synthesis aswell as by employing efficient downstream processingstrategies. Hence, the main factor the determinesfeasibility is the product yield. Maximum product yieldis vital to lower manufacturing costs and to make bio-chemical production competitive against fossil fuel-based production.
Fumaric acid has been identified as one of the keybiobased platform chemicals that have diverse appli-cations. Studies to produce this dicarboxylic acidhave yielded high titers, especially by the fungus R.oryzae, but these high titers obtained are still lowerthan the theoretical expected yield. Moreover, thecomplications associated with the use of this fungalstrain have necessitated the development of an alter-native means for producing this acid. The advancesin metabolic engineering tools have allowed for thedevelopment of novel microorganisms capable toyield higher fumaric concentration compared to theparental strain. Some of these metabolic engineeringstrategies that have been investigated and can beadopted for improving FA yield has been discussed inthis review. The concentrations achieved are stilllower than those obtained by overproducing strainsof R. oryzae obtained from culture collections. Hence,more research particularly the optimization of themetabolic engineering strategies is essential to obtainhigher titers to make bioproduction feasible. Thisis particularly true of the redirection of metabolicpathways via the introduction of modifications thatenable in situ conversion of fumaric acid to its deriva-tives, such as fumarate esters and aspartic acid. Thiscan potentially improve the feasibility of the biopro-duction process.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The authors are sincerely thankful for the financial supportof the Minist�ere des Q12Relations Internationales du Qu�ebec(coop�eration Paran�a-Qu�ebec 2010–2012) and the NaturalSciences and Engineering Research Council of Canada(NSERC) Discovery Grant.
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18 J. SEBASTIAN ET AL.