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Microbial production of amino acids and derived chemicals: Synthetic biology approaches to strain development
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ScienceDirect Microbial production of amino acids and derived chemicals: Synthetic biology approaches to strain development Volker F Wendisch Amino acids are produced at the multi-million-ton-scale with fermentative production of L-glutamate and L-lysine alone being estimated to amount to more than five million tons in the year 2013. Metabolic engineering constantly improves productivities of amino acid producing strains, mainly Corynebacterium glutamicum and Escherichia coli strains. Classical mutagenesis and screening have been accelerated by combination with intracellular metabolite sensing. Synthetic biology approaches have allowed access to new carbon sources to realize a flexible feedstock concept. Moreover, new pathways for amino acid production as well as fermentative production of non-native compounds derived from amino acids or their metabolic precursors were developed. These include dipeptides, a,v-diamines, a,v-diacids, keto acids, acetylated amino acids and v-amino acids. Addresses Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Germany Corresponding author: Wendisch, Volker F ([email protected])
Current Opinion in Biotechnology 2014, 30:51–58 This review comes from a themed issue on Chemical biotechnology Edited by Curt R Fischer and Steffen Schaffer
http://dx.doi.org/10.1016/j.copbio.2014.05.004 0958-1669/# 2014 Published by Elsevier Ltd. All rights reserved.
Introduction L-Amino
acids find various applications in food and feed biotechnology as well as intermediates for the chemical industry [1]. Essential L-amino acids are used in human parenteral nutrition and L-glutamate and its salts are used as flavor enhancers in the food industry [1,2]. Fermentative production of L-amino acids in the million-ton-scale has shaped modern biotechnology [2]. The growth of the amino acid market, which is due a growing world population and a higher demand of animal products, drive strain optimization and amino acid process intensification.
Amino acid producing strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in www.sciencedirect.com
food biotechnology, and of Escherichia coli are continuously improved using metabolic engineering approaches [1,2]. This review on metabolic engineering of amino acid production highlights new methods, new amino acid biosynthesis pathways, biotin-prototroph recombinant C. glutamicum and access to alternative carbon sources. Moreover, products derived from amino acids or their precursors will be described. For production of non-amino acid derived products such as carotenoids [3,4], polymers [5], fuels [6] organic acids such as glycolate [7], the reader is referred to recent reviews, e.g. [8,9,10,11,12].
Strain improvement by classical mutagenesis combined with metabolite sensors C. glutamicum, the workhorse of fermentative amino acid production, has been isolated as L-glutamate-secreting bacterium in auxanographic plate assays using L-glutamate requiring indicator strains [2]. Similarly, an acidophilic Pantoea ananatis strain resistant to high L-glutamate concentrations was isolated and shown to produce Lglutamate to saturating concentrations allowing product recovery by crystallization [13]. On the basis of transcriptional regulatory proteins and their metabolite binding capacities sensor strains have been developed, e.g. high intracellular L-lysine concentrations are sensed by transcriptional activator LysG and the lysE promoter is activated and sensor cells fluoresce due to transcriptional coupling of the lysE promoter to a fluorescent protein gene [14]. After chemical mutagenesis and FACS sorting mutants with high intracellular L-lysine levels could be isolated, some of which also excreted L-lysine [14]. On the basis of the transcriptional regulator Lrp [15], this principle was used to isolate branched-chain amino acid producing mutants [16] (Figure 1). Sensor strains for detection of O-acetyl serine and O-acetyl homoserine, of serine and of arginine have also been developed [14]. Even indirect coupling to transcriptional regulation, e.g. activation of E. coli SoxR by increasing cellular NADPH demand, helped screening for variants of NADPH-dependent enzymes [17,18].
Metabolic engineering to improve L-lysine production and to access alternative carbon sources New variants of aspartokinase beneficial for L-lysine production were identified either based on screening mutant libraries [19] or by rational deregulation of allosteric inhibition guided by co-evolutionary analysis [20]. Current Opinion in Biotechnology 2014, 30:51–58
52 Chemical biotechnology
Figure 1
(a)
(b)
pSensor lrp
negative control
H2 μm
eYFP
– + Lrp
Lrp lrp
– +
brnF
L-leucine, L-isoleucine, L-methionine or L-valine
brnE
BrnFE
+ L-methionine
Current Opinion in Biotechnology
LRP-based fluorescent metabolite sensor. (a) C. glutamicum WT transformed with a sensor plasmid encoding transcriptional regulator Lrp and a eYFP gene driven by the promoter of the brnFE operon [16]. Plasmid encoded genes are given in black, chromosomal genes are given in gray. Lrp is activated by binding to L-methionine or branched-chain amino acids (depicted as pentagons) and represses transcription of its own gene and while activating transcription of the chromosomal brnFE operon [15] or the eYFP gene on the sensor plasmid. L-Methionine and branched-chain amino acids are exported by two-component transport system BrnFE. (b) Phase contrast and fluorescence microscopy images of C. glutamicum WT cells transformed with the sensor plasmid during feeding with L-lysyl-L-alanine dipeptide (upper panel) or with L-alanyl-L-methionine dipeptide (lower panel). Figure (b) is modified from [16] and the photograph was kindly provided by Julia Frunzke, Ju¨lich.
The latter approach was also applied to dihydrodipicolinate synthase and phosphoenolpyruvate carboxykinase, which is subject to complex genetic regulation [21,22,23]. Improving C. glutamicum strains mainly focused L-lysine production from glucose, however, L-lysine production, e.g. from crude glycerol [24], starch [25,26], glucans [27], lignocellulosics [28,29,30], grass juice [31] or components of chitin [32,33] has also been described. Unlike E. coli and S. cerevisiae, C. glutamicum typically does not show sequential carbon source utilization [34], but simultaneous utilization of carbon sources present in blends [35]. This advantage of C. glutamicum prevails even when exogenous carbon utilization pathways were introduced, e.g. for coutilization of glucose with glycerol [36], xylose and arabinose [28], or of cellobiose with xylose and glucose [37].
Metabolic engineering of L-ornithine cyclodeaminase-based L-proline production L-Proline,
the only proteinogenic amino acid with a secondary amine, is used as feed additive, organocatalyst and chemical synthon [1,2]. Besides extraction from protein hydrolysates, fermentative L-proline production is known. L-Proline is synthesized from L-glutamate by gglutamyl kinase, g-glutamyl phosphate reductase, spontaneous cyclization and pyrroline-5-carboxylate reductase (Figure 2), but can also be synthesized from L-ornithine in plants, animals, and some bacteria (Figure 2). Classical strain development targeted proline degradation and/or feedback-deregulation of g-glutamyl kinase [1,2].
Current Opinion in Biotechnology 2014, 30:51–58
Recently, production of about 13 g/L L-proline with a yield of 0.36 g L-proline per g glucose via the L-ornithine pathway was achieved [38]. This required plasmid-borne expression of the ornithine cyclodeaminase gene from Pseudomonas putida for conversion of L-ornithine to Lproline and ammonia. Moreover, conversion of Lornithine toward L-arginine was blocked by deletion of argF, the arg operon was derepressed by deletion of Larginine repressor gene argR and a feedback-deregulated N-acetylglutamate kinase was produced [38]. Overcoming biotin auxotrophy of C. glutamicum
C. glutamicum is auxotrophic for biotin. Biotin limitation elicits L-glutamate production while production of L-lysine requires sufficient biotin [1]. C. glutamicum takes up biotin by the energy-coupling factor transporter BioYMN [39] and deletion of bioY caused biotin hyperauxotrophy [40] (Figure 3). C. glutamicum possesses only two biotinylated proteins, pyruvate carboxylase and acetyl-CoA carboxylase, which are biotinylated by biotin-protein ligase BirA [41] (Figure 3). Surprisingly, biotin auxotrophic C. glutamicum possesses functional biotin transcriptional regulator BioQ [42] and BioA, BioD, and BioB, the enzymes for the final reactions of biotin synthesis [2]. C. glutamicum lacks bioF (Figure 3) and heterologous expression of bioF from E. coli allowed for biotin synthesis only from externally added pimelic acid suggesting its inability for de novo synthesis of pimelate thioester as biotin precursor [43]. In the E. coli BioC–BioH pathway, BioC methylates malonyl-CoA followed by two elongation cycles in fatty acid www.sciencedirect.com
Amino acids and derived products Wendisch 53
Figure 2
NADPH + H+
NADP+
2-oxoglutarate
Acetyl-CoA
L-glutamate NH3+
CoA
N-acetylglutamate
argA
gdh
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ATP argB
proB ADP
ADP
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N-acetylglutamyl phosphate
NADPH + H+
NADPH + H+ argC
proA NADP+ + Pi
NADP+ + Pi
glutamate–γ– semialdehyde
N-acetylglutamyl semialdehyde glutamate argD
spontaneous
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Δ1-pyrroline-5-carboxylate
NADPH + H+
N-acetylornithine
L-ornithine argJ
proC ocd
NADP+
L-proline Current Opinion in Biotechnology
Ornithine cyclodeaminase-based L-proline biosynthesis as compared to endogenous L-proline biosynthesis in Corynebacterium glutamicum. Reactions and enzymes of the proline and ornithine biosynthetic pathways of C. glutamicum are depicted together with the reaction catalyzed by ornithine cyclodeaminase (encoded by ocd): glutamate dehydrogenase (encoded by gdh), g-glutamyl kinase (encoded by proB), g-glutamyl phosphate reductase (encoded by proA), pyrroline-5-carboxylate reductase (encoded by proC), N-acetylglutamate synthase (encoded by cg3035/argA [83]), Nacetylglutamate kinase (encoded by argB), N-acetyl-g-glutamyl-phosphate reductase (encoded by argC), acetylornithine aminotransferase (encoded by argD) and ornithine acetyltransferase (encoded by argJ). Modified from Jensen and Wendisch [38].
synthesis and BioH demethylates methyl-pimeloyl-ACP to pimeloyl-ACP [44]. However, when bioC and bioH from E. coli were expressed in addition to bioF, C. glutamicum remained biotin auxotrophic [43]. The nonfunctionality of the BioC–BioH pathway from E. coli was likely not due to toxic methanol formed by hydrolysis of the methylpimeloyl-ACP ester since C. glutamicum possesses enzymes to degrade toxic methanol and formaldehyde [45,46]. Rather, the BioC–BioH pathway might be incompatible to type-I fatty acid synthase complex (FAS-I) of C. glutamicum. Unlike type-II fatty acid synthase (FAS-II) from E. coli, FAS-I from C. glutamicum may not accept methylmalonyl-CoA as substrate and/or BioH may not gain access to methyl-pimeloyl-ACP in FAS-I from C. glutamicum to liberate pimeloyl-ACP. www.sciencedirect.com
However, biotin-prototrophic C. glutamicum was obtained based on the BioI pathway [40,43] (Figure 3). BioI of Bacillus subtilis yields pimeloyl-ACP by oxidative carboncarbon bond cleavage of an acyl-ACP intermediate of fatty acid biosynthesis [47]. Expression of bioWAFDBI from B. subtilis [43] allowed for stable growth for at least eight serial transfers to biotin-depleted medium while the parental strain stopped growth after the first transfer. Since BioI from B. subtilis complements mutations in either bioC or bioH of E. coli [48] and rendered C. glutamicum biotin prototrophic, BioI appears compatible with both, FAS-I and FASII. Expression of bioWAFDBI from B. subtilis enabled production of the amino acids L-lysine and L-arginine, the Current Opinion in Biotechnology 2014, 30:51–58
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Figure 3
fatty acids
malonyl-CoA
FAS
operating costs in million-ton-scale amino acid production processes such as for media components immediately increases competitiveness and, thus, biotin prototrophy provides an excellent means to improve a wide range of biotechnological processes with C. glutamicum. Products derived from amino acids
O2 bioI octanoic acid
pimeloyl-ACP alanine bioF CoA + H20 7-keto-8-aminopelargonic acid SAM
bioA
AMTOB
7,8-diaminopelargonic acid ATP + CO2
bioD
ADP + Pi
dethiobiotin
Decarboxylation and deamination of amino acids may yield valuable products such as diamines or carboxylates [49]. Glutamate can be deaminated to 2-ketoglutarate [50] or decarboxylated to g-amino-butyrate (GABA), which can be used in pharmaceuticals, functional foods or in polyamide 4. Expression of glutamate decarboxylase genes from E. coli or lactobacilli in glutamate producing C. glutamicum strains was sufficient to enable production of GABA [51,52]. About 12 g/L GABA and about 1 g/L glutamate were produced from 50 g/L glucose in 72 h [51], however, GABA levels decreased upon longer cultivation. Deletion of the endogenous uptake genes improved GABA production [53]. Avoiding glutamate as by-product and degradation of GABA pose challenges for future process development.
ATP ADP+ Pi
bioB
2 desoxyadenosine + 2 methionine
bioYMN biotin
[S] + 2 SAM
biotin birA
biotindependentprotein + ATP AMP+ PPi
biotinylated protein Current Opinion in Biotechnology
Schematic representation of incomplete biotin biosynthesis in C. glutamicum WT (thin arrows) with exogenous bioI and bioF encoded enzyme reactions added to render C. glutamicum biotin prototrophic (fat arrows, rectangular boxes) [40,43]. Absence of biotin uptake system BioYMN (gray oval and arrow) [39] from C. glutamicum WT by deletion of bioY results in biotin hyperauxotrophy [40]. Biotin protein ligase BirA biotinylates two C. glutamicum proteins: pyruvate carboxylase and acetyl-CoA carboxylase [41]. The type-I fatty acid synthase complex (FAS) of C. glutamicum is depicted as tube. Gene names of enzymes are given: Biotin protein ligase (birA), biotin uptake system (bioYMN), biotin synthetase (bioB), dethiobiotin synthetase (bioD), 7,8-diaminopelargonic acid aminotransferase (bioA), 7-keto-8-aminopelargonic acid synthase (bioF), cytochrome P450 hydroxylase (bioI). Abbreviations: AMTOB, Sadenosyl-4-methylthio-2-oxobutanoate; FAS, type-I fatty acid synthase complex; SAH, S-adenosyl-homocysteine; SAM-S-adenosyl-Lmethionine. Modified from [43].
diamine putrescine, and the carotenoid lycopene, respectively, by recombinant C. glutamicum under biotin-depleted conditions [43]. As expected, the biotin-prototrophic recombinants did not excrete L-glutamate, but it remains to be tested if triggers other than biotin limitation elicit Lglutamate production. Future work needs to define a minimal set of heterologous bio genes required for biotin prototrophy, possibly just bioI and bioF, and to integrate these into the corynebacterial genome. Reducing Current Opinion in Biotechnology 2014, 30:51–58
The polyamide 5 precursor 5-aminovalerate can be derived from L-lysine in two reactions involving lysine 2-monooxygenase and D-aminovaleramidase. Expression of the Pseudomonas putida davAB in E. coli entailed production of 5-aminovalerate. In order to produce the alpha-omega diacid glutaric acid, two additional genes, davDT or gabTD, encoding 5-aminovalerate transaminase and glutarate semialdehyde dehydrogenase, were overexpressed and the aminoreceptor of the 5-aminovalerate transaminase reaction, alpha-ketoglutarate, was added externally in stoichiometric concentrations [54,55]. Thus, both precursors for PA5,5 were produced simultaneously. The diamine putrescine or 1,4-diaminobutane can be derived from L-ornithine by decarboxylation [49] and is used in polyamides such as PA4,10 or PA4,6. Introduction of ornithine decarboxylase from E. coli into an L-ornithine producing C. glutamicum strain enabled putrescine production [56]. Since E. coli, unlike C. glutamicum, is able to degrade putrescine E. coli-based putrescine production required deletion of genes for putrescine degradation [57]. The C. glutamicum system was developed further with the highest putrescine yield (about 0.26 g/g) reported in bacteria [58]. Tuning expression of ornithine transcarbamoylase gene argF over 1000 fold through modulation of transcription and translation efficiencies was key to balance low-level ornithine transcarbamoylase for obtaining high productivity and high titer. Moreover, expression of argF and ornithine decarboxylase gene speC from an addiction plasmid obviated addition of antibiotics to the fermentation broth at titers of about 19 g/L and volumetric productivities of about 0.6 g L 1 h 1 [58]. Putrescine was also produced from alternative carbon sources such as crude glycerol [24], hemicellulosic www.sciencedirect.com
Amino acids and derived products Wendisch 55
hydrolysates [29] or amino sugars [32,33] and by a biotinprototrophic putrescine producing strain [43]. The diamine cadaverine or 1,5-diaminopentane is used as monomer for polyamides. Its fermentative production by direct decarboxylation of lysine was first described by Mimitsuka and colleagues who replaced the endogenous homoserine dehydrogenase gene hom by the lysine decarboxylase gene cadA from E. coli and achieved production of 2.6 g/L cadaverine from glucose in 18 hours [59]. Subsequent export engineering and elimination of the acetylated by-product led to titers of about 2 g/L [11]. Cadaverine could also be produced from starch, xylose and cellubiose [26,60,61]. E. coli-based cadaverine production required inactivating cadaverine degradation and led to titers of about 9 g/L [62]. Amino acids can be deaminated to enoic acids [63], e.g. in the well-known example of aspartate ammonia lyase converting L-aspartate to fumarate. Similarly, trans-cinnamate, trans-p-hydroxycinnamate and urocanate, respectively, arise from deamination of L-phenylalanine, L-tyrosine and L-histidine, respectively. Conversion of aamino acids to b-amino acids by aminomutases involves the same cofactor 4-methylideneimidazole-5-one [63]. Enantiomerically pure a-amino acids and b-amino acids were prepared in enzymatic processes with L-phenylalanine and L-tyrosine aminomutases [64]. While biotransformation of fumarate to aspartic acid is a commercial process [65], fermentative processes using aminomutases or ammonia lyases have yet to be developed. L-Glutamate
was acetylated to N-acetyl-glutamate using four different recombinant E. coli strains overproducing one thermostable enzymes each [66]. Thus, at high temperature N-acetylglutamate production from pyruvate and 2-ketoglutarate was possible in a cofactorbalanced and CoA-recycling synthetic pathway combining pyruvate decarboxylase from Acetobacter pasteurianus, CoA-acylating aldehyde dehydrogenase from Thermus thermophiles, glutamate dehydrogenase from T. thermophilus and N-acetylglutamate synthase from Thermotoga maritima [66].
Dipeptides have various functions, e.g. L-glutamyl-Lthreonine or L-leucyl-L-serine enhances the umami taste of L-glutamate, L-arginyl-L-phenylalanine and L-glutamyl-L-tryptophan show antiangiogenic properties [67]. Dipeptides may be obtained by ligation of an amino acid to a second amino acid or an aminoacylester [68]. L-Amino acid ligase, which condenses unprotected amino acids in an ATP-dependent manner, is used for fermentative production of dipeptides [67,68]. Ligation of an amino acid to a N-methyl aminoacylester does not require ATP. About 70 g/L L-alanyl-L-glutamine were produced from L-alanine methyl ester hydrochloride and L-glutamine in www.sciencedirect.com
40 min by a whole cell biotransformation using E. coli overproducing a-amino acid ester acyltransferase from Sphingobacterium siyangensis [69]. Products derived from amino acid precursors
The L-glutamate precursor 2-ketoglutarate is used in dairy industry and to treat chronic renal insufficiency. Glutamate can be deaminated to 2-ketoglutarate, e.g. by whole cell biotransformation with B. subtilis overproducing L-amino acid deaminase from Proteus mirabilis [70]. Fermentative production of 2-ketoglutarate required deleting three genes involved in 2-ketoglutarate conversion (gdh, gltB, and aceA for glutamate dehydrogenase, glutamate synthase and isocitrate lyase, respectively) in Lglutamate overproducing C. glutamicum [71]. A fed-batch process resulted in about 50 g/L of 2-ketoglutarate [72]. The immediate precursor of L-valine, 2-ketoisovalerate (KIV) is used as a substitute for L-valine or L-leucine in parenteral nutrition of chronic kidney disease patients. Deletion of transaminase gene ilvE and overexpression of the ilv genes resulted in KIV production. Increased pyruvate supply in the absence of pyruvate dehydrogenase subunit AceE and pyruvate quinone oxidoreductase Pqo led to about 22 g/L KIV and a productivity of about 0.5 g L 1 h 1 [73]. Following the approach developed by Liao and colleagues [73], KIV could be efficiently converted to isobutanol in aerobic and anaerobic processes [6,74,75]. The immediate precursor of L-leucine, 2-ketoisocapraote (KIC) finds application in infusion solutions for patients with kidney or hepatic diseases and in functional foods for muscle regeneration. C. glutamicum was engineered for production of KIC by abrogation of the conversion of KIC to L-leucine by deletion of transaminase gene ilvE, overexpression of the ilv genes, derepression of the leu genes by deletion of transcriptional regulatory gene ltbR and low levels of citrate synthase [76]. A heterologous feedbackresistant isopropylmalate synthase variant from E. coli boosted KIC production to about 7 g/L from 40 g/l glucose in 24 h [76]. Similarly, an L-leucine overproducing strain was developed [77]. The KIC producing strain has potential to be used for production of 3-methyl-1-butanol, a potential future biofuel [78].
Conclusions Amino acid production is a success story of biotechnology and the industry still shows robust growth. Scientific developments such as synthetic and systems biology have not only led to improved amino acid processes, but have also enabled strain development for novel compounds. This trend gained additional impetus, e.g. by generating prophage-free C. glutamicum strains [79] or by developing multi-use platform strains [80] and is complemented by furthering genomic methodology, e.g. by RNAseq analysis of C. glutamicum [81,82]. We are looking forward Current Opinion in Biotechnology 2014, 30:51–58
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Acknowledgements
14. Binder S, Schendzielorz G, Stabler N, Krumbach K, Hoffmann K, Bott M, Eggeling LA: high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell F level. Genome Biol 2012, 13:R40. Together with Ref. [15] construction, validation and application of fluorescent amino acid sensors for faster single-cell FACS-assisted classical strain optimization by mutagenesis and screening are described.
Work in the lab of VFW is supported in part by PROMYSE (EU, FP7 project 289540), by a ZIM project (KF2969003SB2), and by BMBF projects SysEnCor (0315598E) and Genome reduction (0316017A) and by SynMet, a 09-EuroSYNBIO-FP-023 project, co-funded by DFG through grant no. WE 2320/2-1.
15. Lange C, Mustafi N, Frunzke J, Kennerknecht N, Wessel M, Bott M, Wendisch VF: Lrp of Corynebacterium glutamicum controls expression of the brnFE operon encoding the export system for L-methionine and branched-chain amino acids. J Biotechnol 2012, 158:231-241.
References and recommended reading
16. Mustafi N, Grunberger A, Kohlheyer D, Bott M, Frunzke J: The development application of a single-cell biosensor for the detection of L-methionine branched-chain amino acids. Metab Eng 2012, 14:449-457. See annotation to Ref. [14].
to see more and more examples of transfer of biotechnological production processes of amino acids and derived products from proof-of-concept to economic realization.
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Wendisch VF: Amino Acid Biosynthesis — Pathways, Regulation and Metabolic Engineering. Heidelberg, Germany: Springer; 2007, .
2.
Eggeling L, Bott M: Handbook of Corynebacterium glutamicum. Boca Raton, USA: CRC Press; 2005, .
3.
Heider SA, Peters-Wendisch P, Netzer R, Stafnes M, Brautaset T, Wendisch VF: Production and glucosylation of C50 and C 40 carotenoids by metabolically engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol 2014, 98:1223-1235.
4.
Heider SA, Peters-Wendisch P, Wendisch VF: Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum. BMC Microbiol 2012, 12:198.
5.
Song Y, Matsumoto K, Tanaka T, Kondo A, Taguchi S: Single-step production of polyhydroxybutyrate from starch by using alpha-amylase cell-surface displaying system of Corynebacterium glutamicum. J Biosci Bioeng 2013, 115:12-14.
6.
Blombach B, Riester T, Wieschalka S, Ziert C, Youn JW, Wendisch VF, Eikmanns BJ: Corynebacterium glutamicum tailored for efficient isobutanol production. Appl Environ Microbiol 2011, 77:3300-3310.
7.
Zahoor A, Otten A, Wendisch VF: Metabolic engineering of Corynebacterium glutamicum for glycolate production. J Biotechnol 2014 http://dx.doi.org/10.1016/j.jbiotec.2013.12.020. (in press).
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Wieschalka S, Blombach B, EikmannsF B.J.: Engineering Corynebacterium glutamicum for the production of pyruvate. Appl Microbiol Biotechnol 2012, 94:449-459.
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10. Connor MR, Liao JC: Microbial production of advanced transportation fuels in non-natural hosts. Curr Opin Biotechnol 2009, 20:307-315.
17. Siedler S, Lindner SN, Bringer S, Wendisch VF, Bott M: Reductive whole-cell biotransformation with Corynebacterium glutamicum: improvement of NADPH generation from glucose by a cyclized pentose phosphate pathway using pfkA and gapA deletion mutants. Appl Microbiol Biotechnol 2013, 97:143152. 18. Siedler S, Schendzielorz G, Binder S, Eggeling L, Bringer S, Bott M: SoxR as a single-cell biosensor for NADPH-consuming enzymes in Escherichia coli. ACS Synth Biol 2013. 19. Schendzielorz G, Dippong M, Grunberger A, Kohlheyer D, Yoshida A, Binder S, Nishiyama C, Nishiyama M, Bott M, Eggeling L: Taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways. ACS Synth Biol 2013. 20. Chen Z, Meyer W, Rappert S, Sun J, Zeng AP: Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production. Appl Environ Microbiol 2011, 77:4352-4360. A cluster of interconnected residues of aspartokinase of C. glutamicum linking the inhibitor binding site with other regions of the protein have been identified by statistical coupling analysis of protein sequences. Introduction of mutation Q298G led to feedback desensitation of aspartokinase and to high level L-lysine production when introduced into the genome of C. glutamicum wild type. 21. Klaffl S, Brocker M, Kalinowski J, Eikmanns BJ, Bott M: Complex regulation of the phosphoenolpyruvate carboxykinase gene pck and characterization of its GntR-type regulator IolR as a repressor of myo-inositol utilization genes in Corynebacterium glutamicum. J Bacteriol 2013, 195:42834296. 22. Chen Z, Bommareddy RR, Frank D, Rappert S, Zeng AP: Deregulation of feedback inhibition of phosphoenolpyruvate carboxylase for improved lysine production in Corynebacterium glutamicum. Appl Environ Microbiol 2013. 23. Geng F, Chen Z, Zheng P, Sun J, Zeng AP: Exploring the allosteric mechanism of dihydrodipicolinate synthase by reverse engineering of the allosteric inhibitor binding sites and its application for lysine production. Appl Microbiol Biotechnol 2013, 97:1963-1971.
11. Becker J, Wittmann C: Bio-based production of chemicals, materials and fuels — Corynebacterium glutamicum as versatile cell factory. Curr Opin Biotechnol 2012, 23:631-640.
24. Meiswinkel TM, Rittmann D, Lindner SN, Wendisch VF: Crude glycerol-based production of amino acids and putrescine by Corynebacterium glutamicum. Bioresour Technol 2013, 145:254-258.
12. Heider SA, Peters-Wendisch P, Wendisch VF, Beekwilder J, Brautaset T: Metabolic engineering for the microbial production of carotenoids and related products with a focus on the rare C50 carotenoids. Appl Microbiol Biotechnol 2014, 98:4355-4368.
25. Seibold G, Auchter M, Berens S, Kalinowski J, Eikmanns BJ: Utilization of soluble starch by a recombinant Corynebacterium glutamicum strain: growth and lysine production. J Biotechnol 2006, 124:381-391.
13. Hara Y, Kadotani N, Izui H, Katashkina JI, Kuvaeva TM, Andreeva IG, Golubeva LI, Malko DB, Makeev VJ, Mashko SV et al.: The complete genome sequence of Pantoea ananatis AJ13355 an organism with great biotechnological F potential. Appl Microbiol Biotechnol 2012, 93:331-341. The genome of the low pH tolerant bacterium Pantoea ananatis that produces L-glutamate to saturating concentrations facilitating product recovery by crystallization was sequenced. Current Opinion in Biotechnology 2014, 30:51–58
26. Tateno T, Okada Y, Tsuchidate T, Tanaka T, Fukuda H, Kondo A: Direct production of cadaverine from soluble starch using Corynebacterium glutamicum coexpressing alpha-amylase and lysine decarboxylase. Appl Microbiol Biotechnol 2009, 82:115-121. 27. Adachi N, Takahashi C, Ono-Murota N, Yamaguchi R, Tanaka T, Kondo A: Direct L-lysine production from cellobiose by www.sciencedirect.com
Amino acids and derived products Wendisch 57
Corynebacterium glutamicum displaying beta-glucosidase on its cell surface. Appl Microbiol Biotechnol 2013, 97:7165-7172. 28. Gopinath V, Meiswinkel TM, Wendisch VF, Nampoothiri KM: Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing Corynebacterium glutamicum. Appl Microbiol Biotechnol 2011, 92:985-996. Recombinant C. glutamicum strains are shown to produce amino acids from biotechnologically relevant hemicellulosic hydrolysates for potential use in biorefineries. 29. Meiswinkel TM, Gopinath V, Lindner SN, Nampoothiri KM, Wendisch VF: Accelerated pentose utilization by Corynebacterium glutamicum for accelerated production of lysine, glutamate, ornithine and putrescine. Microb Biotechnol 2013, 6:131-140. 30. Schneider J, Niermann K, Wendisch VF: Production of the amino acids L-glutamate, L-lysine L-ornithine and L-arginine from arabinose by recombinant Corynebacterium glutamicum. J Biotechnol 2011, 154:191-198. 31. Neuner A, Wagner I, Sieker T, Ulber R, Schneider K, Peifer S, Heinzle E: Production of L-lysine on different silage juices using genetically engineered Corynebacterium glutamicum. J Biotechnol 2013, 163:217-224. 32. Uhde A, Youn JW, Maeda T, Clermont L, Matano C, Kramer R, Wendisch VF, Seibold GM, Marin K: Glucosamine as carbon source for amino acid-producing Corynebacterium glutamicum. Appl Microbiol Biotechnol 2013, 97:1679-1687. 33. Matano C, Uhde A, Youn JW, Maeda T, Clermont L, Marin K, Kramer R, Wendisch VF, Seibold GM: Engineering of Corynebacterium glutamicum for growth and L-lysine and lycopene production from N-acetyl-glucosamine. Appl Microbiol Biotechnol 2014 http://dx.doi.org/10.1007/s0025300014-05676-00259. 34. Blombach B, Seibold GM: Carbohydrate metabolism in Corynebacterium glutamicum and applications for the metabolic engineering of L-lysine production strains. Appl Microbiol Biotechnol 2010, 86:1313-1322.
growth and L-lysine production. Appl Microbiol Biotechnol 2012, 93:2493-2502. 42. Brune I, Gotker S, Schneider J, Rodionov DA, Tauch A: Negative transcriptional control of biotin metabolism genes by the TetR-type regulator BioQ in biotin-auxotrophic Corynebacterium glutamicum ATCC 13032. J Biotechnol 2012, 159:225-234. 43. Peters-Wendisch P, Gotker S, Heider SAE, Komati Reddy G, Nguyen AQ, Stansen KC, Wendisch VF: Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production. J Biotechnol 2014 http://dx.doi.org/10.1016/j.jbiotec.2014.01.023. (in press). See annotation to Ref. [40]. 44. Agarwal V, Lin S, Lukk T, Nair SK, Cronan JE: Structure of the enzyme-acyl carrier protein (ACP) substrate gatekeeper complex required for biotin synthesis. Proc Natl Acad Sci U S A 2012, 109:17406-17411. 45. Lessmeier L, Hoefener M, Wendisch VF: Formaldehyde degradation in Corynebacterium glutamicum involves acetaldehyde dehydrogenase and mycothiol-dependent formaldehyde dehydrogenase. Microbiology 2013, 159:2651-2662. 46. Witthoff S, Muhlroth A, Marienhagen J, Bott M: C1 Metabolism in Corynebacterium glutamicum: an endogenous pathway for oxidation of methanol to carbon dioxide. Appl Environ Microbiol 2013, 79:6974-6983. 47. Lin S, Hanson RE, Cronan JE: Biotin synthesis begins by hijacking the fatty acid synthetic pathway. Nat Chem Biol 2010, 6:682-688. 48. Bower S, Perkins JB, Yocum RR, Howitt CL, Rahaim P, Pero J: Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon. J Bacteriol 1996, 178:41224130. 49. Schneider J, Wendisch VF: Biotechnological production of polyamines by bacteria: recent achievements and future perspectives. Appl Microbiol Biotechnol 2011, 91:17-30.
35. Wendisch VF, de Graaf AA, Sahm H, Eikmanns BJ: Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose. J Bacteriol 2000, 182:3088-3096.
50. O¨dman P, Wellborn WB, Bommarius AS: An enzymatic process to 2-ketoglutarate from L-glutamate: the coupled system Lglutamate dehydrogenase/NADH oxidase. Tetrahedron Asymmetry 2004, 15:2933-2937.
36. Rittmann D, Lindner SN, Wendisch VF: Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum. Appl Environ Microbiol 2008, 74:6216-6222.
51. Takahashi C, Shirakawa J, Tsuchidate T, Okai N, Hatada K, Nakayama H, Tateno T, Ogino C, Kondo A: Robust production of gamma-amino butyric acid using recombinant Corynebacterium glutamicum expressing glutamate decarboxylase from Escherichia coli. Enzyme Microb Technol 2012, 51:171-176.
37. Sasaki M, Jojima T, Inui M, Yukawa H: Simultaneous utilization of D-cellobiose, D-glucose, and D-xylose by recombinant Corynebacterium glutamicum under oxygen-deprived conditions. Appl Microbiol Biotechnol 2008, 81:691-699. 38. Jensen JV, Wendisch VF: Ornithine cyclodeaminase-based proline production by Corynebacterium glutamicum. Microb Cell Fact 2013, 12:63. High-level L-proline production has been achieved by engineering a heterologous L-ornithine-based pathway for L-proline biosynthesis into an L-arginine overproducing background.
52. Shi F, Jiang J, Li Y, Xie Y: Enhancement of gamma-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis. J Ind Microbiol Biotechnol 2013, 40:1285-1296. 53. Zhao Z, Ding JY, Ma WH, Zhou NY, Liu SJ: Identification and characterization of gamma-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum. Appl Environ Microbiol 2012, 78:2596-2601.
39. Schneider J, Peters-Wendisch P, Stansen KC, Gotker S, Maximow S, Kramer R, Wendisch VF: Characterization of the biotin uptake system encoded by the biotin-inducible bioYMN operon of Corynebacterium glutamicum. BMC Microbiol 2012, 12:6.
54. Park SJ, Kim EY, Noh W, Park HM, Oh YH, Lee SH, Song BK, Jegal J, Lee SY: Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals. Metab Eng 2013, 16:42-47.
40. Ikeda M, Miyamoto A, Mutoh S, Kitano Y, Tajima M, Shirakura D, Takasaki M, Mitsuhashi S, Takeno S: Development of biotinprototrophic and -hyperauxotrophic Corynebacterium glutamicum strains. Appl Environ Microbiol 2013, 79:4586-4594. Together with Ref. [43], metabolic engineering of the naturally biotin auxotrophic C. glutamicum to biotin prototrophy and subsequent application to production of amino acids, carotenoids and diamines without the need to supplement production media with biotin is described.
55. Adkins J, Jordan J, Nielsen DR: Engineering Escherichia coli for renewable production of the 5-carbon polyamide buildingblocks 5-aminovalerate and glutarate. Biotechnol Bioeng 2013, 110:1726-1734.
41. Peters-Wendisch P, Stansen KC, Gotker S, Wendisch VF: Biotin protein ligase from Corynebacterium glutamicum: role for www.sciencedirect.com
56. Schneider J, Wendisch VF: Putrescine production by engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol 2010, 88:859-868. This paper together with Refs. [57,58] describes fermentative production of the diamine 1,4-diaminobutane (putrescine) and strain optimization. Current Opinion in Biotechnology 2014, 30:51–58
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57. Qian ZG, Xia XX, Lee SY: Metabolic engineering of Escherichia coli for the production of putrescine: a four carbon diamine. Biotechnol Bioeng 2009, 104:651-662. See annotation to Ref. [56].
71. Jo JH, Seol HY, Lee YB, Kim MH, Hyun HH, Lee HH: Disruption of genes for the enhanced biosynthesis of alpha-ketoglutarate in Corynebacterium glutamicum. Can J Microbiol 2012, 58:278-286.
58. Schneider J, Eberhardt D, Wendisch VF: Improving putrescine production by Corynebacterium glutamicum by fine-tuning ornithine transcarbamoylase activity using a plasmid addiction system. Appl Microbiol Biotechnol 2012, 95:169-178. See annotation to Ref. [56].
72. Lee YB, Jo JH, Kim MH, Lee HH, Hyun HH: Enhanced production of alpha-ketoglutarate by fed-batch culture in the metabolically engineered strains of Corynebacterium glutamicum. Biotechnol Bioprocess Eng 2013, 18:770-777.
59. Mimitsuka T, Sawai H, Hatsu M, Yamada K: Metabolic engineering of Corynebacterium glutamicum for cadaverine fermentation. Biosci Biotechnol Biochem 2007, 71:2130-2135. This is the first report on production of the diamine 1,5-diaminobutane (cadaverine) by recombinant C. glutamicum. 60. Ikeda N, Miyamoto M, Adachi N, Nakano M, Tanaka T, Kondo A: Direct cadaverine production from cellobiose using betaglucosidase displaying Escherichia coli. AMB Express 2013, 3:67. 61. Buschke N, Becker J, Schafer R, Kiefer P, Biedendieck R, Wittmann C: Systems metabolic engineering of xylose-utilizing Corynebacterium glutamicum for production of 1,5diaminopentane. Biotechnol J 2013, 8:557-570. 62. Qian ZG, Xia XX, Lee SY: Metabolic engineering of Escherichia coli for the production of cadaverine: a five carbon diamine. Biotechnol Bioeng 2011, 108:93-103. 63. Turner NJ: Ammonia lyases and aminomutases as biocatalysts for the synthesis of alpha-amino and beta-amino acids. Curr Opin Chem Biol 2011, 15:234-240. 64. Heberling MM, Wu B, Bartsch S, Janssen DB: Priming ammonia lyases and aminomutases for industrial and therapeutic applications. Curr Opin Chem Biol 2013, 17:250-260. 65. Schmid A, Hollmann F, Park JB, Buhler B: The use of enzymes in the chemical industry in Europe. Curr Opin Biotechnol 2002, 13:359-366. 66. Krutsakorn B, Imagawa T, Honda K, Okano K, Ohtake H: Construction of an in vitro bypassed pyruvate decarboxylation pathway using thermostable enzyme modules and its application to N-acetylglutamate production. Microb Cell Fact 2013, 12:91. 67. Arai T, Arimura Y, Ishikura S, Kino K: L-Amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides. Appl Environ Microbiol 2013, 79:5023-5029. 68. Yagasaki M, Hashimoto S: Synthesis and application of dipeptides; current status and perspectives. Appl Microbiol Biotechnol 2008, 81:13-22. 69. Hirao Y, Mihara Y, Kira I, Abe I, Yokozeki K: Enzymatic production of L-alanyl-L-glutamine by recombinant E. coli expressing alpha-amino acid ester acyltransferase from Sphingobacterium siyangensis. Biosci Biotechnol Biochem 2013, 77:618-623. 70. Hossain GS, Li J, Shin HD, Chen RR, Du G, Liu L, Chen J: Bioconversion of L-glutamic acid to alpha-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing L-amino acid deaminase from Proteus mirabilis. J Biotechnol 2014, 169:112-120.
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73. Krause FS, Blombach B, Eikmanns BJ: Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production. Appl Environ Microbiol 2010, 76:8053-8061. 74. Smith KM, Cho KM, Liao JC: Engineering Corynebacterium glutamicum for isobutanol production. Appl Microbiol Biotechnol 2010, 87:1045-1055. 75. Yamamoto S, Suda M, Niimi S, Inui M, Yukawa H: Strain optimization for efficient isobutanol production using Corynebacterium glutamicum under oxygen deprivation. Biotechnol Bioeng 2013, 110:2938-2948. 76. Buckle-Vallant V, Krause FS, Messerschmidt S, Eikmanns BJ: Metabolic engineering of Corynebacterium glutamicum for 2ketoisocaproate production. Appl Microbiol Biotechnol 2014, 98:297-311. This paper and Ref. [77] describe metabolic engineering of C. glutamicum for high-level production of L-leucine and its immediate precursor 2ketoisocaproate. 77. Vogt M, Haas S, Klaffl S, Polen T, Eggeling L, Ooyen JV, Bott M: Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction. Metab Eng 2013. See annotation to Ref. [76]. 78. Connor MR, Liao JC: Engineering of an Escherichia coli strain for the production of 3-methyl-1-butanol. Appl Environ Microbiol 2008, 74:5769-5775. 79. Baumgart M, Unthan S, Ruckert C, Sivalingam J, Grunberger A, Kalinowski J, Bott M, Noack S, Frunzke J: Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032 for use as a platform strain for basic research and industrial biotechnology. Appl Environ Microbiol 2013, 79:60066015. 80. Buchholz J, Schwentner A, Brunnenkan B, Gabris C, Grimm S, Gerstmeir R, Takors R, Eikmanns BJ, Blombach B: Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate. Appl Environ Microbiol 2013, 79:5566-5575. 81. Pfeifer-Sancar K, Mentz A, Ruckert C, Kalinowski J: Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique. BMC Genomics 2013, 14:888. 82. Mentz A, Neshat A, Pfeifer-Sancar K, Puhler A, Ruckert C, Kalinowski J: Comprehensive discovery and characterization of small RNAs in Corynebacterium glutamicum ATCC 13032. BMC Genomics 2013, 14:714. 83. Petri K, Walter F, Persicke M, Ruckert C, Kalinowski J: A novel type of N-acetylglutamate synthase is involved in the first step of arginine biosynthesis in Corynebacterium glutamicum. BMC Genomics 2013, 14:713.
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ScienceDirect Microbial production of amino acids and derived chemicals: Synthetic biology approaches to strain development Volker F Wendisch Amino acids are produced at the multi-million-ton-scale with fermentative production of L-glutamate and L-lysine alone being estimated to amount to more than five million tons in the year 2013. Metabolic engineering constantly improves productivities of amino acid producing strains, mainly Corynebacterium glutamicum and Escherichia coli strains. Classical mutagenesis and screening have been accelerated by combination with intracellular metabolite sensing. Synthetic biology approaches have allowed access to new carbon sources to realize a flexible feedstock concept. Moreover, new pathways for amino acid production as well as fermentative production of non-native compounds derived from amino acids or their metabolic precursors were developed. These include dipeptides, a,v-diamines, a,v-diacids, keto acids, acetylated amino acids and v-amino acids. Addresses Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Germany Corresponding author: Wendisch, Volker F ([email protected])
Current Opinion in Biotechnology 2014, 30:51–58 This review comes from a themed issue on Chemical biotechnology Edited by Curt R Fischer and Steffen Schaffer
http://dx.doi.org/10.1016/j.copbio.2014.05.004 0958-1669/# 2014 Published by Elsevier Ltd. All rights reserved.
Introduction L-Amino
acids find various applications in food and feed biotechnology as well as intermediates for the chemical industry [1]. Essential L-amino acids are used in human parenteral nutrition and L-glutamate and its salts are used as flavor enhancers in the food industry [1,2]. Fermentative production of L-amino acids in the million-ton-scale has shaped modern biotechnology [2]. The growth of the amino acid market, which is due a growing world population and a higher demand of animal products, drive strain optimization and amino acid process intensification.
Amino acid producing strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in www.sciencedirect.com
food biotechnology, and of Escherichia coli are continuously improved using metabolic engineering approaches [1,2]. This review on metabolic engineering of amino acid production highlights new methods, new amino acid biosynthesis pathways, biotin-prototroph recombinant C. glutamicum and access to alternative carbon sources. Moreover, products derived from amino acids or their precursors will be described. For production of non-amino acid derived products such as carotenoids [3,4], polymers [5], fuels [6] organic acids such as glycolate [7], the reader is referred to recent reviews, e.g. [8,9,10,11,12].
Strain improvement by classical mutagenesis combined with metabolite sensors C. glutamicum, the workhorse of fermentative amino acid production, has been isolated as L-glutamate-secreting bacterium in auxanographic plate assays using L-glutamate requiring indicator strains [2]. Similarly, an acidophilic Pantoea ananatis strain resistant to high L-glutamate concentrations was isolated and shown to produce Lglutamate to saturating concentrations allowing product recovery by crystallization [13]. On the basis of transcriptional regulatory proteins and their metabolite binding capacities sensor strains have been developed, e.g. high intracellular L-lysine concentrations are sensed by transcriptional activator LysG and the lysE promoter is activated and sensor cells fluoresce due to transcriptional coupling of the lysE promoter to a fluorescent protein gene [14]. After chemical mutagenesis and FACS sorting mutants with high intracellular L-lysine levels could be isolated, some of which also excreted L-lysine [14]. On the basis of the transcriptional regulator Lrp [15], this principle was used to isolate branched-chain amino acid producing mutants [16] (Figure 1). Sensor strains for detection of O-acetyl serine and O-acetyl homoserine, of serine and of arginine have also been developed [14]. Even indirect coupling to transcriptional regulation, e.g. activation of E. coli SoxR by increasing cellular NADPH demand, helped screening for variants of NADPH-dependent enzymes [17,18].
Metabolic engineering to improve L-lysine production and to access alternative carbon sources New variants of aspartokinase beneficial for L-lysine production were identified either based on screening mutant libraries [19] or by rational deregulation of allosteric inhibition guided by co-evolutionary analysis [20]. Current Opinion in Biotechnology 2014, 30:51–58
52 Chemical biotechnology
Figure 1
(a)
(b)
pSensor lrp
negative control
H2 μm
eYFP
– + Lrp
Lrp lrp
– +
brnF
L-leucine, L-isoleucine, L-methionine or L-valine
brnE
BrnFE
+ L-methionine
Current Opinion in Biotechnology
LRP-based fluorescent metabolite sensor. (a) C. glutamicum WT transformed with a sensor plasmid encoding transcriptional regulator Lrp and a eYFP gene driven by the promoter of the brnFE operon [16]. Plasmid encoded genes are given in black, chromosomal genes are given in gray. Lrp is activated by binding to L-methionine or branched-chain amino acids (depicted as pentagons) and represses transcription of its own gene and while activating transcription of the chromosomal brnFE operon [15] or the eYFP gene on the sensor plasmid. L-Methionine and branched-chain amino acids are exported by two-component transport system BrnFE. (b) Phase contrast and fluorescence microscopy images of C. glutamicum WT cells transformed with the sensor plasmid during feeding with L-lysyl-L-alanine dipeptide (upper panel) or with L-alanyl-L-methionine dipeptide (lower panel). Figure (b) is modified from [16] and the photograph was kindly provided by Julia Frunzke, Ju¨lich.
The latter approach was also applied to dihydrodipicolinate synthase and phosphoenolpyruvate carboxykinase, which is subject to complex genetic regulation [21,22,23]. Improving C. glutamicum strains mainly focused L-lysine production from glucose, however, L-lysine production, e.g. from crude glycerol [24], starch [25,26], glucans [27], lignocellulosics [28,29,30], grass juice [31] or components of chitin [32,33] has also been described. Unlike E. coli and S. cerevisiae, C. glutamicum typically does not show sequential carbon source utilization [34], but simultaneous utilization of carbon sources present in blends [35]. This advantage of C. glutamicum prevails even when exogenous carbon utilization pathways were introduced, e.g. for coutilization of glucose with glycerol [36], xylose and arabinose [28], or of cellobiose with xylose and glucose [37].
Metabolic engineering of L-ornithine cyclodeaminase-based L-proline production L-Proline,
the only proteinogenic amino acid with a secondary amine, is used as feed additive, organocatalyst and chemical synthon [1,2]. Besides extraction from protein hydrolysates, fermentative L-proline production is known. L-Proline is synthesized from L-glutamate by gglutamyl kinase, g-glutamyl phosphate reductase, spontaneous cyclization and pyrroline-5-carboxylate reductase (Figure 2), but can also be synthesized from L-ornithine in plants, animals, and some bacteria (Figure 2). Classical strain development targeted proline degradation and/or feedback-deregulation of g-glutamyl kinase [1,2].
Current Opinion in Biotechnology 2014, 30:51–58
Recently, production of about 13 g/L L-proline with a yield of 0.36 g L-proline per g glucose via the L-ornithine pathway was achieved [38]. This required plasmid-borne expression of the ornithine cyclodeaminase gene from Pseudomonas putida for conversion of L-ornithine to Lproline and ammonia. Moreover, conversion of Lornithine toward L-arginine was blocked by deletion of argF, the arg operon was derepressed by deletion of Larginine repressor gene argR and a feedback-deregulated N-acetylglutamate kinase was produced [38]. Overcoming biotin auxotrophy of C. glutamicum
C. glutamicum is auxotrophic for biotin. Biotin limitation elicits L-glutamate production while production of L-lysine requires sufficient biotin [1]. C. glutamicum takes up biotin by the energy-coupling factor transporter BioYMN [39] and deletion of bioY caused biotin hyperauxotrophy [40] (Figure 3). C. glutamicum possesses only two biotinylated proteins, pyruvate carboxylase and acetyl-CoA carboxylase, which are biotinylated by biotin-protein ligase BirA [41] (Figure 3). Surprisingly, biotin auxotrophic C. glutamicum possesses functional biotin transcriptional regulator BioQ [42] and BioA, BioD, and BioB, the enzymes for the final reactions of biotin synthesis [2]. C. glutamicum lacks bioF (Figure 3) and heterologous expression of bioF from E. coli allowed for biotin synthesis only from externally added pimelic acid suggesting its inability for de novo synthesis of pimelate thioester as biotin precursor [43]. In the E. coli BioC–BioH pathway, BioC methylates malonyl-CoA followed by two elongation cycles in fatty acid www.sciencedirect.com
Amino acids and derived products Wendisch 53
Figure 2
NADPH + H+
NADP+
2-oxoglutarate
Acetyl-CoA
L-glutamate NH3+
CoA
N-acetylglutamate
argA
gdh
ATP
ATP argB
proB ADP
ADP
γ-glutamyl phosphate
N-acetylglutamyl phosphate
NADPH + H+
NADPH + H+ argC
proA NADP+ + Pi
NADP+ + Pi
glutamate–γ– semialdehyde
N-acetylglutamyl semialdehyde glutamate argD
spontaneous
2-oxoglutarate
Δ1-pyrroline-5-carboxylate
NADPH + H+
N-acetylornithine
L-ornithine argJ
proC ocd
NADP+
L-proline Current Opinion in Biotechnology
Ornithine cyclodeaminase-based L-proline biosynthesis as compared to endogenous L-proline biosynthesis in Corynebacterium glutamicum. Reactions and enzymes of the proline and ornithine biosynthetic pathways of C. glutamicum are depicted together with the reaction catalyzed by ornithine cyclodeaminase (encoded by ocd): glutamate dehydrogenase (encoded by gdh), g-glutamyl kinase (encoded by proB), g-glutamyl phosphate reductase (encoded by proA), pyrroline-5-carboxylate reductase (encoded by proC), N-acetylglutamate synthase (encoded by cg3035/argA [83]), Nacetylglutamate kinase (encoded by argB), N-acetyl-g-glutamyl-phosphate reductase (encoded by argC), acetylornithine aminotransferase (encoded by argD) and ornithine acetyltransferase (encoded by argJ). Modified from Jensen and Wendisch [38].
synthesis and BioH demethylates methyl-pimeloyl-ACP to pimeloyl-ACP [44]. However, when bioC and bioH from E. coli were expressed in addition to bioF, C. glutamicum remained biotin auxotrophic [43]. The nonfunctionality of the BioC–BioH pathway from E. coli was likely not due to toxic methanol formed by hydrolysis of the methylpimeloyl-ACP ester since C. glutamicum possesses enzymes to degrade toxic methanol and formaldehyde [45,46]. Rather, the BioC–BioH pathway might be incompatible to type-I fatty acid synthase complex (FAS-I) of C. glutamicum. Unlike type-II fatty acid synthase (FAS-II) from E. coli, FAS-I from C. glutamicum may not accept methylmalonyl-CoA as substrate and/or BioH may not gain access to methyl-pimeloyl-ACP in FAS-I from C. glutamicum to liberate pimeloyl-ACP. www.sciencedirect.com
However, biotin-prototrophic C. glutamicum was obtained based on the BioI pathway [40,43] (Figure 3). BioI of Bacillus subtilis yields pimeloyl-ACP by oxidative carboncarbon bond cleavage of an acyl-ACP intermediate of fatty acid biosynthesis [47]. Expression of bioWAFDBI from B. subtilis [43] allowed for stable growth for at least eight serial transfers to biotin-depleted medium while the parental strain stopped growth after the first transfer. Since BioI from B. subtilis complements mutations in either bioC or bioH of E. coli [48] and rendered C. glutamicum biotin prototrophic, BioI appears compatible with both, FAS-I and FASII. Expression of bioWAFDBI from B. subtilis enabled production of the amino acids L-lysine and L-arginine, the Current Opinion in Biotechnology 2014, 30:51–58
54 Chemical biotechnology
Figure 3
fatty acids
malonyl-CoA
FAS
operating costs in million-ton-scale amino acid production processes such as for media components immediately increases competitiveness and, thus, biotin prototrophy provides an excellent means to improve a wide range of biotechnological processes with C. glutamicum. Products derived from amino acids
O2 bioI octanoic acid
pimeloyl-ACP alanine bioF CoA + H20 7-keto-8-aminopelargonic acid SAM
bioA
AMTOB
7,8-diaminopelargonic acid ATP + CO2
bioD
ADP + Pi
dethiobiotin
Decarboxylation and deamination of amino acids may yield valuable products such as diamines or carboxylates [49]. Glutamate can be deaminated to 2-ketoglutarate [50] or decarboxylated to g-amino-butyrate (GABA), which can be used in pharmaceuticals, functional foods or in polyamide 4. Expression of glutamate decarboxylase genes from E. coli or lactobacilli in glutamate producing C. glutamicum strains was sufficient to enable production of GABA [51,52]. About 12 g/L GABA and about 1 g/L glutamate were produced from 50 g/L glucose in 72 h [51], however, GABA levels decreased upon longer cultivation. Deletion of the endogenous uptake genes improved GABA production [53]. Avoiding glutamate as by-product and degradation of GABA pose challenges for future process development.
ATP ADP+ Pi
bioB
2 desoxyadenosine + 2 methionine
bioYMN biotin
[S] + 2 SAM
biotin birA
biotindependentprotein + ATP AMP+ PPi
biotinylated protein Current Opinion in Biotechnology
Schematic representation of incomplete biotin biosynthesis in C. glutamicum WT (thin arrows) with exogenous bioI and bioF encoded enzyme reactions added to render C. glutamicum biotin prototrophic (fat arrows, rectangular boxes) [40,43]. Absence of biotin uptake system BioYMN (gray oval and arrow) [39] from C. glutamicum WT by deletion of bioY results in biotin hyperauxotrophy [40]. Biotin protein ligase BirA biotinylates two C. glutamicum proteins: pyruvate carboxylase and acetyl-CoA carboxylase [41]. The type-I fatty acid synthase complex (FAS) of C. glutamicum is depicted as tube. Gene names of enzymes are given: Biotin protein ligase (birA), biotin uptake system (bioYMN), biotin synthetase (bioB), dethiobiotin synthetase (bioD), 7,8-diaminopelargonic acid aminotransferase (bioA), 7-keto-8-aminopelargonic acid synthase (bioF), cytochrome P450 hydroxylase (bioI). Abbreviations: AMTOB, Sadenosyl-4-methylthio-2-oxobutanoate; FAS, type-I fatty acid synthase complex; SAH, S-adenosyl-homocysteine; SAM-S-adenosyl-Lmethionine. Modified from [43].
diamine putrescine, and the carotenoid lycopene, respectively, by recombinant C. glutamicum under biotin-depleted conditions [43]. As expected, the biotin-prototrophic recombinants did not excrete L-glutamate, but it remains to be tested if triggers other than biotin limitation elicit Lglutamate production. Future work needs to define a minimal set of heterologous bio genes required for biotin prototrophy, possibly just bioI and bioF, and to integrate these into the corynebacterial genome. Reducing Current Opinion in Biotechnology 2014, 30:51–58
The polyamide 5 precursor 5-aminovalerate can be derived from L-lysine in two reactions involving lysine 2-monooxygenase and D-aminovaleramidase. Expression of the Pseudomonas putida davAB in E. coli entailed production of 5-aminovalerate. In order to produce the alpha-omega diacid glutaric acid, two additional genes, davDT or gabTD, encoding 5-aminovalerate transaminase and glutarate semialdehyde dehydrogenase, were overexpressed and the aminoreceptor of the 5-aminovalerate transaminase reaction, alpha-ketoglutarate, was added externally in stoichiometric concentrations [54,55]. Thus, both precursors for PA5,5 were produced simultaneously. The diamine putrescine or 1,4-diaminobutane can be derived from L-ornithine by decarboxylation [49] and is used in polyamides such as PA4,10 or PA4,6. Introduction of ornithine decarboxylase from E. coli into an L-ornithine producing C. glutamicum strain enabled putrescine production [56]. Since E. coli, unlike C. glutamicum, is able to degrade putrescine E. coli-based putrescine production required deletion of genes for putrescine degradation [57]. The C. glutamicum system was developed further with the highest putrescine yield (about 0.26 g/g) reported in bacteria [58]. Tuning expression of ornithine transcarbamoylase gene argF over 1000 fold through modulation of transcription and translation efficiencies was key to balance low-level ornithine transcarbamoylase for obtaining high productivity and high titer. Moreover, expression of argF and ornithine decarboxylase gene speC from an addiction plasmid obviated addition of antibiotics to the fermentation broth at titers of about 19 g/L and volumetric productivities of about 0.6 g L 1 h 1 [58]. Putrescine was also produced from alternative carbon sources such as crude glycerol [24], hemicellulosic www.sciencedirect.com
Amino acids and derived products Wendisch 55
hydrolysates [29] or amino sugars [32,33] and by a biotinprototrophic putrescine producing strain [43]. The diamine cadaverine or 1,5-diaminopentane is used as monomer for polyamides. Its fermentative production by direct decarboxylation of lysine was first described by Mimitsuka and colleagues who replaced the endogenous homoserine dehydrogenase gene hom by the lysine decarboxylase gene cadA from E. coli and achieved production of 2.6 g/L cadaverine from glucose in 18 hours [59]. Subsequent export engineering and elimination of the acetylated by-product led to titers of about 2 g/L [11]. Cadaverine could also be produced from starch, xylose and cellubiose [26,60,61]. E. coli-based cadaverine production required inactivating cadaverine degradation and led to titers of about 9 g/L [62]. Amino acids can be deaminated to enoic acids [63], e.g. in the well-known example of aspartate ammonia lyase converting L-aspartate to fumarate. Similarly, trans-cinnamate, trans-p-hydroxycinnamate and urocanate, respectively, arise from deamination of L-phenylalanine, L-tyrosine and L-histidine, respectively. Conversion of aamino acids to b-amino acids by aminomutases involves the same cofactor 4-methylideneimidazole-5-one [63]. Enantiomerically pure a-amino acids and b-amino acids were prepared in enzymatic processes with L-phenylalanine and L-tyrosine aminomutases [64]. While biotransformation of fumarate to aspartic acid is a commercial process [65], fermentative processes using aminomutases or ammonia lyases have yet to be developed. L-Glutamate
was acetylated to N-acetyl-glutamate using four different recombinant E. coli strains overproducing one thermostable enzymes each [66]. Thus, at high temperature N-acetylglutamate production from pyruvate and 2-ketoglutarate was possible in a cofactorbalanced and CoA-recycling synthetic pathway combining pyruvate decarboxylase from Acetobacter pasteurianus, CoA-acylating aldehyde dehydrogenase from Thermus thermophiles, glutamate dehydrogenase from T. thermophilus and N-acetylglutamate synthase from Thermotoga maritima [66].
Dipeptides have various functions, e.g. L-glutamyl-Lthreonine or L-leucyl-L-serine enhances the umami taste of L-glutamate, L-arginyl-L-phenylalanine and L-glutamyl-L-tryptophan show antiangiogenic properties [67]. Dipeptides may be obtained by ligation of an amino acid to a second amino acid or an aminoacylester [68]. L-Amino acid ligase, which condenses unprotected amino acids in an ATP-dependent manner, is used for fermentative production of dipeptides [67,68]. Ligation of an amino acid to a N-methyl aminoacylester does not require ATP. About 70 g/L L-alanyl-L-glutamine were produced from L-alanine methyl ester hydrochloride and L-glutamine in www.sciencedirect.com
40 min by a whole cell biotransformation using E. coli overproducing a-amino acid ester acyltransferase from Sphingobacterium siyangensis [69]. Products derived from amino acid precursors
The L-glutamate precursor 2-ketoglutarate is used in dairy industry and to treat chronic renal insufficiency. Glutamate can be deaminated to 2-ketoglutarate, e.g. by whole cell biotransformation with B. subtilis overproducing L-amino acid deaminase from Proteus mirabilis [70]. Fermentative production of 2-ketoglutarate required deleting three genes involved in 2-ketoglutarate conversion (gdh, gltB, and aceA for glutamate dehydrogenase, glutamate synthase and isocitrate lyase, respectively) in Lglutamate overproducing C. glutamicum [71]. A fed-batch process resulted in about 50 g/L of 2-ketoglutarate [72]. The immediate precursor of L-valine, 2-ketoisovalerate (KIV) is used as a substitute for L-valine or L-leucine in parenteral nutrition of chronic kidney disease patients. Deletion of transaminase gene ilvE and overexpression of the ilv genes resulted in KIV production. Increased pyruvate supply in the absence of pyruvate dehydrogenase subunit AceE and pyruvate quinone oxidoreductase Pqo led to about 22 g/L KIV and a productivity of about 0.5 g L 1 h 1 [73]. Following the approach developed by Liao and colleagues [73], KIV could be efficiently converted to isobutanol in aerobic and anaerobic processes [6,74,75]. The immediate precursor of L-leucine, 2-ketoisocapraote (KIC) finds application in infusion solutions for patients with kidney or hepatic diseases and in functional foods for muscle regeneration. C. glutamicum was engineered for production of KIC by abrogation of the conversion of KIC to L-leucine by deletion of transaminase gene ilvE, overexpression of the ilv genes, derepression of the leu genes by deletion of transcriptional regulatory gene ltbR and low levels of citrate synthase [76]. A heterologous feedbackresistant isopropylmalate synthase variant from E. coli boosted KIC production to about 7 g/L from 40 g/l glucose in 24 h [76]. Similarly, an L-leucine overproducing strain was developed [77]. The KIC producing strain has potential to be used for production of 3-methyl-1-butanol, a potential future biofuel [78].
Conclusions Amino acid production is a success story of biotechnology and the industry still shows robust growth. Scientific developments such as synthetic and systems biology have not only led to improved amino acid processes, but have also enabled strain development for novel compounds. This trend gained additional impetus, e.g. by generating prophage-free C. glutamicum strains [79] or by developing multi-use platform strains [80] and is complemented by furthering genomic methodology, e.g. by RNAseq analysis of C. glutamicum [81,82]. We are looking forward Current Opinion in Biotechnology 2014, 30:51–58
56 Chemical biotechnology
Acknowledgements
14. Binder S, Schendzielorz G, Stabler N, Krumbach K, Hoffmann K, Bott M, Eggeling LA: high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell F level. Genome Biol 2012, 13:R40. Together with Ref. [15] construction, validation and application of fluorescent amino acid sensors for faster single-cell FACS-assisted classical strain optimization by mutagenesis and screening are described.
Work in the lab of VFW is supported in part by PROMYSE (EU, FP7 project 289540), by a ZIM project (KF2969003SB2), and by BMBF projects SysEnCor (0315598E) and Genome reduction (0316017A) and by SynMet, a 09-EuroSYNBIO-FP-023 project, co-funded by DFG through grant no. WE 2320/2-1.
15. Lange C, Mustafi N, Frunzke J, Kennerknecht N, Wessel M, Bott M, Wendisch VF: Lrp of Corynebacterium glutamicum controls expression of the brnFE operon encoding the export system for L-methionine and branched-chain amino acids. J Biotechnol 2012, 158:231-241.
References and recommended reading
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to see more and more examples of transfer of biotechnological production processes of amino acids and derived products from proof-of-concept to economic realization.
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