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Home , Fumaric acid, Oxaloacetic acid, Pyruvic acid

J Biosci (2019) 44:47 Ó Indian Academy of Sciences

DOI: 10.1007/s12038-019-9853-y (0123456789().,-volV)(0123456789().,-volV) Review

Biosynthesis of some organic acids and lipids in industrially important microorganisms is promoted by pyruvate carboxylases

1,2, 1,3, 1,3 4 1 SHOU-FENG ZHAO ,ZHE CHI ,GUANG-LEI LIU ,ZHONG HU ,LONG-FEI WU 1 and ZHEN-MING CHI * 1College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao 266003, People’s Republic of China 2Central Laboratories, Qingdao Municipal Hospital, Qingdao 266000, Shandong, People’s Republic of China 3Key Laboratory of Marine Genetics and Breeding, Ocean University of China, Yushan Road, No. 5, Qingdao 266003, People’s Republic of China 4Department of , Shantou University, Shantou 515063, People’s Republic of China

*Corresponding author (Email, [email protected], [email protected]) These authors contributed equally to this work. MS received 14 July 2018; accepted 8 February 2019; published online 10 April 2019

Pyruvate carboxylase (Pyc) catalyzes formation of from by fixing one mole of CO2. Many evidences have confirmed that of some different kinds of organic acids and intracellular and extracellular lipids is driven by Pyc and over-expression of the PYC gene in the industrial microorganisms can promote production of the different kinds of organic acids and intracellular and extracellular lipids. Therefore, the Pyc from different sources is regarded as a key in microbial biotechnology and is an important target for of the industrial microbial strains. However, very little is known about the native Pycs and their functions and regulation in the industrial microorganisms.

Keywords. C4 dicarboxylic acids; industrial microorganisms; lipids; metabolic engineering;

1. Introduction (PMLA), , , (CA), a-ketoglutaric acid, lipids and a few amino acids. Pyruvate carboxylase (Pyc: EC6.4.1.7) is a biotin-requir- It has been reported that the Pyc can play its important ing tetrameric enzyme that catalyzes the carboxylation of role in providing oxaloacetate to the tricarboxylic acid pyruvic acid to oxaloacetic acid. Many , archaea (TCA) cycle and to its indirect linkage with meta- and many fungi can synthesize the Pyc. However, the bolism through the interconversion of oxaloacetate-aspartate enteric bacteria, cyanobacteria and streptomycetes do not (Menefee and Zeczycki 2014). As shown below, the Pyc is a have such activity. In most bacteria and yeasts, Pyc limiting factor for , PMLA, succinic acid, fumaric can exist as an a4, but in Pseudomonas,Azo- acid, CA, a-ketoglutaric acid, lipids and some amino acids. tobacteraceae and Methanobacterium the Pycs occur as an For example, after overexpression of the PYC gene, (ab)4 multimeric complex (Jitrapakdee et al. 2008). It has will be fluxed to the biosynthesis of CA and liamocins (Fu been confirmed that each monomer consists of a N-ter- et al. 2016; Tang et al. 2018). Carboxylic acids produced by minal biotin carboxylase (BC) domain, a carboxyltrans- microorganisms include pyruvate, , gluconic acid, ferase (CT) domain, a C-terminal biotin carboxyl carrier malic acid, PMLA, succinic acid, fumaric acid, CA, a-ke- protein (BCCP) domain and a Pyc tetramerization domain toglutaric acid and some amino acids. From figure 1, we can (Menefee and Zeczycki 2014). see that high-yield production of these compounds by However, so far, very little has been known about the microorganisms requires large fluxes through the carboxy- properties of the Pycs in the industrial microorganisms, lating anaplerotic pathways, converting the three-carbon which can produce high levels of malic acid, polymalate intermediates (pyruvates) of into the desired four- http://www.ias.ac.in/jbiosci 1 47 Page 2 of 7 S-F Zhao et al.

Figure 1. The main carbon flows during microbial biosynthesis of carboxylic acids; amino acids, intracellular fatty acids and extracellular liamocins. 1: Pyruvate carboxylase (Pyc1); 2: pyruvate (Pdh); 3: (Cs), 4: ; 5: (Icdh); 6: (Mdh); 7: (Fum); 8: fumarate reductase (Fumr); 9: aspartate (Ast); 10: ligase;11: polymalic acid (PMLA) synthetase, (Pmlas); 12: phosphoenolpyruvate carboxykinase (Pepck); 13: (Icl); 14: , (Ms); 15: (Gdh); 16: ATP-citrate lyase (Acl); 17: acetyl-CoA carboxylase, (Acc); 18: synthase (Fas); 19: polyketide synthase (PKS); 20: malate enzyme (Me). carbon backbones of malate, then into aspartate, succinate, acid, PMLA, succinic acid, fumaric acid, lipids and a few fumarate and alpha-ketoglutaric acid. As such, PMLA, amino acids by the fixation of CO2 under of the succinic acid, fumaric acid, a-ketoglutaric acid and a few Pyc, this will be one way to reduce CO2 emissions and the amino acids among the carboxylic acids produced by greenhouse gas effect (Zheng et al. 2009). Therefore, bio- microorganisms can, at least in theory, be produced from engineering of the industrially important microorganisms glucose with a net gain of CO2. Therefore, the microbial with the PYC genes is also important in environmental biosynthesis of these compounds is closely related to the protection. Pycs (figure 1) and metabolic engineering of the producers of carboxylic acids with the PYC genes from different sources will enhance the biosynthesis of malic acid, PMLA, 2. Role of the Pycs in microbial biosynthesis of C4 succinic acid, fumaric acid, CA, a-ketoglutaric acid and a dicarboxylic acids and PMLA few amino acids in principle. Furthermore, in oleaginous yeasts an ATP-citrate lyase (Acl), which only occurs in C4 dicarboxylic acids produced by microorganisms include oleaginous yeasts catalyzes transformation of the formed CA malic acid, succinic acid and fumaric acid. Among them, into acetyl-CoA, the main precursor for malic acid can be polymerized into PMLA (Chi et al. and this enzyme can play a key role in high lipid production 2016a). Biosynthesis of all the C4 dicarboxylic acids and (figure 2). Therefore, the Pyc is regarded also to be involved PMLA starts from oxaloacetate which is a product from in lipid biosynthesis in the oleaginous yeasts. Recently, it has pyruvate under the catalysis of the Pyc according to figure 1. been shown that all the bioalkane and bioalkene, the main components of oils are also produced from fatty acids syn- thesized by different native organisms and engineered 2.1 Role of Pycs in biosynthesis of malic acid microorganisms (Fu et al. 2015; Zhou et al. 2016). There- and PMLA fore, the Pyc may also have some roles in the biosynthesis of bioalkane and bioalkene (figure 1). Malic acid is mainly used in beverages, candy and (Chi It is reported that CO2 contributes about 50% of the et al. 2016a). Presently, malic acid can be produced mainly greenhouse gas effect. If we can develop methods to effi- by chemical synthesis, enzymatic process, one-step fer- ciently convert CO2 into useful chemicals, such as malic mentation from glucose using native and genetically PYC and Page 3 of 7 47

Figure 2. Citrate biosynthesis in and mitochondria in yeasts. OAA: Oxaloacetic acid; cPDC: cytosolic ; cACS: cytosolic acetyl-CoA synthetase; cPDH: cytosolic ; cPYC: cytosolic Pyc; cCS: cytosolic citrate synthetase; mCS: mitochondrial citrate synthetase; mPDH: mitochondrial pyruvate dehydrogenase; cMDH: cytosolic malate dehydrogenase; cACL: cytosolic ATP-citrate lyase. engineered strains and of PMLA (Chi et al. In some fungi such as Aureobasidium spp., the malic acid 2016b). During the one-step , CaCO3 in the synthesized can be polymerized into PMLA (Li et al. 2015). medium is required for malate production because CaCO3 According to figure 1, the Pyc, malate dehydrogenase, provides CO2 as a substrate (figure 1) and CaCO3 also thiokinase and PMLA synthetase may also be implicated in causes precipitation of calcium salts of the acid so that the the PMLA biosynthesis (Li et al. 2015). Indeed, many reaction can be fluxed towards the acid production (Peleg results have shown that the addition of CaCO3 could sig- et al. 1989). This means that the Pyc can play an important nificantly stimulate the production of PMLA, while the role in the malic acid biosynthesis. For example, overex- biosynthesis of pullulan in Aureobasidium spp. was greatly pression of the genes encoding a native cytosolic Pyc, a reduced (Zhang et al. 2011;Maet al. 2013a). This suggests malate dehydrogenase and a native C4-dicarboxylate trans- that the Pyc may also play an important role in PMLA porter in Aspergillus oryzae NRRL 3488 rendered the biosynthesis and regulation in Aureobasidium spp. engineered fungus to produce a malate titer of 154 g/L (Brown et al. 2013). After the genes RoPYC encoding a Pyc, RoMDH encoding a malate dehydrogenase and SpMAE1 2.2 Role of Pycs in biosynthesis of fumaric acid encoding a malate transporter simultaneously were overex- pressed in the vitamin-auxotroph of Torulopsis glabrata, 8.5 Fumaric acid that is widely used in the food, chemical and malate g/L was accumulated by the engineered strain T. pharmaceutical industry is currently produced by a chemical G-PMS (Chen et al. 2013). Similarly, simultaneous expres- way with many disadvantages (Straathof and van Gulik sion of the PYC2 gene encoding a native Pyc, the MDH3 2012). gene encoding a cytosolic malate dehydrogenase, and a As stated above, CO2 fixation by Pyc leads to oxaloacetic Schizosaccharomyces pombe malate transporter gene acid formation. Then, oxaloacetic acid formed can be SpMAE1 in S. cerevisiae could make the resulting engi- withdrawn for fumaric acid biosynthesis under aerobic neered strain produce 59 g/L L-malic acid (Zelle et al. 2008). conditions. Therefore, the Pyc may also be a key enzyme for All these results demonstrated that the Pyc and other different producers of fumaric acid (figure 1). For example, enzymes involved in L-malic acid biosynthesis and secretion some filamentous fungi which could produce fumaric acid indeed play an important role in L-malic acid production. had higher activities of the cytosolic Pyc during fumaric acid 47 Page 4 of 7 S-F Zhao et al. production (Peleg et al. 1989). In this case, fumarase must be rendered the engineered strain to generate 12.08 g/L succinic also involved in fumaric acid biosynthesis by the filamen- acid from glucose (Yang et al. 2014). In another study (Ma tous fungi Rhizopus oryzae and Rhizopus arrhizus (figure 1) et al. 2013b), co-expression of a pncB gene encoding a (Peleg et al. 1989). nicotinic acid phosphoribosyltransferase (NAPRTase) and a Because native S. cerevisiae has no ability to accumulate a heterologous PYC gene from Lactococcus lactis in E. coli large amount of fumarate in its cytosol, the genes encoding a BA002 deficient in the ldhA and pflB genes made a malate dehydrogenase (RoMDH), a fumarase (RoFUM1) recombinant E. coli BA016 produce 14.08 g/L succinate and the endogenous Pyc (Pyc2) were expressed in S. cere- from glucose under anaerobic conditions. visiae FMME-001 in order to produce high titer (3.18 ± 0.15 g/L) of fumarate from glucose (Xu et al. 2013). In order to improve the low yield of fumarate, overexpression of the 3. Role of the Pycs in microbial biosynthesis of CA heterologous gene RoPYC from R. oryzae NRRL 1526 in S. cerevisiae CEN.PK2-1CDTHI2 with the low Pyc activity As shown in figure 2, CA biosynthesis and secretion are enabled an engineered strain FMME004-6 grown in the connected to malate biosynthesis and Pyc is also involved in presence of 32 lg/L biotin to produce up to 5.64 ± 0.16 g/L CA biosynthesis (Liu et al. 2010). fumaric acid (Xu et al. 2013). In our previous studies (Liu et al. 2010, 2013), it was Furthermore, after the pathways to acetyl-CoA, acetate, found that overexpression of an inulinase gene in Y. lipoly- formate and succinate in the engineered E. coli JM125 were tica SWJ-1b isolated from a sea sample made the recombi- blocked, but the PYC gene was over-expressed, 80 g/L nant yeast strain 30 strain yield 84.0 g/L citric acid from glucose and 15 g/L MgCO3 was anaerobically transformed inulin. Furthermore, expression of PYC gene from Meyer- into 30.7 g/L fumarate by the engineered E. coli JM125 ozyma guilliermondii in the marine derived Y. lipolytica (Jiang et al. 2010). This again indicated that Pyc also can SWJ-1b resulted in a transformant 86 having much higher play an important role in fumarate formation. Pyc activity and producing more CA (101.0 ± 1.3 g/L) than Y. lipolytica SWJ-1b (Tan et al. 2016). In another study (Fu et al. 2016), overexpression of a PYC gene from Penicillium 2.3 Role of Pycs in biosynthesis of succinic acid rubens in the same yeast, also yielded a transformant PR32 producing 111.1 ± 1.3 g/L of CA. This meant that the more Succinic acid has a wide range of applications in chemical, Pyc in the yeast cells, the more citric acid was produced. All food and environmental industries. Although succinic acid these results demonstrate that the Pyc also has positive also can be produced via chemical routes, they have many influence on CA production by the yeast. However, it is still disadvantages. Recently, it has been found that biological unknown if the Pyc can exhibit a similar role in CA production of succinic acid has many advantages over the biosynthesis of A. niger, the common producer of CA in chemical one. In addition, as mentioned above, CO2 fermentation industry (Angumeenal and Venkappayya assimilation during the succinic acid biosynthesis can be 2013). considered as an environmental advantage (Zheng et al. It has been reported that biosynthesis of CA involves at 2009). For example, expression of the PYC gene from Rhi- least one mitochondrial step, i.e., citrate synthase, which is zobium etli in E. coli resulted in an engineered E. coli pro- located exclusively in the mitochondria and excretion of CA ducing succinate from 1.18 to 1.77 g/L (Gokarn et al. 1998). from the mitochondria is one of the rate-limiting steps in CA It also has been reported that the engineered E. coli production (Karaffa and Kubice 2003; Liu et al. 2010). So SBS110MG harboring a plasmid pHL413 carrying a we think if the cytosolic pathway for CA biosynthesis is heterologous PYC gene from Lactococcus lactis, produced constructed in the CA producing yeast Y. lipolytica, the 15.6 g/L succinate from glucose (Sanchez et al. 2005). fermentation period for CA production will be reduced as Meanwhile, expression of a PYC gene and a galactose per- CA synthesized in cytoplasm can be secreted into the mease gene in the ptsG mutant enables the engineered E. coli medium directly according to figure 2. This work is also to have higher succinate yield (1.2 mol/mol of glucose) than under investigation in this laboratory. its wild-type strain (Wang et al. 2009). It has been reported that under the oxygen deprivation, Corynebacterium glu- tamicum was able to transform sugars to organic acids such 4. Role of the Pycs in microbial biosynthesis of a- as L-lactic, succinic and acetic acids. Therefore, overex- ketoglutaric acid pression of a PYC gene in the C. glutamicum strain (DldhApCRA717), led to a succinic acid concentration of a-Ketoglutaric acid (KGA), a key intermediate in the TCA 146 g/L produced by the engineered strain (Okino et al. cycle (figure 1) has wide applications as a and 2008). Co-expression of a NAPRTase gene encoding a in the agrochemical and pharmaceutical industries. nicotinic acid phosphoribosyltransferase and a PYC gene in Because thiamine is a of pyruvate dehydrogenase E. coli that was deficient in pyruvate formate-lyase, lactate and alpha-ketoglutarate dehydrogenase, both of which are dehydrogenase and phosphoenolpyruvate carboxylase the key enzymes in the metabolism of pyruvate and KGA, PYC and metabolism Page 5 of 7 47 the further metabolism of KGA is inhibited under the acids by the transformant P7 was greatly increased compared to conditions of thiamine deficiency. For example, Y. lipolytica that of the secreted citric acids by their wild-type strain ACA- WSH-Z06, a thiamine-auxotrophic yeast produced 39.2 g/L DC 50109. In order to efficiently transform the secreted citric KGA from glycerol. However, the high concentration of acid into fatty acids, both a ACL1 gene encoding an ATP-citrate pyruvate as a by-product was also simultaneously produced lyase and PYC gene was simultaneously over-expressed in the (Yin et al. 2012). As the native Pyc in this yeast has low transformant P7. Finally, the lipid content of the transformant enzyme activity, overexpression of both the ScPYC1 gene PA56 obtained reached 49.6% (w/w) and the amount of from S. cerevisiae and the RoPYC2 gene from R. oryzae in secreted citric acid was only 2.9 g/L. At the same time, the Pyc this thiamine-auxotrophic yeast made the recombinant yeast and Acl1 activities and their gene transcriptional levels in the strains produce 62.5 g/L KGA with the significant decline in transformant PA56 were greatly enhanced compared to those in pyruvate titer from 35.2 to 13.5 g/L during the fermentation its wild-type strain (Wang et al. 2015). The results indicate that (Yin et al. 2012). These results reveal that overexpression of the Pyc and Acl1 indeed can play an important role in lipid the PYC gene in Y. lipolytica also can lead to a carbon flow biosynthesis in the oleaginous yeast Y. lipolytica ACA-DC from pyruvate to KGA. 50109. Bioalkanes or bioalkenes produced by different organisms and genetically modified microorganisms are being thought 5. Role of Pycs in microbial biosynthesis of a few amino to be the advanced biofuels because they are similar to the acids of the aspartate family main components of the petroleum and have high-energy density (Zhou et al. 2016). It has been known that the fatty It is well known that L-aspartate is a precursor of L-, acids synthesized by the organisms and genetically modified L-, L- and purine and pyrimidine bases microorganisms are the precursors of all the bioalkanes or in microorganisms (figure 1) and is mainly synthesized from bioalkenes (Fu et al. 2015; Zhou et al. 2016). As mentioned oxalacetate and L-glutamate under the catalysis of L-aspar- above, the Pyc can play the important role in fatty acids of tate: 2-oxoglutarate aminotransferase (figure 1). Therefore, the oleaginous yeasts, it must have the same role in the the Pyc could play an important role in the biosynthesis of a bioalkanes or bioalkenes biosynthesis. This investigation is few amino acids of the aspartate family by producing being done in this laboratory. oxaloacetate (OAA) supply for L-aspartate and L-lysine The extracellular liamocins and intracellular lipids pro- biosynthesis (figure 1) (Peters-Wendisch et al. 2001). For duced by Aureobasidium spp. have received much attention example, overexpression of the PYC gene in a lysine-pro- because the intracellular lipids can be transformed into ducing strain of Corynebacterium glutamicum resulted in biodiesel (methyl of fatty acids) and the extracellular approximately 50% higher lysine accumulation in the culture liamocins have many potential applications in food, phar- supernatant whereas inactivation of the PYC gene led to a maceutical and cosmetic industries (Garay et al. 2018; Tang decrease by 60% (Peters-Wendisch et al. 2001). In contrast, et al. 2018). It can be seen from figure 1 that biosynthesis of the overexpression of the PYC gene in a threonine-producing the intracellular lipids and the extracellular liamocins is also strain of C. glutamicum led to only 10–20% increase in closely related to Pyc1 activity because their precursors are threonine production (Peters-Wendisch et al. 2001). It was CA which biosynthesis is controlled by Pyc. For example, a also reported that the native PYC gene overexpression in a L- PYC1-over-expressing transformant M39 produced glutamate producer of C. glutamicum CN1021 increased the 43.04 ± 1.2 g/L liamocins and 4.2 g/L lipids while its wild- supply of oxaloacetate for L-glutamate synthesis (Guo et al. type strain 9-1 only produced 27.4 ± 0.3 g/L liamocins 2013). These results identify that the anaplerotic Pyc reac- (Tang et al. 2018). tion can be as a major bottleneck for a few amino acids All the products, titers and the recombinant producers production by C. glutamicum and show that the Pyc is also expressing the PYC gene are summarized in table 1. an important target for the metabolic engineering of the hyper amino acids-producing strains. 7. Future perspectives

6. Role of the Pycs in lipid biosynthesis of oleaginous All the results mentioned above demonstrate that Pyc can play yeasts an important role in the biosynthesis of malic acid, PMLA, succinic acid, fumaric acid, citric acid, a-ketoglutaric acid, lipid As mentioned above, the Pyc may play an important role in and a few amino acids according to figures 1 and 2. However, lipid biosynthesis of the oleaginous yeasts (figure 1). There- it is still a little known about the Pycs and their genes from fore, overexpression of the PYC gene cloned from P. guillier- malic acid, PMLA, succinic acid, fumaric acid, citric acid and mondii Pcla22 in Y. lipolytica ACA-DC 50109 resulted in a lipid-producing microorganisms. Moreover, it is also still a little transformant P7 yielding 38.2% (w/w) lipid in its cells while known about effects of over-expression of each native PYC the lipid content in its wild-type strain ACA-DC 50109 was gene on malic acid, PMLA, succinic acid, fumaric acid and only 30.2% (w/w). However, the amount of the secreted citric citric acid production in its corresponding producer. We expect 47 Page 6 of 7 S-F Zhao et al.

Table 1. The products and titers produced by the recombinant producers expressing the PYC gene and their wild-type strains

Products Titers (g/L) Producers References Malate 154 (0) A. niger Brown et al. (2013) Malate 8.5 (0) T. glabrata Chen et al. (2013) L-Malic acid 59 (0) S. cerevisiae Zelle et al. (2008) Fumarate 3.18 (0.1) S. cerevisiae Xu et al. (2013) Fumaric acid 5.64 (0.1) S. cerevisiae Xu et al. (2013) Fumarate 30.7 (0) E. coli Jiang et al. (2010) Succinate 1.77 (1.18) E. coli Gokarn et al. (1998) Succinate 15.6 (0.5) E. coli Sanchez et al. (2005) Succinic acid 146 (23) C. glutamicum Okino et al. (2008) Succinic acid 12.08 (0) E. coli Yang et al. (2014) Succinate 4.36 (1.57) E. coli Gokarn et al. (2001) Succinate 14.08 (1.57) E. coli Ma et al. (2013b) Citric acid 101.0 (27.3) Y. lipolytica Tan et al. (2016) Citric acid 111.1 (27.3) Y. lipolytica Fu et al. (2016) a-Ketoglutaric acid 62.5 (42.4) Y. lipolytica Yin et al. (2012) Lipids 6.21 (3.1) Y. lipolytica Wang et al. (2015) Liamocin 43.04 (27.4) A. melanogenum Tang et al. (2018) Lipids 4.2 (3.5) A. melanogenum Tang et al. (2018)

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