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 Biology, 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 oxaloacetic acid from pyruvic acid by fixing one mole of CO2. Many evidences have confirmed that biosynthesis 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 enzyme in microbial biotechnology and is an important target for metabolic engineering 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; pyruvate carboxylase 1. Introduction (PMLA), succinic acid, fumaric acid, citric acid (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 bacteria, archaea (TCA) cycle and to its indirect linkage with nitrogen 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 malic acid, PMLA, succinic acid, fumaric enzymes 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, glucose (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, lactic acid, 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 glycolysis 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 dehydrogenase (Pdh); 3: citrate synthase (Cs), 4: aconitase; 5: isocitrate dehydrogenase (Icdh); 6: malate dehydrogenase (Mdh); 7: fumarase (Fum); 8: fumarate reductase (Fumr); 9: aspartate transaminase (Ast); 10: ligase;11: polymalic acid (PMLA) synthetase, (Pmlas); 12: phosphoenolpyruvate carboxykinase (Pepck); 13: isocitrate lyase (Icl); 14: malate synthase, (Ms); 15: glutamate dehydrogenase (Gdh); 16: ATP-citrate lyase (Acl); 17: acetyl-CoA carboxylase, (Acc); 18: fatty acid 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 catalysis 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 fatty acid synthesis 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 food (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 metabolism Page 3 of 7 47 Figure 2. Citrate biosynthesis in cytosol and mitochondria in yeasts. OAA: Oxaloacetic acid; cPDC: cytosolic pyruvate decarboxylase; cACS: cytosolic acetyl-CoA synthetase; cPDH: cytosolic pyruvate dehydrogenase; 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 hydrolysis of PMLA (Chi et al. In some fungi such as Aureobasidium spp., the malic acid 2016b). During the one-step fermentation, 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).