Appl Microbiol Biotechnol (2001) 57:451–459 DOI 10.1007/s002530100804 MINI-REVIEW Y. Li · J. Chen · S.-Y. Lun Biotechnological production of pyruvic acid Received: 23 April 2001 / Received revision: 30 July 2001 / Accepted: 17 August 2001 / Published online: 22 September 2001 © Springer-Verlag 2001 Abstract Pyruvic acid is an important organic acid Table 1 More applications of pyruvic acid or pyruvate widely used in the chemical and drug, as well as agro- chemical, industries. Compared with the chemical meth- Application Reference od, biotechnological production of pyruvic acid is an al- Significantly increases fat and weight loss Stanko et al. 1992 ternative approach because of the low cost. An overview Improves exercise endurance capacity Stanko et al. 1990 of biotechnological production of pyruvate, including di- Effectively reduces cholesterol Stanko et al. 1994 rect fermentative production employing eukaryotic and Serves as a potent antioxidant DeBoer et al. 1993 Reduces anoxic injury and free radical Borle and Stanko prokaryotic microorganisms, production by a resting cell formation 1996 method and an enzymatic method as well as the recovery of pyruvate, is discussed. A multi-vitamin auxotrophic yeast strain, Torulopsis glabrata, has been used in the On an industrial scale, pyruvic acid is produced commercial production of pyruvate; emphasis is there- by dehydration and decarboxylation of tartaric acid fore placed on the mechanism and characteristics of py- (Howard and Fraser 1932). In this process, pyruvic acid ruvate production by this strain. is distilled from a mixture of tartaric acid and potassium hydrogen sulfates at 220°C; the crude acid obtained is then distilled under vacuum. This process is simple to re- Introduction alize but not cost-effective; the total cost is estimated to be about US $8,000–9,000/ton according to the actual Pyruvic acid, also known as 2-oxopropanoic acid, α- market price of raw materials (Li 2000). Pyruvic acid is, ketopropionic acid or acetylformic acid, is the most im- therefore, too expensive to be widely used over a long portant α-oxocarboxylic acid. It plays a central role in period. For example, although pyruvic acid is a potential energy metabolism in living organisms. Industrially, it is flavoring agent that imparts a sour taste to foods, it is used mainly as a starting material in the biosynthesis of hardly ever used to replace other organic acids because pharmaceuticals, such as L-tryptophan, L-tyrosine and of the high price. alanine, as well as L-DOPA, by known methods (Uchio Compared with the chemical method, production of et al. 1976). It is also employed in the production of crop pyruvic acid by using biotechnological methods is an al- protection agents, polymers, cosmetics and food addi- ternative approach to reduce the production cost. There tives. Calcium pyruvate also has a strong effect in reduc- are three methods for biotechnological production of py- ing fat because it can accelerate the metabolism of fatty ruvate: the direct fermentation method, the resting cell acids in the human body (Roufs 1996). Other applica- method, and the enzymatic method. Of these, direct fer- tions of pyruvate are shown in Table 1. As it is widely mentative production of pyruvate from a carbon source used in drug, agrochemical, chemical and food indus- (such as glucose) has merits in terms of both cost-effec- tries, the commercial demand for pyruvic acid has been tiveness and the high purity of the product. However, as expanding (Yonehara and Miyata 1994a). pyruvate is located at a vital junction of cell metabolism, it is usually difficult to obtain strains that can accumulate large amounts of pyruvate extracellularly. Many scholars Y. Li · J. Chen (✉) · S.-Y. Lun have tried to identify microorganisms with high pyru- Key Laboratory of Industrial Biotechnology, vate-producing ability, but it was not until 1989 that sig- Ministry of Education, Southern Yangtze University, Huihe Road, No. 170, Wuxi, 214036, P.R. China nificant progress was achieved in the fermentative pro- e-mail: [email protected] duction of pyruvate. Some yeast strains, belonging to the Tel.: +86-510-5888301, Fax: +86-510-5888301 genus Torulopsis, that produced more than 50 g/l pyru- 452 vate were identified in a screen carried out by the re- duction of pyruvate compared with the chemical method. searchers of Toray Industries, Japan (Miyata et al. Therefore, T. glabrata IFO 0005 was mutagenized to 1989a). This result indicates that the fermentative pro- give different additional genetic characteristics, such as duction of pyruvate can be commercialized. Pyruvate L-arginine auxotrophy (Miyata et al. 1988b), L-valine production using the fermentative method, with a scale and L-isoleucine auxotrophy (Miyata et al. 1989a), ami- of about 400 tons per year, was industrialized by Toray nooxyacetic acid resistance (Miyata et al. 1989b), and Industries in 1992 and the fermentation has now been 2-deoxyglucose resistance (Miyata and Yonehara 1990). scaled-up to 50 m3 fermentors (Yonehara et al. 2000) Yields of 0.52–0.54 g/g could be achieved by mutants This review summarizes the development of biotech- having one or more of these genetic characteristics nological production of pyruvate, with emphasis on the (Table 2). As ethanol is a main by-product when produc- fermentative production of pyruvate by yeast. The litera- ing pyruvate in yeast, a Torulopsis sp. mutant strain with ture cited comprises most of the publications in this field lower PDC activity was selected, by which means a py- since 1976, including many Japanese patents. For de- ruvate yield of 0.58 g/g was achieved (Miyata et al. tailed discussion of pyruvate production using a multi- 1990). vitamin auxotrophic Torulopsis strain, and Escherichia Other yeasts, such as thiamine auxotrophs of Candida coli defective in energy metabolism, reviews written in lipolytica (Uchio et al. 1976), Debaryomyces hansenii Japanese by the groups of Yonehara (Yonehara and (Yanai et al. 1994), and Saccharomyces cerevisiae Miyata 1994b; Yonehara et al. 2000) and Yokota (Yokota (Yonehara and Yomoto, 1987b), also accumulate pyru- and Tomita 1995; Yokota 1997), respectively, can be vate under conditions of thiamine limitation. The pyru- consulted. vate yield, 0.37–0.44 g/g, was not as good as in the case of T. glabrata. However, these strains can use inorganic ammonium as a sole nitrogen source, while T. glabrata Biotechnological production of pyruvate: IFO 0005 and its derivatives cannot (Table 2). Halophilic an overview Torulopsis etchellsii accumulates pyruvate from glucose in a medium supplementedwith 80 g/l salts, but the yield Direct fermentative production of pyruvate (0.02 g/g) is rather low (Kiuchi et al. 1987a, b). by eukaryotic microorganisms For T. glabrata, the difficulty of achieving high yield and high concentration of pyruvate simultaneously Yeast is the most commonly used microorganism that seems to be a drawback. For example, in a report by directly accumulates pyruvate in the medium using Miyata and Yonehara (1996), 67.8 g/l pyruvate was glucose or other carbon source as the substrate. Table 2 achieved at 63 h in a fed-batch culture, but the yield summarizes the development in this field contributed by from glucose was only 0.49 g/g. In another report (Hua Japanese researchers. As can be seen from Table 2, et al. 1999), pyruvate yield of higher than 0.8 g/g was strains belonging to the species of Torulopsis with the achieved in a typical pyruvate production phase. How- phenotype of multi-vitamin auxotrophy, such as T. ever, the overall yield, 0.53 g/g, was still not high despite glabrata IFO 0005, without doubt show excellent ability applying a thiamine-addition strategy to a fed-batch cul- in producing pyruvate (Yonehara and Yomoto 1987a; ture. In order to overcome this problem, Li et al. (2001) Yonehara and Miyata 1994a). The physiological mecha- selected an excellent pyruvate producer from T. glabrata nism for the accumulation of pyruvate by a multi-vita- WSH-IP12 (Li et al. 2000) that could use ammonium min auxotrophic strain of Torulopsis is shown in Fig. 1. chloride as a sole nitrogen source. In this case, both high Thiamine is a co-factor of the pyruvate dehydrogenase pyruvate concentration (69 g/l) and high pyruvate yield (PDH) complex and of pyruvate decarboxylase (PDC). (0.62 g/g) were achieved in a batch culture. Nicotinic acid, biotin, and pyridoxine are co-factors of PDH, pyruvate carboxylase (PC), and transaminase, re- spectively. As a multi-vitamin auxotrophic strain cannot Direct fermentative production of pyruvate synthesize these vitamins itself, under conditions of defi- by prokaryotic microorganisms ciency for these four vitamins, pyruvate is accumulated due to the decreased activity of PDH, PDC, PC, and As shown in Table 2, glucose is the most commonly transaminase. In order to accumulate large amounts of used substrate for pyruvate production in yeast. How- pyruvate when cultivating the multi-vitamin auxotrophic ever, the category of substrates assimilated by prokaryotic strain T. glabrata, it is very important to maintain a con- microorganisms to produce pyruvate seems to be wider centration balance between thiamine, nicotinic acid, py- than in yeast. For example, as shown in Table 3, many ridoxine and biotin (Li et al. 2001). microorganisms use gluconate, 1,2-propanediol or prop- To achieve high yields of pyruvate from glucose is inoic acid as a sole carbon source to accumulate pyru- very important in the commercialization of pyruvate pro- vate. Although these prokaryotic microorganisms (ex- duction by fermentative methods. Under normal culture cept E. coli) have no special genetic characteristics with conditions, the pyruvate yield of T. glabrata IFO 0005 is respect to pyruvate accumulation, the pyruvate yield was 0.41 g/g (Yonehara and Yomoto 1987a); this value is not also comparable (0.3–0.4 g/g).