The research of L-malic acid

Malic acid, a four-carbon dicarboxylic acid, is widely used in the food, chemical and medical industries. As an intermediate of the TCA cycle, malic acid is one of the most promising building block chemicals that can be produced from renewable sources. To date, chemical synthesis or enzymatic conversion of petrochemical feedstocks are still the dominant mode for malic acid production. However, with increasing concerns surrounding environmental issues in recent years, microbial fermentation for the production of L-malic acid was extensively explored as an eco-friendly production process. The rapid development of genetic engineering has resulted in some promising strains suitable for large-scale bio-based production of malic acid. This review offers a comprehensive overview of the most recent developments, including a spectrum of wild-type, mutant, laboratory-evolved and metabolically engineered microorganisms for malic acid production. The technological progress in the fermentative production of malic acid is presented. Metabolic engineering strategies for malic acid production in various microorganisms are particularly reviewed. Biosynthetic pathways, transport of malic acid, elimination of byproducts and enhancement of metabolic fluxes are discussed and compared as strategies for improving malic acid production, thus providing insights into the current state of malic acid production, as well as further research directions for more efficient and economical microbial malic acid production.

Malic acid is a ubiquitous dicarboxylic acid found in all organisms, but its name derives from the fact that it was first isolated from unripe apples in 1785 (Meek, 1975). In 1967, it was classified as a safe food-grade product by the U.S. Food and Drug Administration (FDA). Currently, malic acid is mainly used as an acidulant and flavor enhancer in the food and beverage industries. Due to its more intense acid taste and better taste retention compared with citric acid, L-malic acid is becoming one of the most widely used organic acidulants. In the pharmaceutical industry, L-malic acid is used to improve the absorption of drugs and is used in amino acid infusions for the treatment of liver dysfunction or high blood ammonia (Chi et al., 2014). A mixture of calcium citrate and calcium malate is a commonly used source of calcium for improved bone strength without increasing the risk of kidney stones (Thakker et al., 2015). Other commercial applications include metal cleaning, finishing, animal feed and chemical synthesis of biodegradable polymers, such as polymalic acid (PMA) (Goldberg et al., 2006;Dai et al., 2018). Malic acid was listed as one of the top twelve bio-based building block chemicals by the US Department of Energy (Werpy and Petersen, 2004). The current global malic acid production capacity is estimated between 80,000 and 100,000 tons per year, while the annual market demand is estimated at over 200,000 tons, with a steadily rising market potential (Sauer et al., 2008;Zou et al., 2015).

The primary commercial production of malic acid is currently based on petrochemical routes, such as the hydration of maleic anhydride generated from the oxidation of benzene or butane at high temperature and high pressure, yielding a racemic mixture of D- and L-malic acid (Naude and Nicol, 2018). Malic acid has an asymmetric carbon and therefore it occurs in two isomers. Enantiopure L-malic acid is the physiological form present in all living organisms, ranging from bacteria to humans, while D-malic acid is rare in nature and difficult to assimilate by humans, thus it is not applicable to very young infants and elderly people. In 1970, the U.S. FDA ruled that DL-malic acid could not be used as an additive in infant food. Enzymatic conversion is an alternative process for synthesis of L-malic acid, using either immobilized fumarate hydratase or whole cells (Brevibacterium ammoniagenes or Saccharomyces cerevisiae) containing the enzyme fumarate hydratase to catalyze the conversion of fumarate into malic acid (Chibata et al., 1987;Peleg et al., 1988;Knuf et al., 2014). However, the expensive purification of fumarate hydratase and difficult separation of L-malic acid from the unreacted substrate greatly increased the cost of L-malic acid production. In addition, substrates such as maleic anhydride or fumarate are derived from non-sustainable petroleum, and the upward trend in the cost of finite petroleum resources further hampered the expansion of the malic acid market (Goldberg et al., 2006;Liu et al., 2017a). With the increasingly severe challenges related to the depletion of fossil-based resources as well as environmental issues, ecofriendly sustainable microbial fermentative production of malic acid has been given more attention. A lot of progresses has been made in the development of engineered strains or processes in recent years.