Metabolic engineering has been developed into a powerful tool for understanding the mechanism of malic acid biosynthesis, and also greatly promoted the progresses of engineering in E. coli, yeasts, and filamentous fungi for malic acid production. Since bacteria such as E. coli are generally not good natural malic acid production strains and also accumulate many by-products such as acetate, lactate, ethanol and formate, introduction of heterologous genes or pathways to reconstruct biosynthesis pathways with combinational deletion of genes from competing pathways is the most common strategy. While some yeasts and filamentous fungi can naturally produce high amounts of malic acid, combined enhancement of their native synthetic pathways, generally the rTCA pathway, with increased export of malic acid from the cell could dramatically improve the product titer. Efficient strategies for eliminating by-products such as succinic and citric acid, as well as the enhancement of relevant metabolic fluxes have also been exploited to increase the malic acid yield in A. niger, A. oryzae and M. thermophila. Compared with S. cerevisiae and prokaryotes, the malic acid titers of filamentous fungi were usually higher. Therefore, filamentous fungi are considered the most promising host strains for the microbial fermentation of malic acid. Notably, the engineered A. niger S575 with GRAS status produced the highest malic acid titer reported to date, and after elimination of the by-product citric acid and enhancement of the main metabolic flux, the yield of malic acid from glucose was further improved. The elimination of major by-products can significantly decrease the cost of downstream processing by simplifying separation and purification. Additionally, 50% of the total cost is used for the separation and extraction process of malic acid produced by microbal fermentation. Nevertheless, the industrial success of biosynthesis is ultimately based on rapid and economical conversion of substrates into target products, so from the view of industrialization, the next reconstructive emphasis will concern on: 1) shortening the fermentation period, 2) identifying the limiting factors for the efficient use of cheap carbon feedstocks, 3) exploring the regulatory factors of L-malate synthesis pathway to improve production efficiency, 4) reducing by-product synthesis to increase L-malate yield and reduce the costs of downstream separation and extraction.
Low-cost sugar feedstocks are preferred for large scale fermentation for increase of profit margin. The price of raw materials accounts for a large proportion of the total production cost in industrial fermentation processes. However, the substrate used for microbial fermentation of malic acid is mostly the relatively expensive glucose. Accordingly, the selection of more economical renewable feedstocks for malic acid production, such as lignocellulosic biomass from agricultural waste or crude glycerol from the biodiesel industry, has received increasing attention. Biotechnological processes have shown great potential to utilize these cheap feedstocks for malic acid production. Metabolically engineering the most promising strains to develop versatile processes which can be adapted to cost-effective feedstocks may be another important subject of future research.
In all the current processes of microbial fermentation for malic acid production, large amounts of CaCO3 must be added as a neutralizing agent to keep the culture pH constant at around 6.5. As a consequence, the fermentation end-product is calcium malate formed in the bioreactor, which requires cost-intensive acidification and precipitation for conversion into pure malic acid during downstream processing. Systems biology or the latest genome-scale metabolic models can provide solutions to complex metabolic engineering goals of industrial importance, and further genetic engineering of malic acid-producing strains of Aspergillus, which have extremely high natural acid tolerance, to produce malic acid at low pH values would be a promising approach to avoid the excessive addition of neutralizing agents.