Читать книгу Biomolecular Engineering Solutions for Renewable Specialty Chemicals - Группа авторов - Страница 51

Succinic acid (C₄H₆O₄) is the intermediate metabolite of krebs or TCA cycle. In earlier times, it was formed by distillation of amber and therefore called as amber acid or spirit of amber (Song and Lee, 2006). After that it was produced from butane through maleic anhydride. It is used as a flavoring agent in food industry. It has also a range of applications in dyes, perfumes, and in the manufacturing of clothing, paint, fibers, etc. Succinic acid is widely used as precursor for chemicals such as 1,4‐butanediol, γ‐butyrolactone, N‐methyl‐2‐pyrrolidone, tetrahydrofurane, and 2‐pyrrolidone (Pateraki et al., 2016). These chemicals are further derived into biodegradable polyesters such as polybutyrate succinate, polyamides, pesticides, and green solvents (Sun et al., 2015). This leads to the increased demand of succinic acid which was 50 000 metric tons in 2016 to double by 2025 (Chinthapalli et al., 2018).

Like citric acid, succinic acid is also synthesized in almost all plants, animals, and microorganisms. Various microorganisms like E. coli, Actinobacillus succinogenes, Mannheimia succiniciproducens, etc. produce succinic acid. Among them M. succiniciproducens gives the highest yield of 1.64 mol/mol glucose and productivity of 6.02 g/l/h (Lee et al., 2016). M. succiniciproducens was first isolated from the bovine rumen of Korean cow. Availability of whole genome sequence of M. succiniciproducens makes it easier candidate for genetic engineering (Lee et al., 2005). Glucose‐6‐phosphate 1‐dehydrogenase (zwf) gene is upregulated when succinic acid production is increased. Overexpression of zwf gene in M. succiniciproducens increases succinic acid synthesis (Kim et al., 2017).

Escherichia coli produce succinic acid in a very scarce amount, but is the model organism for genetic manipulation due to its fast growth and availability of genetic toolboxes. One of the strains NZN111 is generated by knocking out pyruvate formate lyase and LDH (Singh et al., 2009). This leads to the inhibition of formic and lactic acid, increasing the succinic acid production. But the strain generated was not able to thrive anaerobically producing lactic acid. This was solved by further overexpressing the gene for malate dehydrogenase in the same strain producing 31.9 g/l of succinic acid (Wang et al., 2009). Spontaneous chromosomal mutation of glucose phosphotransferase generates strain AFP111, which can anaerobically grow on glucose giving productivity of 0.87 g/l/h (Chatterjee et al., 2001). Prolonged anaerobic conditions can hamper cell growth giving low production rates. To produce succinate under aerobic conditions, five genes were inactivated in E. coli, namely, succinate dehydrogenase, pyruvate oxidase, acetate kinase phosphotransacetylase, aceBAK operon repressor, and glucose phosphotransferase (Lin et al., 2005). Apart from inactivation of these genes phosphoenolpyruvate carboxylase gene was overexpressed giving productivity of 1.08 ± 0.06 g/l/h of succinic acid.

Species of yeast like Y. lipolytica and Saccharomyces cerevisiae are also prospective host for succinic acid production. A robust Y. lipolytica PGC01003 strain was developed by deleting the succinate dehydrogenase gene (SDH). This leads to accumulation of succinic acid giving 43 g/l succinic acid from crude glycerol (Gao et al., 2016a). Later acetyl‐CoA hydrolase (Ylach) gene was deleted from Y. lipolytica to inhibit aceitic acid production and giving a yield of 23.8 g/l succinic acid (Yu et al., 2018). Ylach‐deficient strain with overexpression of phosphoenolpyruvate carboxykinase (ScPCK) and endogenous succinyl‐CoA synthase beta subunit (YlSCS2) improved succinic acid titer by 4.3‐fold (Cui et al., 2017). In a recent study, in spite of deleting SDH gene, SDH promoter was truncated with expressing ScPCK from A. succinogenes. It produced 7.8 ± 0.0 g/l succinic acid with a yield of 0.105 g/g glucose (Babaei et al., 2019). S. cerevisiae’s SDH have four subunits SDH1, SDH2, SDH3, and SDH4 (Kubo et al., 2000). For SDH to be inactivated SDH1 and SDH2 is to be disrupted. This leads to increase in succinic acid synthesis by further adding malic transporter from Schizosaccharomyces pombe (Ito et al., 2014).