The reductive branch of the TCA cycle coupled to PEP carboxylase (PPC) for succinic acid formation (rTCA (PPC) pathway), which exists in almost all microbes, is one of the most commonly used processes in bio-based succinic acid production. In this pathway, PEP carboxylase (PPC) converts PEP and CO₂ into oxaloacetate, which is then converted into malate catalyzed by malate dehydrogenase (MDH). Then, malate is transformed into fumarate by fumarate hydratase (FumABC). Finally, fumarate and NADH are turned into succinic acid catalyzed by fumarate reductase (FrdABCD). Through this pathway, succinic acid is synthesized from PEP and CO₂ at a stoichiometric ratio of 1:1. Among the components in this process, PPC (EC 4.1.1.31) plays a critical role in CO₂ fixation. Taking E. coli as an example, PPC is produced during growth on glycolytic substrates (Keseler et al., 2011). PPC catalyzes the carboxylation of PEP with bicarbonate to form oxaloacetate and inorganic phosphate using Mg²⁺ as a cofactor (Millard et al., 1996; Kai et al., 1999). The main function of PPC is to replenish the oxaloacetate consumed by biosynthetic reactions, and under anaerobic fermentation conditions, it can direct a portion of PEP to succinic acid (Millard et al., 1996). Studies have shown that under a CO₂ atmosphere, E. coli Δppc strains grow slower and more poorly than wild-type strains in glucose-based medium (40% reduction in biomass and more than 70% reduction in succinic acid production) (Millard et al., 1996; Ahn et al., 2020). PPC also exhibits high catalytic velocity (the enzymatic activity is 250 μmol/min/mg) and high substrate affinity (the K _m value for HCO₃ ^- is 0.1 mM and the K _m value for PEP is 0.19 mM), which is conducive to succinic acid biosynthesis (Millard et al., 1996; Kai et al., 1999; Tan et al., 2013). Millard et al. evaluated the effect of PPC on the amount of succinic acid produced in E. coli under control of the tac promoter. The engineered strain that overexpressed PPC showed a 3.5-fold increase in the concentration of succinic acid, from 3.0 g/L in a control culture to 10.7 g/L, and the yield showed a 3.75-fold increase, from 0.12 mol/mol glucose to 0.45 mol/mol glucose (Table 3) (Millard et al., 1996). There seemed to be a positive correlation between PPC activity and succinic acid production, but excessive PPC activity was detrimental to cell growth and succinic acid formation (Tan et al., 2013). Tan et al. found higher PPC activity along with higher succinic acid production, when the PPC activity was equal to or less than 0.47 U/mg protein (Tan et al., 2013). The reason may have been that the overexpression of PPC caused a shortage of PEP. PEP is also essential for the PEP-PTS (phosphoenolpyruvate-phosphotransferase) system, which has both a transport function and an extensive regulatory function. And the shortage of PEP will lead to a compromise in glucose uptake and cell growth (Gokarn et al., 2000). Therefore, the regulation of PPC expression was significant when using the PPC dependent biosynthesis pathway for succinic acid production.

The reductive branch of the TCA cycle coupled to PEP carboxykinase (PCK) for succinic acid formation (rTCA (PCK) pathway), is another representative succinic acid biosynthesis route that mainly exists in bacteria and fungi, especially in some natural succinic acid producers (Werf et al., 1997; Lee et al., 2006). This process is almost the same as the PPC-dependent pathway, except for the key CO₂-fixation enzyme. In this pathway, the key CO₂-fixation enzyme is PCK (EC 4.1.1.31) which can catalyze the carboxylation of PEP with bicarbonate and ADP to form oxaloacetate and ATP. Compared with PPC, the PCK catalytic process is accompanied by the production of energy, and as such it is more suitable for cell growth and succinic acid production (Zhu and Tang, 2017). However, the overexpression of PCK in E. coli at low CO₂ concentrations in fact has no effect on succinic acid biosynthesis. PCK can be valuable for succinic acid production but only in the presence of high HCO₃ ^- concentrations (Millard et al., 1996; Kwon et al., 2006). There are two possible explanations for this result: 1) PCK has low HCO₃ ^- affinity (the K _m value for HCO₃ ^- is 13 mM, 130 times larger than that of PPC, the Km value for PEP is 0.07 mM and the Km value for ADP is 0.05 mM); 2) PCK has low catalytic velocity (the enzymatic activity is 28 μmol/min/mg, which is just 11.2% of the PPC activity) (Krebs and Bridger, 1980; Zhang et al., 2009a; Tan et al., 2013). Previous studies have demonstrated that when 20 g/L NaHCO₃ was added to a culture, the succinic acid titer in recombinant E. coli overexpressing PCK was 2.2-fold higher than a wild-type strain, from 6.4 g/L in a control culture to 14.2 g/L (Kwon et al., 2006). Kim et al. found that in wild-type E. coli K12 cells, PCK overexpression had no effect on succinic acid fermentation, but the overexpression of PCK in an engineered E. coli K12 Δppc strain was 6.5-fold higher than E. coli K12 Δppc, and rose from 3.1 g/L to 20.2 g/L (Table 3) (Millard et al., 1996). In addition, the PCK activity was closely related to succinic acid biosynthesis. Tan et al. reported that the succinic acid titer and yield were positively correlated with the PCK activity. A 16-fold increase in PCK activity resulted in a 57-fold increase in succinic acid titer (from 4 to 226 mM) in E. coli Suc-T108 (Tan et al., 2013). In some natural succinic acid producers, such as A. succiniciproducens (Millard et al., 1996), M. succiniciproducens (Hong et al., 2004; Lee et al., 2006), and A. succinogenes (Millard et al., 1996; Werf et al., 1997), PCK is mainly employed in CO₂ fixation and cell growth instead of PPC. Taking M. succiniciproducens as an example, genes encoding PPC, PCK, or MAE were separately disrupted, and fermentation results showed that strains with PPC or MAE deletion did not show much effect on succinic acid productivity or cell growth, while the strain with PCK inactivation showed severe growth retardation and a remarkable decrease in succinic acid productivity (Lee et al., 2006). This result was significant because although PCK has a high K _m value for HCO₃ ^-, it still plays a major role in succinic acid biosynthesis in M. succiniciproducens. Compared with M. succiniciproducens, the indispensable feature of PCK was not observed in wild-type E. coli. It might have been due to the differences in the internal concentration of HCO₃ ^- in these two organisms (Millard et al., 1996). These results also suggests that the optimization of a CO₂ delivery system and the amelioration of the intracellular inorganic carbon concentration may be a good choice for improving the PCK depended succinic acid biosynthesis route (Xiao et al., 2017). Except for PCK, MDH was reported to be another rate-limiting enzyme for succinic acid production (Hong et al., 2004; Ahn et al., 2020). In M. succiniciproducens, MDH reduces oxaloacetate to malate using NADH as a cofactor, but it shows low specific activity and strong uncompetitive inhibition towards oxaloacetate (Ahn et al., 2020). In silico simulation suggested that MDH is instrumental in succinic acid production (Ahn et al., 2020). Jung et al. reported biochemical and structural analyses of various MDH variants and discovered a key amino acid residue (Gly-11) required for high kinetic efficiency in MDH. After screening, a valuable MDH mutant (MDH-G11Q) was obtained, which had high enzyme activity (2.9-fold increase in activity) and less substrate inhibition (6.4-fold increase in k _i ) compared with a wild type MDH. Furthermore, the application of this MDH-G11Q mutant in engineering bacteria led to a 1.4-fold increase in succinic acid production, indicating the significance of enzyme optimization in strain development (Choi et al., 2016; Ahn et al., 2020).

The reductive branch of the TCA cycle coupled to pyruvate carboxylase (PYC) for succinic acid formation (rTCA (PYC) pathway), is widespread in fungi, plants, as well as some bacteria (Modak and Kelly, 1995), but it is absent in Enterobacteriaceae (Gokarn et al., 2000). In this pathway, PEP is first converted into pyruvate by pyruvate kinase (PYK). Then PYC (EC 6.4.1.1) catalyzes the ATP-dependent carboxylation of pyruvate to produce oxaloacetate. Oxaloacetate is further converted into succinic acid through MDH, FumABC and FrdABCD. As the key CO₂-fixation step, the reaction catalyzed by PYC also plays an anaplerotic role in the provision of oxaloacetate for maintaining the dynamic balance of the TCA cycle. In R. etli, the anaplerotic role of PYC has been shown to be essential for its normal growth (Jitrapakdee et al., 2008), and thus PYC can be considered to be one of the most significant regulatory enzymes in this pathway (Modak and Kelly, 1995). In most PYC catalytic reactions, acetyl-CoA acts as a positive allosteric activator (Jitrapakdee et al., 2008; Tong, 2013), except for PYC from L. lactis, which is regulated by the bacterial second messenger c-di-AMP (Choi et al., 2017). The PYC depended route is a promising tool for succinic acid biosynthesis. Yan et al. reconstructed this pathway in S. cerevisiae, and found that the engineered strain showed a 19.3-fold increase in succinic acid production in shaken flasks, rising from 0.32 g/L to 6.17 g/L (Yan et al., 2014). Chen et al. reported that the overexpression of PYC from R. oryzae in a S. cerevisiaeΔsdh2 strain resulted in a 1.2-fold increase in the concentration of succinic acid, from 0.709 g/L to 0.841 g/L (Table 3) (Chen et al., 2019). In addition, the functions of PYC, PPC and MAE on the carboxylation processes for high succinic acid productivity in E. coli were analyzed in silico. According to analysis results, carboxylation reactions catalyzed by PYC were the most suitable ones for obtain high productivity in E. coli. Furthermore, based on these analysis results, PYC from C. glutamicum ATCC13032 was overexpressed in E. coli ZJG13 and the final succinic acid production was 36.1 g/L, with a specific productivity and yield of 2.75 mmol g CDW⁻¹ h⁻¹ and 0.72 mol mol⁻¹ glucose, respectively (Table 3) (Yang et al., 2014). Sánchez et al. overexpressed the L. lactis pyc gene in E. coli MG1655 (ΔadhE, ΔldhA) and found that a 25-fold increasein succinic acid production, from 0.6 g/L to 15.6 g/L, as well as a 6-fold increase in the yield, from 0.13 g/g glucose to 0.78 g/g glucose (Table 3) (Sánchez et al., 2005).

The reductive branch of the TCA cycle coupled to malic enzyme (MAE) for succinic acid formation (rTCA (MAE) pathway), is shorter than the PYC dependent pathway. In the MAE dependent pathway, pyruvate and CO₂ are directly converted into malate by malic enzyme, and malate is further converted into succinic acid through FumABC and FrdABCD. There are two forms of malic enzymes: MaeA (EC 1.1.1.38) and MaeB (EC 1.1.1.40), which can use NADH and NADPH as electron donors, respectively. Stols et al. overexpressed MAE from A. suum in E. coli NZN111 to produce succinic acid, resulting in a 2.9-fold increase, from 2.45 g/L to 7.07 g/L (Table 3) (Stols et al., 1997). Kwon et al. cloned MaeA and MaeB, derived from E. coli W3110, and overexpressed them in E. coli W3110 under anaerobic conditions. They found that the enzyme activities of overexpressed MaeA and MaeB were 2.34 and 1.69 μmol min⁻¹mg_protein ⁻¹, whereas their activities were only 0.82 and 0.01 μmol min⁻¹mg_protein ⁻¹ in the wild-type W3110 strain, respectively (Kwon et al., 2007). Furthermore, the succinic acid yields were measured in different strains. MaeB overexpression resulted in a 2.4-fold increase in succinic acid production, to 15.43 mmol/L, compared with 6.41 mmol/L in wild-type E. coli W3110. However, MaeA overexpression resulted in just 6.69 mmol/L succinic acid, which was similar to a wild-type strain under the same anaerobic fermentation conditions (Kwon et al., 2007). In this study, there was a negative correlation between succinic acid production and the activities of MaeA and MaeB. The main reason for this result might have been related to the differences in the intracellular co-substrate availability of MaeA and MaeB. The intracellular concentration of NADPH was 146 μmol/L, which was 9.12-fold higher than the 16 μmol/L of NADH intracellular concentration (Bochner and Ames, 1982; Kwon et al., 2007). Correspondingly, NADPH-dependent MaeB showed an advantage over the NADH-dependent MaeA, which would encourage CO₂ fixation by MaeB over by MaeA in wild-type E. coli (Kwon et al., 2007). In addition, the reaction catalyzed by MAE is reversible and this enzyme prefers to form pyruvate, which is unfavorable for succinic acid formation (Hong and Lee, 2002). The K _m values of MAE for malate and pyruvate in E. coli are 0.4 and 16 mM, respectively. Similarly, the K _m values of MAE in C. glutamicumare 3.8 mM for malate and 13.8 mM for pyruvate, respectively (Stols and Donnelly, 1997; Gourdon et al., 2000). This indicates that sufficiently high intracellular pyruvate concentrations may have a significant influence to overcome the unfavorable equilibrium of MAE catalytic processes. However, the wild-type strain is unable to accumulate a sufficiently high pyruvate concentration to allow MAE to be effective for producing excessive malate (Stols et al., 1997). Therefore, the enhancement of the PYK catalytic process, which can convert PEP and ADP into pyruvate and ATP, maybe a good choice for intracellular pyruvate enrichment. In some natural succinic acid producers, this reaction is also closely related to normal metabolic activity. Taking M. succiniciproducens as an example, Lee found that PYK operates by providing the pyruvate and ATP required for cell growth, as its pyruvate concentration increases with cell growth and remains constant after cell growth has stopped (Lee et al., 2006).

Although high productivities and yields of succinic acid can be achieved through anaerobic biosynthesis pathways, some drawbacks still exist with using these pathways, such as their slow carbon throughput, limitation of NADH availability (2 mol of NADH are required for 1 mol succinic acid formation) and poor cell growth (Yang et al., 2014). One solution is to produce succinic acid under aerobic conditions, which can generate higher biomass and extensive energy with O₂ as the electron acceptor (Lin et al., 2005a; 2005b; Yang et al., 2014). The oxidative branch pathway of the TCA cycle (oTCA pathway) is one of the candidate aerobic biosynthesis routes for succinic acid production. In this pathway, acetyl-CoA and oxaloacetate are converted into succinic acid through citrate synthetase (GltA), aconitase (AcnAB), isocitrate dehydrogenase (ICD), and Succinyl-CoA synthetase (SucABCD), accompanied by the synthesis of NADH at a stoichiometric ratio of 2:1. Although succinic acid can be generated by oTCA under aerobic conditions, most of it will be further converted in the TCA cycle or to biomass and cannot be excreted from the cells. Thus, the metabolic engineering studies are necessary to improve its yield. Taking E. coli as an example, succinic acid is a minor fermentation product under anaerobic conditions. Under aerobic conditions, however, succinic acid is only an intermediate of the TCA cycle and cannot be detected in the extracellular medium (Clark, 1989; Lin et al., 2005b). Correspondingly, acetate is the main byproduct under the same aerobic conditions (Lin et al., 2005b). In order to produce succinic acid as a major product aerobically, Lin et al. created a mutant E. coli HL27659k (pKK313) strain that overexpressed of S. vulgare pepc and had five enzymes inactivated: ΔsdhAB, Δ(ackA-pta), ΔpoxB, ΔiclR, andΔptsG. This mutant strain could use both the oxidative branch pathway of the TCA cycle and the glyoxylate shunt pathway to produce succinic acid under aerobic conditions, and its maximum theoretical succinic acid yield could reach 1.0 mol/mol glucose consumed (Lin et al., 2005c). Fed-batch fermentation results showed that this strain could produce 58.3 g/L of succinic acid under complete aerobic conditions with a yield of 0.94 mol/mol glucose consumed (Table 3) (Lin et al., 2005b; 2005d). Arikawa et al. constructed a Saccharomyces cerevisiae mutant with fumarate reductase (FRDS) and succinate dehydrogenase (SDH1) inactivated. However, under aerobic conditions, this mutant strain could synthesize succinic acid through the oxidative branch pathway of the TCA cycle and the succinic acid yield showed a 2.7-fold increase compared with the parental strain (Arikawa et al., 1998). S. cerevisiae exhibits a high tolerance to low pH values, which makes it superior for succinic acid production (Qiang et al., 2010). These results were significant and Lin et al. inferred the potential of the oTCA pathway for aerobic succinic acid biosynthesis (Lin et al., 2005b).

The glyoxylate shunt pathway (GAC pathway) is another aerobic succinic acid biosynthesis route. In this process, 2 mol of acetyl-CoA and 1 mol of oxalacetate are converted into 1 mol of malate and 1 mol of succinic acid by citrate synthetase (GltA), aconitase (AcnAB) and isocitrate lyase (AceAB). Compared with the oxidative branch pathway of the TCA cycle, this process bypasses the two oxidative steps where CO₂ is released, and thus it is considered to be an atom-economic aerobic pathway (Lin et al., 2004). Additionally, malate is produced as the main byproduct in the glyoxylate shunt pathway. Malate can be catalyzed by malate dehydrogenase (MDH) to generate NADH and regeneration of oxaloacetate. Under anaerobic conditions, 1 mol of malate can also be further transformed into 1 mol of succinic acid by FumABC and FrdABCD by consuming 1 mol of NADH. Compared with the traditional rTCA pathway (formation of 1 mol of succinic acid requires 2 mol of NADH), the glyoxylate shunt pathway does not contain NADH consumption process. Therefore, its application will be beneficial for solving the limitation of NADH availability during succinic acid biosynthesis (Sánchez et al., 2005). Vemuri et al. found that in the absence of an additional electron acceptor under dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase), the maximum theoretical succinic acid yield was up to 1.714 mol/mol glucose, when 28.6% of the carbon flows to the glyoxylate shunt pathway and 71.4% of the carbon flows to the reductive branch pathway of the TCA cycle. It is worth noting that the maximal yield of succinic acid cannot be achieved without an active glyoxylate shunt pathway (Vemuri et al., 2002). It should be noticed that C2 substrates like acetate or fatty acids are very prominent activators for the glyoxylate shunt pathway under aerobic conditions (Gui et al., 1996). The aceBAK operon, which encodes enzymes of the glyoxylate cycle, is controlled by IclR. Under aerobic conditions, acetate can induce aceBAK and reduce the repressor activity of IclR, resulting in activation of the glyoxylate cycle (Gui et al., 1996; Sánchez et al., 2005). Meanwhile, studies have also revealed that the disruption of iclR could dramatically induce the expression of the aceBAK operon even when growing on glucose (Gui et al., 1996; Lin et al., 2004; Hendrik et al., 2011). Thus, the inactivation of iclR is significant for activating the glyoxylate shunt pathway for succinic acid production (Sánchez et al., 2005). Lin et al. constructed an engineered E. coli HL27615k strain with mutations in the TCA cycle (ΔiclR, Δicd, and ΔsdhAB) and acetate pathways (ΔpoxB, and ΔackA-pta) for maximal aerobic succinic acid production through the glyoxylate shunt pathway. The results of aerobic batch fermentation showed that the succinic acid production reached 43 mM with a yield of 0.7, which was close to the maximum theoretical yield of 1 mol/mol glucose (Lin et al., 2004). Except for the absence of the iclR mutation, Zhu et al. demonstrated that the glyoxylate shunt pathway could also be activated by deleting other relevant genes in E. coli under anaerobic conditions. The key genes included ackA (a gene encoding an acetate kinase) and pta (a gene encoding a phosphotransacetylase). Metabolic flux analysis reflected that the carbon flow shunted to the glyoxylate pathway from oxalacetate in wild-type E. coli DY329 was 0 and 31% in the mutant YJ003 (ΔackA, Δpta, ΔldhA, and ΔpstG). In addition, the succinic acid production also showed a 6-fold increase, from 25.13 mM in DY329 to 150.78 mM in YJ003, implying the importance of the activation of the glyoxylate shunt pathway (Zhu et al., 2014).

The 3-hydroxypropionate cycle is one of the natural aerobic CO₂-fixation pathways, and mainly exists in photosynthetic green nonsulfur bacteria (Herter et al., 2001). This cycle is complex, containing 16 enzymatic reaction steps that are catalyzed by 13 enzymes (Gong et al., 2016). Liu et al. introduced part of this cycle into recombinant E. coli for succinic acid production (3HP pathway) (Liu et al., 2020). In this process, acetyl-CoA is carboxylated by acetyl-CoA carboxylase (ACC) to generate malony-CoA. Malony-CoA is converted into propionyl-CoA by malonyl-CoA reductase (MCR) and propionyl-CoA synthase (PCS). Then propionyl-CoA is carboxylated by propionyl-CoA carboxylase (PCC) to generate (S)-methylmalonyl-CoA. Next, methylmalonyl-CoA epimerase (MmcE) and methylmalonyl-CoA mutase (MmcM) convert (S)-methylmalonyl-CoA into its isomer succinyl-CoA. Finally, succinyl-CoA is deesterificated by succinyl-CoA synthetase (SucCD) to generate succinic acid (Menendez et al., 1999; Liu et al., 2020). The conversion of 1 mol of acetyl-CoA into 1 mol of succinic acid via the 3-hydroxypropionate cycle can fix 2 mol of CO₂, which was confirmed by isotope labeling experiments with NaH¹³CO₃ (Liu et al., 2020). Compared with succinic acid production based on carboxylation of PEP or pyruvate, this route showed a higher CO₂ fixation efficiency. Among these pathway, ACC (EC 6.4.1.2) and PCC (EC 6.4.1.3) are the key CO₂-fixing and rate-limiting enzymes. Both ACC and PCC belong to a subgroup of biotin-dependent short-chain acyl-CoA carboxylases that contain biotin carboxyl carrier protein domain (BCCP), biotin carboxylase domain (BC), and carboxyltransferase domain (CT), and utilize a covalently bound biotin as a cofactor (Wongkittichote et al., 2017; Liu and Jiang, 2021). The CT domain determines the specificity of the substrate, and these proteins can even work with other BCCP and BC domains from different enzymes (Lombard and Moreira, 2012; Tong, 2013). To obtain PCC with high enzymatic activity, Liu et al. tested different CT subunits of the PCC homologs from diverse bacterial species and developed a direct evolution strategy to further optimize this protein. A highly active PCC mutant (PccB_BS-N220I/I391T) was obtained, which showed a 94-fold increase in overall catalytic efficiency indicated by k _cat/K _m compared with a wild-type PCC. In addition, the application of this PCC mutant resulted in a 1.5-fold increase in succinic acid production compared to the engineered strain with wild-type PCC, which indicated the significance of this rate-limiting enzyme engineering strategy for microbial production (Liu et al., 2020). ACC acts as another CO₂-fixing enzyme and ACC is essential for most living organism’s growth (Baba et al., 2006). The intracellular concentration of its carboxylation product (malonyl-CoA) is tightly regulated to be very low, leading to limited production of ACC-derived compounds (Zha et al., 2009; Liu et al., 2010). It has been reported that ACC is negatively regulated by AMP-activated serine/threonine protein kinase (Snf1) in S. cerevisiae when glucose is depleted. Moreover, the phosphorylation triggered by Snf1 at one or more serine residues results in the deactivation of ACC (Woods et al., 1994; Scott et al., 2002). Jin et al. identified a critical amino acid (Ser-1157) responsible for deactivation via phosphorylation and mutated it to an alanine. The in vitro activity results showed that this ACC mutant (ACC-S1157A) resulted in 9-fold higher specific activity relative to wild-type ACC (Choi and Silva, 2014). Shi et al. demonstrated that introduction of this S659A mutation in ACC-S1157A could also lead to an enhanced enzyme activity with 1.72-fold increase (Shi et al., 2014). Further utilization of these modified acetyl-CoA carboxylase will be beneficial for product biosynthesis built from malonyl-CoA, such as succinic acid (Choi and Silva, 2014). Except for ACC and PCC, malonyl-CoA reductase (MCR), a characteristic enzyme of the 3-hydroxypropionate cycle, is regarded as another rate-limiting enzyme (Fixation et al., 2002). MCR catalyzes a two-step NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate, and malonate semialdehyde has been suggested to be the likely free intermediate (Fixation et al., 2002; Liu et al., 2013). Liu et al. demonstrated that MCR, a natural fusion protein of two short-chain dehydrogenase/reductases, has higher enzyme activity when dissected into two functionally individual fragments, including an MCR-C fragment (amino acids 550–1,219) and MCR-N fragment (amino acids 1–549) (Liu et al., 2013). Further studies indicated that the initial reduction of malonyl-CoA to malonate semialdehyde catalyzed by MCR-C was the rate-limiting step of MCR (Liu et al., 2013; Liu et al., 2016). Liu et al. developed a direct evolution strategy to further optimize MCR-C. A triple MCR-C mutant (N940V/K1106W/S1114R) was obtained, which showed a 5.54-fold increase in enzyme activity. Combined with fine tuning of the MCR-N expression levels and culture condition optimization, the 3-hydroxypropionate yield showed a 270-fold increase, which could relieve the pressure of being the rate-limiting step in succinic acid biosynthesis (Liu et al., 2016). Although some progress has been achieved in this succinic acid pathway, some problems are still worthy of attention. The main challenge is that significant amounts of ATP and reducing power (3ATP and 3NADPH) are consumed from one molecule acetyl-CoA to one molecule succinic acid. Using fatty acids as carbon sources or feeding electricity to bacteria to produce intracellular energy may be optional promising approaches (Chen et al., 2018; Liu et al., 2019).

Whether under anaerobic or aerobic conditions, succinic acid is not the main product of many microbes. In general, microbes preferentially accumulate substances such as acetate, lactate, and ethanol etc. These by-product pathways compete with the succinic acid pathway for ATP or reducing power. Therefore, it is necessary to redirect the carbon flux from these competitive pathways into succinic acid production (Li et al., 2020). The redirection methods often used mainly include blocking these competitive pathways and regulating succinic acid biosynthesis pathways.

The intracellular redox ratio is closely related to metabolite profiles, membrane transport, microbial fitness and cellular functions (Berríos-Rivera et al., 2004; Singh et al., 2009). The redox ratio is mainly reflected by the intracellular NADH/NAD⁺ ratio, which is influenced by several factors, such as the physiological state of a given strain, the oxidation state of carbon sources, and the expression levels of NADH/NAD⁺-related enzymes (such as formate dehydrogenase, membrane-bound transhydrogenase, NAD⁺ dehydrogenase, terminal oxidases and lactic dehydrogenase) (Lin et al., 2005a; Singh et al., 2009). In microbes, both NADH and NAD⁺ are kept in balance. NADH is oxidized to NAD⁺ through the oxidative phosphorylation process or fermentation reactions. NADH provides the reducing power for reductive product formation and NAD⁺ serves as an electron acceptor during substrate degradation (Li et al., 2017). In addition, the appropriate NADH/NAD⁺ ratio is a major prerequisite of many biosynthetic processes, and it is also crucial for succinic acid production (Zhang Y. et al., 2009; Li et al., 2017).

Taking E. coli as an example, lactate dehydrogenase (encoded by ldhA), which drives the formation of lactate, and pyruvate formate lyase (encoded by pflB), which drives the formation of formate and acetyl-CoA derivants (acetate, ethanol), were deleted to redirect the carbon flux towards succinic acid under anaerobic conditions (Singh et al., 2010). This mutant was termed E. coli NZN111, and it has been considered an excellent candidate as a succinic acid producer (Hui et al., 2009). However, E. coli NZN111 lost its ability to ferment glucose anaerobically and accumulates high levels of pyruvate and NADH intracellularly (Wu et al., 2007; Ma et al., 2013). It has been reported that E. coli NZN111 growth was impaired mainly due to the anomalously high NADH/NAD⁺ ratio in vivo, which was shown to be three times higher than a wild-type E. coli strain (Singh et al., 2009; Singh et al., 2010). To reduce this NADH/NAD⁺ ratio, an effective genomic library-based screening approach was employed, and genes including pfkB (encoding phosphofructokinase II), marA (encoding DNA-binding transcriptional repressor), and moaE (encoding the subunit of molybdopterin synthase), were shown to be significant for balancing the redox ratio. Overexpression of these genes was beneficial for succinic acid production, especially pfkB, and led to a 7.8-fold increase in M9+10 g/L glucose medium under microaerobic conditions (that means controlled dosing of small amount of air or oxygen into reactor).

Conversely, deficiency in NADH has a negative impact on succinic acid biosynthesis as well. Except for the glyoxylate shunt pathway, conversion of phosphoenolpyruvate, pyruvate or acetyl-CoA to one molecule of succinic acid requires a minimum of two molecules of reducing equivalents (Lin et al., 2005c). However, only two molecules of NADH can be obtained from one molecule of glucose through the glycolytic pathway. Thus, the shortage of NADH limits the succinic acid production to a theoretical yield of 1 mol/mol glucose (Zhu et al., 2014). One solution is to use the glyoxylate shunt pathway (mentioned in section 2), which has a theoretical yield of 1.7 mol/mol glucose (Vemuri et al., 2002). Another solution is to provide more NADH. NAD⁺-dependent formate dehydrogenase can convert one molecule of formate into one molecule of CO₂ and NADH. In this reaction, formate is one of the main by-products produced by succinic acid producers, while CO₂ and NADH are necessary for succinic acid synthesis. When using glucose as a carbon source, the expression of formate dehydrogenase can double the maximum yield of NADH, from 2 to 4 mol NADH/mol glucose consumed (Berríos-Rivera et al., 2004). In Grant et al., fdh (encoding NAD⁺-dependent formate dehydrogenase) from C. boidiniiwas and pycA (encoding pyruvate carboxylase) from L. lactis were co-expressed in E. coli MG1655 (ΔadhE, ΔldhA, ΔiclR, Δack-pta) under the control of the same promoters (Thakker et al., 2011). Compared with the overexpression of pyc alone, this co-expression strain had better succinic acid production capacity. The succinic acid yield at 24 h showed a 1.9-fold increase, from 176 to 334 mM, and the succinic acid productivity showed a 2-fold increase, from 1 to 2 g/L/h. Meanwhile the byproduct formate concentration showed a significant decrease, from 17 mM to 0–3 mM. This indicated that higher NADH availability conditions significantly changed the final metabolite concentration pattern and promoted an increase in the contents of succinic acid. Additionally, providing carbon sources with different oxidation states is also an optional method. Sorbitol, for instance, has a higher NADH maximum theoretical yield (3 mol NADH/mol sorbitol) than glucose (Berríos-Rivera et al., 2004). It has been reported that using sorbitol can also generate higher yield and productivity of succinic acid (Chatterjee et al., 2001).

In succinic acid biosynthesis, HCO₃ ^- is the major carboxylation substrate. During fermentation processes, CO₂ that is dissolved in the liquid medium, first crosses the cell membrane through passive diffusion, and is then converted into HCO₃ ^- in the cytoplasm to participate in carboxylation (Lu et al., 2009). However, the low concentration of CO₂ in the growth medium is the critical limiting step in succinic acid production (Zhu et al., 2015). To date, several strategies have been attempted to improve succinic acid production by optimizing CO₂ supplementation and several satisfying results have been achieved.