CHO cells cultured with galactose, instead of glucose as the sole carbon source, showed a low survival rate (Neermann and Wagner, 1996). In contrast, when cultured in a medium containing both glucose and galactose, the cells could utilize lactate and reduce lactate generation, probably because CHO cells metabolize galactose slower than glucose, reducing the glycolytic rate and avoiding a large accumulation of pyruvate. However, in this case, the galactose-related metabolism, which is much slower than that of glucose, also causes a decrease in the specific cell growth rate (Kelly et al., 2018). To improve galactose metabolism, CHO cells overexpressing galactose kinase (GALK1) were cultured in a medium containing galactose, which increased their specific growth rate by 39% and maintained their growth with galactose as the main carbon source. The slightly lower density of cells overexpressing GALK1 compared to controls may be related to metabolic stress due to GALK1 overexpression (Gu et al., 1995); however, lactate accumulation was less and the parameter ΔL/ΔGal increased by 54% indicating improved cellular galactose metabolism (Jimenez et al., 2019). Metabolic flux analysis during the glucose depletion phase versus the galactose depletion phase in CHO cells overexpressing GALK1 and control CHO cells showed that inefficient glucose metabolism led to pyruvate accumulation during the glucose depletion phase. This excess pyruvate is directed to the synthesis of alanine and lactate, which is characteristic of cells cultured with glucose as a carbon source (Altamirano et al., 2006; Wilkens et al., 2011). Furthermore, cells overexpressing GALK1 exhibited a lower glycolytic flux during the glucose depletion phase and a higher TCA cycle flux during the galactose depletion phase than the controls (Jimenez et al., 2019). Moreover, culturing CHO cells using galactose increases the sialic acid glycosylation modification of recombinant proteins (Liu et al., 2015). However, when CHO cells were cultured in a galactose medium, some amino acids, such as histidine and glutamine, were depleted in the early stages of cell growth and became growth limiting factors. Therefore, this method of culturing CHO cells with galactose as a carbon source can still be improved. In addition, overexpression of galactose transporter protein (GLUT8) can also increase the uptake and metabolism of galactose by cells (Jiménez et al., 2011).

The affinity of fructose transporter protein (GLUT5) for fructose is lower than that of glucose transporter protein (GLUT1) for glucose. Thus, replacing glucose in the medium with fructose while CHO cells express GLUT5 can also reduce lactate accumulation by reducing the glycolytic flux (Wlaschin and Hu, 2007). CHO cells usually do not grow well in media containing fructose but not glucose due to low or possibly no expression of GLUT5 transporter protein. Cells express GLUT5 show a good lactate metabolism profile when fructose is present, indicating an increase in lactate consumption efficiency (Wlaschin and Hu, 2007; Le et al., 2013; Wilkens and Gerdtzen, 2015). In 2013, Le et al. used the ability of the promoter of the thioredoxin-interacting protein (TXNIP) gene to drive GLUT5 expression in late cell culture by driving the expression of the GOI as the cells grow, allowing glucose to stimulate cell growth in early culture and later shifting the cells to metabolize fructose, which facilitates the overall cellular metabolic balance (Le et al., 2013).

By stably expressing both GLUT5 and pyruvate carboxylase (PYC), energy metabolism was improved, and lactate production was reduced, resulting in increased cell density and prolonged cell life span. Cellular metabolic flux analysis showed that CHO cells subjected to double gene editing had higher metabolic fluxes in glycolysis and TCA cycles, were able to consume more fructose, and maintained higher cell density (Wilkens and Gerdtzen, 2015).

Pyruvate is the main product of the glycolytic process in CHO cells, and the concentration of intracellular pyruvate is mainly influenced by its production rate. If production rate exceeds consumption rate, a higher glycolytic flux leads to the accumulation of pyruvate. The rapid production and accumulation of pyruvate lead to the accumulation of lactate (Wilkens et al., 2011) and alanine (Ma et al., 2009) early in the course of fed-batch cultures. This was verified in the metabolic profiling data (Zhang et al., 2004; Sheikholeslami et al., 2014). One idea is to slow down glycolysis to avoid pyruvate accumulation, which will inevitably affect the normal metabolism, growth, and protein expression of CHO cells. For example, inhibition of GLUT1 (Paredes et al., 1999) expression will lead to a decrease in both cell growth rate and maximum cell density (Altamirano et al., 2013). Another idea is to increase the rate of glycolysis and simultaneously promote pyruvate depletion using gene editing techniques. However, feedback inhibition exists for key enzymes in glycolysis, such as hexokinase, phosphofructokinase (PFK), and pyruvate kinase; for example, PFK can be inhibited by ATP, low pH, and lactate, whereas hexokinase is inhibited by glucose 6-phosphate (Halestrap and Price, 1999; Costa Leite et al., 2007), suggesting that it is difficult to genetically edit the glycolytic pathway.

The site of pyruvate production is primarily the cytoplasm, but its metabolism is in the mitochondrial matrix. Although pyruvate can freely cross the outer mitochondrial membrane (through pores or non-selective channels), it needs to cross the inner membrane and enter the mitochondrial matrix with the help of the mitochondrial pyruvate carrier (MPC), which participates in the TCA cycle, gluconeogenesis, and the metabolism of lipids and amino acids to provide energy to the organism (Vanderperre et al., 2015). Therefore, MPC can regulate the energy metabolism of the organism by regulating the flux of pyruvate into the mitochondrial matrix.

MPC are pyruvate transport proteins located on the inner mitochondrial membrane, which were identified by Papa and Halestrap (Papa et al., 1971; Halestrap, 1975) in the 1970s and further characterized in mammals in 2012 (Brivet, 2003; Bricker et al., 2012; Herzig et al., 2012). In mammalian cells, MPC is a dimeric complex consisting of two subunits, MPC1 and MPC2, and loss of activity of either subunit results in loss of activity of the MPC complex. It has been demonstrated that deletion or transcriptional repression of MPC1 results in defective mitochondrial pyruvate uptake and accumulation of glycolytic intermediates (Bricker et al., 2012; Herzig et al., 2012). Therefore, it can be assumed that the presence of MPC is a key factor in determining the occurrence of pyruvate transport or accumulation. Researchers (Bulte et al., 2020) constructed CHO cell lines stably overexpressing two subunits of the MPC complex to facilitate pyruvate entry into mitochondria and participate in the aerobic oxidation. Compared to controls, CHO cells overexpressing MPC produced up to 50% less lactate, had increased specific cell growth rates and maximum live cell densities, and transiently expressed a 40% higher maximum concentration of two recombinant model proteins, alkaline phosphatase, and monoclonal antibody. The results of the cell metabolism model showed that overexpression of MPC increased the metabolic flux of pyruvate across the mitochondrial membrane and promoted cell growth. However, the accelerated cell growth resulted in faster nutrient consumption in the medium, making it difficult to maintain a high-density culture of cells. Therefore, fed-batch culture is an optional method to develop the potential of this cell line. Regarding lactate production, the MPC overexpressing cell line showed faster lactate production during the first 12 h of culture, which was associated with higher growth and glycolysis rates. However, the rate of lactate production in this cell line rapidly decreased and was significantly lower than that of the control group, and the point in time at which lactate consumption began was earlier than the control group, a metabolic feature largely attributable to the translocation of pyruvate from the cytoplasm to the mitochondria.

Pyruvate consumption is inseparable from the metabolism of lactate, nearly two-thirds of pyruvate generated by glycolysis is converted to lactate (Jimenez et al., 2019). Lactate dehydrogenase (LDH) catalyzed lactate production and consumption is a reversible reaction; therefore, the LDH-catalyzed reaction in CHO cells is often in equilibrium, and it is the relative concentration of the substrate for this reversible reaction that is the key factor in determining the direction of the reaction (Quistorff and Grunnet, 2011). For example, elevated levels of pyruvate or NADH favor the production of lactate for the forward reaction, whereas elevated levels of lactate or NAD⁺ favor the consumption of lactate for the reverse reaction. Typically, the ratio of NAD⁺/NADH in the cytoplasm is ∼700:1, whereas the ratio of lactate/pyruvate is ∼20:1 (Wilkens and Gerdtzen, 2015). Changing the product/substrate ratio can shift the equilibrium position, which can drive the reversal of the LDH reaction, leading to lactate depletion. The production of lactate is limited to LDH catalysis, and the extent of its accumulation depends almost entirely on this reversible reaction, but pyruvate can participate in multiple metabolic pathways, suggesting that various factors can influence its concentration. When pyruvate is consumed by enzymes other than LDH, there is a large reduction in intracellular pyruvate, which also upsets the balance of the LDH reversible reaction, making the reaction favorable to lactate consumption. Therefore, editing key genes as GOIs for CHO metabolic engineering in other depletion pathways of pyruvate could provide a new idea to solve problems, such as lactate accumulation.

In CHO cells, the main metabolic route for pyruvate consumption other than lactate production is through MPC into the mitochondria, and acetyl coenzyme A is produced by the action of the dehydrogenase complex. Therefore, to promote pyruvate consumption, GOIs for editing in this metabolic route is warranted. For example, in the early stages of CHO cell culture, the pyruvate dehydrogenase (PDH) complex may be inactivated by pyruvate dehydrogenase kinase (PDK) phosphorylation, resulting in the inability to metabolize pyruvate (Jeong et al., 2006). Therefore, knockdown of PDK can increase the activity of PDH (Zhou et al., 2011), which is beneficial for directing pyruvate to acetyl coenzyme A, promoting the oxidation of pyruvate, and solving the problem of lactate production due to the massive accumulation of pyruvate. Reducing the expression of LDH-A alone can also reduce lactate production. However, despite a 45–79% decrease in lactate levels, specific productivity (Qp) and protein production did not increase significantly, suggesting that the knockdown of LDH-A alone in CHO cells is insufficient to effectively increase Qp and protein production (Kim and Lee, 2007). In contrast, simultaneous inhibition of LDH-A expression with PDK can successfully reduce lactate formation and simultaneously increase volumetric productivity (Zhou et al., 2011). In addition, pyruvate can be directly carboxylated to oxaloacetate catalyzed by PYC, which reduces pyruvate content while directly replenishing the carbon source for the TCA cycle and accelerating the cycle rate. It has been demonstrated that CHO cells overexpressing yeast pyruvate carboxylase (PYC2) showed an approximately fourfold reduction in lactate accumulation and a 70% increase in monoclonal antibody production compared to controls (Gupta et al., 2017).

The TCA cycle is the metabolic center of mammalian cells. To determine the key metabolic responses of the TCA cycle in CHO cells, researchers (Dhami et al., 2018) transiently downregulated the expression of each TCA cycle gene in CHO cells using siRNA and examined its effects on cell growth and energy production. The results showed that the silencing of at least four TCA cycle genes was detrimental to the growth of CHO cells. Notably, the reaction catalyzed by mitochondrial aconitase (ACO2) is one of the key points of the TCA cycle in CHO cells. Cell growth was significantly decreased (p ≤ 0.0001) after 48–72 h of aconitase silencing using siRNA alone, demonstrating that the downregulation of aconitase genes has the most lethal effect among TCA cycle genes. Aconitase catalyzes the reversible conversion of citric acid to cis-aconitic acid and then cis-aconitic acid to isocitric acid (Krebs and Holzach, 1952). In mammalian cells, aconitase has a higher activity rate than other TCA cycle reactions, and it maintains the balance between citric acid, cis-aconitate, and isocitric acid (Costello and Franklin, 2013). Gene expression analysis and metabolic profiling of CHO cells with silenced aconitase gene showed that downregulation of aconitase gene expression caused oxidative stress and significantly reduced ATP and NAD production. High levels of oxidative stress reduce protein production, so amino acids such as pyruvate and proline can be added during fed-batch culture to reduce oxidative stress. In addition, reduced aconitase gene expression compromised the function of the entire TCA cycle and CHO cells were unable to replenish other intermediates to maintain the normal function of this cycle. This is the first demonstration of the regulation of the TCA cycle in CHO cells by the aconitase gene. Elucidation of the critical role of the aconitase gene in CHO cells allows the gene to be applied to future cell engineering strategies for efficient expression of recombinant proteins in response to oxidative stress or to regulate the rate of TCA cycle.

The search for corresponding target genes for editing has proven to be effective in improving cellular metabolic capacity at various points of the CHO cell gluconeogenesis pathway with the aim of promoting metabolism. However, focusing on the mitochondria themselves may allow us to regulate energy metabolism in CHO cells more accurately and efficiently. Study of mitochondrial content in CHO cells showed no significant linear correlation between mitochondrial content and cell growth and recombinant protein synthesis (O'Callaghan et al., 2015), suggesting that mitochondrial efficiency rather than abundance plays a more important role in the specific productivity of CHO cells. Therefore, for the optimization of mitochondria in CHO cells, efforts should focus on gene editing related to mitochondrial efficiency rather than just increasing the amount of mitochondria in the cell.

After depleting miR-23 using the miR-23 sponge (m23sp) in CHO cells, the researchers found that the expression of an inner mitochondrial membrane protein called Leucine Zipper and EF-Hand Containing Transmembrane Protein 1 (LETM1) improved. Therefore, LETM1 was identified as a potential target of miR-23. Meanwhile, due to the depletion of miR-23, although the growth of CHO cells was not changed, the specific productivity was increased, leading to a three-fold increase in the secreted alkaline phosphatase (SEAP) volume productivity (Kelly et al., 2015). Further studies showed that LETM1 is a Ca²⁺/H⁺ antiporter that can channel Ca²⁺ into the mitochondrial matrix, and the elevated Ca²⁺ concentration in mitochondria can activate the rate-limiting enzymes of the TCA cycle, such as PDH and ATP synthase, and subsequently increase mitochondrial activity (Osellame et al., 2012) and increase SEAP production. Mitochondrial editing using transcription activator-like effector nucleases (MitoTALENs) and zinc finger nucleases (MtZFNs) has been successfully applied in the treatment of mitochondrial diseases and other applications, while targeted editing of CHO cell mtDNA has not been reported. Although clustered regularly interspaced short palindromic repeats (CRISPR) technology has been widely used for CHO nuclear genome engineering, it is more difficult to introduce nucleic acids such as guide RNAs into the mitochondrial matrix, which becomes a bottleneck for the application of CRISPR technology on mitochondrial engineering (Jo et al., 2015; Gammage et al., 2018).

In addition, the TCA cycle is dependent on the synthesis of NADH for energy production in mitochondria; therefore, the level of NAD⁺ is important in mitochondria as a raw material for the synthesis of NADH. In mitochondrial reactions related to glucose metabolism, NAD⁺ is involved in the TCA cycle, oxidative phosphorylation, and oxidative respiratory chain (Anderson et al., 2017), and elevated NAD⁺ levels promote the overall metabolic capacity and energy production of CHO cells. Multiple NAD⁺ synthesis pathways exist in CHO cells, among which the salvage pathway is considered to be critical in controlling intracellular NAD⁺ levels (Yaku et al., 2018). Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme regulating the salvage pathway, has elevated expression in several malignant tumor cells to meet the latter’s large energy requirements, and it has been shown that inhibition of NAMPT blocks glycolysis (Tan et al., 2013). Nicotinamide (NAM) added to the cell culture medium, and the NAM generated upon NAD⁺ depletion can be catalyzed by NAMPT to synthesize nicotinamide mononucleotide (NMN) (Mori et al., 2014; Opitz and Heiland, 2015), which in turn synthesizes NAD⁺ from NMNAT. Although NAMPT is localized intracellularly in the cytosol, NAD⁺ synthesized by the salvage pathway can be imported into the mitochondria with the help of a recently discovered mitochondrial NAD⁺ transporter protein, SLC25A51 (Girardi et al., 2020; Kory et al., 2020; Luongo et al., 2020), thus suggesting that overexpression of NAMPT and SLC25A51 in CHO cells would be a worthwhile approach to try, although it has not been reported. Besides, nicotinamide mononucleotide adenyltransferase (NMNAT) is the only enzyme responsible for NAD⁺ production (Croft et al., 2020) and is a rate-limiting factor in the NAD⁺ synthesis pathway, as is NAMPT. It has been demonstrated that overexpression of nicotinamide/nicotinic acid mononucleotide adenyltransferase 1 leads to a significant increase in total cellular NAD⁺ content (Croft et al., 2018) and regulates the concentration of NAD⁺ in relation to ATP (Pinson et al., 2019). In 2009, NMNAT3 overexpressing mice were constructed by microinjection of cDNA of NMNAT3 protein into mouse oocytes (Yahata et al., 2009), and NMNAT3 is thought to be localized in the mitochondria (Berger et al., 2005). The results showed increased levels of NAD⁺ in mitochondria and enhanced mitochondrial energy metabolism in mice (Gulshan et al., 2018). This experiment has important implications for the optimization of mitochondrial metabolism in CHO cells and provides new ideas for metabolic engineering of CHO cells.