Hyaluronic acid (HA), composed of alternating β-1,3-N-acetyl-d-glucosamine (GlcNAc) and β-1,4-d-glucuronic acid (GlcUA), belongs to the glycosaminoglycan family (Westbrook et al., 2018; Cheng et al., 2019b; Wang et al., 2020) and mainly exists in animal tissues such as chicken crowns. HA has already been applied in the clinical, cosmetic, pharmaceutical, and food industry due to its excellent biocompatibility and extraordinary water-retaining properties (Stecco et al., 2011; Graca et al., 2020; Yu et al., 2020). Generally, HAs with different molecular weights (M_Ws) have different applications (Qiu et al., 2021). High-molecular-weight HA (HMW-HA, M_W ≥ 1 × 10⁶ Da) is mainly used for joint cavity injection and cartilage degeneration repair, owing to its good viscoelasticity, moisture retention, and lubrication properties. Low-molecular-weight HA (LMW-HA, 1 × 10⁴–1 × 10⁶ Da) usually plays an important role in the cosmetics field (Qiu et al., 2021), since it can improve skin elasticity and reduce wrinkles (Pavicic et al., 2011), as well as regulate skin metabolism and delay aging (Camacho et al., 2016). In addition, HA oligosaccharides (M_W ≤ 1 × 10⁴ Da) may have significant application prospects in the food health field (Boltje et al., 2009), as they have been widely used in fruit juice, soy milk, jelly, and other food. In the future, HAs with different M_Ws will have more and better development prospects in different fields (Qiu et al., 2021).

Nowadays, industrial HA production has already been achieved through fermentation of group C Streptococcus (Streptococcus equisimilis and Streptococcus zooepidemicus) (Liu et al., 2011) and has gradually replaced the traditional animal issue extraction methods. However, Streptococcus sp. may produce some exotoxins and immunogens during HA production (Cheng et al., 2019b; Wang et al., 2020). Considering the potential hazards, adoption of GRAS (generally recognized as safe) strains to produce HA is required urgently. Up to now, HA has been biosynthesized successfully in Escherichia coli (Yu and Stephanopoulos 2008; Mao et al., 2009; Woo et al., 2019), Lactococcus lactis (Prasad et al., 2010; Sunguroglu et al., 2018; Jeeva et al., 2019), Bacillus subtilis (Westbrook et al., 2018a; Westbrook et al., 2018b; Li et al., 2019), Agrobacterium sp. (Mao and Chen 2007), and Corynebacterium glutamicum (Hoffmann and Altenbuchner 2014; Cheng et al., 2016; Cheng et al., 2017; Cheng et al., 2019b; Wang et al., 2020; Zheng et al., 2020). Even though the HA titers obtained from heterologous hosts are in general still lower than those achieved by natural, pathogenic producers. In the biosynthesis of HA, HA polymer is synthesized by an enzyme called HA synthase (HAS), which is grouped into two classes (Agarwal et al., 2019). Class I HAS is a kind of integral membrane protein containing a single domain, while Class II HAS is a soluble/membrane anchored protein with two domains (Weigel 2015). Most of the HAS found so far belong to Class I, such as HAS from S. equisimilis (seHAS) and Streptococcus pyogenes (spHAS); however, Class II enzyme is found only in Pasteurella multocida (pmHAS). It was reported that seHAS and spHAS contained a single HAS protein associated with an additional component with a mass of about 23 kDa, which was identified as cardiolipin (CL), one of the common bacterial membrane phospholipids (Weigel 2002). Thus, the active HAS enzyme contains a HAS protein monomer and about 14–18 CL molecules as a complex, in which CL is essential for enzymatic activity. It has not been studied whether enhanced CL synthesis would have an impact on HA production.

In order to meet the requirements of industrial production, researchers have made lots of efforts to increase the yield of HA in recombinant strains. Some hosts naturally harbor an almost complete metabolic route for HA synthesis, just lacking the HAS gene (hasA), such as B. subtilis, E. coli, and C. glutamicum (Manfrao-Netto et al., 2021). Based on the heterologous expression of hasA, different artificial has operons containing a combination of different genes have been constructed in various hosts to increase HA titer. Examples are spABC and sseAB in E. coli (Yu and Stephanopoulos 2008), pmhasA-tuaD-gtaB (operon hasABC) in B. subtilis 168 (Jia et al., 2013), and ssehasA-hasB operon in C. glutamicum (Cheng et al., 2016), which in general obtained about 1–7 g/L of HA. Besides, engineering metabolic pathways promoted the yield of HA as well in different strains (Jin et al., 2016; Westbrook et al., 2018a). Wang et al. (2020) coupled HA degradation with HA production through adding leech hyaluronidase, which led to 74.1 g/L of HA accumulation with super-low M_W (∼53 kDa). In addition, some researchers also found that the cell-morphology engineering strategies, for example, downregulating or overexpressing the cell division initiator protein FtsZ in B. subtilis or C. glutamicum, can further enhance the HA titer (Westbrook et al., 2016; Zheng et al., 2020). For the overexpression of Vitreoscilla sp. hemoglobin (VHb), however, contrary results were reported in different strains: in B. subtilis, expression of VHb improved the HA titer from 0.9 to 1.8 g/L (Chien and Lee 2007); but in C. glutamicum, co-expression of the VHb gene (vgb) with hasA lowered HA yield by about 1.5-fold (Hoffmann and Altenbuchner 2014). In view of this, the effect of VHb varies case by case, which needs to be further investigated. Except for the genetic strategies, some researchers also enhanced the HA yield through optimization of the culture medium. Important functions of trace element and different kinds of carbon and nitrogen sources such as corn syrup powder and glucose were highlighted (Cheng et al., 2016; Chahuki et al., 2019).

Herein, we proposed four indirect pathway metabolic strategies to further enhance the HA titer in engineered C. glutamicum, i.e., enhancing the carbon substrate uptake via genetically activating the PTS-independent uptake system; regulating HA synthesis through the intensified synthesis of CL (an auxiliary molecule of HAS); duly expressing VHb via integration of the gene vgb into the genome of C. glutamicum, thereby promoting cell oxygen transfer, energy metabolism, and finally HA synthesis; and lastly finding the key inorganic nitrogen source (glutamine) through transcriptome analyses and then enhancing the HA titer via its supplementation. Combination strategies were further evaluated, and HA titer was significantly enhanced by optimal combinations.

As mentioned above, various efforts have been made to promote HA production in recombinant C. glutamicum, for example, optimization of has operon, knocking out competitive metabolic pathways, membrane engineering to enlarge the availability of cell membrane, and coupling HA synthesis with HA hydrolysis. Besides these, here, we tried to find out some strategies, indirect, non-regular, but effective as well, to further improve the HA titer in engineered C. glutamicum. Figure 1 showed the overview of the indirect metabolic strategies to promote HA production in this work.

In this study, we strengthened the biosynthesis of HA in recombinant C. glutamicum through four indirect, non-regular metabolic strategies. We assumed the HA-producing strain as a “whole block factory” and enhanced the product synthesis via intensified bioprocesses, such as substrate uptake, HAS activity improvement, and oxygen transfer.

Firstly, glucose uptake was intensified by IolR knockout, leading to a 6.9% increase of HA titer. This is a common effective strategy for enhancing the titer of different products synthetized by C. glutamicum. For example, deletion of IolR and overexpression of IolT1 also increased the yield of l-ornithine by 10% (Zhang B. et al., 2019). Similarly, Zhang X. et al. (2019) found enhanced cell growth and l-serine production of 3.9-fold and 5.9-fold, respectively, via inactivation of IolR. Based on the same metabolic regulation strategy, some other alternative strategies can also be tried to strengthen carbon utilization and thereby enhance the product yield, such as overexpression of the glucose transporters IolT1/T2 and EII_glc.

CLs can bind HAS to form complexes and play an essential role in maintaining the biological activity of HAS. In a cell-free system, Weigel et al. reported that addition of CL into purified HAS increased the K _m for UDP-GlcUA and decreased the K _m for UDP-GlcNAc, finally giving an overall stimulation of V_max. Both the seHAS and spHAS could maintain ∼60% initial activity by addition of bovine CL after being stored at −80°C for 2 months (Tlapak-Simmons et al., 1999). Based on the literature, we analyzed the CL synthesis pathway in C. glutamicum and focused on three key enzymes, PgsA1, PgsA2, and Cls.

We found that overexpression of pgsA1/pgsA2/cls can all accelerate cell growth to some degree, especially in the first 24 h. As reported previously, the pgsA gene codes for phosphatidylglycerophosphate synthase, which catalyzes the committed step of biosynthesis of phosphatidylglycerol (Dowhan 1997). And the cls gene codes for CLS, which condenses two molecules of phosphatidylglycerol to form CL in the prokaryote. As the major acidic phospholipids of the organism, phosphatidylglycerol and CL play important roles in bacterial cell structure and diverse physiological processes, such as DNA replication, cell division, respiration, and osmotic stress response (Wood 2018). Yasuhiro Shiba et al. (2004) reported that a pgsA-null mutation is lethal for E. coli. Xia et al. confirmed that E. coli are dependent on phosphatidylglycerol for cell growth, which cannot be substituted with phosphatidylinositol (Xia and Dowhan 1995). It can be seen that phosphatidylglycerol is essential for cell growth. As for CL, Kazuhisa Sekimizu et al. found that CL could activate the dnaA protein, which serves as the initiation of the protein of replication (Sekimizu and Kornberg 1988). Amer H. Asseri et al. (2021) reported that CL can also enhance the enzymatic activity of cytochrome bd for C. glutamicum, which is a terminal oxidase of respiratory pathways and can also enhance the oxygen consumption activity by twofold. Satomi Nishijima et al. (1988) came to the conclusion that the cls gene may confer growth or survival advantages for E. coli. In summary, pgsA1/pgsA2/cls genes are vital for cell growth and other physiological processes, and overexpression of these genes may promote cell growth to a certain degree.

In addition, we also found that pgsA1 overexpression had a negative effect on HA titer, while pgsA2 and cls could significantly promote the HA synthesis. We measured the concentration of free CLs in Cg-0 and Cg-CLS (Supplementary Table S2) and found that overexpression of cls led to an obvious increase of intracellular CLs. As for the negative effect of pgsA1 on the HA titer, we assumed that pgsA1 and pgsA2 might have different activities towards conversion of CDP-DAG, thus leading to different concentrations of CL. In addition, Triscott and Vanderijn (1986) reported that the optimal activity of HAS occurred at a CL/protein ratio (μg/μg) of 5:1. In the future, we can clone pgsA1/pgsA2/cls and HAS under different inducible promoters and then accurately adjust the ratio of CL/HAS, thereby further enhancing the HA synthesis.

The HA synthesis process requires high-oxygen and high-energy conditions. It was observed that a higher HA titer was achieved in aerobic conditions than in anaerobic conditions (Huang et al., 2006; Prasad et al., 2010). According to our previous study, the recombinant C. glutamicum can produce HA with M_w ranging from 0.2 to 0.3 MDa (Cheng et al., 2019b). It was reported that when the M_w of HA was above 5.7 × 10⁴ Da, the broth viscosity increased with the HA concentration increasing, which would result in a great obstacle for nutrient and oxygen transfer. Nowadays, the industrialized HA titer was 6–10 g/L with a M_w of 2 MDa in Streptococcus (Cheng et al., 2019b). The high viscosity of fermentation media made the elevation of the HA titer in Streptococcus much more difficult. Therefore, to duly express VHb is probably an effective solution for this. Zhao et al. (2017) found that expression of VHb in E. coli resulted in a 94.4% increase of trans-4-hydroxy-l-proline production in a 100 ml shaking flask culture compared to the same strain without VHb expression. Wang et al. (2018) cloned and expressed VHb in a surfactin-producing strain B. subtilis THY-15, leading to a 24% increase in flask. But surprisingly, in C. glutamicum, co-expression of the VHb gene (vgb) with hasA based on plasmids lowered HA yield by 1.5-fold (Hoffmann and Altenbuchner 2014). Therefore, we deduced that duly expressing VHb is important for its positive function. We introduced VHb into the host via a genome-integrated strategy. After introduction of VHb, intracellular ATP and NAD⁺ increased dramatically, indicating that the energy metabolism was improved during fermentation. And it was assumed that more oxygen strengthened the oxidative phosphorylation process, thus improving NAD⁺ replenishment as well as the ratio of NAD⁺/NADH (Garrigues et al., 1997; Lan et al., 2006; Guez et al., 2008).

Besides, expression of the vgb gene increased the total concentration of NADH and NAD⁺. This could be attributed to the increased activity of the tricarboxylic acid (TCA) cycle (Pablos et al., 2014). It was reported that VHb can capture oxygen and transfer it to the terminal oxidases, and the dissociation rate constant of VHb is significantly higher than other hemoglobins. E. coli-expressing vgb would direct a higher fraction of glucose through the pentose phosphate pathway (ppp) and channel less acetyl-CoA through TCA than the wild-type strain, which generated an excess amount of NADPH, and resulted in a transhydrogenation reaction, leading to an H⁺-flux from NADPH to NAD⁺. To sum up, the effective delivery of oxygen to the cytochromes would regenerate NAD⁺ faster, activating the TCA cycle as well. During the dynamic balance of NADH and NAD⁺, the generation rate of NAD⁺ got faster, which probably led to the increase of the total concentration of NAD⁺ and NADH. As for the HA titer, we found that the HA titer of Cg-VHb was highly increased by 26%. Therefore, we supposed that integration of the VHb gene into the host genome is a promising strategy for industrialized HA bioproduction.

Finally, we investigated the influence of another important factor, amino group carrier (glutamine), on HA synthesis. We analyzed the transcriptome differences between HA high-producing strain and HA low-producing strain and identified that five genes relating to the metabolism of glutamate or glutamine were upregulated significantly. This indicated that when producing HA, the bacteria would enhance their native glutamic acid and glutamine synthesis to enhance the supplement of inorganic nitrogen for HA synthesis. To confirm this deduction, we tested the effectiveness of the exogenous addition of glutamine. Correspondingly, we found that the titer of HA was increased significantly. These results also implied that deficient supplement of glutamine will limit HA bioproduction to some degree. Aside from addition of glutamate/glutamine into the medium, we can also strengthen the synthesis of glutamine or glutamic acid in the engineered strains, for example, by overexpression of glutamine or glutamate synthase.

To conclude, four indirect metabolic engineering strategies for HA titer enhancement in engineered C. glutamicum were proposed and investigated in this work. In general, the enhancement of carbon uptake efficiency (specifically glucose), the auxiliary factor titer of HAS (CL), oxygen-transfer efficiency via duly expressing VHb, and supplement of glutamine all played positive and significant roles for enhanced HA synthesis. Combination strategies further elevated the HA titer, and the optimal strains showed a 30% increase in HA production under present conditions. It can be expected that after overall optimization and accurate regulation of different strategies next, the HA titer will be further increased significantly and thereby enable the scaled-up production of HA via the engineered C. glutamicum.