Abstract
Hyaluronic acid (HA) is composed of alternating d-glucuronic acid and N-acetyl-d-glucosamine, with excellent biocompatibility and water retention capacity. To achieve heterologous biosynthesis of HA, Corynebacterium glutamicum, a safe GRAS (generally recognized as safe) host, was utilized and metabolically engineered previously. In this work, to achieve further enhancement of HA yield, four strategies were proposed and performed separately first, i.e., (1) improvement of glucose uptake via iolR gene knockout, releasing the inhibition of transporter IolT1/IolT2 and glucokinases; (2) intensification of cardiolipin synthesis through overexpression of genes pgsA1/pgsA2/cls involved in cardiolipin synthesis; (3) duly expressed Vitreoscilla hemoglobin in genome, enhancing HA titer coupled with more ATP and improved NAD+/NADH (>7.5) ratio; and (4) identification of the importance of glutamine for HA synthesis through transcriptome analyses and then enhancement of the HA titer via its supplement. After that, we combined different strategies together to further increase the HA titer. As a result, one of the optimal recombinant strains, Cg-dR-CLS, yielded 32 g/L of HA at 60 h in a fed-batch culture, which was increased by 30% compared with that of the starting strain. This high value of HA titer will enable the industrial production of HA via the engineered C. glutamicum.
Highlights
Hyaluronic acid (HA), composed of alternating β-1,3-N-acetyl-D-glucosamine (GlcNAc) and β-1,4D-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
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
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
Summary
Hyaluronic acid (HA), composed of alternating β-1,3-N-acetyl-D-glucosamine (GlcNAc) and β-1,4D-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. HAs with different molecular weights (MWs) have different applications (Qiu et al, 2021). High-molecular-weight HA (HMW-HA, MW ≥ 1 × 106 Da) is mainly used for joint cavity injection and cartilage degeneration repair, owing to its good viscoelasticity, moisture. 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). HAs with different MWs will have more and better development prospects in different fields (Qiu et al, 2021)
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