As environmental problems have rapidly become more serious in recent years, many industries worldwide have been forced to use sustainable materials and processes (Jung et al., 2023, Kate et al., 2022). Because existing methods for synthesizing high-value chemicals require considerable energy and emit environmentally toxic pollutants, there have been many attempts to establish a bio-based synthesis method for high-value chemicals (AliAkbari et al., 2021, Singh, 2017).

5-Hydroxyvaleric acid (5-HV) is especially considered an important intermediate of several high-value materials, such as polyhydroxyalkanoate (PHA) monomer subunit, 1,5-pentanediol, δ-valerolactone, and tetrahydropyran; hence, many trials have been conducted to design bio-based synthetic pathway that include 5-HV (Adeleye et al., 2020, Cen et al., 2022, Chuah et al., 2013, Lakshmanan et al., 2019, Wang et al., 2020). In previous studies, lysine has been suggested as the starting material for 5-HV production (Kim et al., 2023b). First, lysine is metabolized into 5-aminopentamide through lysine monooxygenase and 5-aminopentamide is deaminated to 5-aminovaleric acid (5-AVA) via δ-aminovaleramidase (Cen et al., 2022, Kim et al., 2023b). Overexpressed 4-aminobutyrate aminotransferase (GabT) and aldehyde reductase (YqhD) then convert 5-AVA into glutarate semialdehyde and 5-HV (Cen et al., 2022, Kim et al., 2023b, Sohn et al., 2021).

Although many 5-HV-related genes have been studied extensively, there are critical shortcomings related to the supply of cofactors (Cen et al., 2022, Kim et al., 2023b, Sohn et al., 2021). First, supplementation of NADPH, which is consumed as a reducing agent when YqhD is activated, needs to be improved (Cen et al., 2022, Kim et al., 2023b, Sohn et al., 2021). This was improved our previous study by introducing an NADPH regeneration system with glucose dehydrogenase (GDH) derived from B. subtilis 168, which successfully achieved high 5-HV productivity of 1.708 g/(L∙h) (Kim et al., 2023). However, the second issue, α-ketoglutaric acid (α-KG), still remains the biggest hurdle (Cen et al., 2022, Hong et al., 2018, Kim et al., 2023b, Yang et al., 2020). In the reaction of GabT from 5-AVA to glutarate semialdehyde, pyridoxal 5’-phosphate monohydrate (PLP) and α-KG are required as cofactors (Hong et al., 2018, Yang et al., 2020). The commercial methods to produce PLP are being actively conducted for several years and involve halogenation of alanine or amination of propionic acid (He et al., 2022, Tanase et al., 1979); however, the research on α-KG is lacking despite its importance. α-KG acts as an amine acceptor by being aminated into L-glutamate during the reaction of GabT, therefore, large amounts of α-KG are required for high production of 5-HV (Hong et al., 2018, Kim et al., 2023b, Sohn et al., 2021, Yang et al., 2020). In theory, α-KG should be provided in equivalents of 5-AVA; however, it is economically unfeasible due to its high cost (Song et al., 2016). In addition, using a same equivalent of α-KG and 5-AVA lowers pH so that requires various buffering agents to maintain proper pH (Yang et al., 2020).

Therefore, in this study, we designed α-KG regeneration system by introducing L-glutamate oxidase (GOX) (EC 1.4.3.11), which catalyzes the oxidative deamination of L-glutamate into α-KG (Lee et al., 2022, Song et al., 2022, Yang et al., 2020, Zhang et al., 2020a). Since GOX exhibits remarkable α-KG conversion with its low Km value and does not require additional cofactors (Zhang et al., 2020b), continuous research has been conducted to apply GOX to α-KG production as final products or regeneration as cofactors (Table 1). Herein, we determined that the α-KG regeneration system using GOX could be successfully applied to 5-HV production and introduced GOX derived from Streptomyces sp. X119-6 as it boasts a high conversion rate.

Although GOX effectively reduces the input of α-KG for 5-HV production, it produces H2O2 as byproduct, which induces toxicity to the whole cell system and can degrade α-KG into succinic acid (Ham et al., 2021, Lee et al., 2022, Song et al., 2022). Accordingly, we overexpressed of catalase (KatE) (EC 1.11.1.6), which was derived from Escherichia coli K-12 to prevent exogenous catalase from removing H2O2 (Ham et al., 2021, Lee et al., 2022). KatE detoxifies reactive oxygen species (ROS) by degrading 2 mol H2O2 into 1 mol O2 and 2 mol H2O (Ham et al., 2021, Lee et al., 2022).

Thus, a two-cell biotransformation system for high 5-HV production was established. By simultaneously applying GOX and KatE for regeneration of α-KG, we successfully produced up to 11.3 g/L titer of 5-HV from 200 mM 5-AVA, showing a 1.60-fold faster turnover compared to that of without GOX. In addition, this two-cell system was expanded into 2-step strategy for the synthesis of poly(5-hydroxyvaleric acid) (P(5HV)) homopolymer, and 412 mg/L of P(5HV) was obtained with the supernatant directly harvested from the biotransformation. Considering that 5-HV and P(5HV) production has several difficulties related to cofactors, especially α-KG, our study would provide a practical and economical alternative method to produce 5-HV and P(5HV) (Kim et al., 2023b, Yang et al., 2020).