Exploring l-isoleucine riboswitches for enhancing 4-hydroxyisoleucine production in Corynebacterium glutamicum

To explore an l-isoleucine (Ile)-induced biosensor for down-regulation of Ile synthesis pathway and enhancement of 4-hydroxyisoleucine (4-HIL) production in Corynebacterium glutamicum SN01. Four Ile-induced riboswitches (IleRSN) with different strength were screened from mutation library based on TPP riboswitch. Firstly, IleRSN were integrated into the chromosome of strain SN01 immediately upstream of ilvA gene. The 4-HIL titer of strains carrying PtacM-driven IleRS1 or IleRS3 (14.09 ± 1.07, 15.20 ± 0.93 g 4-HIL L−1) were similar with control strain S-D5I (15.73 ± 2.66 g 4-HIL L−1). Then, another copy of IleRS3-ilvA was integrated downstream of the chromosomal cg0963 gene in SN01-derived strain D-RS with down-regulated l-lysine (Lys) biosynthesis. The Ile supply and 4-HIL titer increased in ilvA two-copy strains KIRSA-3-D5I and KIRSA-3-9I, and Ile concentration was maintained less than 35 mmol L−1 under the control of IleRS3 during fermentation. The resulting strain KIRSA-3-9I produced 22.46 ± 0.96 g 4-HIL L−1. The screened IleRS was effective in the dynamic down-regulation of Ile synthesis pathway in C. glutamicum, and IleRSN with different strength can be applied in various conditions.


Introduction
Diabetes is a common chronic disease in the world. There have been 537 million adults living with diabetes, and majority were undiagnosed. It was predicted that the number of people living with diabetes would reach 783 million in 2045 (International Diabetes Federation 2021). The amino acid 4-hydroxyisoleucine (4-HIL) was firstly found and isolated from fenugreek seeds (Fowden et al. 1973;Sauvaire et al. 1998). 4-HIL with (2S,3R,4S) configuration is the major isomer in the seeds, which possesses biological activity of stimulating glucose-induced insulin secretion (Sauvaire et al. 1998) and enhancing insulin sensitivity of acceptor (Broca et al. 2004). Thereby, it is considered as a potential drug for diabetes treatment.
In order to produce 4-HIL on a large scale, several methods to synthesize 4-HIL were reported Smirnov et al. 2007;Wang et al. 2002). Along with the identification of isoleucine dioxygenase (IDO) from Bacillus thuringiensis (Hibi et al. 2011;Kodera et al. 2009), the microbial fermentation methods are gradually developed (Kivero et al. 2012;Shi et al. 2015;Zhang et al. 2018). As we known, l-isoleucine (Ile) is the direct precursor of 4-HIL. The synthesis of Ile should be enhanced in Corynebacterium glutamicum to supply Ile for 4-HIL production. However, when Ile synthesis is extremely strengthened, IDO activity will be inhibited by excessive Ile, which resulted in the decrease of 4-HIL titer (Lai et al. 2022;Tan et al. 2020). In order to dynamically modulate the biosynthesis of Ile, the Ile synthesis pathway should be down-regulated when Ile is excessively accumulated. Due to the absence of down-regulation biosensor in response to Ile, it is important to develop a novel biosensor.
Riboswitch, a non-coding cis element located in 5′ untranslated region (UTR) of mRNA, can be used as a dynamic regulation tool (Barrick et al. 2004;Bédard et al. 2020;Zhou and Zeng 2015). It is composed of two domains, aptamer domain and expression platform (Winkler and Breaker 2003). The natural riboswitches found in bacterium so far respond to few kinds of ligands. The riboswitch with much more research is thiamine pyrophosphate (TPP) riboswitch which exists in many bacteria. The riboswitch in response to amino acid is much fewer, mainly including l-lysine riboswitch (LysRS), l-glycine riboswitch (GlyRS) and S-adenosine methionine riboswitch. Up to now, there is no natural or modified Ile-induced riboswitch developed. In order to modify the existing riboswitches for desired characterization, a dual genetic selection approach with tetA gene (coding tetracycline/H + antiporter) as selection marker can be used (Muranaka et al. 2009;Nomura and Yokobayashi 2007). The cells were resistant to tetracycline (Tc) by expressing tetA, while they were resistant to nickel ion (Ni 2+ ) by inhibiting the tetA expression. It is able to screen the functional elements or circuits based on the tetA dual genetic selection model (Jang et al. 2018;Page et al. 2018).
In this study, riboswitches in response to Ile (IleRS) were screened from mutation libraries of TPP riboswitch (TPPRS) (Winkler et al. 2002) by tetA dual genetic selection. The selected riboswitches were applied to dynamically modulate Ile biosynthesis pathway, avoiding excess Ile to inhibit IDO activity in fermentation. The Ile riboswitches were integrated into the chromosome of strain SN01, immediately upstream of ilvA gene, to attempt dynamic regulation of Ile biosynthesis (Fig. 1). In addition, the IleRS-ilvA dynamic expression cassettes were integrated downstream of strong expressing gene cg0963, getting another copy of ilvA expression, to increase the dynamic supply of Ile. The effect of Ile-induced riboswitch on 4-HIL production of C. glutamicum was subsequently explored.

Materials and methods
Bacterial strains, media and culture conditions Strains used in this study are listed in Supplementary  Table 1. Escherichia coli JM109 was used for plasmid construction and propagation. E. coli strains were inoculated into Luria-Bertani medium and cultured on a rotary shaker at 37 °C and 200 r min −1 . C. glutamicum ssp. lactofermentum SN01, an l-isoleucine (Ile)-producing strain, was used for genetic manipulation and 4-hydroxyisoleusine (4-HIL) production. C. glutamicum strains were cultivated at 30 °C and 200 r min −1 in LBB medium (5 g tryptone L −1 , 2.5 g yeast extract L −1 , 5 g NaCl L −1 , and 18.5 g brain heart infusion powder L −1 ). If required, 30 mg kanamycin L −1 or 30 mg chloramphenicol L −1 was added to the media for E. coli, and 10 mg chloramphenicol L −1 was added for C. glutamicum.

Construction of library and test plasmids and strains
Primers are listed in Supplementary Table 2. To obtain the library plasmid, pTPPRS lib -tetA, the plasmid pTPPRS-tetA was constructed at first. The P tacM promoter was amplified from pJYW-5, tetA gene was amplified from pIL-tetA, and thiamine pyrophosphate riboswitch (TPPRS) fragment (Supplementary Table 3) was amplified from the genome of E. coli MG1655. After overlap extension PCR, the P tacM -TPPRS-tetA fragment was ligated into pDTW107 by one step cloning, generating pTPPRS-tetA. The linear DNA of full-length plasmid was amplified from pTPPRS-tetA, introducing a randomized and discontinuous 15 bp region (N15) in aptamer of TPPRS or a randomized 10 bp region (N10) of TPPRS1 (Fig. 2). After self-ligation, the library plasmid pTPPRS lib -tetA was generated.
The dual genetic selection of pTPPRS lib -tetA was conducted according to the previous report of Muranaka et al. (2009) with some modifications. Briefly, four rounds of dual genetic selection were conducted, and each round processed negative selection and positive selection in order (Fig. 3). After selection, the bacterial culture was plated on LBB agar, and clones were randomly selected to test the growth in the Ni 2+ -contained medium with or without Ile. The mutated riboswitches of strains whose growth were recovered by Ile addition were selected to sequence and renamed as IleRSN. To characterize The aptamer of TPPRS is underlined and the ligand-binding region is marked red, where a randomized mutation region was introduced IleRSN, the test plasmids pIleRSN-egfp were constructed. The test plasmids were transformed into C. glutamicum ATCC 13032 for characterizing fluorescence intensity.

Fluorescence assays
The strains carrying different test plasmids were incubated in LBB medium with 0-60 mmol Ile L −1 . After incubating for 12 h, the culture was taken every 4 h, and the cells were harvested. The cells were resuspended in 0.9% NaCl solution, and the cell suspension was added into a black 96-well plate with clear bottom (Corning-Costar plates) and measured with a Cytation5Microplate Reader (BioTek). Fluorescence was detected with an excitation filter of 479/20 nm and an emission filter of 520/20 nm, using the fluorescence of strain SN01/pJYW-5 as the blank.

4-HIL fermentation
4-HIL fermentation of control strain and dynamic C. glutamicum strains in shake flasks were conducted as described previously, using optimized fermentation medium (Shi et al. 2019). The cell density, residual glucose, amino acid and 4-HIL concentrations in the fermentation broth were measured every 24 h by the methods described previously. The residual glucose concentration was measured by an SBA-40C biosensor (Institute of Biology, Shandong Academy of Science, China). The pH was measured by a pH Fig. 3 The mechanism of dual genetic selection. A The response of mutated riboswitches to Ile. B The dual genetic selection of riboswitches in response to Ile by tetA. The expression of tetA gene is activated in the absence of Ile and repressed in the presence of Ile. The cells are resistant to tetracycline (Tc) and sensitive to Ni 2+ , as tetA gene is expressed. When tetA gene isn't expressed, the cells are resistant to Ni 2+ and sensitive to Tc. Thereby, during positive selection in the absence of Ile, only strains expressing TetA normally can survive in the medium containing Tc; during negative selection in the presence of Ile, only strains carrying Ile-induced riboswitch can survive in the medium containing Ni 2+ electrode (Mettler-Toledo, Germany). The amino acids and 4-HIL concentrations were detected by Agilent 1260 HPLC detector equipped with a Thermo ODS-2 HYPER-SIL C18 column (250 mm × 4.6 mm, USA) using the ortho-phthalaldehyde precolumn derivatization method. The HPLC samples processed as described previously (Lai et al. 2022).

Statistical analysis
The results were expressed as the means ± SD. A oneway analysis of variance (ANOVA) was performed for data analysis using SPSS (Statistical Package for Social Sciences) (version 27) software. Significant differences were indicated at p < 0.05.

Determination of riboswitch selection conditions
Riboswitches were commonly used to dynamically regulate gene expression at RNA level, further modulating metabolic flux (Chen et al. 2020;Hong et al. 2022;Pang et al. 2020). In order to avoid excess l-isoleucine (Ile) inhibiting isoleucine dioxygenase (IDO) activity, down-regulation riboswitch in response to Ile should be developed. We firstly tried to obtain the Ileinduced riboswitch mutant from natural riboswitches in response to other amino acids, such as l-glycine riboswitch (GlyRS) and l-lysine riboswitch (LysRS). In the pre-experiments detecting the resistance of cells carrying GlyRS-and LysRS-controlled tetA to Tc and Ni 2+ , it was found that amino acids component in the selection medium greatly interfered the determination. The thiamine pyrophosphate riboswitch (TPPRS) is the most widely spread riboswitch in bacteria (Pavlova et al. 2019), especially in E. coli and Bacillus subtilis. Therefore, the TPPRS was subsequently selected to construct mutation library, and further for screening the Ile-induced riboswitch. To optimize and determine the selection conditions of TPPRS mutant library, different concentrations of NiCl 2 or Tc were added into medium. It was found that the growth of strain SN01/pTPPRS-tetA was significantly repressed when the concentration of NiCl 2 was more than 0.5 mmol L −1 or the concentration of Tc was more than 1 mg L −1 (Fig. 4). Thus, the concentration of 0.5 mmol NiCl 2 L −1 or 1 mg Tc L −1 was chosen as the original concentration added in the selection medium for negative or positive selection of mutant library (Fig. 3). Since the Ile-induced downregulating mutants were enriched in the negative selection, the concentration of Ni 2+ was increased by 0.1 mmol L −1 after each round of negative selection, while the concentration of Tc was remained at 1 mg L −1 in each round of positive selection.

The dual genetic selection of riboswitches library
To change the specificity of TPPRS, a randomized and discontinuous 15 bp mutation was introduced into the ligand-binding central ring region of the TPPRS (Winkler et al. 2002), using degenerate base N (including A, T, C and G). The riboswitch library selection process was designed based on the report of Muranaka et al. (2009). In the negative selection, only the cells with the repressed tetA expression in the presence of 20 mmol Ile L −1 could survive, making the Ile-downregulated riboswitches to be selected; Fig. 4 The natural resistance of strain SN01/pTP-PRS-tetA to Ni 2+ (A) and Tc (B). The ΔOD 562 means the difference between OD 562 at 8 h and 22 h during incubation in the positive selection, only the cells with the activated tetA expression in the absence of 20 mmol Ile L −1 could survive, making the unexpressed riboswitches to be eliminated (Fig. 3B). After 4 rounds of dual genetic selection, which included negative selection and positive selection in a round, 120 clones were randomly selected to test the growth recovery in the Ni 2+ -contained medium with Ile compared with growth in medium without Ile. Fifty-six riboswitches were sequenced, and 29 of them had mutated. Two kinds of mutations occurred, 28 riboswitches were mutated to TPPRS1, and the last one was mutated to TPPRS2 (Fig. 5A). It was found that the mutations in both two types were focused on first 6 degenerate Fig. 5 The sequence (A) and fitted dose-response curve of IleRS1 (B), IleRS2 (C), IleRS3 (D) and IleRS4 (E). RFU represents relative fluorescence unit, the ratio of fluorescence intensity and OD 562 . The RFU of strain SN01/pJYW-5 was used as the blank bases instead of evenly distributed in 15 bp region. Thus, additional riboswitch library was constructed based on TPPRS1 and TPPRS2, separately, introducing a randomized and continuous 10 bp mutation region in the end of P5 stem of TPPRS (Winkler et al. 2002), generating TPPRS1 library and TPPRS2 library (Fig. 2). After 3 rounds of dual genetic selection, 40 clones were randomly selected to test growth recovery from TPPRS2 library, and 19 clones were sequenced among them. Two of them mutated to TPPRS3, and only one riboswitch mutated to TPPRS4 (Fig. 5A). However, not any new mutation occurred in TPPRS1 library.

Characterization of riboswitches in response of Ile
The Ile-induced TPPRSN riboswitches selected were named IleRSN (N = 1, 2, 3, 4) (Supplementary Table 3). In order to characterize the IleRSN, the test plasmids pIleRSN-egfp were transformed into C. glutamicum ATCC 13032 for characterizing fluorescence intensity. To characterize the sensitivity and dynamic range of IleRSN to Ile, different concentrations of Ile (0-60 mmol L −1 ) were added in the LBB medium. After incubating for 12 h, the culture was taken every 4 h for fluorescence assays. Among these riboswitches, the IleRS1 showed the highest global expression level, and the fluorescence intensity was down-regulated by 21.08% after induced by 60 mmol Ile L −1 (Fig. 5). The IleRS3 showed the highest repression rate (29.59%), and the expression level was about a tenth of IleRS1 (Fig. 5). The repression rate of IleRS2 and IleRS4 were 24.20% and 8.97%, respectively, under the induction of 40 mmol Ile L −1 . The expression level of both IleRS2 and IleRS4 were weaker than IleRS3, about half of it (Fig. 5). As a result, the mutated riboswitches IleRSN could be applied for dynamic regulation of gene expression and metabolic pathway.

Dynamic regulation of chromosomal ilvA expression and 4-HIL biosynthesis by IleRS
As it mentioned previously, excess Ile would inhibit the activity of IDO which catalyzes the 4-hydroxyisoleucine (4-HIL) synthesis from Ile (Fu et al. 2014). Therefore, the synthesis of Ile is expected to be downregulated when the inhibition occurs. However, Ile is the direct substrate of 4-HIL production. When Ile concentration is low, the Ile synthesis shall be enhanced. As a consequence, riboswitch biosensor IleRS was applied to dynamically down-regulate Ile synthesis when the concentration of Ile was extremely high, and when the concentration of Ile was lower, the synthetic pathway would be normally expressed for supply of Ile precursor.
In order to use IleRSN riboswitches for dynamic regulation of Ile synthesis pathway, the chromosomal ilvA gene was targeted. The single IleRSN fragment and IleRSN under the control of constitutive strong promoter P tacM were inserted immediately upstream of ilvA gene, generating strains IRSA-N and IRSA-PN (N = 1, 2, 3), respectively. To synthesize 4-HIL, the producing-plasmid pI-D5 I with Ile-induced dynamically upregulated ido U gene was transformed into strains IRSA-N and IRSA-PN with Ile-induced dynamically downregulated ilvA gene, generating strains IRSA-N-D5 I and IRSA-PN-D5 I. The fermentation of these 6 dynamic strains was conducted then. The strain SN01/pI-D5 I (S-D5 I) was used as control.
Integration of another copy of ilvA expression cassette As the results shown (Fig. 6), all Ile was converted and the Ile anabolic flux in IRSA-PN-D5 I was insufficient to support the further increase of 4-HIL production. Thereby, we tried to improve the biosynthesis of Ile by integrating another copy of ilvA expression cassette in chromosome. To decrease the production of main by-product Lys in C. glutamicum, the LysRS-integrated strain D-RS constructed previously was used as original strain here. The chromosomal dapA gene was down-regulated by LysRS (Supplementary Table 3) in D-RS. In previous work of Lai et al. (2022), bidirectional regulation was conducted by plasmids harboring Lrp-P brnFE N-upregulated ido U gene and P ilvBNC -downregulated ilvA gene in strains D-N IPA. Some of the D-N IPA strains performed well in 4-HIL titer, such as D-1 IPA and D-D5 IPA, while some of them performed poorly, such as D-0 IPA and D-9 IPA. However, the 4-HIL titer of corresponding unidirectionally-regulated strains D-0 I and D-9 I was substantial, similar with the control strain SI. It was speculated that the dynamic regulation of ilvA gene by plasmid resulted in the substantial enhancement of Ile biosynthesis, while the degree of dynamic down-regulation of ilvA by attenuator P ilvBNC was insufficient to release the inhibition of IDO caused by excessive Ile. Therefore, here we tried to integrate another copy of ilvA expression cassette in chromosome of D-RS, and use the more flexible dynamic tool, IleRS, to regulate the expression of the second copy of ilvA. According to the 4-HIL titer of IRSA-N-D5 I and IRSA-PN-D5 I, the second copy ilvA gene should be expressed to synthesis Ile for 4-HIL production. Thus, IleRS3-ilvA was integrated downstream of cg0963 gene, generating strain KIRSA-3. The dynamically-regulated producing-plasmids, pI-D5 I and pI-9 I were transformed into KIRSA-3, generating strain KIRSA-3-D5 I and KIRSA-3-9 I, respectively. The fermentation of these 2 strains was conducted then. The strains S-D5 I and S-9 I were used as control.
The growth of dynamic strains KIRSA-3-N I was similar with the control S-N I (Fig. 7A), and the culture pH of dynamic strains was steadier than the control (Fig. 7B). Compared with previously constructed strain D-9 IPA (8.59 ± 1.41 g L −1 ) where ilvA gene was dynamically down-regulated in plasmid (Lai et al. 2022), the strain KIRSA-3-9 I where ilvA gene was dynamically down-regulated in chromosome produced much more 4-HIL (22.46 ± 0.96 g L −1 ) (Fig. 7C), close to or even more than unidirectionallyregulated strain S-9 I (20.58 ± 3.80 g L −1 ). The 4-HIL titer of strain KIRSA-3-D5 I (19.89 ± 2.62 g L −1 ) was also higher than that of S-D5 I (15.73 ± 2.66 g L −1 ). It indicated that the integration of the second copy of IleRS-induced ilvA gene in chromosome was beneficial to the enhancement of 4-HIL production, likely due to the appropriately increased Ile  , 9). The data represent the means ± SD. *p < 0.05 compared with S-D5 I. The names of strains were abbreviated as S-D5 (S-D5 I), S-9 (S-9 I), K-D5 (KIRSA-3-D5 I), K-9 (KIRSA-3-9 I) in C biosynthesis that did not trigger the inhibition of IDO activity. The strategy to decrease the synthesis of Lys by LysRS-induced dapA gene also worked well (Fig. 7C). The Lys concentration in strains KIRSA-3-D5 I (1.36 ± 0.36 g L −1 ) and KIRSA-3-9 I (1.49 ± 0.18 g L −1 ) were lower than strains S-D5 I (3.57 ± 1.66 g L −1 ) and S-9 I (4.95 ± 1.37 g L −1 ). Taking together in the fermentation of these 4 strains, Ile didn't accumulate extensively, but almost converted to 4-HIL.

Discussion
In order to achieve the overproduction of 4-hydroxyisoleucine (4-HIL), variety of approaches were conducted in bacteria (Du et al. 2020;Wei et al. 2022;Zhang et al. 2018). Kivero et al. (2012) firstly introduced ido (coding isoleucine dioxygenase, IDO) into E. coli to produce 4-HIL. However, the production of 4-HIL in E. coli need the addition of extra l-isoleucine (Ile) as substrate. Afterwards, Ile-producing C. glutamicum was used to construct the synthesis pathway of 4-HIL (Shi et al. 2015;Zhang et al. 2018). As the advance of metabolic engineering, it is realized that the cell resource competition between biomass and production limited the overproduction of target metabolites. Compared with inducible promoter induced by exogenous inducer (e.g. pBAD/ arabinose), biosensors auto-induced by endogenous metabolites are more convenient and economical for regulation. In our previous work, several biosensors were applied to balance the cell growth and production in C. glutamicum SN01 to overproduce 4-HIL, including Lrp-P brnFE , l-lysine riboswitch (LysRS) and P ilvBNC attenuator (Lai et al. 2022;Tan et al. 2020). As the direct precursor of 4-HIL and inhibitor of IDO, Ile synthesis is usually modulated. However, the developed Ile-induced biosensors only includes Ile-activated Lrp-P brnFE and Ile-repressed P ilvBNC attenuator, and P ilvBNC attenuator is the only one for down-regulation. There are few reports to modify the dynamic range and sensitivity of P ilvBNC attenuator. In addition, the regulation of P ilvBNC attenuator is irreversible, which limits its application in complex conditions (e.g. the targeted metabolite was toxic but necessary for production). Therefore, it is important to explore a novel Ile-induced biosensor to down-regulate Ile synthesis pathway in C. glutamicum.
Riboswitch is a kind of dynamic regulation tool with general applicability in bacteria (Kamiura et al. 2019;Qian and Cirino 2016). Kinds of strategies to modify the riboswitches have been proposed (Hallberg et al. 2017;Wu et al. 2023). Here, we screened Ile-induced riboswitch (IleRS) in mutation library based on thiamine pyrophosphate riboswitch (TPPRS) from E. coli MG1655 using tetA dual genetic selection (Figs. 3 and 5). In 4-HIL-producing C. glutamicum constructed, Ile always accumulated less than 60 mmol L −1 in fermentation. Therefore, the concentration of 0-60 mmol Ile L −1 was chosen as inducer range for fluorescence characterization. The obtained IleRSN with various strength were firstly used to directly regulate chromosomal ilvA gene in C. glutamicum SN01. Two of the resulting strains, IRSA-P1-D5 I and IRSA-P3-D5 I, performed similarly with the control strain S-D5 I (Fig. 6C), suggesting that the expression level of P tacM -IleRS1-and P tacM -IleRS3-controlled ilvA was same with the natural P ilvA -ilvA. The rest of strains performed poorly, especially in which the expression of ilvA gene was controlled by P ilvA -driven IleRSN. Thus, another copy of ilvA expression cassette (IleRS3-ilvA) was integrated downstream of strongly expressed cg0963 gene, and the producing plasmids pI-N I were transformed into the strains. In this way, both Ile synthesis and titer of 4-HIL were increased (Fig. 7C).
It was mentioned that the weak expression of ilvA was unbeneficial to the synthesis of Ile, further decreased 4-HIL production (Fig. 6C). Whereas, excessively strong expression of ilvA was also harmful to the cell. The accumulation of α-ketobutyrate, product of threonine dehydrase (coded by ilvA), results in severe cellular toxicity (Fang et al. 2021), which led to the failure of construction of strain KIRSA-1. It was considered that the corporately and appropriately strong expression of ilvA and downstream genes would recover the damage and direct the metabolic flux towards the synthesis of Ile and 4-HIL. According to the Ile accumulation in the fermentation of strains KIRSA-3-N I (Fig. 7D), the concentration of Ile was far from the concentration that would severely inhibit IDO activity, which may be resulted from IleRS dynamic regulation. Compared with strain IRSA-P3-D5 I (15.20 ± 0.93 g L −1 ), the integration of the second copy of ilvA expression cassette (strain KIRSA-3-D5 I) increased the 4-HIL titer (19.89 ± 2.62 g L −1 ) by 30.8% (Fig. 7C), and this titer increased by 24.1% compared with previously constructed strain D-RS-D5 IPA with P ilvBNC -controlled ilvA (16.03 ± 0.04 g L −1 ) (Lai et al. 2022). In addition, the 4-HIL titer of strain KIRSA-3-9 I (22.46 ± 0.96 g L −1 ) even increased by 266.4% compared with previously constructed strain D-RS-9 IPA (6.13 ± 0.41 g L −1 ) (Lai et al. 2022). It is indicated that the Ile synthesis pathway in strains KIRSA-3-N I was promoted and well balanced with 4-HIL conversion pathway. In another research which engineered C. glutamicum for the 4-HIL producing, the best strain produced 6.15 g L −1 4-HIL after 48-h fermentation , the strain KIRSA-3-9 I in this study had the greater 4-HIL titer (6.95 g L −1 ) in the same fermentation period.

Data availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Consent to publication
All authors have read the manuscript and agreed to publish.