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. 2026 Feb 6;13:163–171. doi: 10.1016/j.synbio.2026.01.024

Multistep metabolic engineered Klebsiella oxytoca for efficient l-leucine production

Weikang Sun a,1, Qiaoyue Yang a,1, Shuo Wang a,1, Lingru Gong a, Zhi Zhou b, Mingyuan Liu a, Xiaoxu Tan a, Qianjin Kang c, Wensi Meng a, Yidong Liu a, Zhaoqi Kang a, Ping Xu c, Cuiqing Ma a, Chao Gao a,⁎, Chuanjuan Lü a,⁎⁎
PMCID: PMC12907686  PMID: 41704463

Abstract

l-Leucine is widely applied in food, feed and medical industries. In this work, an efficient l-leucine producing strain Klebsiella oxytoca LKO-14 was constructed based on an l-valine producer K. oxytoca VKO-9. The exogenous l-leucine biosynthesis pathway was introduced to achieve l-leucine accumulation and decrease l-valine production. Modifying l-leucine transport system and optimizing copy numbers of genes including CgleuAM encoding l-leucine insensitive isopropylmalate synthase, EcleuCD encoding isopropylmalate isomerase, EcleuB encoding isopropylmalate dehydrogenase, NopheDH encoding phenylalanine dehydrogenase, were conducted to increase l-leucine production. The expression of budB encoding α-acetolactate synthase and EcilvD encoding dihydroxyacid dehydratase were also enhanced to improve precursor supply of l-leucine. In addition, the dihydroxy acid dehydratase from Streptococcus mutans containing an oxygen-tolerant [2Fe–2S] cluster was introduced to further enhance l-leucine production. Finally, the plasmid free and inducer independent K. oxytoca LKO-14 produced 70.1 g/L l-leucine, with a yield of 0.347 g/g and a productivity of 1.46 g/L/h, respectively.

Keywords: l-Leucine, Klebsiella oxytoca, Metabolic engineering, l-Valine, Metabolic flux redirection

Graphical abstract

Image 1

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1. Introduction

l-Leucine is an important branched-chain amino acid with diverse applications and broad market demand [[1], [2], [3], [4], [5]]. The vast majority of industrial generated l-leucine has been applied as the feed additive [6]. Its addition can improve the disease resistance and growth performance of poultry, swine and aquatic animals [[7], [8], [9], [10]]. Nowadays, the industrial production of l-leucine predominantly relies on microbial fermentation [11]. Escherichia coli and Corynebacterium glutamicum are the two main microorganisms employed for l-leucine fermentation [[12], [13], [14], [15], [16]]. The highest concentrations of l-leucine produced by C. glutamicum and E. coli were 54.3 g/L and 63.29 g/L, respectively [12,17].

The biosynthesis of l-leucine begins with the condensation of 2-ketoisovalerate with acetyl-CoA, and proceeds via the catalytic actions of isopropylmalate synthase (IPMS), isopropylmalate isomerase (IPMI), and isopropylmalate dehydrogenase (IPMDH) to form 2-ketoisocaproate. Then, 2-ketoisocaproate can be transformed into l-leucine by branched-chain amino acid transaminase or leucine dehydrogenase (LeuDH) [18]. 2-Ketoisovalerate, the vital precursor for l-leucine production, is generated from pyruvate by the action of α-acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase (AHAIR), and dihydroxyacid dehydratase (DHAD). Importantly, AHAS contains a regulatory subunit and is feedback inhibited by l-leucine and l-valine [16].

Klebsiella oxytoca is a Risk Group 2 organism with rapid growth speed and wide substrate spectrum. It has been utilized for various bio-based products generation [[19], [20], [21]]. Besides AHAS, K. oxytoca expresses α-acetolactate synthase (BudB), another enzyme catalyzing the condensation of pyruvate to produce α-acetolactate. BudB is responsible for 2,3-butanediol production in K. oxytoca and lacks a regulatory subunit [22]. Thus, it is not feedback inhibited by l-leucine and l-valine. The insensitivity characteristic of BudB makes K. oxytoca a promising candidate for l-valine and l-leucine production. Recently, Cao et al. obtained a recombinant K. oxytoca strain VKO-9 through rearranging the metabolic flux from 2,3-butanediol manufacture to l-valine generation [23]. K. oxytoca VKO-9 can produce up to 122 g/L l-valine, which is higher than those obtained with E. coli and C. glutamicum.

In this study, K. oxytoca VKO-9 was further metabolically engineered for efficient l-leucine synthesis. A recombinant strain K. oxytoca LKO-14 was constructed via overexpressing key enzymes for l-leucine production, blocking branch pathways, rebalancing of cofactor, and modifying l-leucine transport system (Fig. 1). l-Leucine at a high concentration of 70.1 g/L was manufactured by K. oxytoca LKO-14, with a yield of 0.347 g/g and a productivity of 1.46 g/L/h. Importantly, the key genes for l-leucine synthesis were integrated at genome of K. oxytoca LKO-14 and controlled under constitutive promoters. K. oxytoca LKO-14 may be a promising alternative for stable industrial l-leucine production.

Fig. 1.

Fig. 1

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Metabolic engineering of l-valine-producing strain K. oxytoca VKO-9 for l-leucine production. Red crosses indicated blocked pathways and green arrows indicate overexpressed pathways in the metabolic engineered strain. PTS, phosphotransferase system; TCA cycle, tricarboxylic acid cycle; NOD, non-enzymatic oxidative decarboxylation; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; BPG, 1,3-bisphosphoglycerate; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; R5P, ribose-5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrose-4-phosphate; DHIV, 2,3-dihydroxyisovalerate; ptsG, glucose-specific phosphotransferase system IIBC component encoding gene; zwf, glucose-6-phosphate dehydrogenase encoding gene; gnd, 6-phosphategluconate dehydrogenase encoding gene; gapA, glyceraldehyde-3-phosphate dehydrogenase encoding gene; mgsA, methylglyoxal synthase encoding gene; ldhD, d-lactate dehydrogenase encoding gene; ldhL, l-lactate dehydrogenase encoding gene; poxB, pyruvate oxidase encoding gene; pflB, pyruvate formate-lyase encoding gene; pta, phosphate acetyltransferase encoding gene; adhE, alcohol dehydrogenase encoding gene; frdA, α-subunit of fumarate reductase encoding gene; budB, α-acetolactate synthase encoding gene; BsalsS, α-acetolactate synthase encoding gene from Bacillus subtilis 168; budA, α-acetolactate decarboxylase encoding gene; budC, meso-2,3-butanediol dehydrogenase encoding gene; gldA, glycerol dehydrogenase encoding gene; bkdAA, α-subunit of branched-chain keto acid dehydrogenase complex encoding gene; BsleuDH, leucine dehydrogenase encoding gene from B. subtilis 168; EcilvC, acetohydroxyacid isomeroreductase encoding gene from E. coli W3110; EcilvCM, mutant acetohydroxyacid isomeroreductase (L67E, R68F, and K75E) encoding gene from E. coli W3110; EcilvD, dihydroxyacid dehydratase encoding gene from E. coli W3110; SmilvD, dihydroxyacid dehydratase encoding gene from Streptococcus mutans; panE, α-hydroxyacid dehydrogenase encoding gene; CgleuAM, mutant isopropylmalate synthase (R529H and G532D) encoding gene from C. glutamicum ATCC13032; EcleuCD, 2-isopropylmalate isomerase encoding gene from E. coli W3110; EcleuB, isopropylmalate dehydrogenase encoding gene from E. coli W3110; NopheDH, phenylalanine dehydrogenase encoding gene from Nocardia sp. strain 239; BrnFE, branched-chain amino acid transporter from C. glutamicum ATCC13869; LeuE, l-leucine exporter; LivK, l-leucine importer.

2. Materials and methods

2.1. Strains, plasmids, and medium

The strains and plasmids used in this study are listed in Table S1. E. coli and K. oxytoca culture were carried out in LB medium for strain construction and seed preparation. Spectinomycin (50 mg/L), chloramphenicol (40 mg/L), and kanamycin (50 mg/L) were added as appropriate. The fermentation medium (1 L) for l-leucine production contained 60 g glucose, 2 g KH2PO4, 10 g K2HPO4·3H2O, 0.1 g MgSO4·7H2O, 5 g yeast extract, and 10 g (NH4)2SO4, and 1 mL metal ion stock solution [24].

2.2. Genetic engineering of Klebsiella oxytoca

Genetic engineering at genome of K. oxytoca were performed using pEcCasCm carrying the Cas9 nuclease and pEcgRNA carrying the guide RNA (gRNA) [25]. Primers gRNA-BsleuDH-1 and gRNA-BsleuDH-2 were annealed to generate double-stranded DNA targeting BsleuDH (Table S2). The double-stranded DNA was ligated to pEcgRNA plasmid to construct pEcgRNA-ΔBsleuDH. Primers ΔBsleuDH::NopheDH-1/ΔBsleuDH::NopheDH-2 and ΔBsleuDH::NopheDH-5/ΔBsleuDH::NopheDH-6 were used to amplify upstream and downstream homologous arms of BsleuDH from K. oxytoca VKO-9 genome, while primers ΔBsleuDH::NopheDH-3/ΔBsleuDH::NopheDH-4 were used to amplify NopheDH from plasmid pET28a-NopheDH. The three fragments were assembled to generate the donor DNA, which was then co-transformed with plasmid pEcgRNA-ΔBsleuDH into K. oxytoca harboring plasmid pEcCasCm via electroporation. Positive clones with replacement of BsleuDH by NopheDH were selected on plates with 40 μg/mL chloramphenicol and 50 μg/mL spectinomycin. Elimination of plasmids pEcgRNA and pEcCasCm was carried out as described previously [24].

2.3. Protein expression and purification

The recombinant plasmids pET28a-BsleuDH and pET28a-NopheDH were transformed into E. coli BL21 (DE3) to obtain expression strains for leucine dehydrogenase (LeuDH) and phenylalanine dehydrogenase (PheDH), respectively. Expression of the target proteins was induced at 16 °C and 160 rpm for 12 h. The collected cells were resuspended in binding buffer (20 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole, pH 7.4) and then lysed using a high pressure homogenizer (AH-Basic, China) to obtain crude enzyme extracts. The crude enzyme extracts were centrifuged at 4 °C and 12,000 rpm for 30 min to remove cell debris. The supernatants containing the target proteins were filtered through a 0.22 μm membrane, loaded onto a 5 mL HisTrap HP column (GE Healthcare, USA) using an ÄKTA-purifier system, and eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCl, and 500 mM imidazole, pH 7.4). An Amicon Ultra 15 mL 10K column (Merck Millipore, USA) was used for the desalting and concentration of the proteins. The purity of the proteins was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentrations of proteins were measured with a bradford protein assay kit (Sangon, China).

2.4. Assays of PheDH and LeuDH activities

The enzymatic activities of PheDH and LeuDH towards α-ketoisovalerate or α-ketoisocaproate were determined using a SpectraMax Plus384 microplate reader. The change in absorbance of NADH at 340 nm was monitored at 37 °C. The reaction mixture contained 100 mM Tris-HCl (pH 7.8), 5 mM α-ketoisovalerate or α-ketoisocaproate, 0.25 mM NADH, 200 mM NH4Cl, and an appropriate amount of PheDH or LeuDH.

2.5. l-Leucine fermentation in bioreactor

Batch fermentation was conducted in a 1-L bioreactor with a working volume of 0.8 L. Fed-batch fermentation and repeated fed-batch fermentation were performed in a 7.5-L bioreactor with a working volume of 5 L. The l-leucine fermentation was conducted at 37 °C, 500 rpm, 1.6 vvm, pH 6.8, and glucose concentration of 60 g/L. Glucose powder was supplemented when its concentration in the fermentation broth fell below 20 g/L, restoring the concentration to approximately 40–45 g/L. The pH of the fermentation broth was controlled at 6.8 by automatically feeding 25 % ammonia solution. Each cycle of repeated fed-batch fermentation was stopped before crystallization of l-leucine. A total of 4.3 L of broth was discharged from the bioreactor, and an equal volume of fresh medium was replenished using a peristaltic pump before the next cycle of fed-batch fermentation.

2.6. Analytical methods

The concentrations of acetoin and 2,3-butanediol were determined by gas chromatograph (GC2014C, Shimadzu, Japan) equipped with an Agilent HP-5 capillary column (0.32 mm inner diameter, 30 m length, 1.0 μm film thickness). The detector (FID) temperature was set at 280 °C, the injection volume was 1 μL, the column oven temperature was maintained at 80 °C, with an analysis time of 4 min [22]. The concentrations of organic acids like pyruvate, 2,3-dihydroxyisovalerate, 2-isopropylmalate, 3-isopropylmalate, and α-ketoisocaproate were detected using HPLC [26]. Glucose concentration was analyzed with an SBA-40D bioanalyzer. Biomass was quantified by measuring OD600nm. The concentrations of l-leucine and l-valine, derivatized with triethylamine and phenylisothiocyanate, were determined by HPLC (Agilent 1100, Agilent, USA) using a ZORBAX SB-C18 column (250 × 4.6 mm, Agilent, USA) as described previously [27].

3. Results and discussion

3.1. Introducing exogenous l-leucine biosynthesis pathway to achieve l-leucine accumulation

K. oxytoca VKO-9, an efficient producer of l-valine, was metabolic engineered for l-leucine production [23]. The isopropylmalate synthase (IPMS) catalyzes the condensation between acetyl-CoA and 2-oxoisovalerate to generate 2-isopropylmalate. Its activity determines the metabolic flux distribution between l-valine and l-leucine synthesis and is subject to feedback inhibition by l-leucine [28]. The CgleuAM gene encoding l-leucine insensitive IPMS mutant (R529H and G532D) from C. glutamicum was inserted at the poxB site in K. oxytoca VKO-9 genome and controlled by trc promoter, resulting in K. oxytoca LKO-1 (Fig. 2a). K. oxytoca LKO-1 produced 0.149 g/L l-leucine but accumulated 1.84 g/L 2-isopropylmalate during shake flask fermentation (Fig. 2b). 2-Isopropylmalate can be converted into α-ketoisocaproate, the direct precursor of l-leucine, through isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH) [29]. Thus, EcleuCD and EcleuB genes encoding IPMI and IPMDH from E. coli W3110 and CgleuAM gene were assembled into an artificial operon with trc promoter, and then integrated into the poxB locus of VKO-9, generating K. oxytoca LKO-2 (Fig. 2a). The concentration of 2-isopropylmalate produced by K. oxytoca LKO-2 decreased to 1.06 g/L, while the production of l-leucine increased to 0.340 g/L (Fig. 2b).

Fig. 2.

Fig. 2

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Introducing exogenous l-leucine biosynthesis pathway to accumulate l-leucine. (a) K. oxytoca LKO-1, LKO-2, and LKO-3 construction. (b) l-Leucine production by K. oxytoca VKO-9, LKO-1, LKO-2, and LKO-3 in medium with 38.0 g/L glucose. Values are the average ± SD (n = 3 independent experiments). (c) Detection of activities of PheDH and LeuDH toward different substrates. Control 1‌, LeuDH catalytic system without addition of α-ketoisocaproate or α-ketoisovalerate; Control 2‌, PheDH catalytic system without addition of α-ketoisocaproate or α-ketoisovalerate. (d) Specific activities of PheDH and LeuDH towards α-ketoisocaproate and α-ketoisovalerate. Values are the average ± SD (n = 3 independent experiments). (e) HPLC analysis of l-leucine, l-valine standards, and produced amino acids in fermented broth of strain VKO-9, LKO-1, LKO-2 and LKO-3. (f) OD600nm, carbon source consumption, and l-leucine production during batch fermentation of K. oxytoca LKO-3.

K. oxytoca LKO-2 produced 5.50 g/L l-valine, the major by-product accumulated by the strain. The BsleuDH encoding leucine dehydrogenase (LeuDH) from B. subtilis 168 was previously inserted in K. oxytoca VKO-9 genome to catalyze the reductive amination of α-ketoisovalerate to generate l-valine. LeuDH was overexprssed in E. coli BL21 (DE3) and then purified (Fig. S1). As shown in Fig. 2c and d, the purifed LeuDH also catalyzes the generation of l-leucine from α-ketoisocaproate, but its specific activity toward α-ketoisocaproate is lower than that toward α-ketoisovalerate. Phenylalanine dehydrogenase (PheDH) from Nocardia sp. 239 was reported to exhibit high enzymatic activity toward α-ketoisocaproate. Importantly, its specific activity toward α-ketoisocaproate is much higher than that toward α-ketoisovalerate (Fig. 2c and d) [30]. The NopheDH encoding PheDH was inserted into K. oxytoca LKO-2 genome to replace BsleuDH (Fig. 2a). The obtained strain K. oxytoca LKO-3 produced 0.679 g/L l-leucine and 4.32 g/L l-valine (Fig. 2b). Chromatographic analysis also revealed that the introduction of l-leucine synthesis pathway can increase l-leucine production and decrease l-valine generation (Fig. 2e). Batch cultivation of K. oxytoca LKO-3 in a 1-L bioreactor resulted in 4.73 g/L l-leucine production from 60.0 g/L glucose (Fig. 2f).

3.2. Enhancing the conversion of α-ketoisovalerate into α-ketoisocaproate for improvement of l-leucine generation

The leuABCD operon for l-leucine production is negatively regulated by l-leucine-mediated transcription attenuation [16]. Thus, the promoter and transcription attenuation region of leuABCD operon in K. oxytoca LKO-3 were replaced with trc promoter. The endogenous leuA of K. oxytoca LKO-3 was also replaced with the second copy of CgleuAM, generating K. oxytoca LKO-4 (Fig. 3a). Batch fermentation of K. oxytoca LKO-4 was then conducted in a 1-L bioreactor. K. oxytoca LKO-4 produced 10.7 g/L l-leucine from 63.0 g/L glucose (Fig. 3b). Distribution of metabolic flux in K. oxytoca LKO-3 and K. oxytoca LKO-4 was also illustrated by comparison of the extracellular‌ product distribution. The carbon ratio directing to by-products decreased from 41.6 % to 26.0 %, and the carbon ratio directing to l-leucine or its precursors (2-isopropylmalate and pyruvate) increased from 49.3 % to 52.1 % (Fig. 3c and d). Theoretically, quantitative analysis of flux changes at key intracellular metabolic nodes is more intuitive for characterizing the effects of genetic engineering and thus worth attempting.

Fig. 3.

Fig. 3

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Enhancing the expression of IPMSM, IPMDH, and IPMI to increase l-leucine generation. (a) K. oxytoca LKO-4, LKO-5, and LKO-6 construction. (b) OD600nm, carbon source consumption, and l-leucine production during batch fermentation of K. oxytoca LKO-4. Values are the average ± SD (n = 3 independent experiments). (c) Carbon ratio of by-products to glucose consumption of K. oxytoca LKO-3, LKO-4, LKO-5, and LKO-6. (d) Carbon ratio of l-leucine or its precursors to glucose consumption of K. oxytoca LKO-3, LKO-4, LKO-5, and LKO-6. Values are the average of independent experiments. (e) OD600nm, carbon source consumption, and l-leucine production during batch fermentation of K. oxytoca LKO-5. (f) OD600nm, carbon source consumption, and l-leucine production during batch fermentation of K. oxytoca LKO-6. The experiments were conducted in triplicate. Two representative time-courses of K. oxytoca LKO-5 (e) and K. oxytoca LKO-6 (f) are reported herein.

To enhance the conversion of 2-isopropylmalate to α-ketoisocaproate, the second copy of EcleuB and EcleuCD were successively integrated into the genome of K. oxytoca LKO-4, resulting in LKO-5 and LKO-6, respectively. As shown in Fig. 3e, 15.3 g/L of l-leucine was produced by K. oxytoca LKO-5 from 63.0 g/L glucose within 33 h. As shown in Fig. 3f, 16.0 g/L l-leucine was produced by K. oxytoca LKO-6 from 63.0 g/L glucose within 30 h. Compared to K. oxytoca LKO-4, the carbon ratio directing to 2-isopropylmalate in K. oxytoca LKO-6 decreased from 11.5 % to 6.4 %, and the carbon ratio directing to l-leucine increased to 52.4 % (Fig. 3d).

3.3. Modifying l-leucine transport system and increasing CgleuAM expression to enhance l-leucine synthesis

Transmembrane export of l-leucine can alleviate feedback inhibition and cytotoxicity caused by high intracellular l-leucine accumulation [12,31]. To improve the l-leucine efflux in K. oxytoca LKO-6, the promoter of leuE (encoding l-leucine exporter LeuE) was replaced with the trc promoter, yielding K. oxytoca LKO-7 (Fig. 4a). To prevent l-leucine reabsorption, the livK (encoding l-leucine importer LivK) in K. oxytoca LKO-7 was deleted, resulting in K. oxytoca LKO-8.

Fig. 4.

Fig. 4

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Modifying l-leucine transport and increasing copy number of CgleuAM to enhance l-leucine synthesis. (a) K. oxytoca LKO-7, LKO-8, and LKO-9 construction. (b) OD600nm, carbon source consumption, and l-leucine production during batch fermentation of K. oxytoca LKO-7. (c) OD600nm, carbon source consumption, and l-leucine production during batch fermentation of K. oxytoca LKO-8. (d) Carbon ratio of l-leucine or its precursors to glucose consumption of K. oxytoca LKO-6, LKO-7, LKO-8, and LKO-9. (e) Carbon ratio of by-products to glucose consumption of K. oxytoca LKO-6, LKO-7, LKO-8, and LKO-9. Values are the average of independent experiments. (f) OD600nm, carbon source consumption, and l-leucine production during batch fermentation of K. oxytoca LKO-9. The experiments were conducted in triplicate. Three representative time-courses of K. oxytoca LKO-7 (b), K. oxytoca LKO-8 (c), and K. oxytoca LKO-9 (f) are reported herein.

K. oxytoca LKO-7 produced 16.8 g/L l-leucine from 59.0 g/L glucose, with a yield of 0.285 g/g (Fig. 4b). After further knockout of livK, the l-leucine production and yield of K. oxytoca LKO-8 increased to 17.7 g/L and 0.300 g/g, respectively (Fig. 4c). The carbon ratio directing to l-leucine in K. oxytoca LKO-8 increased from 52.4 % to 61.6 % (Fig. 4d).

As shown in Fig. 4e, 11.0 % of the carbon ratio was still directed to l-valine in K. oxytoca LKO-8. α-Ketoisovalerate, the substrate of IPMS, is also the direct precursor of l-valine. The accumulation of l-valine indicated that the expression of CgleuAM (encoding IPMSM) in K. oxytoca LKO-8 was still insufficient to fully redirect the metabolic flux at the α-ketoisovalerate node towards l-leucine production. Thus, the third copy of CgleuAM was inserted into K. oxytoca LKO-8 genome at mgsA site to enhance IPMSM expression (Fig. 4a). The resulting strain K. oxytoca LKO-9 produced 19.0 g/L l-leucine within 30 h, with a yield of 0.322 g/g (Fig. 4f). Introducing the third copy of CgleuAM in K. oxytoca LKO-9 further raised the carbon ratio directing to l-leucine, and the carbon ratio directing to l-valine decreased to 9.18 % (Fig. 4d and e).

3.4. Strengthening pyruvate condensation and 2,3-dihydroxyisovalerate dehydration to promote l-leucine production

Pyruvate was another l-leucine precursor accumulated by K. oxytoca LKO-9 (Fig. 4d). The α-acetolactate synthase (BudB) catalyzes the condensation of pyruvate, the initial reaction in l-leucine biosynthesis. The expression of budB in K. oxytoca is activated by acetate activation [32]. However, the pta gene responsible for acetate production was replaced by EcleuB in K. oxytoca LKO-9, which may result in insufficient BudB expression and pyruvate accumulation. Shake flask fermentation of K. oxytoca LKO-9 was conducted in media with or without 2 g/L acetate addition. Acetate addition obviously decreased pyruvate accumulation (from 6.01 g/L to 0.945 g/L), and increased l-leucine production (from 3.11 g/L to 4.38 g/L) of K. oxytoca LKO-9 (Fig. S2). Thus, the promoter of budB was replaced by trc promoter to get rid of its dependence on acetate, resulting in K. oxytoca LKO-10 (Fig. 5a).

Fig. 5.

Fig. 5

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Enhancing the conversion of precursors to improve l-leucine production. (a) K. oxytoca LKO-10, LKO-11, LKO-12, and LKO-13 construction. (b) OD600nm, concentration and yield of l-leucine of K. oxytoca LKO-10, LKO-11, LKO-12, and LKO-13. (c) Carbon ratio of l-leucine and its precursors to glucose consumption of K. oxytoca LKO-9, LKO-10, LKO-11, LKO-12, and LKO-13. (d) Carbon ratio of by-products to glucose consumption of K. oxytoca LKO-9, LKO-10, LKO-11, LKO-12, and LKO-13. Values are the average of independent experiments. (e) OD600nm, carbon source consumption, and l-leucine production during fed-batch fermentation of K. oxytoca LKO-13. The experiments were conducted in triplicate. One representative time-course of K. oxytoca LKO-13 is reported herein. (f) Quantification of l-leucine before and after re-dissolution of crystal in broth after fed-batch fermentation of strain LKO-13.

Batch fermentation of K. oxytoca LKO-10 produced 14.2 g/L l-leucine, with a yield of 0.233 g/g (Fig. 5b). As expected, K. oxytoca LKO-10 no longer accumulated pyruvate (Fig. 5c). However, 2,3-dihydroxyisovalerate accumulation in the fermentation broth was observed (Fig. 5c). 2,3-Dihydroxyisovalerate can be dehydrated to α-ketoisovalerate by dihydroxyacid dehydratase (DHAD) [23]. The gene EcilvD encoding EcDHAD from E. coli W3110 was previously inserted at ldhD site of strain VKO-9. The α-hydroxyacid dehydrogenase encoded by panE in K. pneumoniae has been reported to reduce α-ketoisovalerate to α-hydroxyisovalerate [33]. To decrease the accumulation of 2,3-dihydroxyisovalerate and prevent the reduction of α-ketoisovalerate to α-hydroxyisovalerate, the second copy of EcilvD was integrated into the panE locus in K. oxytoca LKO-10 genome and controlled by trc promoter (Fig. 5a). The obtained strain K. oxytoca LKO-11 produced 20.4 g/L l-leucine, with a yield of 0.334 g/g (Fig. 5b). Neither pyruvate nor 2,3-dihydroxyisovalerate accumulation was detected in fermentation broth.

3.5. Increasing the expression of isopropylmalate synthase and phenylalanine dehydrogenase to facilitate l-leucine biosynthesis

Slight accumulation of α-ketoisovalerate and α-ketoisocaproate was observed during batch fermentation of K. oxytoca LKO-11 (Fig. 5c). To reduce the accumulation of α-ketoisovalerate, the fourth copy of CgleuAM was integrated into the ldhL locus of K. oxytoca LKO-11 (Fig. 5a), generating K. oxytoca LKO-12. The concentration and yield of l-leucine produced by K. oxytoca LKO-12 increased to 21.5 g/L and 0.352 g/g, respectively (Fig. 5b). α-Ketoisovalerate accumulation was eliminated in K. oxytoca LKO-12, while the carbon ratio of α-ketoisocaproate increased from 0.82 % to 2.66 % (Fig. 5c).

α-Ketoisocaproate can be transformed into l-leucine via reductive amination by PheDH [30]. Thus, the second copy of NopheDH was integrated at livK site of strain LKO-12, resulting in K. oxytoca LKO-13 (Fig. 5a). As shown in Fig. 5b, the l-leucine production of K. oxytoca LKO-13 in a 1-L bioreactor reached 21.7 g/L, and the yield of l-leucine increased to 0.368 g/g.

3.6. Introduction of oxygen-tolerant dihydroxyacid dehydratase to enhance l-leucine production

Fed-batch fermentation of K. oxytoca LKO-13 was carried out in a 7.5-L bioreactor. K. oxytoca LKO-13 consumed 179 g/L glucose in 60 h (Fig. 5e). l-Leucine concentration reached 28.4 g/L at 21 h and it began to crystallize after 24 h. After collecting and redissolving the crystal in fermentation broth, the actual l-leucine production by K. oxytoca LKO-13 was determined to be 40.3 g/L (Fig. 5f). The yield of l-leucine in 7.5-L bioreactor was 0.225 g/g, which was significantly lower than that in the 1-L bioreactor (0.368 g/g).

The decrease in l-leucine yield in the 7.5-L bioreactor for K. oxytoca LKO-13 may be caused by the generation of 2,3-dihydroxyisovalerate, which began to accumulate at 24 h (Fig. S3). The genome of K. oxytoca LKO-13 harbored two copies of EcilvD encoding EcDHAD, which were controlled by ldhD and trc promoters, respectively. EcDHAD exhibits a high specific activity of 63 U/mg but its active site contains a [4Fe–4S] cluster, which is susceptible to inactivation under conditions of high oxidative stress [34]. The accumulation of 2,3-dihydroxyisovalerate may result from oxygen-induced inactivation of EcDHAD. The dihydroxy acid dehydratase from Streptococcus mutans (SmDHAD) exhibits a specific activity of 7.9 U/mg but its active site contains a [2Fe–2S] cluster and thus is more oxygen-tolerant [35]. As demonstrated by Flint et al., SmDHAD is stable (maintaining >80 % of its initial activity) after 72 h of incubation in air, while EcDHAD rapidly loses its activity in 24 h under identical conditions [35]. Except for preventing deactivation of EcDHAD through process optimization like precise oxygen control, directly introducing oxygen-tolerant SmDHAD may also decrease the accumulation of 2,3-dihydroxyisovalerate and increase l-leucine production. Therefore, the EcilvD controlled by the trc promoter in K. oxytoca LKO-13 was replaced with SmilvD encoding SmDHAD, resulting in K. oxytoca LKO-14 (Fig. 6a). Batch fermentation of K. oxytoca LKO-14 in 1-L bioreactor resulted in 22.4 g/L l-leucine production, with a yield of 0.379 g/g and a productivity of 0.827 g/L/h (Fig. 6b).

Fig. 6.

Fig. 6

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Batch fermentation, fed-batch fermentation, and repeated fed-batch fermentation of K. oxytoca LKO-14. (a) K. oxytoca LKO-14 construction strategy. (b) OD600nm, carbon source consumption, and l-leucine production of K. oxytoca LKO-14 during batch fermentation. The experiments were conducted in triplicate. One representative time-course of K. oxytoca LKO-14 is reported herein. (c) OD600nm, carbon source consumption, and l-leucine production of K. oxytoca LKO-14 during fed-batch fermentation. The experiments were conducted in triplicate. One representative time-course of K. oxytoca LKO-14 is reported herein. (d) Quantification of l-leucine before and after re-dissolution of crystal in broth after fed-batch fermentation of strain LKO-14. (e) OD600nm, carbon source consumption, and l-leucine production of K. oxytoca LKO-14 during repeated fed-batch fermentation.

3.7. Fed-batch fermentation and repeated fed-batch fermentation for l-leucine production

Fed-batch fermentation of K. oxytoca LKO-14 was then carried out in 7.5-L bioreactor. It consumed 202 g/L glucose and produced 70.1 g/L l-leucine within 48 h (Fig. 6c), with a yield of 0.347 g/g and a productivity of 1.46 g/L/h (Fig. 6d). The concentrations of byproducts including 2-isopropylmalate, acetate, acetoin, and 2,3-butanediol were 4.45 g/L, 2.40 g/L, 1.52 g/L, and 1.68 g/L, respectively (Fig. S4). Importantly, the introduce of SmDHAD prevented the accumulation of 2,3-dihydroxyisovalerate, while the final concentration of l-valine was 0.887 g/L (Fig. S4). To exclude the possible damage of crystallized l-leucine toward fermentation equipment, three cycles of repeated fed-batch fermentation of strain LKO-14 was also conducted (Fig. 6e). The average concentration, yield, and productivity of l-leucine were 30.6 g/L, 0.353 g/g, and 1.70 g/L/h, respectively. A total of 30 serial subcultures of strain LKO-14 were performed in shake flask with a gap of every 12 h, and shake flask fermentation was conducted at every 10th subculture. No significant change in glucose consumption and l-leucine production in shake flask fermentation was detected (Fig. S5), indicating the stability of the production performance of K. oxytoca LKO-14.

Both E. coli and C. glutamicum have been metabolic engineered for l-leucine production (Table 1). Ding et al. achieved high production of l-leucine by multistep metabolic engineering of E. coli. The obtained strain E. coli LXH-21 produced 63.29 g/L l-leucine, which is the highest l-leucine production among previous reports [12]. Recently, Hao et al. constructed a plasmid-free l-leucine producing strain E. coli LEU27. E. coli LEU27 produced 55 g/L of l-leucine in 48 h, with a yield of 0.23 g/g and a productivity of 1.15 g/L/h [13]. In this study, K. oxytoca LKO-14 was constructed through redirecting the metabolic flux in K. oxytoca VKO-9, from l-valine production to l-leucine synthesis. K. oxytoca LKO-14 produced 70.1 g/L l-leucine within 48 h, with a yield of 0.347 g/g and a productivity of 1.46 g/L/h. The key genes for l-leucine generation were inserted into K. oxytoca LKO-14 genome and constitutively expressed, making it a promising candidate for low cost and stable l-leucine production. Importantly, the final concentration of l-valine of K. oxytoca LKO-14 was only 0.887 g/L, which may reduce the separation cost and be beneficial for downstream process of industrial l-leucine production.

Table 1.

Comparison of l-leucine production by different microorganisms.

Strain Titer (g/L) Yield (g/g) Productivity (g/L/h) Plasmid Inducer Reference
E. coli LXH-21 63.29 0.37 2.64 pTrc99aΔlacI-leuACPBCD NR [12]
E. coli LEU-27 55 0.23 1.15 NRb NR [13]
C. glutamicum MV-LeuF2 24 0.19 0.428 NR NR [29]
C. glutamicum JL-51 40.11 0.25 0.59 pECXK99E-acscobB IPTG [36]
C. glutamicum Leu-9 54.3 NMa 0.754 pECXK99E-leuAilvBNCE IPTG [17]
K. oxytoca LKO-14 70.1 0.347 1.46 NR NR This study
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a

Not mentioned.

b

Not required.

4. Conclusions

In summary, the metabolic flux in K. oxytoca VKO-9, an l-valine producing strain, was redirected to l-leucine synthesis. The obtained strain K. oxytoca LKO-14 efficiently produced l-leucine from glucose via fed-batch fermentation, with a concentration, yield and productivity of 70.1 g/L, 0.347 g/g and 1.46 g/L/h, respectively. The average concentration, yield, and productivity of l-leucine in repeated fed-batch fermentation were 30.6 g/L, 0.353 g/g, and 1.70 g/L/h, respectively. The plasmid free, inducer independent, and low l-valine generation characteristics of K. oxytoca LKO-14 made it a promising alternative for industrial l-leucine production.

CRediT authorship contribution statement

Weikang Sun: Writing – original draft, Investigation, Formal analysis, Data curation. Qiaoyue Yang: Writing – original draft, Investigation, Data curation. Shuo Wang: Investigation, Formal analysis. Lingru Gong: Investigation. Zhi Zhou: Resources, Project administration. Mingyuan Liu: Formal analysis. Xiaoxu Tan: Formal analysis. Qianjin Kang: Resources, Project administration. Wensi Meng: Project administration, Formal analysis. Yidong Liu: Resources, Project administration, Formal analysis. Zhaoqi Kang: Project administration, Data curation. Ping Xu: Validation, Supervision. Cuiqing Ma: Validation, Supervision, Resources, Project administration, Data curation, Conceptualization. Chao Gao: Validation, Supervision, Resources, Project administration, Formal analysis, Data curation, Conceptualization. Chuanjuan Lü: Validation, Supervision, Resources, Project administration, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (32300045, 32300029), Youth Program of Natural Science Foundation of Shandong Province (ZR2022QC092, ZR2023QC237), China Postdoctoral Science Foundation (2023M742085, 2024M751800), Youth Program of Natural Science Foundation of Qingdao City (23-2-1-31-zyyd-jch), Postdoctoral Innovation Program of Shandong Province (No. SDCX-ZG-202400150), Qingdao Postdoctoral Research Project (QDBSH20230201011), Open Funding Project of State Key Laboratory of Microbial Metabolism (MMLKF24-07), and State Key Laboratory of Microbial Technology (SKLMTFCP-2023-03).

Footnotes

Peer review under the responsibility of Editorial Board of Synthetic and Systems Biotechnology.

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.synbio.2026.01.024.

Contributor Information

Weikang Sun, Email: 18861824910@163.com.

Qiaoyue Yang, Email: yangqiaoyue2001@163.com.

Shuo Wang, Email: sduwangshuo@mail.sdu.edu.cn.

Lingru Gong, Email: 18822722433@163.com.

Zhi Zhou, Email: zhouzhi6090@126.com.

Mingyuan Liu, Email: sdulmy@163.com.

Xiaoxu Tan, Email: 17863920092@163.com.

Qianjin Kang, Email: qjkang@126.com.

Wensi Meng, Email: mengwensi123@126.com.

Yidong Liu, Email: liuyidong94@163.com.

Zhaoqi Kang, Email: kangzhaoqi_sdu@163.com.

Ping Xu, Email: pingxu@sjtu.edu.cn.

Cuiqing Ma, Email: macq@sdu.edu.cn.

Chao Gao, Email: jieerbu@sdu.edu.cn.

Chuanjuan Lü, Email: chuanjuanlv@sdu.edu.cn.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.doc (2.1MB, doc)

References

  • 1.Vellai T. How the amino acid leucine activates the key cell-growth regulator mTOR. Nature. 2021;596(7871):192–194. doi: 10.1038/d41586-021-01943-7. [DOI] [PubMed] [Google Scholar]
  • 2.Maykish A., Sikalidis A.K. Utilization of hydroxyl-methyl butyrate, leucine, glutamine and arginine supplementation in nutritional management of sarcopenia-implications and clinical considerations for type 2 diabetes mellitus risk modulation. J Pers Med. 2020;10(1):19. doi: 10.3390/jpm10010019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Holeček M. Branched-chain amino acids in health and disease: metabolism, alterations in blood plasma, and as supplements. Nutr Metab. 2018;15:33. doi: 10.1186/s12986-018-0271-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Yamamoto K., Tsuchisaka A., Yukawa H. Branched-chain amino acids. Adv Biochem Eng Biotechnol. 2017;159:103–128. doi: 10.1007/10_2016_28. [DOI] [PubMed] [Google Scholar]
  • 5.Pedroso J.A., Zampieri T.T., Donato J. Reviewing the effects of L-leucine supplementation in the regulation of food intake, energy balance, and glucose homeostasis. Nutrients. 2015;7(5):3914–3937. doi: 10.3390/nu7053914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Luise D., Correa F., Stefanelli C., Simongiovanni A., Chalvon-Demersay T., Zini M., et al. Productive and physiological implications of top-dress addition of branched-chain amino acids and arginine on lactating sows and offspring. J Anim Sci Biotechnol. 2023;14(1):40. doi: 10.1186/s40104-022-00819-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Han G., Yang H., Wang Y., Haraguchi S., Miyazaki T., Bungo T., et al. L-Leucine increases the daily body temperature and affords thermotolerance in broiler chicks. Asian-Australas J Anim Sci. 2019;32(6):842–848. doi: 10.5713/ajas.18.0677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Fan Q., Long B., Yan G., Wang Z., Shi M., Bao X., et al. Dietary leucine supplementation alters energy metabolism and induces slow-to-fast transitions in longissimus dorsi muscle of weanling piglets. Br J Nutr. 2017;117(9):1222–1234. doi: 10.1017/S0007114517001209. [DOI] [PubMed] [Google Scholar]
  • 9.Huo Y.W., Jin M., Sun P., Hou Y.M., Li Y., Qiu H., et al. Effect of dietary leucine on growth performance, hemolymph and hepatopancreas enzyme activities of swimming crab, Portunus trituberculatus. Aquac Nutr. 2017;23(6):1341–1350. doi: 10.1111/anu.12509. [DOI] [Google Scholar]
  • 10.Nakashima K., Yakabe Y., Ishida A., Yamazaki M., Abe H. Suppression of myofibrillar proteolysis in chick skeletal muscles by alpha-ketoisocaproate. Amino Acids. 2007;33(3):499–503. doi: 10.1007/s00726-006-0404-0. [DOI] [PubMed] [Google Scholar]
  • 11.Hao Y., Pan X., You J., Li G., Xu M., Rao Z. Microbial production of branched chain amino acids: advances and perspectives. Bioresour Technol. 2024;397 doi: 10.1016/j.biortech.2024.130502. [DOI] [PubMed] [Google Scholar]
  • 12.Ding X., Yang W., Du X., Chen N., Xu Q., Wei M., et al. High-level and -yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering. Metab Eng. 2023;78:128–136. doi: 10.1016/j.ymben.2023.06.003. [DOI] [PubMed] [Google Scholar]
  • 13.Hao Y., Pan X., Li G., You J., Zhang H., Yan S., et al. Construction of a plasmid-free L-leucine overproducing Escherichia coli strain through reprogramming of the metabolic flux. Biotechnol Biofuels Bioprod. 2023;16(1):145. doi: 10.1186/s13068-023-02397-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wang Y., Xu J., Jin Z., Xia X., Zhang W. Improvement of acetyl-CoA supply and glucose utilization increases L-leucine production in Corynebacterium glutamicum. Biotechnol J. 2022;17(8) doi: 10.1002/biot.202100349. [DOI] [PubMed] [Google Scholar]
  • 15.Wang Y.Y., Shi K., Chen P., Zhang F., Xu J.Z., Zhang W.G. Rational modification of the carbon metabolism of Corynebacterium glutamicum to enhance L-leucine production. J Ind Microbiol Biotechnol. 2020;47(6–7):485–495. doi: 10.1007/s10295-020-02282-8. [DOI] [PubMed] [Google Scholar]
  • 16.Wang Y.Y., Xu J.Z., Zhang W.G. Metabolic engineering of L-leucine production in Escherichia coli and Corynebacterium glutamicum: a review. Crit Rev Biotechnol. 2019;39(5):633–647. doi: 10.1080/07388551.2019.1577214. [DOI] [PubMed] [Google Scholar]
  • 17.Chen S.L., Liu T.S., Zhang W.G., Xu J.Z. An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain. Int Microbiol. 2023;26(1):11–24. doi: 10.1007/s10123-022-00270-9. [DOI] [PubMed] [Google Scholar]
  • 18.Park J.H., Lee S.Y. Fermentative production of branched chain amino acids: a focus on metabolic engineering. Appl Microbiol Biotechnol. 2010;85(3):491–506. doi: 10.1007/s00253-009-2307-y. [DOI] [PubMed] [Google Scholar]
  • 19.Phosriran C., Wong N., Jantama K. An efficient production of bio-succinate in a novel metabolically engineered Klebsiella oxytoca by rational metabolic engineering and evolutionary adaptation. Bioresour Technol. 2024;393 doi: 10.1016/j.biortech.2023.130045. [DOI] [PubMed] [Google Scholar]
  • 20.Khunnonkwao P., Jantama S.S., Jantama K., Joannis-Cassan C., Taillandier P. Sequential coupling of enzymatic hydrolysis and fermentation platform for high yield and economical production of 2,3-butanediol from cassava by metabolically engineered Klebsiella oxytoca. J Chem Technol Biotechnol. 2021;96(5):1292–1301. doi: 10.1002/jctb.6643. [DOI] [Google Scholar]
  • 21.In S., Khunnonkwao P., Wong N., Phosiran C., Jantama S.S., Jantama K. Combining metabolic engineering and evolutionary adaptation in Klebsiella oxytoca KMS004 to significantly improve optically pure D-(-)-lactic acid yield and specific productivity in low nutrient medium. Appl Microbiol Biotechnol. 2020;104(22):9565–9579. doi: 10.1007/s00253-020-10933-0. [DOI] [PubMed] [Google Scholar]
  • 22.Yang T.H., Rathnasingh C., Lee H.J., Seung D. Identification of acetoin reductases involved in 2,3-butanediol pathway in Klebsiella oxytoca. J Biotechnol. 2014;172:59–66. doi: 10.1016/j.jbiotec.2013.12.007. [DOI] [PubMed] [Google Scholar]
  • 23.Cao M., Sun W., Wang S., Di H., Du Q., Tan X., et al. Efficient L-valine production using systematically metabolic engineered Klebsiella oxytoca. Bioresour Technol. 2024;395 doi: 10.1016/j.biortech.2024.130403. [DOI] [PubMed] [Google Scholar]
  • 24.Sun W., Wang S., Tan X., Guo L., Liu W., Tian W., et al. Production of α-ketoisovalerate with whey powder by systemic metabolic engineering of Klebsiella oxytoca. Microb Cell Fact. 2024;23:264. doi: 10.1186/s12934-024-02545-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Li Q., Sun B., Chen J., Zhang Y., Jiang Y., Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin. 2021;53(5):620–627. doi: 10.1093/abbs/gmab036. [DOI] [PubMed] [Google Scholar]
  • 26.Cao M., Jiang T., Li P., Zhang Y., Guo S., Meng W., et al. Pyruvate production from whey powder by metabolic engineered Klebsiella oxytoca. J Agric Food Chem. 2020;68(51):15275–15283. doi: 10.1021/acs.jafc.0c06724. [DOI] [PubMed] [Google Scholar]
  • 27.Guo S., Tan X., Zhu J., Lu C., Yang C., Wang Q., et al. Enhanced L-serine production from glycerol by integration with thermodynamically favorable D-glycerate oxidation. ACS Sustainable Chem Eng. 2022;10(8):2587–2592. doi: 10.1021/acssuschemeng.2c00040. [DOI] [Google Scholar]
  • 28.Yu B., Zheng B., Chen Z., Huo Y.X. Metabolic engineering of Corynebacterium glutamicum for producing branched chain amino acids. Microb Cell Fact. 2021;20:230. doi: 10.1186/s12934-021-01721-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Vogt M., Haas S., Klaffl S., Polen T., Eggeling L., Ooyen J., et al. Pushing product formation to its limit: Metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction. Metab Eng. 2014;22:40–52. doi: 10.1016/j.ymben.2013.12.001. [DOI] [PubMed] [Google Scholar]
  • 30.De Boer L., Van Rijssel M., Euverink G.J., Dijkhuizen L. Purification, characterization and regulation of a monomeric L-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239. Arch Microbiol. 1989;153(1):12–18. doi: 10.1007/BF00277534. [DOI] [Google Scholar]
  • 31.Kutukova E.A., Livshits V.A., Altman I.P., Ptitsyn L.R., Zyiatdinov M.H., Tokmakova I.L., et al. The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression. FEBS Lett. 2005;579(21):4629–4634. doi: 10.1016/j.febslet.2005.07.031. [DOI] [PubMed] [Google Scholar]
  • 32.Meng W., Zhang Y., Cao M., Zhang W., Lü C., Yang C., et al. Efficient 2,3-butanediol production from whey powder using metabolically engineered Klebsiella oxytoca. Microb Cell Fact. 2020;19:162. doi: 10.1186/s12934-020-01420-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Wang Q., Jiang W., Cai Y., Tišma M., Baganz F., Shi J., et al. 2-Hydroxyisovalerate production by Klebsiella pneumoniae. Enzym Microb Technol. 2024;172 doi: 10.1016/j.enzmictec.2023.110330. [DOI] [PubMed] [Google Scholar]
  • 34.Flint D.H., Emptage M.H., Finnegan M.G., Fu W., Johnson M.K. The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase. J Biol Chem. 1993;268(20):14732–14742. doi: 10.1016/S0021-9258(18)82394-8. [DOI] [PubMed] [Google Scholar]
  • 35.Flint D., Rothman S.C., Suh W., Tomb J.F., Ye R.W. Identification and use of bacterial [2Fe-2S] dihydroxy-acid dehydratases. US Patent US8951937B2. 2015 [Google Scholar]
  • 36.Wang Y., Xu J., Jin Z., Xia X., Zhang W. Improvement of acetyl-CoA supply and glucose utilization increases L-leucine production in Corynebacterium glutamicum. Biotechnol J. 2022;17(8) doi: 10.1002/biot.202100349. [DOI] [PubMed] [Google Scholar]

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