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Production of α-ketoisovalerate with whey powder by systemic metabolic engineering of Klebsiella oxytoca

Microbial Cell Factories volume 23, Article number: 264 (2024) Cite this article

Abstract

Background

Whey, which has high biochemical oxygen demand and chemical oxygen demand, is mass-produced as a major by-product of the dairying industry. Microbial fermentation using whey as the carbon source may convert this potential pollutant into value-added products. This study investigated the potential of using whey powder to produce α-ketoisovalerate, an important platform chemical.

Results

Klebsiella oxytoca VKO-9, an efficient L-valine producing strain belonging to Risk Group 1 organism, was selected for the production of α-ketoisovalerate. The leucine dehydrogenase and branched-chain α-keto acid dehydrogenase, which catalyzed the reductive amination and oxidative decarboxylation of α-ketoisovalerate, respectively, were inactivated to enhance the accumulation of α-ketoisovalerate. The production of α-ketoisovalerate was also improved through overexpressing α-acetolactate synthase responsible for pyruvate polymerization and mutant acetohydroxyacid isomeroreductase related to α-acetolactate reduction. The obtained strain K. oxytoca KIV-7 produced 37.3 g/L of α-ketoisovalerate from lactose, the major utilizable carbohydrate in whey. In addition, K. oxytoca KIV-7 also produced α-ketoisovalerate from whey powder with a concentration of 40.7 g/L and a yield of 0.418 g/g.

Conclusion

The process introduced in this study enabled efficient α-ketoisovalerate production from low-cost substrate whey powder. Since the key genes for α-ketoisovalerate generation were integrated in genome of K. oxytoca KIV-7 and constitutively expressed, this strain is promising in stable α-ketoisovalerate fermentation and can be used as a chassis strain for α-ketoisovalerate derivatives production.

Introduction

α-Ketoisovalerate is a branched-chain α-keto acid with diversified applications. For example, it can be used in treatment of hyperphosphatemia in uremic patients [1, 2]. In addition, α-ketoisovalerate can also be used in synthesis of functional substances such as L-valine [3], L-leucine [4] and vitamin B5 [5]. Currently, α-ketoisovalerate is primarily produced from potassium cyanide and formaldehyde via Heine’s method [6]. However, this process involves cumbersome steps and use of toxic raw materials. The green production of α-ketoisovalerate from renewable resources by microbial fermentation is thus highly desirable. α-Ketoisovalerate serves as the precursor for L-valine biosynthesis and it cannot be accumulated in most of the natural microorganisms. Various microorganisms including Escherichia coli [7, 8], Corynebacterium glutamicum [9, 10], Pseudomonas putida [11], Klebsiella pneumoniae [12], and Streptomyces cinnamonensis [13] have been metabolic engineered for fermentative production of α-ketoisovalerate.

K. oxytoca is a Risk Group 1 organism exhibiting high growth rate and wide substrate spectrum [14,15,16]. It has been used in the fermentative production of many platform chemicals including 1,3-propanediol [17], 2,3-butanediol [18], D-lactate [17, 19], succinate [20], and L-valine [21]. For example, Cao et al. recently redirected the metabolic flux of K. oxytoca from 2,3-butanediol production to L-valine synthesis [21]. Briefly, the key enzymes responsible for 2,3-butanediol generation from α-acetolactate were deleted while the enzymes related to L-valine production from α-acetolactate including acetohydroxyacid isomeroreductase (AHAIR), dihydroxyacid dehydratase (DHAD), and leucine dehydrogenase (LeuDH) were overexpressed. The obtained strain K. oxytoca VKO-9 was able to generate 122 g/L of L-valine with a yield of 0.587 g/g [21].

Whey is a by-product of cheese and yogurt production. Its disposal without appropriate treatment may lead to serious pollution problems due to high biochemical oxygen demand (BOD) and high chemical oxygen demand (COD) [22]. Whey powder typically contains approximately 77% lactose [23], which is a utilizable carbon source of many microorganisms. Extensive research has explored the use of whey powder in fermentative production of pyruvate [14], citrate [24], fatty acids [25], and isobutanol [26]. Recently, Darwin et al. constructed a recombinant E. coli strain W1262 to produce α-ketoisovalerate from whey powder but the final concentration of α-ketoisovalerate was only 3.22 g/L [8].

In this study, the L-valine producing strain K. oxytoca VKO-9 (referred to as KIV-0) was metabolic engineered to produce α-ketoisovalerate from whey powder. The leucine dehydrogenase, which catalyzes the amination of α-ketoisovalerate into L-valine, was inactivated in K. oxytoca KIV-0 to accumulate α-ketoisovalerate. Then, branched-chain α-keto acid dehydrogenase catalyzing the oxidative decarboxylation of α-ketoisovalerate was inactivated to prevent α-ketoisovalerate degradation. Mutant AHAIR (AHAIRM) catalyzing α-acetolactate reduction and α-acetolactate synthase (ALS) for α-acetolactate synthesis were also overexpressed to further enhance the α-ketoisovalerate production (Fig. 1). Efficient production of α-ketoisovalerate with a concentration of 40.7 g/L and a yield of 0.418 g/g from whey powder was realized with the obtained strain K. oxytoca KIV-7.

Fig. 1
figure 1

Metabolic engineering strategies for α-ketoisovalerate production from lactose by recombinant K. oxytoca. NOD, non-enzymatic oxidative decarboxylation; GALT, galactose-1-phosphate uridylyltransferase; LacY, lactose permease; GALK, galactokinase; GK, glucose kinase; GALE, UDP-galactose-4-epimerase; PGM, phosphoglucomutase; Pta, phosphate acetyltransferase; PFL, pyruvate formate-lyase; LdhD, D-lactate dehydrogenase; PoxB, pyruvate oxidase; AdhE, ethanol dehydrogenase; FrdA, α-subunit of fumarate reductase. BudB, α-acetolactate synthase of K. oxytoca; AlsS, α-acetolactate synthase of B. subtilis 168; DHAD, dihydroxyacid dehydratase from E. coli W3110; AHAIR, acetohydroxyacid isomeroreductase of E. coli W3110; AHAIRM, mutant acetohydroxyacid isomeroreductase of E. coli W3110; BudA, α-acetolactate decarboxylase; BudC, meso-2,3-butanediol dehydrogenase; BkdAA, α-subunit of branched-chain α-keto acid dehydrogenase complex; LeuDH, leucine dehydrogenase of B. subtilis 168

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Methods

Chemicals

α-Ketoisovalerate was purchased from Sigma-Aldrich (Louis, Missouri, USA). Whey powder containing 77.0% lactose, 11.2% protein, 1.1% fat, 1.9% moisture and 8.2% ash was purchased from Kuoquan Biotech (Jinan, China). Crude whey containing 4.0% lactose, 0.66% protein, 0.01% fat, 94% moisture and 0.6% ash was purchased from BASHIKE MILK SHOP (Tianjin, China). Restriction enzymes were acquired from Thermo Fisher (USA). Polymerase chain reaction (PCR) primers were supplied by Beijing Tsingke Biotech Co., Ltd (Qingdao, China). T4 DNA ligase and DNA polymerase were procured from Thermo Fisher (USA) and Vazyme Biotech Co., Ltd (Nanjing, China), respectively. All other chemicals were of analytical grade and commercially available.

Bacterial strains, plasmids and medium

The strains and plasmids used in this study are listed in Table 1. E. coli and K. oxytoca were generally cultured at 37 °C in LB medium. Plasmids pEcgRNA and pEcCasCm were employed for genes knock-in, knock-out, and promoter replacement in K. oxytoca. Chloramphenicol and spectinomycin were added at a concentration of 40 and 50 µg/mL, respectively. The α-ketoisovalerate production capabilities of different K. oxytoca strains were compared in medium containing 5 g/L yeast extract, 2 g/L KH2PO4, 3.3 g/L (NH4)2SO4, 10 g/L K2HPO4⋅3H2O, 0.1 g/L MgSO4⋅7H2O, and 1 mL/L multicomponent metal ion solution [14]. Glucose, lactose, or whey powder were added in the medium with appropriate amounts to achieve the desired concentrations. Crude whey supplemented with 5 g/L yeast extract, 2 g/L KH2PO4, 3.3 g/L (NH4)2SO4, 10 g/L K2HPO4⋅3H2O, 0.1 g/L MgSO4⋅7H2O, and 1 mL/L multicomponent metal ion solution [14] was also used as a culture medium for α-ketoisovalerate production.

Table 1 Bacterial strains and plasmids used in this study
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DNA manipulation in K. oxytoca

The primers are listed in Table S1. Knock-in, knock-out, and promoter replacement of genes in K. oxytoca were conducted through CRISPR/Cas9 gene editing [27]. Herein, the replacement of pox encoding pyruvate oxidase with budB encoding α-acetolactate synthase under the trc promoter was taken as an example. The primer pair (gRNA-pox-1 and gRNA-pox-2) was annealed to form dsDNA targeting pox. Then, the dsDNA was ligated into linearized pEcgRNA using the T4 DNA ligase, resulting in plasmid pEcgRNA-Δpox. The donor DNA, comprising homologous arms of pox and Ptrc-budB, was obtained with the primer pairs Δpox::Ptrc-budB-1/Δpox::Ptrc-budB-2, Δpox::Ptrc-budB-3/Δpox::Ptrc-budB-4, and Δpox::Ptrc-budB-5/Δpox::Ptrc-budB-6 by overlap PCR. The donor DNA and the plasmid pEcgRNA-Δpox were co-transformed into K. oxytoca harboring the plasmid pEcCasCm via electrotransformation. Positive colonies were selected on LB agar plates with spectinomycin and chloramphenicol and identified by colony PCR. The verified positive colonies were then inoculated in LB medium containing chloramphenicol and rhamnose to cure the plasmid pEcgRNA-Δpox. The appropriately diluted bacterial solution was then coated on LB agar plates containing 100 g/L sucrose and incubated at 37 °C overnight to eliminate the plasmid pEcCasCm. Antibiotic marker-free strains were identified as those growing only on LB agar plates. Other mutants of K. oxytoca were constructed by using the same procedure.

Batch fermentation and fed-batch fermentation

Batch fermentation with glucose as the carbon source and fed-batch fermentation with lactose or whey powder as the carbon source were both conducted under aerobic condition in a 1-L bioreactor (Infors AG, Bottmingen, Switzerland) with 0.8 L medium. The cultivation was conducted at 37 °C, with a stirring speed of 500 rpm and an aeration rate of 1.6 vvm. The pH was controlled at 6.8 by 10 M NaOH via a program-controlled peristaltic pump. In batch fermentation, the initial glucose concentration was controlled at 60.0 g/L. In fed-batch fermentation, the initial lactose concentration was about 45 g/L. When its concentration dropped below 5.0 g/L, lactose or whey powder was added to maintain the concentration at around 20 g/L.

Analytical methods

The concentrations of α-ketoisovalerate, pyruvate, acetate, and lactose were detected through high performance liquid chromatography (HPLC) [14, 21]. Glucose concentration was detected by an SBA-40D bioanalyzer (Shandong Academy of Sciences, China). The concentrations of acetoin and 2,3-butanediol were determined by gas chromatography (GC) (Shimadzu, GC2014c) with a capillary GC column [21].

Enzymatic assays

The α-acetolactate synthase activities in the crude extracts of different recombinant K. oxytoca strains were detected followed the procedure of Gao et al. with some modifications [28]. The reaction solution contained 40 mM pyruvate, 0.1 mM TPP, 0.1 mM FAD, 10 mM MgCl2, and 50 µL appropriately diluted crude extract. The reaction was conducted at 37 °C and 120 rpm for 15 min and then pyruvate consumption was measured using HPLC. One unit of activity is defined as the amount of enzyme required to catalyze the condensation of 1 µmol of pyruvate per minute.

Results and discussion

Blocking L-valine synthesis to accumulate α-ketoisovalerate

K. oxytoca KIV-0 is a recombinant strain genetically engineered to produce L-valine from glucose [21]. α-Ketoisovalerate is the direct precursor of L-valine. Thus, K. oxytoca KIV-0 was chosen as the starting strain for α-ketoisovalerate production. The leuDH gene encoding an NADH-dependent leucine dehydrogenase (LeuDH) in B. subtilis 168 was previously integrated into genome of K. oxytoca KIV-0 to transform α-ketoisovalerate into L-valine [21]. This gene was knocked out to prevent the consumption of α-ketoisovalerate, resulting in strain KIV-1.

As expected, the inactivation of leuDH gene in strain KIV-0 resulted in accumulation of α-ketoisovalerate. K. oxytoca KIV-1 produced 8.09 g/L of α-ketoisovalerate from 60.0 g/L of glucose within 24 h, with a yield of 0.135 g/g (Fig. 2a). As shown in Fig. 2b, the main by-product generated during α-ketoisovalerate production by K. oxytoca KIV-1 was pyruvate (24.9 g/L). Actually, 42.5% of the carbon ratio was diverted to pyruvate production while only 20.9% of the carbon ratio was directed to α-ketoisovalerate accumulation (Fig. 2c). Other by-products, including acetate (0.686 g/L), acetoin (1.06 g/L), and 2,3-butanediol (0.266 g/L), were accumulated at rather low concentrations (Fig. 2b and c).

Fig. 2
figure 2

Blocking L-valine synthesis to accumulate α-ketoisovalerate. (a) Biomass, substrate consumption, and α-ketoisovalerate generation of K. oxytoca KIV-1. (b) By-products generation by K. oxytoca KIV-1. (c) Metabolic flux of K. oxytoca KIV-1. Solid circles represent accumulated products; hollow circles indicate non-accumulated products; the area of the circles represents the carbon ratio of different products to glucose. The carbon ratio (%) was defined as the ratio of the theoretical amount of glucose required for production of different products to the total consumed glucose

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Increasing the expression of α-acetolactate synthase BudB to enhance α-ketoisovalerate generation

The initial step in α-ketoisovalerate biosynthesis is the α-acetolactate synthase catalyzed α-acetolactate generation from pyruvate. The endogenous α-acetolactate synthase BudB encoded by budB in K. oxytoca KIV-0 is responsible for polymerization of pyruvate in this L-valine producing strain [21]. Accumulation of pyruvate by K. oxytoca KIV-1 suggested that the activity of the endogenous BudB may not be sufficient to convert pyruvate into α-acetolactate. The pox gene in K. oxytoca encodes pyruvate oxidase, which catalyzes the oxidization of pyruvate with oxygen to produce acetate. Thus, the second copy of budB with constitutive trc promoter was introduced in K. oxytoca KIV-1 at the location of pox to simultaneously strengthen pyruvate condensation and decrease acetate generation.

As shown in Fig. 3a, K. oxytoca KIV-2 produced 9.77 g/L of α-ketoisovalerate from 60.0 g/L of glucose, with a yield of 0.163 g/g within 24 h. The production of α-ketoisovalerate of K. oxytoca KIV-2 increased by 20.8%. Pyruvate was still the major by-product of K. oxytoca KIV-2, but its concentration decreased from 24.9 g/L to 20.2 g/L (Fig. 3b). The carbon ratio diverted into pyruvate decreased from 42.5 to 34.5%, while the carbon ratio diverted into α-ketoisovalerate increased from 20.9 to 25.2% (Fig. 3c).

Fig. 3
figure 3

Enhancing α-ketoisovalerate production through increasing α-acetolactate synthase expression. (a) Biomass, substrate consumption, and α-ketoisovalerate generation of K. oxytoca KIV-2. (b) By-products generation by K. oxytoca KIV-2. (c) Metabolic flux of K. oxytoca KIV-2. Solid circles represent accumulated products; hollow circles indicate non-accumulated products; the area of the circles represents the carbon ratio of different products to glucose. The carbon ratio (%) was defined as the ratio of the theoretical amount of glucose required for production of different products to the total consumed glucose

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Inactivating branched-chain α-keto acid dehydrogenase to enhance α-ketoisovalerate accumulation

The concentration of α-ketoisovalerate decreased at the end of fermentation by K. oxytoca KIV-1 and KIV-2, suggesting the α-ketoisovalerate degradation by these two strains (Figs. 2a and 3a). The branched-chain α-keto acid dehydrogenase (BCKDH) complex catalyzes oxidative decarboxylation of different branched-chain α-keto acids including α-ketoisovalerate, α-ketoisocaproate and α-ketomethylvalerate [29]. In previous reports, the bkdAA gene encoding the α-subunit of BCKDH complex was studied in P. putida [11], P. aeruginosa [29], Listeria monocytogenes [30], B. subtilis [31] and Staphylococcus aureus [32]. Comparative genomics revealed that homolog of BkdAA was also present in K. oxytoca KIV-0 (Additional file 1: Figure S1). To prevent the degradation α-ketoisovalerate through BCKDH complex, the bkdAA gene in K. oxytoca KIV-2 was deleted, resulting in strain KIV-3.

As shown in Fig. 4a, K. oxytoca KIV-3 exhibited a slight reduction in biomass generation and a significantly increase in α-ketoisovalerate production (12.7 g/L). The yield of α-ketoisovalerate of K. oxytoca KIV-3 increased to 0.212 g/g. Pyruvate, the structural analog of α-ketoisovalerate, may also serve as a substrate of BkdAA. Thus, the pyruvate production of K. oxytoca KIV-3 also slightly increased (Fig. 4b). The carbon ratio diverted to α-ketoisovalerate increased to 32.9% after bkdAA deletion (Fig. 4c).

Fig. 4
figure 4

Enhancing α-ketoisovalerate production through bkdAA deletion. (a) Biomass, substrate consumption, and α-ketoisovalerate generation of K. oxytoca KIV-3. (b) By-products generation by K. oxytoca KIV-3. (c) Metabolic flux of K. oxytoca KIV-3. Solid circles represent accumulated products; hollow circles indicate non-accumulated products; the area of the circles represents the carbon ratio of different products to glucose. The carbon ratio (%) was defined as the ratio of the theoretical amount of glucose required for production of different products to the total consumed glucose

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Enhancing the expression of mutant acetohydroxyacid isomeroreductase to improve α-ketoisovalerate synthesis

The second step in α-ketoisovalerate biosynthesis is the acetohydroxyacid isomeroreductase (AHAIR) catalyzed α-acetolactate reduction. AHAIR of E. coli W3110 mainly utilizes NADPH as the cofactor, while the L67E, R68F, and K75E mutant of AHAIR (AHAIRM) prefers NADH over NADPH as the cofactor [33, 34]. Previously, the ilvC encoding AHAIR and ilvCM encoding AHAIRM were integrated into the genome of K. oxytoca KIV-0 and under the control of promoters gld (the promoter of gldA gene) and bud (the promoter of budA gene), respectively [21]. Since K. oxytoca generally metabolizes glucose through glycolytic pathway and generates NADH, the expression of NADH utilizing AHAIRM was further enhanced through replacing the bud promoter of ilvCM with trc promoter.

Compared to K. oxytoca KIV-3, the obtained strain K. oxytoca KIV-4 exhibited significant increased substrate consumption and biomass generation (Figs. 4a and 5a). It produced 18.8 g/L of α-ketoisovalerate from 60.0 g/L of glucose in 21 h, with a yield of 0.313 g/g. The production of α-ketoisovalerate by K. oxytoca KIV-4 increased by 48% (Fig. 5a). In addition, the concentration of pyruvate accumulated by K. oxytoca KIV-4 decreased to 11.6 g/L (Fig. 5b). Enhancing the expression of AHAIRM increased the carbon ratio diverted to α-ketoisovalerate from 32.9 to 48.4% (Fig. 5c).

Fig. 5
figure 5

Enhancement of α-ketoisovalerate production through increasing mutant acetohydroxyacid isomeroreductase expression. (a) Biomass, substrate consumption, and α-ketoisovalerate generation of K. oxytoca KIV-4. (b) By-products generation by K. oxytoca KIV-4. (c) Metabolic flux of K. oxytoca KIV-4. Solid circles represent accumulated products; hollow circles indicate non-accumulated products; the area of the circles represents the carbon ratio of different products to glucose. The carbon ratio (%) was defined as the ratio of the theoretical amount of glucose required for production of different products to the total consumed glucose

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Introducing different α-acetolactate synthases to decrease pyruvate accumulation

Pyruvate was still the major by-product of K. oxytoca KIV-4 and its accumulation may potentially limit the production of α-ketoisovalerate. The frdA gene in K. oxytoca encodes α-subunit of fumarate reductase, which participates in reduction of fumarate to produce succinate. A third copy of budB with trc promoter was inserted into the frdA locus of stain KIV-4 to simultaneously enhance pyruvate condensation and decrease byproduct succinate generation, resulting in stain KIV-5. The α-acetohydroxyacid synthase IlvBN from E. coli W3110 and α-acetolactate synthase AlsS from B. subtilis 168 were reported to efficiently condense pyruvate into α-acetolactate [4, 7, 21]. The Q487S mutation of AlsS exhibited reduced decarboxylation activity toward α-ketoisovalerate, which may be beneficial for accumulation of α-ketoisovalerate [7]. The genes ilvBN (encoding IlvBN), alsS (encoding AlsS), and alsSM (encoding AlsSM) were inserted into genome of strain KIV-4 under the control of trc promoter at the position of frdA, resulting in the strains KIV-6, KIV-7, and KIV-8, respectively.

Then, K. oxytoca KIV-5, KIV-6, KIV-7, and KIV-8 were cultured in a 1-L bioreactor with 60.0 g/L of glucose and the biomass, α-ketoisovalerate production and by-products generation were assayed (Additional file 1: Figure S2). Compared with K. oxytoca KIV-5, KIV-6, and KIV-8, the strain K. oxytoca KIV-7 overexpressing AlsS exhibited a significant increase in α-ketoisovalerate production (Fig. 6a-d). It consumed 60.0 g/L of glucose and produced 24.8 g/L of α-ketoisovalerate within 18 h. The pyruvate accumulation of K. oxytoca KIV-7 decreased to 2.97 g/L while the yield of α-ketoisovalerate increased to 0.413 g/g. The carbon ratio diverted to pyruvate and its oxidative decarboxylation product acetate in K. oxytoca KIV-7 decreased to 5.6% (Fig. 6e), while the carbon ratio diverted to downstream products of α-acetolactate, including α-ketoisovalerate, acetoin, and 2,3-butanediol, increased to 76.1% (Fig. 6f). These results indicated that the introduction of AlsS from B. subtilis 168 was more favorable for the pyruvate condensation and α-ketoisovalerate production in K. oxytoca. The reduction in pyruvate concentration significantly improved the biomass, glucose consumption, and α-ketoisovalerate production of K. oxytoca KIV-7 (Fig. 6c).

The activities of α-acetolactate synthase in K. oxytoca KIV-4, KIV-5, KIV-6, KIV-7, and KIV-8 were also measured (Additional file 1: Figure S3). The results indicated that the α-acetolactate synthase activity was highest in strain K. oxytoca KIV-7 expressing AlsS. α-Ketoisovalerate is a structural analogue of pyruvate and can competitively inhibit the activity of α-acetolactate synthase [9]. As expected, the activities of α-acetolactate synthase in K. oxytoca KIV-4, KIV-5, KIV-6, KIV-7, and KIV-8 decreased in the presence of α-ketoisovalerate. The α-acetolactate synthase activity of KIV-7 was still the highest among these five strains. The lower pyruvate accumulation and higher α-ketoisovalerate production by K. oxytoca KIV-7 might be due to the high activity and the weak inhibition by α-ketoisovalerate of AlsS in the strain.

Fig. 6
figure 6

Introducing different α-acetolactate synthases to increase α-ketoisovalerate production. (a) Biomass, substrate consumption, pyruvate and α-ketoisovalerate generation of K. oxytoca KIV-5. (b) Biomass, substrate consumption, pyruvate and α-ketoisovalerate generation of K. oxytoca KIV-6. (c) Biomass, substrate consumption, pyruvate and α-ketoisovalerate generation of K. oxytoca KIV-7. (d) Biomass, substrate consumption, pyruvate and α-ketoisovalerate generation of K. oxytoca KIV-8. (e) The carbon ratio of pyruvate and acetate to consumed carbon source during batch fermentation of K. oxytoca KIV-4, KIV-5, KIV-6, KIV-7, and KIV-8. (f) The carbon ratio of downstream products of α-acetolactate to consumed carbon source during batch fermentation of K. oxytoca KIV-4, KIV-5, KIV-6, KIV-7, and KIV-8. The carbon ratio (%) was defined as the ratio of the theoretical amount of glucose required for production of different products to the total consumed glucose

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Utilization of lactose or whey for α-ketoisovalerate production

Whey is a by-product in dairying [35]. It contains lactose as the utilizable carbon source of many microorganisms. Batch fermentations using K. oxytoca KIV-7 with glucose, lactose, or crude whey as the carbon source were conducted in a 1-L bioreactor. α-Ketoisovalerate at a concentration of 14.5 g/L was produced from 38.0 g/L lactose in crude whey. The concentration and yield of α-ketoisovalerate with crude whey were slightly lower than those of with glucose or lactose as the substrate (Additional file 1: Figure S4). Fed-batch fermentation using K. oxytoca KIV-7 with lactose as the substrate was also performed in a 1-L bioreactor. As shown in Fig. 7a, K. oxytoca KIV-7 consumed 94.9 g/L of lactose and produced 37.3 g/L of α-ketoisovalerate in 33 h. The yield of α-ketoisovalerate produced by K. oxytoca KIV-7 with lactose was 0.393 g/g.

Whey powder can be obtained by spray drying of crude whey. Batch fermentation using K. oxytoca KIV-7 with whey power as the carbon source was conducted in a 1-L bioreactor. α-Ketoisovalerate at a concentration of 16.0 g/L was produced from 38.8 g/L lactose in whey power. The yield of α-ketoisovalerate with whey power (0.412 g/g lactose) were slightly higher than that of with crude whey as the substrate (0.382 g/g lactose) (Additional file 1: Figure S4). Then, fed-batch fermentation was also conducted with K. oxytoca KIV-7 using whey powder as the substrate in a 1-L bioreactor. As shown in Fig. 7b, K. oxytoca KIV-7 produced 40.7 g/L of α-ketoisovalerate with a yield of 0.418 g/g lactose.

Many microorganisms have been used for the fermentative biosynthesis of α-ketoisovalerate (Table 2). For example, Zhou et al. constructed a recombinant strain E. coli 050TY/pCTSDTQ487S-RBS55 by overexpressing the key enzymes involved in α-ketoisovalerate biosynthesis, regulating NADPH supply, and dynamically regulating activity of pyruvate dehydrogenase complex. E. coli 050TY/pCTSDTQ487S-RBS55 produced 55.8 g/L of α-ketoisovalerate from glucose with a yield of 0.550 g/g [7]. In this study, K. oxytoca KIV-0, an efficient L-valine producing strain, was metabolic engineered to produce α-ketoisovalerate [21]. The oxidative decarboxylation of α-ketoisovalerate was blocked by inactivating BkdAA in the resultant strain K. oxytoca KIV-7. BkdAA is required for the production of branched-chain fatty acids [36], which plays a vital role in maintenance of appropriate membrane fluidity and stress tolerance of industrial microorganisms [37]. Thus, the directly inactivation of BkdAA may decrease the performance of K. oxytoca KIV-7 under industrial stress. Dynamic control of BkdAA expression may be beneficial for balancing stress tolerance and α-ketoisovalerate formation by the strain K. oxytoca KIV-7 [38]. The LeuDH encoding gene leuDH in K. oxytoca KIV-7 was deleted to block NADH-dependent reductive amination of α-ketoisovalerate. Then, the expression of NADH utilizing AHAIRM was enhanced in K. oxytoca KIV-7 to consume excessive NADH and increase α-ketoisovalerate production. K. oxytoca metabolizes glucose into pyruvate mainly through glycolysis and generates two NADH, while α-ketoisovalerate production from pyruvate with AHAIRM only consumes one NADH [21, 33]. Thus, fine tuning the redox balance in K. oxytoca KIV-7 through control of the dissolved oxygen level or overexpression of NADH oxidase is still needed for further increasing α-ketoisovalerate generation.

Pyruvate is the major byproduct during α-ketoisovalerate fermentation by most of the derivative strains of K. oxytoca KIV-0. Thus, the α-acetolactate synthase BudB and AlsS were overexpressed to reduce pyruvate accumulation in K. oxytoca KIV-7. The significant improvements in final concentration and yield of α-ketoisovalerate were achieved, indicating the overexpression of BudB and AlsS is beneficial for the production of α-ketoisovalerate. Acetoin and 2,3-butanediol can be generated by non-enzymatic oxidative decarboxylation of α-acetolactate and subsequent diacetyl reduction (Fig. 1). Besides α-ketoisovalerate, these two chemicals are also the major downstream products of α-acetolactate. Decreased pyruvate accumulation and increased α-ketoisovalerate, acetoin and 2,3-butanediol production indicated that the carbon flux was successfully diverted to downstream products of α-acetolactate in K. oxytoca KIV-7 (Fig. 6f). In addition, the increased α-ketoisovalerate production in K. oxytoca KIV-7 was accompanied by increased biomass and substrate consumption, implying the overall metabolic balance of strain was also improved (Fig. 6c). Importantly, all of the key genes for α-ketoisovalerate production were integrated into the genome of K. oxytoca KIV-7 and constitutively expressed under the control of the trc promoter. The plasmid and inducer free characteristic made K. oxytoca KIV-7 a promising strain for stable α-ketoisovalerate fermentation with low cost.

Approximately 180–190 million tonnes of whey is annually generated [39]. More than 50% of whey is directly disposed to natural water bodies, which results in a loss of resource and causes serious environmental pollution [40]. In this work, production of α-ketoisovalerate from crude whey was acquired using recombinant strain K. oxytoca KIV-7 (Additional file 1: Figure S4). Due to the low concentration of lactose in crude whey (38.0 g/L), the final concentration of α-ketoisovalerate was 14.5 g/L. Liquid crude whey can be transformed into solid whey powder by spray drying [41]. Whey powder has been widely used in the fermentative production of biochemicals such as pyruvate [14], citrate [24], isobutanol [26] and butyl acetate [42]. Darwin et al. recently inactivated the pyruvate dehydrogenase and malate dehydrogenase in E. coli W and introduced the α-ketoisovalerate-producing plasmid pKIV. The obtained strain E. coli W1262 produced 3.22 g/L of α-ketoisovalerate with whey powder [8]. In this work, K. oxytoca KIV-7 produced α-ketoisovalerate from whey powder with a concentration of 40.7 g/L and a yield of 0.418 g/g lactose, which was close to that of with glucose as the substrate (0.413 g/g glucose). The fermentative process is thus a promising alternative for both biotechnological α-ketoisovalerate production and resource utilization of whey. In addition, α-ketoisovalerate serves as the precursor for many important chemicals, such as L-leucine [4], vitamin B5 [5], and isobutyrate [43]. These chemicals might also be generated from whey powder by expressing different biosynthesis pathways into K. oxytoca KIV-7.

Fig. 7
figure 7

α-Ketoisovalerate production through fed-batch fermentation with K. oxytoca KIV-7 with lactose or whey powder as carbon source. (a) Biomass, lactose consumption, and α-ketoisovalerate generation of K. oxytoca KIV-7 with lactose as the substrate. (b) Biomass, lactose consumption, and α-ketoisovalerate generation of K. oxytoca KIV-7 with whey powder as the substrate

Full size image
Table 2 Comparison of α-ketoisovalerate production by different microorganisms
Full size table

Conclusion

In summary, an efficient α-ketoisovalerate producing strain K. oxytoca KIV-7 was acquired through engineering of the L-valine producer K. oxytoca VKO-9. α-Ketoisovalerate at a concentration of 40.7 g/L and a yield of 0.418 g/g was generated by K. oxytoca KIV-7 with whey powder as the substrate. This work provides a promising process for both efficient α-ketoisovalerate production and industrially produced low-value whey utilization. The recombinant strain K. oxytoca KIV-7 could also be used as the chassis for producing other derivatives of α-ketoisovalerate.

Data availability

All data generated or analyzed during this study are included in this published article.

Abbreviations

AHAIR:

Acetohydroxyacid isomeroreductase

AHAIRM :

Mutant acetohydroxyacid isomeroreductase

DHAD:

Dihydroxyacid dehydratase

LeuDH:

Leucine dehydrogenase

ALS:

α-Acetolactate synthase

BCKDH:

Branched-chain α-keto acid dehydrogenase

BOD:

Biochemical oxygen demand

COD:

Chemical oxygen demand

HPLC:

High performance liquid chromatography

References

  1. Schaefer K, Erley CM, Herrath D, Stein G. Calcium salts of ketoacids as a new treatment strategy for uremic hyperphosphatemia. Kidney Int Suppl. 1989;27(5):136–9.

    Google Scholar 

  2. Schaefer K, Herrath D, Erley CM, Asmus G. Calcium ketovaline as new therapy for uremic hyperphosphatemia. Miner Electrolyte Metab. 1990;16(6):362–4.

    CAS  PubMed  Google Scholar 

  3. 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;397:130502.

    Article  CAS  PubMed  Google Scholar 

  4. Wang YY, Xu JZ, Zhang WG. Metabolic engineering of L-leucine production in Escherichia coli and Corynebacterium glutamicum: a review. Crit Rev Biotechnol. 2019;39(5):633–47.

    Article  CAS  PubMed  Google Scholar 

  5. Guo J, Sun X, Yuan Y, Chen Q, Ou Z, Deng Z, Ma T, Liu T. Metabolic engineering of Saccharomyces cerevisiae for vitamin B5 production. J Agric Food Chem. 2023;71(19):7408–17.

    Article  CAS  PubMed  Google Scholar 

  6. Luo Z, Yu S, Zeng W, Zhou J. Comparative analysis of the chemical and biochemical synthesis of keto acids. Biotechnol Adv. 2021;47:107706.

    Article  CAS  PubMed  Google Scholar 

  7. Zhou L, Zhu Y, Yuan Z, Liu G, Sun Z, Du S, Liu H, Li Y, Liu H, Zhou Z. Evaluation of metabolic engineering strategies on 2-ketoisovalerate production by Escherichia coli. Appl Environ Microbiol. 2022;88(17):e0097622.

    Article  PubMed  Google Scholar 

  8. Carranza-Saavedra D, Torres-Bacete J, Blázquez B, Sánchez Henao CP, Zapata Montoya JE, Nogales J. System metabolic engineering of Escherichia coli W for the production of 2-ketoisovalerate using unconventional feedstock. Front Bioeng Biotechnol. 2023;11:1176445.

    Article  PubMed  PubMed Central  Google Scholar 

  9. Krause FS, Blombach B, Eikmanns BJ. Metabolic engineering of Corynebacterium glutamicum for 2-ketoisovalerate production. Appl Environ Microbiol. 2010;76(24):8053–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Buchholz J, Schwentner A, Brunnenkan B, Gabris C, Grimm S, Gerstmeir R, Takors R, Eikmanns BJ, Blombach B. Platform engineering of Corynebacterium glutamicum with reduced pyruvate dehydrogenase complex activity for improved production of L-lysine, L-valine, and 2-ketoisovalerate. Appl Environ Microbiol. 2013;79(18):5566–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Batianis C, van Rosmalen RP, Major M, van Ee C, Kasiotakis A, Weusthuis RA. Martins Dos Santos VAP. A tunable metabolic valve for precise growth control and increased product formation in Pseudomonas putida. Metab Eng. 2023;75:47–57.

    Article  CAS  PubMed  Google Scholar 

  12. Gu J, Zhou J, Zhang Z, Kim CH, Jiang B, Shi J, Hao J. Isobutanol and 2-ketoisovalerate production by Klebsiella pneumoniae via a native pathway. Metab Eng. 2017;43:71–84.

    Article  CAS  PubMed  Google Scholar 

  13. Pospíšil S, Kopecký J, Přikrylová V, Spížek J. Overproduction of 2-ketoisovalerate and monensin production by regulatory mutants of Streptomyces cinnamonensis resistant to 2-ketobutyrate and amino acids. FEMS Microbiol Lett. 1999;172(2):197–204.

    Article  Google Scholar 

  14. Cao M, Jiang T, Li P, Zhang Y, Guo S, Meng W, Lü C, Zhang W, Xu P, Gao C, Ma C. Pyruvate production from whey powder by metabolic engineered Klebsiella oxytoca. J Agric Food Chem. 2020;68(51):15275–83.

    Article  CAS  PubMed  Google Scholar 

  15. Park JM, Song H, Lee HJ, Seung D. Genome-scale reconstruction and in silico analysis of Klebsiella oxytoca for 2,3-butanediol production. Microb Cell Fact. 2013;12(1):20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Wilson DJ. NIH guidelines for research involving recombinant DNA molecules. Account Res. 1993;3(2–3):177–85.

  17. Xin B, Tao F, Wang Y, Liu H, Ma C, Xu P. Coordination of metabolic pathways: enhanced carbon conservation in 1,3-propanediol production by coupling with optically pure lactate biosynthesis. Metab Eng. 2017;41:102–14.

    Article  CAS  PubMed  Google Scholar 

  18. Khunnonkwao P, Jantama SS, 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):1292–301.

    Article  CAS  Google Scholar 

  19. In S, Khunnonkwao P, Wong N, Phosiran C, Jantama SS, 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):9565–79.

    Article  CAS  PubMed  Google Scholar 

  20. 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;393:130045.

    Article  CAS  PubMed  Google Scholar 

  21. Cao M, Sun W, Wang S, Di H, Du Q, Tan X, Meng W, Kang Z, Liu Y, Xu P, Lü C, Ma C, Gao C. Efficient L-valine production using systematically metabolic engineered Klebsiella oxytoca. Bioresour Technol. 2024;395:130403.

    Article  CAS  PubMed  Google Scholar 

  22. Macwan SR, Dabhi BK, Parmar SC, Aparnathi KD. Whey and its utilization. Int J Curr Microbiol App Sci. 2016;5(8):134–55.

    Article  CAS  Google Scholar 

  23. Li P, Wang M, Di H, Du Q, Zhang Y, Tan X, Xu P, Gao C, Jiang T, Lü C, Ma C. Efficient production of 1,2,4-butanetriol from corn cob hydrolysate by metabolically engineered Escherichia coli. Microb Cell Fact. 2024;23(1):49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Arslan NP, Aydogan MN, Taskin M. Citric acid production from partly deproteinized whey under non-sterile culture conditions using immobilized cells of lactose-positive and cold-adapted Yarrowia Lipolytica B9. J Biotechnol. 2016;231:32–9.

    Article  CAS  PubMed  Google Scholar 

  25. Mano J, Liu N, Hammond JH, Currie DH, Stephanopoulos G. Engineering Yarrowia Lipolytica for the utilization of acid whey. Metab Eng. 2020;57:43–50.

    Article  CAS  PubMed  Google Scholar 

  26. Novak K, Baar J, Freitag P, Pflügl S. Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material. J Ind Microbiol Biotechnol. 2020;47(12):1117–32.

    Article  CAS  PubMed  Google Scholar 

  27. 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):620–7.

    Article  CAS  PubMed  Google Scholar 

  28. Gao C, Li Z, Zhang L, Wang C, Li K, Ma C, Xu P. An artificial enzymatic reaction cascade for a cell-free bio-system based on glycerol. Green Chem. 2015;17(2):804–7.

    Article  CAS  Google Scholar 

  29. McCully V, Burns G, Sokatch JR. Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO. Biochem J. 1986;233(3):737–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhu K, Bayles DO, Xiong A, Jayaswal RK, Wilkinson BJ. Precursor and temperature modulation of fatty acid composition and growth of Listeria monocytogenes cold-sensitive mutants with transposon-interrupted branched-chain alpha-keto acid dehydrogenase. Microbiology. 2005;151(2):615–23.

    Article  CAS  PubMed  Google Scholar 

  31. Lowe PN, Hodgson JA, Perham RN. Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis. Biochem J. 1983;215(1):133–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Singh VK, Hattangady DS, Giotis ES, Singh AK, Chamberlain NR, Stuart MK, Wilkinson BJ. Insertional inactivation of branched-chain alpha-keto acid dehydrogenase in Staphylococcus aureus leads to decreased branched-chain membrane fatty acid content and increased susceptibility to certain stresses. Appl Environ Microbiol. 2008;74(19):5882–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Hasegawa S, Uematsu K, Natsuma Y, Suda M, Hiraga K, Jojima T, Inui M, Yukawa H. Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions. Appl Environ Microbiol. 2012;78(3):865–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hao Y, Ma Q, Liu X, Fan X, Men J, Wu H, Jiang S, Tian D, Xiong B, Xie X. High-yield production of L-valine in engineered Escherichia coli by a novel two-stage fermentation. Metab Eng. 2020;62:198–206.

    Article  CAS  PubMed  Google Scholar 

  35. Prazeres AR, Carvalho F, Rivas J. Cheese whey management: a review. J Environ Manage. 2012;110:48–68.

    Article  CAS  PubMed  Google Scholar 

  36. Kader Chowdhury QMM, Islam S, Narayanan L, Ogunleye SC, Wang S, Thu D, Freitag NE, Lawrence ML, Abdelhamed H. An insight into the role of branched-chain α-keto acid dehydrogenase (BKD) complex in branched-chain fatty acid biosynthesis and virulence of Listeria monocytogenes. J Bacteriol. 2024;206(7):e0003324.

    Article  PubMed  Google Scholar 

  37. Mohedano MT, Konzock O, Chen Y. Strategies to increase tolerance and robustness of industrial microorganisms. Synth Syst Biotechnol. 2021;7(1):533–40.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Shen X, Wang J, Li C, Yuan Q, Yan Y. Dynamic gene expression engineering as a tool in pathway engineering. Curr Opin Biotechnol. 2019;59:122–9.

    Article  CAS  PubMed  Google Scholar 

  39. Buchanan D, Martindale W, Romeih E, Hebishy E. Recent advances in whey processing and valorisation: Technological and environmental perspectives. Int J Dairy Technol. 2023;76(2):291–312.

    Article  CAS  Google Scholar 

  40. Tesfaw A, Oner ET, Assefa F. Evaluating crude whey for bioethanol production using non-Saccharomyces yeast, Kluyveromyces marxianus. SN Appl Sci. 2021;3(42):1–8.

    Google Scholar 

  41. Domínguez-Niño A, Cantú-Lozano D, Ragazzo-Sanchez JA, Andrade-González I, Luna-Solano G. Energy requirements and production cost of the spray drying process of cheese whey. Dry Technol. 2018;36(5):597–608.

    Article  Google Scholar 

  42. Ma Y, Guo N, Wang S, Wang Y, Jiang Z, Guo L, Luo W, Wang Y. Metabolically engineer Clostridium saccharoperbutylacetonicum for comprehensive conversion of acid whey into valuable biofuels and biochemicals. Bioresour Technol. 2024;400:130640.

    Article  CAS  PubMed  Google Scholar 

  43. Zhang K, Woodruff AP, Xiong M, Zhou J, Dhande YK. A synthetic metabolic pathway for production of the platform chemical isobutyric acid. ChemSusChem. 2011;4(8):1068–70.

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Acknowledgements

We thank Chengjia Zhang and Nannan Dong from Core Facilities for Life and Environmental Sciences (State Key Laboratory of Microbial Technology, Shandong University) for assistance in microbial fermentation.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32300045, 32300029), the National Key R&D Program of China (2022YFC2106000), Youth Program of Natural Science Foundation of Shandong Province (ZR2022QC092, ZR2023QC237), China Postdoctoral Science Foundation (2023M742085), 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), State Key Laboratory of Microbial Technology Open Projects Fund (M2023-02), and State Key Laboratory of Microbial Technology (SKLMTFCP-2023–03).

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Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Shandong University, NO.72 Binhai Road, Qingdao, 266237, China

    Weikang Sun, Shuo Wang, Xiaoxu Tan, Leilei Guo, Wei Liu, Wenjia Tian, Hui Zhang, Wensi Meng, Yidong Liu, Zhaoqi Kang, Chuanjuan Lü, Chao Gao & Cuiqing Ma

  2. School of Municipal and Environmental Engineering, Shandong Jianzhu University, Jinan, 250101, China

    Tianyi Jiang

  3. State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai, 200240, China

    Ping Xu

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Contributions

Weikang Sun: Writing original draft, Investigation, Data curation. Shuo Wang: Formal analysis, Software, Validation. Xiaoxu Tan: Software, Validation. Leilei Guo: Software, Validation. Wei Liu: Software, Validation. Wenjia Tian: Software, Validation. Hui Zhang: Software, Validation. Tianyi Jiang: Software, Funding acquisition. Wensi Meng: Funding acquisition. Yidong Liu: Software, Validation. Zhaoqi Kang: Software, Validation. Chuanjuan Lü: Software, Funding acquisition. Chao Gao: Conceptualization, Software, Funding acquisition. Ping Xu: Software, Supervision. Cuiqing Ma: Conceptualization, Supervision, Writing – review and editing, Funding acquisition. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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Sun, W., Wang, S., Tan, X. et al. Production of α-ketoisovalerate with whey powder by systemic metabolic engineering of Klebsiella oxytoca. Microb Cell Fact 23, 264 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12934-024-02545-4

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Microbial Cell Factories

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