Deletion of Pyruvate Formate Lyase in Klebsiella pneumoniae for. Improvement of 2,3-Butanediol Yield

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1 AEM Accepts, published online ahead of print on 1 August 2014 Appl. Environ. Microbiol. doi: /aem Copyright 2014, American Society for Microbiology. All Rights Reserved. 1 2 Deletion of Pyruvate Formate Lyase in Klebsiella pneumoniae for Improvement of 2,3-Butanediol Yield Moo-Young Jung, a Suman Mazumdar, a Sang Heum Shin, b Kap-Seok Yang, b Jinwon Lee, c Min- Kyu Oh a# a Department of Chemical and Biological Engineering, Korea University, Seoul, Republic of Korea b Macrogen Inc., Gasan-dong, Seoul, Republic of Korea c Department of Chemical and Biological Engineering, Sogang University, Seoul, Republic of Korea Running Head: 2,3-Butanediol yield of PflB-deficient K. pneumoniae #Address correspondence to Min-Kyu Oh, mkoh@korea.ac.kr

2 ABSTRACT Klebsiella pneumoniae is considered to be a good host strain for the production of 2,3- butanediol, which are promising platform chemical with various industrial applications. In this study, three genes, including glucosyltransferase (wabg), lactate dehydrogenase (ldha), and pyruvate formate lyase (pflb), were disrupted in K. pneumoniae to reduce both its pathogenic characteristics and the production of several byproducts. In flask cultivation with minimal medium, the yield of 2,3-butanediol from rationally engineered K. pneumoniae ( wabg ldha pflb) reached 0.461g/g glucose, which was 92.2% of the theoretical maximum yield with a significant reduction in byproduct formation. However, the growth rate of the pflb mutant was slightly reduced compared to its parental strain. Comparison with similar mutants of Escherichia coli suggested that the growth defect of pflb deleted K. pneumoniae was caused by redox imbalance rather than reduced level of intracellular acetyl-coa. From an analysis of the transcriptome, it was confirmed that the removal of pflb from K. pneumoniae significantly repressed the expression of genes involved in the formate hydrogen lyase (FHL) system. 2

3 INTRODUCTION The microbial production of 2,3-butanediol and 1,3-propanediol has been intensively studied in the last few years owing to the potential of these diols as platform chemicals in industrial applications such as the production of polymers, fuels, and solvents (1-5). These diols can be produced through microbial fermentation, and several microorganisms such as Klebsiella (6, 7), Enterobacter (8, 9), Bacillus (10, 11), Clostridium (12, 13), and Serratia (14, 15) species have been reported to produce up to150 g/l of 2,3-butanediol from glucose or 80 g/l of 1,3-propanediol from glycerol. Klebsiella pneumoniae has fermentative pathways that can synthesize both 2,3-butanediol and 1,3-propanediol from a wide range of carbon sources and has shown superior performance with respect to diol production (16, 17). However, for industrial application, it will be necessary to reduce byproduct formation in K. pneumoniae because significant amounts of byproducts including acetate, lactate, ethanol, succinate, and formate are produced during diol fermentation, resulting in reduced diol yield (18). Therefore, various genetic modifications of K. pneumoniae have been conducted. Chen et al. (19) conducted the overexpression of NADPH-dependent alcohol dehydrogenase (HOR) to enhance carbon flux toward pentose phosphate pathway, resulting in 72.2% enhancement of 2,3-butanediol production with significant reduction in byproduct formation. Xu et al. (20) reported that the final concentrations of 1,3-propanediol and 2,3-butanediol produced by the lactate dehydrogenase (ldha) mutant K. pneumoniae were increased by 6.99% and 95.93%, respectively, compared to those of its parental strain at 48 h of fedbatch fermentation. However, removal of multiple genes in an attempt to reduce the 3

4 production of several byproducts caused unexpected problems due to the complex mechanisms in the cell (21). In this study, by using minimal gene modifications, we attempted to reduce byproduct production and increase 2,3-butanediol production while maintaining a stable cellular network in K. pneumoniae. Although K. pneumoniae is known to be an efficient strain for 2,3-butanediol production, its pathogenic characteristics limit the industrial application of this strain (22). Therefore, wabg, which is involved in the synthesis of outer core lipopolysaccharides (LPS), was disrupted to reduce the pathogenicity of K. pneumoniae (23). Then, ldha was disrupted because lactate is a main byproduct of 2,3- butanediol production and its production pathway competes with 2,3-butanediol for NADH as a cofactor (24). Finally, pflb was inactivated to reduce the amount of carbon flux directed toward other byproducts including formate, acetate, and ethanol, which are derived from the pathway from pyruvate to acetyl-coa (25). The production of byproducts from the rationally engineered K. pneumoniae strain ( wabg ldha pflb) was significantly reduced, resulting in an increased 2,3-butanediol yield. We further examined the reason for the reduced growth seen in pflb-deficient K. pneumoniae by measuring levels of intracellular acetyl-coa and nicotinamide nucleotides. In addition, transcriptome analysis was performed to reveal the global gene expression level changes caused by pflb inactivation. 4

5 79 MATERIALS AND METHODS Strain development. All K. pneumoniae strains were derived from the wild-type strain KCTC 2242 (Korean Collection for Type Culture), for which the genome has been sequenced (26). The λ Red recombination method was used for gene deletion (27). To construct the wabg-deleted mutant, K. pneumoniae KCTC 2242 containing predet was cultured at 30 C, and 10% L-arabinose in deionized water was added to a final concentration of 24 mm when the optical density at 600 nm (OD 600 ) reached 0.2. A kanamycin resistance gene flanked with FRT sites was amplified from pkd4 with the primers wabg-fkf-fw and wabg -FKF-rv (Table 1). The polymerase chain reaction (PCR) product was transformed into K. pneumoniae containing the predet plasmid and selected on Luria-Bertani (LB) plates containing 25 μg/ml kanamycin at 37 C. The homologous recombination of wabg was confirmed by colony PCR with the primers wabg-con-a and wabg-con-b. Then, the707-flp plasmid was transformed into the wabg-deficient K. pneumoniae to remove the kanamycin selection marker between FRT sites. The removal of the kanamycin selection marker was confirmed by colony PCR with the primers wabg-con-a and wabg-con-b and by observing the kanamycin-sensitive phenotype of the resulting mutant. Using the same method, ldha was deleted from the genome of KMK-01, and the resulting strain was named KMK-02. Finally, pflb was deleted from KMK-02, and the resulting wabg ldha pflb triple-knockout mutant was named KMK-05. The wild-type E. coli K12 strain MG1655 was obtained from the Coli Genetic Stock Center (CGSC; Yale University, New Haven, CT, USA). Consecutive gene 5

6 deletions were performed in MG1655 and its derivatives by P1 phage transduction. Single-gene knockout mutants (Keio strains) were obtained from the CGSC and were used as donors for specific mutations. Details of the protocol used have been described elsewhere (28). All mutations constructed in the host E. coli were also confirmed by PCR using the verification primers. All resulting strains, along with primers and plasmids used in this study, are listed in Table 1 and Table S Media and culture conditions. The 1-L culture medium consisted of 3 g KH 2 PO 4, 6.8 g Na 2 HPO 4, 0.75 g KCl, 5.35 g (NH 4 ) 2 SO 4, 0.28 g Na 2 SO 4, 0.26 g MgSO 4 7H 2 O, 0.42 g citric acid, 5 g yeast extract, 10 g casamino acid, and 0.3 ml of a microelement solution containing 34.2 g/l ZnCl 2, 2.7 g/l FeCl 3 6H 2 O, 10 g/l MnCl 2 4H 2 O, 0.85 g/l CuCl 2 2H 2 O, and 0.31 g/l H 3 BO 3, as described previously (24). The composition of the minimal medium was the same as that of the culture medium but did not include yeast extract and casamino acid. As described below, glucose (80 g/l) or glycerol (60 g/l) was added to the medium as a carbon source. The cultures, in 50 ml medium in 250-mL flasks sealed with silicon stoppers, were incubated micro-aerobically at 250 rpm and 37 C Measurement of intracellular acetyl-coa. To prepare cell lysates, 3 ml of culture in the mid-exponential phase (6 h) was added to 12 ml of cold ( 40 C) 60% MeOH (aq.) followed by centrifugation at 5000 g for 3 minutes at 20 C. The supernatant was removed, 2 ml of boiling 75% EtOH (aq.) was immediately added, and then the reaction was incubated at 95 C for 5 minutes. Cell debris was removed by centrifugation at

7 g for 3 minutes. The supernatant was then transferred to new tubes and dried under vacuum. The levels of acetyl-coa in the cell lysates were measured using the PicoProbe acetyl-coa assay kit (BioVision Research Products; Mountain View, CA, USA) according to the manufacturer s instruction. For calculation of intracellular acetyl-coa concentration, the dry cell weight (DCW) of E. coli and K. pneumoniae was set to 0.44 g/ l and intracellular aqueous volume (V in ) to 1.9 μl /mg (29). The experiment was repeated three times independently Determination of intracellular nicotinamide nucleotide concentration. To measure the intracellular concentration of nicotinamide nucleotides, the NAD/NADH assay kit (Promega; Wisconsin, USA) was used according to the manufacturer s instructions. Briefly, cells were harvested at the mid-exponential phase (6 h) and lysed with 1 ml of bicarbonate base buffer containing 1% DTAB (Dodecyltrimethylammonium bromide). The 50 μl of supernatant was transferred equally into two tubes, and 25 μl of 0.4 N HCl was added to one tube. The samples were incubated in a heating block at 60 C for 15 minutes to selectively decompose NAD + and NADH in basic and acid solutions, respectively. After the enzyme reaction for NADH conversion from NAD + was performed with the two samples, the luminescence from the standards and the samples in a 96-well plate was measured using a microplate reader (Infinite M200 Pro; Tecan, Switzerland). The experiment was repeated three times independently Total RNA extraction and mrna library preparation. To prepare mrna library, 5 ml of culture broth containing either KMK-02 or KMK-05 in mid-exponential phase (6 h) 7

8 was harvested, and then the total RNA in the cell was stabilized using RNAprotect Bacteria Reagent (Qiagen; Hilden, Germany). Cell lysis and total RNA extraction were conducted using RNeasy Mini Kit (Qiagen) following the manufacturer s protocol. Total RNA quality and quantity were measured using a RNA 6000 Nano Kit with the Agilent Bioanalyzer (Agilent Technologies; CA, USA). Then, rrna was removed using Ribo- Zero rrna Removal Kits (Gram-Negative Bacteria; Epicentre, Madison, WI, USA). RNA samples were then washed with 70% EtOH (aq.) for mrna clean up, and mrna integrity was confirmed using the RNA 6000 Pico Kit on an Agilent Bioanalyzer. The concentrations of the mrna samples were 100 ng/μl Transcriptome sequencing and statistical analysis. The cdna library was constructed from purified mrna according to the manufacturer s instructions (TruSeq RNA Sample Preparation Kits v2; Illumina). Briefly, transcriptome sequencing was conducted using Illumina/Hiseq-2000 RNA-seq. After evaluation of the quality of the raw sequence data with FastQC ( short DNA sequences were aligned on the K. pneumoniae genome using bowtie ( Mapping was done by counting reads that belong to genes and normalizing the count to mapable reads per kilobase per million reads (RPKM) with an in-house script. Following the data handling procedure, a total of 4923 open reading frames (ORF) and expression levels were confirmed. The normality of the expression of each gene was confirmed using the F-test (p-value > 0.05). An independent t-test was used with a total of six RPKM values per gene from two strains to classify 8

9 genes whose expression levels were significantly changed by pflb deletion (fold change >2, p-value <0.05) Metabolite assays. To measure metabolites concentration, 1 ml of culture broth was transferred to a microcentrifuge tube and centrifuged for 5 minutes at 13,000 rpm. The amounts of 2,3-butanediol, glucose, pyruvate, succinate, lactate, formate, acetate, acetoin, and ethanol in the supernatant were analyzed by high-performance liquid chromatography (Waters HPLC 1500 series; MA, USA) equipped with a Sugar SH1011 column (Shodex; Tokyo, Japan) and an RI detector at 45 C. The column temperature was maintained at 75 C, and 0.01 M sulfuric acid was used as the mobile phase at a flow rate of 0.5 ml/min. The optical density of the cell was monitored at 600 nm (OD 600 ) using an ultraviolet-visibility spectrophotometer (Shimazu UV mini 1240; Tokyo, Japan) RESULTS Strain development of K. pneumoniae for 2,3-butanediol production. The major metabolic pathway of K. pneumoniae KCTC 2242 for the production of 2,3-butanediol is shown in Fig. 1. First, wabg, which encodes a glucosyltransferase that plays a key role in the synthesis of the outer core LPS, was removed to reduce the strain s pathogenicity (29), and the resulting strain was named KMK-01. LPS, the outer component of the bacterial surface, is considered an important pathogenic determinant in K. pneumoniae. The wabgdeficient K. pneumoniae was found to be avirulent when tested in different animal models (30). In addition, genetic engineering in K. pneumoniae could be conducted easily 9

10 following the removal of a dense LPS mass (23). Because of these advantages, KMK-01 was used as a control strain in this study. Then, ldha was deleted, and the resulting strain was named KMK-02 (wabg ldha). The deletion of ldha is beneficial for 2,3- butanediol production as it leads to increased NADH availability and the redirection of the carbon flux (18, 31). In addition, reduced lactate production may reduce the acidification of cultivation media and thus cause increased biomass production (24). Finally, to reduce carbon flux toward other byproducts including formate, acetate, succinate, and ethanol, pflb, which encodes one of the enzymes that converts pyruvate to acetyl-coa, was deleted, and the resulting strain was named KMK-05 (wabg ldha pflb) Metabolite production from developed K. pneumoniae Flask cultivation of constructed K. pneumoniae mutants was performed with culture medium for 36 h (Fig. 2). The deletion of ldha (strain KMK-02) abolished lactate synthesis and enhanced ethanol production, in accordance with a previous report (32). The significant decrease in lactate production caused increases of succinate and ethanol production by 25.5% and 36.4%, respectively. In addition, significant enhancements of cell mass and 2,3-butanediol production were observed due to the reduced rate of medium acidification (24). Knockout of the pflb gene reduced the growth rate of KMK-05 in comparison with its parental strain, KMK-02. Interestingly, the production of 2,3-butanediol in KMK-05 did not decrease noticeably, but the amount of byproducts produced, particularly ethanol (88.8%) and acetate (~99%), was dramatically decreased. Although the 2,3-butanediol titer (g/l) of KMK-05 was lower than that of its parental strain until 32 h of cultivation, the 10

11 concentration of 2,3-butanediol at 36 h was 16.8% higher than its parental strain in spite of having a 43.48% reduced optical density. Reduction of byproducts formation in KMK-05 greatly improved the yield of 2,3- butanediol. However, it is not possible to determine the 2,3-butanediol yield based on consumed glucose in rich medium because some extra carbon sources, such as yeast extract or casamino acids, were also used for 2,3-butanediol production. Therefore, the 2,3-butanediol production was conducted in minimal medium to calculate the exact yield derived from glucose. For this purpose, strains were initially grown in the culture medium for 12 h to obtain enough cell mass, washed with PBS twice, and transferred to minimal medium. The initial optical density and glucose concentration were adjusted to 6.5 and 70 g/l, respectively. In all flask cultivations, formate was not detected in extracellular medium. The produced formate was probably converted to hydrogen and carbon dioxide by formate hydrogen lyase system. Forty-eight hours after medium transfer, the OD 600 of the strains was reduced, but not significantly (~10% for KMK-01 and KMK-02 and ~20% for KMK-05; Fig. 3A). The KMK-02 strain consumed 70 g/l glucose and completed the conversion of glucose to 2,3-butanediol in 30 h, whereas KMK-01 and KMK-05 took 48 h for the conversion (Fig. 3B and C). Both KMK-01 and KMK-02 had similar yields, which were approximately 70% of the maximum theoretical yield, and the remaining glucose must have been converted to other byproducts (Table 2). As in flask cultivation with culture medium, KMK-05 exhibited a significantly higher yield of 2,3-butanediol than other strains and produced 92.2% of the maximum theoretical yield. The 2,3-butanediol yields of all the strains did not change significantly from 12 h to 48 h in minimal medium cultivation (Fig. 3D). Pyruvate was observed only 11

12 in cultivation of KMK-05 primarily due to the unbalance of carbon flux at pyruvate node by the deletion of ldha and pflb (Fig. 1 and Table 2). The accumulation of intracellular pyruvate could be a driving force for higher 2,3-butanediol yield in KMK-05. This experiment successfully demonstrated that pflb deletion improves 2,3-butanediol yield significantly, with a significant reduction in byproduct formation Comparison of the effects of deleting pflb in E. coli and K. pneumoniae. In the glucose metabolism of E. coli and K. pneumoniae, pyruvate formate lyase (PFL) and pyruvate dehydrogenase complex (PDHc) are the two main routes for pyruvate dissimilation (33, 34). Under aerobic conditions, acetyl-coa is generated from pyruvate by PDHc, whereas PFL substitutes for PDHc in anaerobic conditions to generate formate, carbon dioxide, and hydrogen as co-products (Fig. 1). In E. coli, the pflb deletion and its metabolic effects have been intensively investigated to enhance the yield of target products (35-37). Therefore, flask cultivation of pflb-deficient E. coli was compared with that of K. pneumoniae under microaerobic conditions for 24 h (Fig. 4). Interestingly, the cell mass production of pflb-deficient K. pneumoniae was reduced by 49.2% compared to that of its parental strain, whereas that of E. coli did not change significantly. The effect of pflb deletion on byproduct production was also significantly different between the two species. The deletion of pflb reduced the specific production of succinate, acetate, and ethanol in E. coli by 15.7%, 15.6%, and 32.4%, respectively, whereas the same genetic perturbation caused a 50.8%, 100%, and 92.0% reduction, respectively of the same byproducts in K. pneumoniae. The deletion also significantly increased specific 2,3- butanediol production in K. pneumoniae (85.1%). These results demonstrate that the 12

13 effect of the removal of pflb was much greater in K. pneumoniae than in E. coli under microaerobic conditions Redox balance and acetyl-coa level of pflb-deficient K. pneumoniae. Knocking out ldha and pflb in K. pneumoniae made it possible to obtain high yields of 2,3-butanediol but also impaired cell growth, which decreased the rate of 2,3-butanediol production. Therefore, we tried to determine the causes of the growth defect seen in K. pneumoniae pflb mutants. There were two possible reasons for the growth defect caused by the pflb deletion: the induction of a high intracellular redox ratio (NADH/NAD + ) or a reduction in the size of the acetyl-coa pool (21, 38). Not only many essential metabolic precursors, but also byproducts such as acetate and ethanol are derived from acetyl-coa (Fig. 1). Byproduct production was significantly reduced in KMK-05, indicating that the acetyl- CoA pool was significantly reduced under microaerobic conditions by the pflb deletion in K. pneumoniae. To verify this hypothesis, the levels of intracellular acetyl-coa in E. coli and K. pneumoniae mutants were measured quantitatively. As shown in Fig. 5, a significant reduction of acetyl-coa concentration by 90.9 % and 88.0% were observed by pflb mutation in both K. pneumoniae and E. coli, respectively, under microaerobic conditions. However, the mutation did not cause a growth rate decrease in E. coli, suggesting that the reduction of intracellular acetyl-coa pool alone was not a major factor of the reduced cell growth in pflb-deficient K. pneumoniae. The concentration of intracellular nicotinamide nucleotides were also measured (Fig. 5 and table 3). Generally, the level of redox ratio (NADH/NAD + ) was higher in K. pneumoniae strains compared to that of E. coli (Fig. 5). Interestingly, the removal of ldha 13

14 and pflb genes did not cause any change in the redox ratio (NADH/NAD + ) in E. coli, while those mutations caused 42.9% and 46.7% increases of the ratios, respectively, in K. pnuemoniae. As suggested in previous reports, redox imbalance caused by reduced byproduct formation might also have been responsible for the decrease in the growth rate (38). In E. coli, pflb deletion did not cause significant change in the level of fermentative products (Fig. 4). However, in K. pneumoniae, the level of lactate and ethanol was greatly reduced by ldha and pflb disruptions (Fig. 2 and 4), which resulted in redox imbalance and growth retardation. To verify this hypothesis, 20 mm of sodium nitrate (NaNO 3 ) was added to the culture medium of K. pneumoniae strains as an electron acceptor. Under oxygen limited condition, unusually high redox ratio can be partially ameliorated by the addition of electron acceptor because nitrate is reduced by nitrate reductase with NADH oxidation (39). As expected, the cultivation with nitrate reduced redox ratio in KMK-01, KMK-02, and KMK-05 by 26.1%, 22.7%, and 50.5% respectively. Interestingly, the addition of nitrate caused a growth rate increase only in KMK-05 culture by 37.2%, but not in KMK- 01 and KMK-02 (Table 3). It suggested that the unusually high ratio of NADH/NAD + regenerated by deletion of ldha and pflb was the primary cause of growth defect in KMK-05 under microaerobic condition. In addition, the elevated redox ratio observed by ldha deletion in K. pneumoniae did not seem to be high enough to cause the growth inhibition Transcriptome analysis for pflb-deleted mutant. To understand the global effect of pflb deletion in K. pneumoniae, transcriptome profiling was performed with RNA-seq 14

15 technology. Transcriptome profiles of KMK-02 and KMK-05 were compared in the midexponential growth phase (6 h) at optical densities of 4.3 and 4.1, respectively (Fig. S1). The experiment was repeated three times independently. As shown in Table S2, there were no significant differences in the expression levels of housekeeping genes including gapa, pgi, mdh, and infb in both strains (40). In addition, the expression levels of genes in the 2,3-butanediol pathway operon did not change significantly in response to the deletion of pflb (data not shown). The number of downregulated genes was much higher than that of upregulated genes. The most significantly downregulated genes were those involved in the formate hydrogen lyase (FHL) system (Table S3 and S4). Formate is produced by PflB and oxidized to hydrogen and carbon dioxide by the FHL system, which consists of hydrogenase and specific formate dehydrogenase (FDH H ) (41). The system is activated by the formate hydrogen lyase transcriptional activator (fhla), which is induced in the presence of formate (42). Therefore, a several hundreds to thousands-fold reduction of the gene expression involved in the FHL system was achieved by the removal of pflb. Other pathway genes significantly repressed or induced by pflb mutation were listed in Table S4 and S DISCUSSION With the aim of enhancing the yield of target products, the deletion of byproduct production pathways in host microorganisms has been conducted frequently as a metabolic engineering approach. In many cases, however, the approach caused unexpected problems because of the complex mechanisms in the cell. For instance, 15

16 alcohol dehydrogenase (AdhE) plays a role not only in ethanol formation from acetyl- CoA but also in the alleviation of oxidative stress (43). Therefore, it was reported that the knockout of adhe in E. coli hampered cell growth and caused morphologic defects due to the accumulation of reactive oxygen species. Another example is the removal of phosphate acetyltransferase (pta), which is often selected for disruption of acetate production. Acetyl phosphate, which is known to act as an energy source for transport systems utilizing periplasmic binding protein, can be formed from acetyl-coa by phosphate acetyltransferase and is further converted to acetate via acetate kinase (ack) (Fig. 1) (44). Therefore, pta deletion is known to cause acetate auxotrophs because its mutation halts the production of acetyl phosphate as well as acetate. Indeed, when we constructed adhe- or pta-deficient K. pneumoniae mutants, a significant inhibition of growth was observed, resulting in significantly reduced final 2,3-butanediol titer (data not shown). Thus, the purpose of this study was to find target genes that, when disrupted, would cause little effect on microbial growth while reducing byproduct production to improve 2,3-butanediol yield. During 2,3-butandiol production, K. pneumoniae produces a significant amount of byproducts including ethanol, succinate, formate, acetate, and lactate as byproducts. It was hypothesized that pflb, the gene for PFL, would be a desired gene because various byproducts including ethanol, acetate, and formate are derived from the pathway from pyruvate to acetyl-coa (Fig. 1). As expected, byproduct production of rationally engineered K. pneumoniae ( wabg ldha pflb) was significantly reduced with only a mild reduction in cell mass production. In particular, the 2,3-butanediol yield reached g/g glc, which was 92.2% of the theoretical maximum yield. Another benefit of the 16

17 pflb deletion was a reduction in the speed of the change in ph over time in the culture, which might have been caused by reduction in acid production. According to Biebl et al. (16), the optimum ph value for 2,3-butanediol production by K. pneumoniae was observed when the ph was not controlled but allowed to change gradually from the initial ph value of 7.0 to the final ph value of 5.5. Indeed, the ph change observed with flask cultivation of KMK-05 was almost within the optimal ph condition, whereas the final ph of the other K. pneumoniae mutants was acidified to below ph 5. Thus, the knockout of ldha and pflb in K. pneumoniae provided a number of advantages for the production of 2,3-butanediol. The reduction of growth seen in response to the pflb deletion has been reported previously. Singh et al. (38) constructed the ldha- and pflb-deleted E. coli mutant NZN111 for the overproduction of succinate, and Park et al. (21) reported the deletion of the same genes in Klebsiella oxytoca for 2,3-butanediol overproduction. Both strains resulted in the inhibition of cell growth. Singh et al. (38) suggested that the growth is impaired primarily due to an unusually high redox ratio, whereas Park et al. (21) suggested that it was due to the reduction of the acetyl-coa pool. With the K. pneumoniae and E. coli mutants in this study, a considerable reduction of the acetyl-coa pool was observed by pflb disruption in both strains, but an unchanged growth rate in the E. coli mutant suggested that the limitation of acetyl-coa was not responsible for the growth defect. Meanwhile, the removal of ldha and pflb resulted in unusually high redox ratio (NADH/NAD + ) only in K. pneumoniae mutant and the addition of nitrate improved growth of KMK-05 due to an ameliorated redox ratio. These results suggest that the 17

18 reduction in growth rate seen in K. pneumoniae mutant was caused mainly by ineffective NAD + regeneration under microaerobic condition. The most notable result from the transcriptome analysis was the significant downregulation by pflb inactivation of most of the genes involved in the FHL system. In E. coli, a minimum of 15 genes whose products comprise the FHL system have been identified, and they are organized in two divergently transcribed operons, hyp and hyc (41, 42). The hyc operon comprises genes that function in the synthesis of the redox carriers, and the hydrogenase (hydrogenase 3) and hyp operon genes encode proteins required for the formation of all three hydrogenase isoenzymes. In the absence of external electron acceptors such as oxygen and nitrate, formate is oxidized by FDH H. The expression of the two divergent operons and fdhf, which is a gene coding for the seleno-polypeptide of FDH H, is activated by fhla. In addition, the transcription of fhla and fdhf is activated in the presence of formate. Therefore, the disruption of formate synthesis from pyruvate by the pflb deletion may be the main reason for the significant reduction observed in the expression of genes involved in the FHL complex. Indeed, the expression levels of fhla and the formate dehydrogenase α-subunit (66% protein sequence identity to fdhf in E. coli) in KMK-05 decreased by and fold, respectively, compared to its parental strain. However, the growth of KMK-05 was not restored even when the medium was supplemented with 10 mm of formate (data not shown). These results indicate that either the transcription of genes involved in the FHL system in KMK-05 could not be induced by the addition of formate to the medium or that the inactivation of the FHL system was not directly related to the growth defect; this is currently under investigation in our laboratory. 18

19 399 In summary, a metabolically engineered strain of K. pneumoniae ( wabg ldha 400 pflb) was constructed via minimal gene knockout to reduce virulence and byproduct formation and was able to produce a high 2,3-butanediol yield. The reduction in the growth rate of pflb-deficient K. pneumoniae was mainly due to ineffective NAD + regeneration. In addition, transcriptome analysis revealed the global effects of pflb inactivation on gene expression levels ACKNOWLEDGMENTS This work was supported by the R&D Program of MOTIE/KEIT (No , Development of 2,3-butanediol and derivative production technology for C-Zero bioplatform industry) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2012M1A2A ) REFERENCES Celinska E, Grajek W Biotechnological production of 2,3-butanediol- Current state and prospects. Biotechnol Adv 27: Saxena RK, Anand P, Saran S, Isar J Microbial production of 1,3- propanediol: Recent developments and emerging opportunities. Biotechnol Adv 27: Ji XJ, Huang H, Ouyang PK Microbial 2,3-butanediol production: A state-of-the-art review. Biotechnol Adv 29:

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22 Overexpression of Synthesis-Related Genes. J Microbiol Biotechnol 22: Kim DK, Rathnasingh C, Song H, Lee HJ, Seung D, Chang YK Metabolic engineering of a novel Klebsiella oxytoca strain for enhanced 2,3- butanediol production. J Biosci Bioeng 116: Chen Z, Liu HJ, Liu DH Metabolic pathway analysis of 1,3-propanediol production with a genetically modified Klebsiella pneumoniae by overexpressing an endogenous NADPH-dependent alcohol dehydrogenase. Biochem Eng J 54: Xu YZ, Guo NN, Zheng ZM, Ou XJ, Liu HJ, Liu DH Metabolism in 1,3-Propanediol Fed-Batch Fermentation by a D-Lactate Deficient Mutant of Klebsiella pneumoniae. Biotechnol Bioeng 104: Park JM, Song H, Lee HJ, Seung D In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production. J Ind Microbiol Biotechnol 40: Podschun R, Ullmann U Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 11: Jung SG, Jang JH, Kim AY, Lim MC, Kim B, Lee J, Kim YR Removal of pathogenic factors from 2,3-butanediol-producing Klebsiella species by inactivating virulence-related wabg gene. Appl Microbiol Biotechnol 97:

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26 554 Figure Legends and Tables FIG 1 Fermentation pathways in Klebsiella pneumoniae and the strategies for constructing the 2,3-butanediol producing base strain. The dotted line is the deleted gene in this study FIG 2 Results of flask cultivation with culture medium. Comparison of (A) optical density at 600 nm (OD600), (B) 2,3-butanediol production, and (C) byproduct production by K. pneumoniae mutants. Error bars represent the standard deviation from three independent experiments FIG 3 Results of flask cultivation with minimal medium. Comparison of (A) optical density at 600 nm (OD600), (B) glucose consumption, (C) 2,3-butanediol production, and (D) 2,3-butanediol yield of K. pneumoniae mutants. Error bars represent the standard deviation from three independent experiments FIG 4 Comparison of metabolites and biomass production obtained from (A) E. coli and (B) K. pneumoniae. Symbols:, EMK-01;, EMK-02;, KMK-02;, KMK-05. Error bars 572 represent the standard deviation from three independent experiments FIG 5 Comparison of intracellular acetyl-coa concentration and redox ratio (NADH/NAD + ) obtained from E. coli and K. pneumoniae mutants. Symbols: gray bar, acetyl-coa 26

27 576 concentration; black bar, redox ratio (NADH/NAD + ). Error bars represent the standard 577 deviation from three independent experiments 27

28 578 TABLE 1 Strains and plasmids used in this study. 579 Strains or plasmids Genotype, relevant characteristics, or sequence Source and reference Strains K. pneumoniae KCTC 2242 wild-type K. pneumoniae, Ap r, trpr Korean Collection for Type Culture KMK-01 K. pneumoniae KCTC 2242 wabg This study KMK-02 K. pneumoniae KCTC 2242 wabg ldha This study KMK-05 K. pneumoniae KCTC 2242 wabg ldha pflb This study MG1655 E. coli K-12, F k ilvg negative rfb50 rph1 Coli Genetic Stock Center, Yale University EMK-01 MG1655 ldha::frt This study EMK-02 MG1655 pflb::frt ldha::frt This study Plasmids predet λ phage red γ,β,α-producing vector, pbad_promoter, ori101, tet R Gene Bridges 707-FLP FLP recombinase producing vector, psc101 ori, ci578, Tet R Gene Bridges pkd4 FRT flanked resistance cassette involved vector, orirγ, Km R (27) 28

29 TABLE 2 Comparison of glucose consumption, metabolite yield, 2,3-butanediol production, and ph obtained from K. pneumoniae mutants after 48 h of flask cultivation with minimal medium. Strain name KMK-01 KMK-02 KMK-05 Concentration of initial glucose (g/l) Consumed glucose for 48 h (g/l) ,3-Butanediol yield a (g/g glc) Acetoin yield a (g/g glc) Succinate yield a (g/g glc) Lactate yield a (g/g glc) 3.04 ND b 0.48 Acetate yield a (g/g glc) ND b Ethanol yield a (g/g glc) Pyruvate yield a (g/g glc) ND b ND b 0.76 Initial ph Final ph a Yield calculation in table = metabolite production (g/l)/consumed glucose (g/l) * 100. b ND : Not detect The experiment was repeated three times independently

30 TABLE 3 Comparison of optical density (OD 600) and intracellular concentration of nicotinamide nucleotides in K. pneumoniae mutants by addition of sodium nitrate (NaNO 3) after 6 h of flask cultivation. Nitrate Optical density (OD 600) Intracellular concentration (μmol/ l) NAD + NADH Total concentration (μmol/ l) Redox ratio ( NADH/NAD + ) KMK-01 KMK a a KMK-05 + a a means that culture media contains 20 mm of sodium nitrate (NaNO 3) The experiment was repeated three times independently. 30

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