Appl Microbiol Biotechnol DOI 10.

1007/s00253-009-2222-2

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Engineering Klebsiella oxytoca for efficient 2, 3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene
Xiao-Jun Ji & He Huang & Jian-Guo Zhu & Lu-Jing Ren & Zhi-Kui Nie & Jun Du & Shuang Li

Received: 18 July 2009 / Revised: 25 August 2009 / Accepted: 25 August 2009 # Springer-Verlag 2009

Abstract Ethanol was a major byproduct of 2,3-butanediol (2,3-BD) fermentation by Klebsiella oxytoca ME-UD-3. In order to achieve a high efficiency of 2,3-BD production, K. oxytoca mutants deficient in ethanol formation were successfully constructed by replace the aldA gene coding for aldehyde dehydrogenase with a tetracycline resistance cassette. The results suggested that inactivation of aldA led to a significantly improved 2,3-BD production. The carbon flux to 2,3-BD was enhanced by eliminating the byproducing ethanol and at the same time reducing the accumulation of another byproduct acetoin. At last, by fed-batch culturing of the mutant, the final 2,3-BD titer up to 130 g/l with the productivity of 1.63 g/l.h and the 2,3-BD yield relative to glucose of 0.48 g/g was obtained. Keywords 2,3-butanediol . Klebsiella oxytoca . Aldehyde dehydrogenase . Ethanol . Acetoin

Introduction Development of biorefineries has recently attracted increasing attention as a means to provide sustainable alternative solutions to depleting petroleum resources and environmental pollution. Many chemicals, which could only be produced by chemical processes in the past, could potentially be generated biologically from annually renewX.-J. Ji : H. Huang (*) : J.-G. Zhu : L.-J. Ren : Z.-K. Nie : J. Du : S. Li State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, No. 5 Xinmofan Road, Nanjing 210009, Peoples Republic of China e-mail: biotech@njut.edu.cn

able resources (Ragauskas et al. 2006). Microbial production of 2,3-butanediol (2,3-BD) is one of the examples. Interest in this bioprocess has been increasing recently due to that 2,3-BD has large number of industrial applications and this course would alleviate the dependence on oil supply for the production of platform chemicals. The dehydration of 2,3-BD yields the industrial solvent methyl ethyl ketone (Tran and Chambers 1987). Further dehydration produces 1,3-butadiene, which is the building block of synthetic rubber (van Haveren et al. 2007). And the high octane rating of 2,3-BD makes it a potential aviation fuel (Celiska and Grajek 2009; Wu et al. 2008). Besides, 2,3BD has potential applications in the manufacture of printing inks, perfumes, fumigants, moistening and softening agents, explosives, plasticizers, foods, and pharmaceuticals (Garg and Jain 1995; Syu 2001). 2,3-BD could be produced from carbohydrates by enteric bacteria of the genera Enterobacter and Klebsiella and by bacilli such as Paenibacillus polymyxa via the mixed acidbutanediol fermentation pathway (Kosaric et al. 1992; Syu 2001). In our previous study, a low acid-producing Klebsiella oxytoca ME-UD-3 was obtained by altering the mixed acid fermentation pathway (Ji et al. 2008). However, the strain still relatively highly accumulated ethanol which was another byproduct of 2,3-BD fermentation. The analysis of the metabolic pathways showed that the formation of ethanol competed with the biosynthesis of 2,3-BD for pyruvate and nicotinamide adenine dinucleotide coenzyme (NADH), resulting in a decrease of 2,3-BD yield (Converti et al. 2003). Also, the previous study showed that ethanol had a strong inhibition to the Klebsiella cell growth and thus affected the 2,3-BD production (Cheng et al. 2005; Zeng et al. 1994). Therefore, the straightforward method to improve 2,3-BD yield was to eliminate the biosynthesis of ethanol. In Klebsiella species, biosynthesis of ethanol from

Appl Microbiol Biotechnol

acetyl-CoA was considered as a two-step process (Zeng and Biebl 2002). Acetyl-CoA was converted to acetaldehyde by NAD + -dependent aldehyde dehydrogenase (ALDH, encoded by the gene of aldA) and the acetaldehyde was subsequently converted to ethanol by NAD+-dependent alcohol dehydrogenase (ADH, encoded by the gene of adh). For improving 2,3-BD yield by blocking the formation of ethanol, there are two conceivable approaches including inactivation of ALDH or ADH. Considering that ALDH played a predominant role in ethanol formation, and aldA was shown to be highly expressed in Klebsiella species (Ma et al. 2005), inactivation of ALDH was hypothesized to block ethanol formation effectively. In the present study, aldA of K. oxytoca ME-UD-3 was disrupted by inserting a tetracycline resistance marker, and a mutant deficient in ALDH activity was thus constructed. The fermentation results of both the parent and mutant strain suggested that inactivation of aldA resulted in a nearly abolished ethanol formation, but a significantly improved 2,3-BD production. The final titer, the productivity of 2,3-BD and the yield of 2,3-BD relative to glucose of the mutant were much higher than those of the parent strain.

fermentation was carried out in a 3-l stirred fermenter (BioFlo 100; New Brunswick Scientific Co., NJ, USA) with a working volume of 2 l. All cultivation was carried out at 37 C with the aeration rate and agitation speed at 1.0 vvm and 200 rpm, respectively, and pH was controlled at 6.5 automatically by adding 3 M NaOH. Fed-batch fermentation was conducted by feeding 1000 g/l glucose when the residual glucose in the medium was below 10 g/l. Construction of aldA knock-out mutants Total genomic DNA of K. oxytoca ME-UD-3 cells was extracted with a MiniBEST Bacterial Genomic DNA Extraction kit (TaKaRa, Dalian, China). The aldA of K. oxytoca ME-UD-3 (GenBank accession number GQ359324) was amplified by polymerase chain reaction (PCR) using total genome DNA as template and primers PA1 and PA2, which were designed using the sequence information of aldA from K. pneumoniae MGH 78578 (GenBank accession number CP000647). The PCR mixture consisted of 1 ng genomic DNA, 0.2 mmol each dNTP, 0.2 mol each primer, 2.5 l Ex Taq PCR buffer and 1.5 unit Ex Taq DNA polymerase (TaKaRa, Dalian, China) in a total volume of 25 l. The PCR programs were performed on thermocycler (Biometra, Gttingen, Germany), and consisted of: 95 C for 5 min, followed by 30 cycles at 95 C for 30 s, 62.5 C for 30 s, 72 C for 90 s, as well as final extension step of 72 C for 10 min. The 1.45 kb PCR product was purified and ligated into pMD18-T Simple vector (TaKaRa, Dalian, China) yielding pMDT-aldA for both sequencing and subsequent plasmid construction (Fig. 1A). The tetracycline-resistant gene (Tcr) was amplified by PCR using plasmid pHY300PLK (Makino et al. 1989) as template DNA and primers PT1 and PT2. The PCR mixture consisted of 0.5 ng plasmid pHY300PLK, 0.2 mM each dNTP, 0.2 M each primer, 1.5 unit Ex Taq DNA polymerase, and 2.5 l Ex Taq buffer in a total volume of 25 l. The PCR reaction was carried out at 95 C for 5 min, followed by 30 cycles at 95 C for 30 s, 50.8 C for 45 s, 72 C for 90 s, as well as final extension step of 72 C for 10 min. The 1.56-kb purified PCR product was then inserted into pMDT-aldA using BsrG I and Age I sites that were introduced by primers PT1 and PT2, yielding the plasmid pJXJ-3 (Fig. 1B). The structure of pJXJ-3 extracted from E. coli DH5 was confirmed by restriction analysis (EcoR I+ Hind III, BsrG I+Age I). The inserted Tcr was sequenced by using sequencing primers flanking the Tcr. Sequencing results confirmed that no mutations were introduced. The DNA fragment containing the disrupted aldA was amplified by PCR using pJXJ-3 as template DNA and primers PA1 and PA2. The PCR mixture consisted of 0.5 ng

Materials and methods Strains, plasmids, primers, and culture methods K. oxytoca ME-UD-3, a mutant from K. oxytoca CCTCC M207023 (China Center for Type Culture Collection), was used for the parent strain for 2,3-BD production (Huang et al. 2007, Ji et al. 2008). The other strains, the plasmids and primers used in this study were listed in Table 1. Luria Bertani (LB) medium was used as the seed culture of Escherichia coli, K. oxytoca and their derivatives. When K. oxytoca was used for electroporation, EDTA was added to the LB medium to a final concentration of 0.7 mM (Zhu et al. 2009). After the electroporation, the seed culture for activating the recombinant K. oxytoca was SOC medium (Joseph and David 2001). When necessary, ampicillin (60 g/ml) and/or tetracycline (12 g/ml) was added to either the medium as selection markers. For seed preparation, a full loop of K. oxytoca from a fresh slant tube was inoculated into an Erlenmeyer flask (500 ml) containing 100 ml fresh seed medium and cultivated on a rotary shaker at 200 rpm for 24 h. Seed culture (5%, v/v) was then inoculated into the fermentation medium which consisted of (g/l): glucose, 200; K2HPO4, 13.7; KH2PO4, 2.0; (NH4)2HPO4, 3.3; (NH4)2SO4, 6.6; MgSO47H2O, 0.25; FeSO47H2O, 0.05; ZnSO47H2O, 0.001; MnSO4H2O, 0.001; CaCl22H2O, 0.01; EDTA, 0.05 and pH 6.5 (Ji et al. 2009), Batch and fed-batch

Appl Microbiol Biotechnol Table 1 Bacterial strains, plasmids and primers used in this study Genotype, properties, or sequencea

Strain, plasmid or primer Strains K. oxytoca ME-UD-3 K. oxytoca ME-XJ-8 E. coli DH5

Source or reference

Apr, parent strain Apr, ME-UD-3 aldA::Tcr supE44 lacU169 (80 lacZ M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Apr, Tcr Apr, 2692 bp Apr, pMD18-T-Simple derivative carrying a 1440 bp DNA fragment containing the gene of aldA Apr, Tcr, pMD18-T-aldA derivative, where a 1578 bp DNA fragment containing the gene of Tcr was inserted into aldA 5-GTAGAATTCATGACAGCACCCGTTCAACAC-3 (EcoR I) 5-GTCAAGCTTCAGGCCTGCAGATAGACCACC-3 (Hind III) 5-GCCTGTACACTTAACGATTTAGAAA-3 (BsrG I) 5-GACACCGGTATGAAATACTGAATTT-3 (Age I)

Ji et al. 2008 This work TaKaRa

Plasmids pHY300PLK pMD18-T Simple pMD18-T-aldA Makino et al. 1989 TaKaRa This work

pJXJ-3
b

This work

Primers PA1
a

This work This work This work This work

Apr ampicillin resistance, Tcr tetracycline resistance. The underlined letters indicate the restriction sites.
b

PA2 PT1 PT2

pJXJ-3, 0.2 mmol each dNTP, 0.2 mol each primer, 1.5 unit Ex Taq DNA polymerase, and 2.5 l Ex Taq buffer in a total volume of 25 l. The PCR reaction was carried out at 95 C for 5 min, followed by 30 cycles at 95 C for 30 s,

(A)
pMDT-aldA
4151 bp

EcoR I 427

BsrG I 811 Age I 938

aldA

Hind III 1873

62.5 C for 60 s, 72 C for 90 s, and then a final elongation at 72 C for 10 min. The 2.9-kb PCR product was purified and transformed into the competent cells of K. oxytoca MEUD-3 by standard transformation protocol using the method of electroporation (Fournet-Fayard et al. 1995; Jeong et al. 1998). About 3.0 ml overnight cultured K. oxytoca cells were collected at 12,000 rpm when OD600 =1.01.2, and proper ddH2O (about 200240 l) was added to make the cell concentration 1.0 mg/200 l. Then, the mixtures were treated with 500 ng DNA and about 100 tetracyclineresistant colonies were obtained. Ten of the tetracyclineresistant colonies were subjected to verification of correct recombination and six colonies were proved to be correct. Preparation of cell-free extracts and ALDH activity assays Cells grown under 2,3-BD production conditions were collected and washed twice with 50 ml of 0.85% NaCl solution. Harvested cells were suspended in 10 ml standard buffer (0.1 mol TrisHCl, pH 8.0, 0.1 mmol/l phenylmethanesulfonyl fluoride), and then disrupted by sonication at 4 C. The cell debris was removed by centrifugation at 4 C for 10 min, at 10,000 rpm. The activity measurement of ALDH in cell extracts followed the method described previously with minor modification (Zhang et al. 2006), except that the reaction temperature was 37 C, as K. oxytoca was incubated under this temperature. One unit

EcoR I 427

(B)
pJXJ-3
5595 bp

5'-aldA
BsrG I 811

Tcr

Hind III 3318

3'-aldA

Age I 2371

Fig. 1 Structure of the plasmids constructed in this study. pMDTaldA (A); pJXJ-3 (B)

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bp M 1 2 3 4 5 6 7

5000 3000 2000 1500 1000 750 500 250 100

Fig. 2 Agarose gel electrophoresis of PCR products generated from colonies of K. oxytoca ME-UD-3 and its mutants using primers PA1 and PA2. M, DNA Marker 5.0 kb; 1, ME-UD-3; 2, ME-XJ-1; 3, MEXJ-3; 4, ME-XJ-4; 5, ME-XJ-5; 6, ME-XJ-8; 7, ME-XJ-9

then disrupted by inserting Tcr into the BsrG I and Age I sites of aldA. The restriction sites BsrG I and Age I were located in the 380th bp and the 507th bp of aldA, respectively. After inserting Tcr, the length of aldA fragments flanking the Tcr was 380 bp upstream of the 5Tcr and 935 bp downstream of the Tcr-3. This ensured homologous recombination in K. oxytoca could occur in a relatively high efficiency. The PCR-amplified 2.9-kb DNA fragment, corresponding to aldA-5-Tcr-3-aldA, was transformed into K. oxytoca ME-UD-3. Tetracycline-resistant colonies were screened by colony PCR using primers PA1 and PA2. A 1.45-kb amplicon and a 2.9-kb amplicon represent parent strain and putative mutant, respectively. After screening for tetracycline-resistant colonies followed by colony PCR using primers PA1 and PA2, six putative aldA knock-out mutants, K. oxytoca ME-XJ-1, 3, 4, 5, 8, and 9 were obtained. The PCR product shown in lane 2 to 7 of Fig. 2 was purified and subjected to restriction analysis and sequencing analysis, confirming that Tcr has inserted into aldA of K. oxytoca (data not shown). ALDH activity assays The ALDH activities of the parent strain and six mutants were determined. As shown in Table 2, the ALDH activities of the six mutants were from 2.69 to 7.96% of the parent strain. This result further proved that aldA had been knocked out by insertion, resulting in an inactivation of ALDH. The mutant ME-XJ-8, whose ALDH activity was the lowest, was used in the fermentation after mentioned. Effects of ALDH inactivation on cell growth and metabolic profiles in batch culture Growth of the parent strain ME-UD-3 and the mutant strain ME-XJ-8, was monitored in parallel batch cultures (Fig. 3). The maximum growth yield (3.40 g/l) of the mutant strain
Table 2 ALDH activity of K. oxytoca ME-UD-3 and its mutants Strains ALDH activity U/mg protein ME-UD-3 ME-XJ-1 ME-XJ-3 ME-XJ-4 ME-XJ-5 ME-XJ-8 ME-XJ-9 0.45 0.0272 0.0358 0.0259 0.0235 0.0121 0.0174 Percentage of wild type 100 6.03 7.96 5.76 5.21 2.69 3.87

was defined as the amount of enzyme that catalyzed the formation of 1 pmol NADH per minute. Total protein concentration was determined by means of Bradford (1976) using bovine serum albumin as a standard. Analytical methods Biomass was determined by measuring turbidity at 600 nm with appropriate dilution using a UV-visible spectroscopy system (DU-640, Beckman, USA). Dry cell weight (DCW) was estimated from the absorbance, and one unit of optical density was determined to be equivalent to 0.35 g DCW per liter. The specific cell growth rate (, h1) of K. oxytoca was estimated from the experimental data of DCW using the method indicated in our previous study (Ji et al. 2009). Glucose, 2,3-BD and the two byproducts (acetoin and ethanol) were measured by high-performance liquid chromatography (Summit P 680 HPLC, Dionex, USA; Shodex RI-101 Refractive Index Detector, Showa Denko, Japan; Aminex HPX-87 H Ion Exclusion Column 300 mm 7.8 mm, Bio-Rad, USA) under the following conditions: sample volume 10 l; mobile phase 0.005 M H2SO4; flow rate 0.2 ml/min; column temperature 60 C.

Results Construction and assay of the aldA knock-out mutants Homologous recombination technique was used to inactivate the ALDH in K. oxytoca ME-UD-3. The aldA from K. oxytoca ME-UD-3 was cloned into pMD18-T Simple, and

Strains were grown for 12 h in the 500-ml flasks at 37 C with the agitation speed at 200 rpm.

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h and 0.48 g/g, which were higher than that of the parent strain and close to the maximum theoretical yield of 0.50 g/g (Syu 2001). Production of 2,3-BD by fed-batch culture of K. oxytoca ME-XJ-8 As shown in Fig. 5B, it was observed that the 2,3-BD titer could not increase when the glucose was exhausted. A fedbatch culture by feeding glucose solution was carried out. As shown in Fig. 6, glucose was added to fermentation culture when the substrate was below 10 g/l. After 80 h, the dry cell weight was as high as 6.15 g/l which was almost double the value of batch culture, and the final 2,3-BD titer reached 130 g/l with the yield and productivity up to 0.48 g/g and 1.63 g/l.h respectively. In the process, no ethanol was detected in the final fermentation broth under the fed-batch condition, and the acetoin concentration was only 1.43 g/l.

Fig. 3 Time course of cell growth of the parent strain K. oxytoca MEUD-3 and the mutant strain K. oxytoca ME-XJ-8. The symbols were used: dry cell weight of K. oxytoca ME-UD-3 (filled square), dry cell weight of K. oxytoca ME-XJ-8 (unfilled square), specific cell growth rate of K. oxytoca ME-UD-3 (solid line), and specific cell growth rate of K. oxytoca ME-XJ-8 (dashed line)

ME-XJ-8 was lower than that of the parent strain ME-UD-3 (4.49 g/l). This indicated that the cell growth of K. oxytoca was somewhat impaired due to inactivation of ALDH. However, as shown in Fig. 3, the specific cell growth rate () of the mutant was nearly the same as the parent stain during the batch fermentation. This clearly indicated that ALDH inactivation almost could not affect the specific 2,3BD production rate, as 2,3-BD biosynthesis was a partially growth-associated process (Ji et al. 2009; Qureshi and Cheryan 1989). Furthermore, the ALDH activity of the parent strain ME-UD-3 and the mutant strain ME-XJ-8 was determined during the batch fermentation every 12 h. The specific activity of ALDH of K. oxytoca ME-XJ-8 was around the lowest detection threshold (Fig. 4). This confirmed that aldA in K. oxytoca ME-XJ-8 was disrupted, resulting in an inactivation of ALDH. As shown in Table 3, inactivation of ALDH resulted in a nearly abolished ethanol formation but a significantly improved 2,3-BD production, the 2,3-BD yield of the mutant increased 6.67% and ethanol concentration decreased 92.21% compared with the parent strain. The final titer of 2,3-BD and ethanol were 90.75 and 0.45 g/l, respectively, which were 103.51% and 7.79% of those of the parent strain. Apart from the decreased ethanol titer, the final titer of another byproduct acetoin of the mutant strain ME-XJ-8 was 35.9% of that of the parent strain. The growth yield and glucose consumption of the mutant strain ME-XJ-8 were only 75.72% and 96.47% of those of the parent strain (Table 3). Due to its lower growth yield, the specific 2,3-BD-producing capability (2,3-BD produced per gram of cells) of the mutant strain ME-XJ-8 was 136.66% of that of the parent strain. The productivity of 2,3-BD and the yield of 2,3-BD of the mutant strain reached 1.51 g/l.

Discussion In 2,3-BD fermentation, the ethanol pathway would contest the common precursors pyruvate and NADH with the 2,3BD pathway, and high ethanol yield would certainly reduce the conversion ratio from glucose to 2,3-BD (Converti et al. 2003). Acetoin was not given much importance in former studies maybe because it was not so toxic to the cells and so little acetoin was formed that it could be negligible to 2,3BD yield. However, in this work, acetoin was the main byproduct of K. oxytoca ME-UD-3 apart from ethanol (Fig. 5A). It always reached 3 g/l in the broth and even exceeded 10 g/l in some individual fermentation (data not

Fig. 4 Time course of ALDH activity determination in the parent strain K. oxytoca ME-UD-3 (blank bar) and the mutant strain K. oxytoca ME-XJ-8 (filled bar)

Appl Microbiol Biotechnol Table 3 Comparing of the batch fermentation of the mutant strain K. oxytoca ME-XJ-8 with those of the parent strain K. oxytoca ME-UD-3a Strains Fermentation time (h) Average specific activity of ALDH (U/mg protein) Biomass (g/l) Glucose consumed (g/l) 2,3-Butanediol (g/l) Acetoin (g/l) Ethanol (g/l) Specific 2,3-butanediol producing capability (g/g)b 2,3-Butanediol productivity (g/l.h) 2,3-Butanediol yield (g/g)c
a

K. oxytoca ME-UD-3 60 0.45 4.49 194.69 87.67 2.73 5.78 19.53 1.46 0.45

K. oxytoca ME-XJ-8 60 0.02 3.40 187.82 90.75 0.98 0.45 26.69 1.51 0.48

Ratio of ME-XJ-8 to ME-UD-3 (%) 4.44 75.72 96.47 103.51 35.90 7.79 136.66 103.42 106.67

Batch fermentation was carried out in a 3-l stirred fermenter with a working volume of 2 l at 37 C with the aeration rate and agitation speed at 1.0 vvm and 200 rpm, respectively, and pH was controlled at 6.5 automatically by adding 3 M NaOH. Specific 2,3-butanediol-producing capability was expressed as 2,3-butanediol produced per gram of cells. 2,3-Butanediol yield was expressed as 2,3-butanediol produced per gram of glucose utilized.

b c

shown). Therefore, we had to take action to prevent the byproduct production to ensure high 2,3-BD yield. Moreover, this action will bring facilitation and cost reduction to the downstream processes.

Hereby, in this paper, efficient 2,3-BD production was fulfilled by engineering K. oxytoca through insertional inactivation of aldA. The mutant strain ME-XJ-8 which entirely lost ALDH activity exhibited a significantly improved 2,3-BD production with the yield of 2,3-BD to the consumed glucose up to 96% of the maximum theoretical value, while the two byproducts were at a very low level. This alleviated the multiple inhibition to cell growth and led to the improvement of 2,3-BD production (Table 3). In our previous study, we had successfully reduced the byproducing ethanol and acetoin through two-stage agitation control strategy based on kinetic analysis of the batch culture using the parent strain (Ji et al. 2009). However, the strategy was only suit for the batch process, and it could not be used in the fed-batch process for further improving 2,3BD production. In this study, the intent to obtain high 2,3-

Fig. 5 Metabolite profiles of the parent strain K. oxytoca ME-UD-3 (a) and the mutant strain K. oxytoca ME-XJ-8 (b) in the batch culture. The symbols were used: glucose (filled diamond), 2,3-butanediol (unfilled circle), acetoin (unfilled triangle), and ethanol (unfilled inverted triangle)

Fig. 6 Time course of 2,3-butanediol production of by fed-batch culture of K. oxytoca ME-XJ-8. The symbols were used: dry cell weight (filled square), glucose (filled diamond), 2,3-butanediol (unfilled circle), acetoin (unfilled triangle), and ethanol (unfilled inverted triangle)

Appl Microbiol Biotechnol

BD production through eliminating the two main byproducts was fulfilled by inactivating aldA, and the carbon flux was mainly redirected to 2,3-BD triggered by aldA inactivation. Also, the 2,3-BD production of the mutant strain ME-XJ-8 was superior to the parent strain not only in the batch process but also in the fed-batch process with the two byproducts at a very low level. All the above indicated that the enhancement of 2,3-BD production was at the expense of both ethanol and acetoin production, and these two byproduct elimination was fulfilled only by a solely gene aldA inactivation. This was some different from the previous related reports (Xu et al. 2009; Yang et al. 2007; Zhang et al. 2006; Zhu et al. 2007), in which a gene inactivation only lead to a corresponding metabolite reduction. The results in the present study demonstrated that aldA inactivation could redirect cellular metabolism in K. oxytoca. This might be explained as follows. Firstly, the high yield of 2,3-BD was achieved by eliminating the byproducing ethanol accumulation. Due to aldA inactivation, ALDH which was the upriver enzyme in the ethanol formation pathway lost its activity, and the formation of ethanol was blocked. This would also make the carbon flux from pyruvate to acetyl-CoA eliminate and thus improve the precursor availability of pyruvate for 2,3-BD production. Furthermore, the production of another byproduct acetoin was weakened at the same time. The dramatically decreased formation of ethanol due to aldA inactivation in K. oxytoca would lead to a significantly increased ratio of NADH to NAD+ in vivo. And sufficient NADH supply would drive the reversible reaction from acetoin to 2,3-BD forward for the fulfillment of the redox balance in vivo, and thus the acetoin accumulation decreased with 2,3-BD yield increased (Converti et al. 2003; Ji et al. 2009; Johansen et al. 1975). In conclusion, in the present study, the carbon flux to 2,3-BD was enhanced by eliminating the byproducing ethanol and at the same time reducing the accumulation of another byproduct acetoin. The 2,3-BD yield of the mutant increased by 6.67% with ethanol and acetoin concentration decreased by 92.21% and 64.1% ,respectively, suggesting that aldA insertional inactivation in K. oxytoca was an effective method for improving 2,3-BD production. The elimination of byproducts not only enhanced the efficiency of substrate utilization, but also would bring facilitation and cost reduction to the downstream processes for 2,3-BD production. At last, by fed-batch culturing of the mutant, the final 2,3-BD titer up to 130 g/l with the productivity of 1.63 g/l.h and the 2,3-BD yield relative to glucose of 0.48 g/g was obtained, with the byproducts at a very low level. Up till now, the highest 2,3-BD production up to 150 g/l with a yield of 0.48 g/g was using K. pneumoniae as strain (Ma et al. 2009). However, as for the strain of K. oxytoca, Afschar et al. (1991) had the highest 118 g/l 2,3BD with a yield of 0.42 g/g. To the best of our knowledge,

the result in the present study was a new record on 2,3-BD fermentation by K. oxytoca. Also, this is another example showing the advantage of gene inactivation technology in terms of metabolic pathway analysis to redirect cellular metabolism. The idea developed in this paper could be applied to the other similar industrial biotechnological process to achieve high product concentration.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant No. 20606018), the Key Program of National Natural Science Foundation of China (Grant No. 20936002), the National Basic Research Program of China (Grant No. 2007CB707805), and the National High Technology Research and Development Program of China (Grant No. 2006AA02Z244). X.-J. Ji was supported by the Innovation Fund for Doctoral Dissertation of Nanjing University of Technology (Grant No. BSCX200808).

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