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Enzyme and Microbial Technology 45 (2009) 305–309

Contents lists available at ScienceDirect

Enzyme and Microbial Technology

journa l homepage: www.e lsev ier .com/ locate /emt

egulation of 3-hydroxypropionaldehyde accumulation in Klebsiella pneumoniaey overexpression of dhaT and dhaD genes

hen Chena, Hongjuan Liub,∗, Dehua Liua,∗

Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR ChinaInstitute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, PR China

r t i c l e i n f o

rticle history:eceived 26 March 2009eceived in revised form 22 April 2009ccepted 22 April 2009

eywords:haThaD

a b s t r a c t

3-Hydroxypropionaldehyde (3-HPA), an important intermediary metabolite of 1,3-propanediol (PDO)production, would be toxic to the cell growth and led to the abnormal cessation of the fermentationprocess. In this study, the dhaD gene encoding glycerol dehydrogenase (GDH) and dhaT gene encoding 1,3-propanediol oxidoreductase (PDOR) were overexpressed in Klebsiella pneumoniae ACCC 10082 to decreasethe 3-HPA accumulation and increase the coenzyme NADH supply. By the construction of pTD plasmid,GDH and PDOR were both overexpressed and their enzyme activities were increased by 2.6- and 3.2-fold,respectively. The enzyme activity ratio of PDOR/GDHt (glycerol dehydratase) also was increased. On theother hand, NADH production was enhanced and the ratio of NADH/NAD+ exceeded 1 after the inducement

-Hydroxypropionaldehyde

,3-Propanediollebsiella pneumoniae

of IPTG for the constructed strain. The two factors enhanced the transformation of 3-HPA to PDO. In thebatch and fed-batch fermentation by the constructed strain, the peak of 3-HPA accumulation reducedby 52.2% and 33.3%, respectively, compared with the control. The PDO concentration and yield reached59.2 g/L and 0.48 mol/mol, respectively. Furthermore, the fed-batch fermentation process appeared easierto be regulated. This work is considered helpful for the further understanding on the PDO metabolic

niae a

mechanism of K. pneumo

. Introduction

1,3-Propanediol (PDO) is a promising chemical especially ashe monomer of a novel polymer polytrimethylene terephathalatePTT). For the microbial production of PDO, several microorgan-sms such as Klebsiella pneumoniae [1,2], Clostridium butyricum [3,4]nd Citrobacter freundii [5] can transfer glycerol into PDO. Theechanism for the glycerol conversion involves two branch path-ays: the reductive branch and the oxidative branch (Fig. 1). In the

eductive branch, glycerol dehydratase (GDHt), encoded by threeenes dhaB-alpha, dhaB-beta and dhaB-gamma, catalyzes the reac-ion from glycerol to 3-hydroxypropionaldehyde (3-HPA) and theatter is further reduced to PDO by 1,3-propanediol oxidoreductasePDOR) encoded by the dhaT gene under the consumption of NADH.n the other hand, glycerol is oxidized to dihydroxyacetone (DHA)nd produce NADH by glycerol dehydrogenase (GDH) encoded by

haD gene in the oxidative branch [6,7].

3-HPA is the most important intermediate during the transfor-ation of glycerol to PDO. During the microbial production of PDO,

he accumulation of 3-HPA has been observed at the early phase of

∗ Corresponding authors. Tel.: +86 10 62772130; fax: +86 10 62785475.E-mail addresses: liuhongjuan@tsinghua.edu.cn (H.J. Liu),

hliu@tsinghua.edu.cn (D.H. Liu).

141-0229/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2009.04.005

nd also useful for the PDO fermentation in a large-scale bioreactor.© 2009 Elsevier Inc. All rights reserved.

the fermentation by Enterobacterial agglomerans and K. pneumoniae[8,9]. 3-HPA accumulation can cause an irreversible cessation of thefermentation process, which leads to the serious economic loss inthe large-scale production of PDO. The toxicity of 3-HPA has beendeduced that the reactivity of the aldehyde group of 3-HPA causedDNA damage and inhibit the DNA synthesis [10]. The kinetic andenzymatic analysis of Barbirato’s study suggested that the accu-mulation of 3-HPA was due to the imbalance between conversion ofglycerol to 3-HPA and its further conversion to PDO [11]. It could beexpected to reduce the accumulation of 3-HPA by either decreasingthe conversion rate from glycerol to 3-HPA or increasing the conver-sion rate from 3-HPA to PDO. Since 3-HPA also is an intermediatefor the PDO synthesis, the conversion rate decreased from glycerolto 3-HPA would lead to the lower PDO concentration. So, the latterpathway is a feasible alternative. The conversion of 3-HPA to PDO iscatalyzed by PDOR with the association of coenzyme NADH, there-fore, increasing the activity of PDOR and enhancing the supply ofNADH would enhance the 3-HPA transformation.

In our previous work, the dhaT gene encoding PDOR was indi-vidual overexpressed in K. pneumoniae to reduce the accumulation

of 3-HPA [12]. As a result, the 3-HPA accumulation decreased tosome extent. However, the cell growth rate of the constructed strainwas much slower compared to the wild type strain and the finalPDO concentration decreased, especially in the fed-batch fermenta-tion. The shortage of NADH was assumed one of the reasons, which

306 Z. Chen et al. / Enzyme and Microbial Technology 45 (2009) 305–309

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ig. 1. The metabolic pathway for the conversion of glycerol to PDO. Abbreviations:DO, 1,3-propanediol; 3-HPA, 3-hydroxypropionaldehyde; DHA, dihydroxyacetone;DHt, glycerol dehydratase; GDH, glycerol dehydrogenase; PDOR, 1,3-propanediolxidoreductase.

ffected the cell growth and the PDO production. To overcome theseroblems, in this study, the dhaD gene encoding GDH and dhaTene encoding PDOR were both overexpressed in K. pneumoniaeCCC 10082 to decrease the 3-HPA accumulation and supply moreADH for the cell growth and PDO production at the same time.he associated dynamic enzyme activity and the NADH synthesisere analyzed. The batch and fed-batch fermentation of PDO for

he wild type and the constructed strain also were compared. Sincet is very important to reduce the 3-HPA accumulation and theneduce the risk of abnormal cessation in the large-scale produc-ion of PDO. This work is considered useful for the further strainmprovement and metabolic regulation in the PDO production by. pneumoniae.

. Materials and methods

.1. Strains, plasmids and culture media

Strains and plasmids used in this study were shown in Table 1. LB medium wassed as a rich medium for the routine growth of K. pneumoniae. The compositionf the seed and fermentation media was described as Zheng et al. [13]. The initiallycerol concentration for the batch and fed-batch fermentation was 50 and 30 g/L,espectively.

For the batch and fed-batch fermentation of constructed K. pneumoniae/pTD,0 �g/mL kanamycin (km) was added in the culture media. 0.2 mmol/L isopropyl--d-thiogalactoside (IPTG) was added to the media after 4 and 8 h in batch and

ed-batch fermentation, respectively.

.2. Cultivation conditions

The seeds were grown in a 500-mL shake flask containing 100 mL media at 37 ◦Cnd 120 rpm for 12 h. The batch and fed-batch cultivation were carried out in a 5 Ltirring bioreactor (B. Braun Biotech International) with a working volume of 4 L.emperature, pH and agitation speed were maintained at 37 ◦C, 6.8 and 250 rpm,espectively. The pH was controlled by adding a solution of 50% (mass fraction)aOH. The inoculated volume was 10% (v v−1). An aerobic environment in the biore-ctor was controlled by aerating 0.5 vvm air.

.3. Plasmid pTD construction

dhaT gene was amplified from the genomic DNA of K. pneumoniae ACCC0082 by PCR using the forward primer (5′-CCGGAATTCATGAGCTATCGTATGTTTG-′) and reverse primer (5′-CGGGATCCTCAGAATGCCTGGCGGAAAATC-3′) withcoRI and BamHI restriction sites, respectively. dhaD gene also was ampli-ed by using forward (5′-CGGGATCCATGCGCACTTATTTGAG-3′) and reverse5′-CCCAAGCTTTTAACGCGCCAGCCACTGCTG-3′) primers with the restriction sites ofamHI and HimdIII. The plasmid pTD was constructed with a kanamycin resistanceector pDK6 as Fig. 2.

.4. K. pneumoniae/pTD construction and gene expression

The recombinant strain K. pneumoniae/pTD was cultivated in LB media andermentation medium, respectively, in shaker flask at 37 ◦C and 150 rpm. After0 h, 1 mmol/L isopropyl-�-d-thiogalactoside (IPTG) was added to culture media

Table 1Strains and plasmids used in this work.

Strains or plasmids Genotype/phenotype

K. pneumoniae ACCC 10082 Wild type, Ampr

K. pneumoniae/pTD Contructed, Ampr, KmpDK6 Expressing vector, KmpTD pDK6 containing dhaT

Fig. 2. Construction of plasmid pTD.

to induce the gene expression and the wild type K. pneumoniae ACCC 10082 was asthe control.

2.5. Analytical method

The cell concentration was measured as absorbance at 650 nm using a BeckmanDU640 UV/vis spectrophotometer. Glycerol, PDO, ethanol, acetate, lactate, succi-nate, and 2,3-butanediol were determined by HPLC with a refractive index detector(Shimadzu 10AVP HPLC system, Shimadzu, Japan). An Aminex HPX-87H column(300 mm × 7.8 mm) (Bio-Rad, USA) was used with 5 mM H2SO4 as mobile phaseat 0.8 mL min−1. The column was controlled at 65 ◦C [13,14]. The concentration of3-HPA was measured according to the method described by Cirde et al. [15].

2.6. Enzyme assays

The enzyme assays of PDOR, GDH and GDHt were carried out according to themethods described by Forage and Lin [16] and Ahrens et al. [17]. Protein concentra-tion was determined according to Bradford’s method [18].

One unit of enzyme activity is defined as the amount of enzyme required toreduce 1 �mol of substrated per minute.

2.7. Coenzyme assays

The coenzyme assay of NADH and NAD+ were performed as the method ofBernofsky and Swan [19].

3. Results and discussion

3.1. Gene expression of dhaT and dhaD

dhaT and dhaD gene of K. pneumoniae ACCC 10082 were clonedand sequenced. The open reading frames (ORF) of dhaT gene anddhaD gene were 1164 and 1125 bp, respectively. The two genes wereblasted in NCBI and the highest homologous gene was dhaT geneand dha D gene from K. pneumoniae MGH 78578, respectively. Theconstructed K. pneumoniae/pTD was cultivated in LB media and fer-

mentation medium in shake flasks to detect the gene expression ofdhaT and dhaD, respectively. The wild type K. pneumoniae was asthe control. The results were shown in Table 2.

In LB media, almost no activity of PDOR and GDH were detectedin K. pneumoniae ACCC10082, while it reached 0.362 and 1.156 U/mg

Reference

Tsinghua University, Chinar This workr, 5.1kb Kleiner et al. [12]and dhaD genes This work

Z. Chen et al. / Enzyme and Microbial Technology 45 (2009) 305–309 307

Table 2Fermentation of wild type K. pneumoniae and constructed K. pneumoniae/pTD in shake flask.

Strains PDOR (U/mg) GDH (U/mg)

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LB medium Ferme

. pneumoniae ACCC 10082 0.024 ± 0.014 0.253

. pneumoniae/pTD 0.362 ± 0.045 0.911

n K. pneumoniae/pTD, respectively. In the fermentation media withlycerol as sole carbon source, the specific activities of PDOR andDH in the constructed K. pneumoniae/pTD increased by 3.2- and.6-fold higher than that of the control, respectively. The enzymessay indicated that the dhaT and dhaD genes were successfullyverexpressed in the constructed strain K. pneumoniae/pTD in bothB and fermentation media. For K. pneumoniae ACCC 10082, theative dhaT and dhaD genes located in dha regulon need the induce-ent of glycerol or DHA for transcription [6], thus, almost no

nzymatic activities of PDOR and GDH were detected in LB medium,hile it was detected higher in the fermentation medium. However,

or the constructed K. pneumoniae/pTD, the dhaT and dhaD genexpression were induced by IPTG and it resulted in the increasedctivity of PDOR and GDH in LB medium.

.2. Batch fermentation in 5 L bioreactors

Batch fermentation of PDO by the constructed K. pneumo-iae/pTD was performed in 5 L bioreactors and the wild type K.neumoniae ACCC 10082 was used as the control. The results werehown in Fig. 3.

As indicated in Fig. 3, the accumulation rate of 3-HPA for the con-rol was higher and the 3-HPA concentration reached the peak value

f 11.43 mmol/L at 12 h, which led to the cell growth ceasing, thelycerol consumption and PDO production stopping. The maximumDO concentration just reached 15.2 g/L. It has been reported that-HPA with the concentration above 10 mmol/L would inhibit theell growth [13]. DNA synthesis can be inhibited by the reactivity of

ig. 3. Batch fermentation of the wild type and constructed strains in 5 L bioreactors. Symbo

n medium LB medium Fermentation medium

5 0.042 ± 0.022 0.885 ± 0.1112 1.156 ± 0.143 3.719 ± 0.322

the aldehyde group of 3-HPA [10]. Compared with the control, the3-HPA accumulation obviously declined and the maximum concen-tration of 3-HPA was only 5.46 mmol/L which decreased by 52.2%for the constructed K. pneumoniae/pTD. Although the specific cellgrowth rate and glycerol consumption rate of K. pneumoniae/pTDwere lower at the initial 8 h, the glycerol can be completely con-sumed and the final concentration of PDO reached 23.6 g/L whichincreased by 56.3% compared with that of the control. The resultsindicated that the 3-HPA accumulation was successfully decreasedand the risk of fermentation cease has been reduced at the sametime by overexpression of PDOR and GDH.

3.3. Dynamic enzyme and coenzyme analysis

Since 3-HPA synthesis and transformation to PDO was cat-alyzed by GDHt and PDOR, respectively, the activity ratio of thesetwo enzymes appeared more important to the 3-HPA accumula-tion and PDO production. The associated dynamic enzyme GDH,GDHt and PDOR were analyzed (Fig. 4). As a result of overex-pression, the enzyme activities of PDOR and GDH were increasedby 2.6- and 3.2-fold, respectively, under the inducement of IPTGafter the inducement of IPTG for the constructed strain. For thecontrol, the enzyme activity ratio of PDOR/GDHt was less than 1

during the whole process. However, the ratio increased after theinducement of IPTG and above than 1 after 6 h for the constructedstrain, which enhanced the transformation of 3-HPA to PDO. So,the lower 3-HPA concentration and higher PDO concentration wereobtained.

ls: (�) K.p (wild type K. pneumoniae); (�) K.p/pTD (constructed K. pneumoniae/pTD).

308 Z. Chen et al. / Enzyme and Microbial Technology 45 (2009) 305–309

Fig. 4. Dynamic enzyme activities of PDOR, GDH and GDHt in the batch fermentation by wild type K. pneumoniae (a) and constructed K. pneumoniae/pTD (b). Symbols: (�)GDH; (�) GDHt; (�) PDOR.

Fig. 5. Dynamic concentration of NADH and NAD+ in the batch fermentation by wild type K. pneumoniae (a) and constructed K. pneumoniae/pTD (b). Symbols: (�) NADH; (�)NAD+; (�) NADH/NAD+.

Fig. 6. Fed-batch fermentation of the wild type K. pneumoniae (a and c) and the constructed K. pneumoniae/pTD (b and d) in 5 L bioreactors. Abbreviations: SUC, succinate;LAC, lactate; ETH, ethanol; ACE, acetate; BDO, 2,3-butanol. Symbols: (�) glycerol; (�) PDO; (�) OD; (�) 3-HPA; (♦) SUC; (�) LAC; (�) ACE; (×) BDO; (ж) ETH.

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On the other hand, the overexpression of dhaD gene resultedn the increase of the concentration ratio of NADH/NAD+ (Fig. 5),ecause GDH catalyzes the reaction from glycerol to DHA whichroduces the largest proportion of NADH in the whole network of. pneumoniae. From Fig. 5 we can see that the ratio of NADH/NAD+

eached 1 at 4 h and then began to decline keeping lower than 1uring the fermentation process for the control. However, the ratiof NADH/NAD+ increased after the inducement of ITPG at 4 h andept higher than 1 until 12 h of the fermentation for the constructedtrain. Since NADH was essential for the conversion of 3-HPA to PDO,he increased NADH concentration also enhanced the transforma-ion of 3-HPA to PDO.

.4. Fed-batch fermentation in 5 L bioreactors

Based on the above results, the further study of fed-batch fer-entation was performed in 5 L bioreactors. The results were

hown in Fig. 6. From Fig. 6 we can see that the accumulation of-HPA for the constructed K. pneumoniae/pTD was obviously loweruring all the fermentation process and the maxium concentrationecreased 33.3% compared with the control (wild type K. pneumo-iae). However, the PDO synthesis was not affected and the finalDO concentration reached 59.2 g/L at 48 h, which was almost theame with that of the control (58.8 g/L). The final acetate concen-ration decreased by 74.6% while the ethanol and 2,3-butanediolncreased by 36.8% and 18.2%, respectively, compared with theontrol. Actually, during the fermentation process, the regulationf the glycerol feeding rate was very important. For wild type. pneumoniae, the glycerol concentration should be controlledtrictly under 20 g/L during the exponential phase of cell growtho avoid the abnormal cessation of fermentation caused by the-HPA accumulation, which made the regulation of fed-batch pro-ess very complicated. The glycerol feeding rate should be adjustedonstantly according to the glycerol consumption by the cell. How-ver, for the constructed K. pneumoniae/pTD, it would not resultn the cessation of fermentation even that the glycerol concen-ration exceeded 50 g/L (data not shown). That is to say the PDOermentation by the constructed K. pneumoniae/pTD was easier toontrol for the practical fermentation application. On the otherand, the more rational regulation strategies for the constructed. pneumoniae/pTD could be designed for the further fermentationptimization, such as increasing the initial glycerol concentrationr keeping a higher glycerol concentration in the broth during theed-batch fermentation.

. Conclusion

The toxicity of 3-HPA has been confirmed as the main reason ofhe abnormal cessation of PDO fermentation. This work enhancedhe transformation of 3-HPA to PDO. Not only the enzyme activityatio of PDOR/GDHt was increased, but also the NADH synthesisas enhanced. The above two factors led to the obviously decrease

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Technology 45 (2009) 305–309 309

of 3-HPA accumulation and increase of NADH production. By theconstructed K. pneumoniae/pTD, the abnormal cessation of PDOfermentation with the initial glycerol concentration of 50 g/L wasavoided and the easier regulation process was achieved in thefed-batch fermentation. The results are considered useful for fur-ther investigation on the PDO metabolism of K. pneumoniae. Theinformation also is beneficial to establish a rational strategy forefficient production of PDO by K. pneumoniae in a large-scale biore-actor.

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