Enhanced production of dihydroxyacetone from glycerol by overexpression

Enhanced production of dihydroxyacetone from glycerol by overexpression of glycerol dehydrogenase in an alcohol dehydrogenase-de?cient mutant of Gluconobacter oxydans

Ming-hua Li,Jian Wu,Xu Liu,Jin-ping Lin *,Dong-zhi Wei **,Hao Chen

State Key Laboratory of Bioreactor Engineering,Institute of Newworld Biotechnology,East China University of Science and Technology,Shanghai 200237,China

a r t i c l e i n f o Article history:

Received 1December 2009

Received in revised form 11May 2010Accepted 21May 2010

Available online 23June 2010Keywords:

Gluconobacter oxydans Glycerol dehydrogenase Dihydroxyacetone Biotransformation Glycerol

a b s t r a c t

Gluconobacter oxydans can rapidly and incompletely oxidize glycerol to dihydroxyacetone (DHA),a ver-satile product extensively used in cosmetic,chemical and pharmaceutical industries.To improve DHA production,the glycerol dehydrogenase (GDH)responsible for DHA formation was overexpressed in G.oxydans M5AM,in which the gene coding for the membrane-bound alcohol dehydrogenase (ADH)was interrupted.Real-time PCR and enzyme activity assay revealed that the absence of ADH together with the overexpression of GDH gene resulted in an increased GDH activity in the resulting strain M5AM/GDH,which led to a substantially enhanced production of DHA in a resting cell system.In a batch bio-transformation process,M5AM/GDH exhibited a 2.4-fold increased DHA productivity of 2.4g/g CDW/h from 1.0g/g CDW/h,yielding 96g/L DHA from 100g/L glycerol.When 140g/L glycerol was supplied,a ?nal DHA concentration of 134g/L was accumulated within 14h.In four repeated batch runs,385g DHA over a time period of 34h was achieved from 400g glycerol with an average productivity of 2.2g/g CDW/h.These results indicated that this newly developed strain G.oxydans M5AM/GDH with high productivity and increased tolerance against product inhibition has potential for DHA production in an industrial bioconversion process.

ó2010Elsevier Ltd.All rights reserved.

1.Introduction

As a by-product of biodiesel production,glycerol has become a relatively cheap and readily available commodity for which new used have to be explored.Conversion into higher-value products such as 1,3-propanediol,2,3-butanediol,succinic acid,citric acid,ethanol,3-hydroxypropionic acid and glyceric acid by microbial fermentation has already been explored (Zhao et al.,2006;Zhang et al.,2010;Imandi et al.,2007;Easterling et al.,2009;Mohan Raj et al.,2009;Habe et al.,2009),and microbial production of dihydroxyacetone (DHA)has also been described (Mishra et al.,2008).

DHA,which serves as a sunless tanning agent,a precursor of pharmaceuticals and a building block for the synthesis of various ?ne chemicals (Hekmat et al.,2003;Mishra et al.,2008),is cur-rently being produced via incomplete oxidation of glycerol by Gluconobacter oxydans in a fermentation process (Claret et al.,1994;Hekmat et al.,2003;Bauer et al.,2005;G?tgens et al.,2007).The oxidative reaction is catalyzed by the membrane-bound,pyrroloquinoline quinone (PQQ)-dependent glycerol dehy-drogenase (GDH)which transfers electrons from glycerol to the ?-nal acceptor,oxygen,via an electron transport chain without NADH involvement (Claret et al.,1994;Shinjon et al.,2002).Since the active site of the GDH is oriented towards the periplasm,glyc-erol can be oxidized in the periplasmic space without the need to enter the cytoplasm (Matsushita et al.,1994),which facilitates re-lease and accumulation of DHA in the medium.

Attempts at increasing DHA yield have been made by improving oxygen supply (Hoist et al.,1985;Adlercreutz and Mattiasson,1982;Leung et al.,1997),optimizing media components (Wethmar and Deckwer,1999;Wei et al.,2007a;Albin et al.,2007;Wei et al.,2009)and modifying fermentation patterns (Hekmat et al.,2003;Bauer et al.,2005).In addition,G.oxydans has been genetically modi?ed to enhance production of DHA (G?tgens et al.,2007).So far,the highest DHA production of 220g/L was achieved in a two-stage repeated fed-batch fermentation process which lasted as long as 77h and gave a productivity of 2.9g/L h (Bauer et al.,2005).

Recently,Wei (2008)showed that alcohol dehydrogenase (ADH)and acetaldehyde dehydrogenase (ALDH)mutants of G.oxy-dans M5displayed higher DHA production from glycerol than the wild type.A similar observation was made with a gluconate-2-dehydrogenase (GA2DH)mutant of G.oxydans DSM 2343in shake ?ask culture (G?tgens et al.,2007).

0960-8524/$-see front matter ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2010.05.065

*Corresponding author.Tel.:+862164252981;fax:+862164250068.**Corresponding author.Tel.:+862164252981;fax:+862164250068.

E-mail addresses:jplin@1fe2215bbe23482fb4da4c39 (J.-p.Lin),dzhwei@1fe2215bbe23482fb4da4c39 (D.-z.Wei).

In the current study,glycerol dehydrogenase(GDH)was over-expressed in the ADH-de?cient mutant,G.oxydans M5AM,(Wei, 2008)and DHA production of this recombinant strain was deter-mined in a resting cell system.

2.Methods

2.1.Strains,plasmids,and culture conditions

Escherichia coli DH5a(Hanahan,1983)was used as a host for plasmid construction and maintenance.E.coli HB101(Boyer and Roulland-Dussoix,1969)harboring helper plasmid pRK2013(Fig-urski and Helinski,1979)was used for transformation experi-ments.G.oxydans M5(Yang et al.,2008)and G.oxydans M5AM were used for GDH expression and DHA production.The broad host range vector pBBR1MCS-5(Kovach et al.,1995)was used for expression of GDH in G.oxydans.

All E.coli strains were cultivated in Luria–Bertani(LB)medium (yeast extract,5g/L;tryptone,10g/L;NaCl,10g/L;pH7.0)at37°C with the addition of10l g/mL gentamicin or50l g/mL kanamycin to select for the presence of plasmids when necessary.Recombi-nant G.oxydans strains were cultivated at30°C in sorbitol medium (sorbitol,80g/L;yeast extract,20g/L;(NH4)2SO4,5g/L;KH2PO4,

1.5g/L;MgSO4á7H2O,0.5g/L)containing50l g/mL gentamicin.

2.2.Vector construction

General molecular biological techniques were used for the con-struction,puri?cation,and analysis of plasmid DNA and for the transformation of E.coli(Sambrook and Russell,2000).Based on the available sequence information for G.oxydans ATCC621H(Prust et al.,2005),oligonucleotide primers were designed to amplify the GDH gene by PCR.The GDH gene was ampli?ed with LA Taq poly-merase(TaKaRa,Dalian)from genomic DNA of G.oxydans M5,using oligonucleotides(50-CATAAGCTTGGCGTTTACGATGGTGC-30)as a forward primer and(50-CTAGGTACCGTGAACGAAAACAGCCTG-30) as a reverse primer.The resulting fragment was digested with restriction enzymes Hin d III and Kpn I(MBI Fermentas)and ligated into the prepared vector pBBR1MCS-5using T4DNA ligase(MBI Fer-mentas),generating plasmid pBBR-GDH.The recombinant plasmid was transformed into competent E.coli DH5a.Transformants were selected on LB agar plates containing10l g/mL gentamicin,and fur-ther con?rmed by colony PCR and restriction enzyme digestions.

2.3.Conjugational plasmid transfer into G.oxydans

The expression vector pBBR-GDH was transferred into G.oxy-dans M5and G.oxydans M5AM by triparental mating as described previously(H?lscher and G?risch,2006).E.coli DH5a bearing the expression vector pBBR-GDH and E.coli HB101harboring the mobilizing plasmid pRK2013were used as the donor and helper strain,respectively.The three strains were grown to late exponen-tial phase,pelleted,washed and resuspended in sorbitol medium, then mixed at a1:1:1ratio.100l L of this mixture was spreaded onto sorbitol agar plates without antibiotics and incubated over-night at30°C.The cultures were then scraped from the plates and spread onto selective sorbitol agar plates containing cefoxitin (50l g/mL)and gentamicin(50l g/mL).Plates were incubated at 30°C for2–4days until cefoxitin-and gentamicin-resistant colo-nies appeared.Because the recombinant G.oxydans strains exhib-ited a slightly lower cell growth rate than the wild strain in gentamicin-containing medium,we transformed the plasmid pBBR1MCS-5into G.oxydans M5and G.oxydans M5AM to generate M5/pBBR and M5AM/pBBR which were used as control strains dur-ing the experiments.2.4.Preparation of membrane fractions

To prepare membrane fractions,single colonies of the G.oxy-dans strains were inoculated into20mL sorbitol medium and grown at30°C and200rpm for20h on a rotary shaker.Then1% (v/v)of the precultures were inoculated into1000-mL shake?asks containing200mL sorbitol medium and cultivated under the same conditions for24h.The G.oxydans cells were harvested by centri-fugation(8000g,10min,4°C)and then resuspended in50mM so-dium phosphate buffer(pH6)at a cell wet weight(CWW) concentration of0.1g/mL.Cell disruption was carried out with an ultrasoni?er(JY92–2D,Xinzhi,Ningbo)for50cycles(3s sonica-tion,5s pause on ice).After centrifugation at10,000g for10min to remove the cell debris,the resulting supernatants were centrifuged at100,000g and4°C for60min.The sediments were collected and designated the membrane fractions,which were resuspended into the above sodium phosphate buffer on ice subsequently.

2.5.Enzyme activity assay and protein determination

The activity of membrane-bound GDH was determined at30°C by measuring the initial reduction rate of2,6-dichlorophenolindo-phenol(2,6-DCIP,Sigma)at600nm as described by Sugisawa and Hoshino(2002).The basal reaction mixture consisted of50mM so-dium phosphate buffer(pH6),0.25mM DCIP and0.325mM phen-azine methosulphate(PMS,Sigma),which was prepared just before the assay.A cuvette with1cm light path containing0.8mL basal reaction mixture and10l L diluted enzyme was incubated at 30°C for5min.The reaction was started by adding200l L of 0.2M pre-warmed glycerol.One unit of GDH activity was de?ned as the amount of the enzyme that catalyzes the reduction of 1l M of DCIP per min at30°C.Enzyme activity was calculated using the molar extinction coef?cient of DCIP,which was9.45, 10.8,13.0,and15.0mMà1cmà1at pH5.5,6.0,6.5,and7.0–7.6, respectively.Protein concentration was determined by the method of Bradford(1976),using bovine serum albumin as a standard.

2.6.Quantitative real-time PCR

For real-time reverse transcription PCR(RT-PCR)experiments, 1%(v/v)of the precultures of the G.oxydans strains described in Sec-tion2.4were inoculated into250-mL shake?asks containing50mL sorbitol medium and grown to their late exponential phase(16h)at 30°C and200rpm on a rotary shaker.Then the cells were harvested by centrifugation at10,000g and4°C for1min for total RNA isola-tion.Total RNA was extracted by RNAiso reagent(TaKaRa,Dalian) and then treated with DNase I(TaKaRa,Dalian).Each RNA sample was quanti?ed using NanoDrop2000spectrophotometer(Thermo Scienti?c)and adjusted to the same concentration based on the absorbance value.For transcriptional analysis of GDH gene,RT-PCR was carried out with a?rst-strand cDNA synthesis kit(Prome-ga,USA),using total RNA as template.Quantitative gene expression analysis was performed by quantitative real-time PCR,which was performed with the StepOnePlus?Real-Time PCR System(Applied Biosystems,USA)using primers50-CCTGCGTAGCCCTGAAGAAAAC-30and50-CGAGCCGATGTCATAGTCCC-30.All measurements were done in two independent experiments.The16S rRNA gene was used as internal standard,which was obtained based on the primers 50-GCGGTTGTTACAGTCAGATG-30and50-GCCTCAGCGTCAGTATCG-30.

2.7.Preparation of resting cells

Cultivations of G.oxydans were performed either in1000-mL shake?asks containing150mL sorbitol medium at30°C and 200rpm on a rotary shaker,or in a5-L fermentor(Baoxing Biotech

M.-h.Li et al./Bioresource Technology101(2010)8294–82998295

Ltd.,Shanghai)containing 2.5L sorbitol medium with the pH and temperature maintained at 6.0and 30°C automatically.Agitation and aeration were controlled at 900rpm and 2vvm,respectively.Cell cultivations were initiated by inoculating 1%(v/v)of 20-h pre-cultures of G.oxydans strains.After 24h,the cells reached early stationary phase and were harvested by centrifugation at 8000g and 4°C for 10min,washed twice with 50mM sodium phosphate buffer (pH 6),and resuspended in reaction solution at a ?nal con-centration of 20g CWW/L [corresponding to 5.05g/L cell dry weight (CDW)]to start the biotransformation of glycerol to DHA.2.8.Biotransformation of glycerol to DHA

Biotransformation of glycerol to DHA was performed in shake ?asks and a 5-L fermentor.For shake-?ask experiments,resting cells were resuspended in reaction solution containing 200mM so-dium phosphate buffer (pH 6),40g/L glycerol,and incubated at 30°C and 250rpm on a rotary shaker.To further investigate the DHA production of different strains,experiments in a 5-L fermen-tor with a working volume of 2.5L were performed at 30°C and 900rpm.The reaction solution consisted of 100–180g/L glycerol dissolved in distilled water and the reaction was started by adding the resting cells into the fermentor.Dissolved oxygen (DO)was provided by injecting ?ltered air at a ?ow of 5L/min and the pH was controlled at 6.0by automatic addition of an aqueous solution of 2.0M NaOH.Repeated-batch biotransformation was also carried out in a 5-L fermentor supplied with 100g/L glycerol under the above conditions.When the reaction reached equilibrium,the cells were recovered by centrifugation and resuspended in fresh reac-tion buffer for next run of bioconversion.The composition of the reaction buffer was the same as that used in the ?rst cycle.Samples were collected at regular intervals for concentration determination of glycerol and DHA.

to 2.2U/mg protein was observed.When the GDH-expressing plas-mid pBBR-GDH was introduced into M5AM,however,the resultant strain M5AM/GDH displayed a signi?cant increase in GDH activity which was 3.0U/mg protein,57.9%and 76.5%more than those of M5AM/pBBR and M5/pBBR.These results implied that there were synergistic effects between the ADH absence and GDH overexpres-sion on the improvement of GDH activity.Similar effects were ob-served in GA2DH disruptant G.oxydans MF1when the gene coding for glucose dehydrogenase or gluconate-5-dehydrogenase was overexpressed (Merfort et al.,2006).

3.2.Real-time RT-PCR analysis of GDH gene transcription in different G.oxydans strains

To demonstrate the expression of GDH gene in different G.oxy-dans strains,detection of the mRNA was carried out with real-time RT-PCR.The absence of ADH showed no effects on cell growth of G.oxydans ,as well as the overexpression of GDH gene (Fig.1).As gene expression often varied signi?cantly in different growth phases (Quintero et al.,2009),all the cells used for RNA isolation were cul-tured for constant period to minimize its effect on RT-PCR analysis.Taking 16S rRNA as the internal standard,it was clearly observed that the transcription level of GDH gene in M5AM/pBBR was much higher than that of the control strain M5/pBBR (Fig.1).The overex-pression of GDH exhibited a similar improvement in GDH gene transcription to that of ADH disruption.In M5AM/GDH,the tran-script of GDH gene was about 120times more abundant than that in M5/pBBR,which was due to both the ADH disruption and GDH overexpression.However,the increase in GDH activity was much less distinct in contrast to the transcript level (Table 1).3.3.Biotransformation of glycerol to DHA in shake ?asks

As the GDH activity was increased when the GDH gene was 8296M.-h.Li et al./Bioresource Technology 101(2010)8294–8299

constant thereafter.Results also indicated that the amounts of accumulated by the four tested strains were in good agreement the results obtained from the GDH activity measurements.

disruption of the GDH gene in G.oxydans M5increased production to some extent,as observed in the GA2DH disruptant oxydans MF1which displayed an enhanced production of compared to its parental strain DSM2343(G?tgens et al.,2007). sides,the overexpression of GDH gene also improved the oxidation glycerol to DHA in M5/GDH,leading to a slight increase in production compared with M5/pBBR.However,when the was overexpressed in M5AM,the resulting strain M5AM/ produced the highest level of DHA which reached up

g/L after24h,42.2%and60.4%more than those of M5AM/ pBBR(18.5g/L)and M5/pBBR(16.4g/L),respectively.Therefore, was the absence of ADH gene and the GDH overexpression together resulted in the distinct improvement of DHA in M5AM/ GDH.

Biotransformation of glycerol to DHA in a5-L fermentor

To assess the potential use of M5AM/GDH for industrial produc-of DHA,scaled-up biotransformations were performed

pH-controlled conversion process in combination with a continu-high oxygen supply.As can be seen in Fig.3,glycerol oxidation started immediately at a high rate whereas the DO concentration

dropped sharply from100%air saturation to less than23%when the cells were added to the fermentor.For M5AM/GDH,there was only10g/L glycerol left in the reaction solution and88g/L DHA was produced after6h of biotransformation.After a further 2h of bioconversion,all of the supplied glycerol(100g/L)was ex-hausted and96g/L DHA was obtained,giving a yield of96% (Fig.3a).In contrast to M5AM/GDH,M5/pBBR showed a much slower bioconversion rate when incubated in the same reaction solution.As can be calculated from the time course of glycerol oxi-dation,the DHA productivity of M5AM/GDH was signi?cantly in-creased to2.4g/g CDW/h,whereas it was only1.0g/g CDW/h for M5/pBBR.Thus,overexpression of GDH in M5AM accelerated the oxidative reaction and resulted in an increase in DHA productivity of about140%compared with M5/pBBR.In addition,the productiv-ity achieved by M5AM/GDH cells was much higher than that by immobilized cells(Wei et al.,2007b)and by growing cells in a batch and fed-batch fermentation process(Bories et al.,1991).

Taking into account its high productivity and yield of DHA, M5AM/GDH was used for further batch biotransformation experi-ments with initial glycerol concentrations of140and180g/L.As shown in Fig.3b,140g/L glycerol was completely exhausted and 134g/L DHA was accumulated by M5AM/GDH within14h,giving a yield of96%and a productivity of1.9g/g CDW/h.In a previous study,G.oxydans cells were able to tolerate DHA concentrations in the range of80–120g/L for several hours without irreversible damage(Hekmat et al.,2003).In the present work,M5AM/GDH exhibited an increased tolerance against higher DHA concentration for a prolonged time period.When incubated in reaction solution containing180g/L glycerol,M5AM/GDH resting cells accumulated an approximately maximum DHA concentration of153g/L after 18h,and extended incubation for another10h had few effects on DHA yield.These results indicated that the GDH overexpressing strain M5AM/GDH could tolerate no less than130g/L DHA and the DHA productivity decreased with the increase in the initial glycerol concentration in a batch biotransformation.

3.5.Repeated-batch biotransformation of glycerol to DHA by

G.oxydans M5AM/GDH

In repetitive batch experiments with M5AM/GDH,four runs were repeated in a5-L fermentor under de?ned conditions.As re-vealed in Fig.4,the cells could be recycled three times without obvious decline in the DHA productivity,and only a small decrease in the fourth cycle was observed.Within this period,the speci?c productivity of DHA decreased from 2.5g/g CDW/h to 2.0g/g

1fe2215bbe23482fb4da4c39parison of DHA production among different G.oxydans strains

resting cell system in shake?asks.Error bars represent standard deviation

for three batches.

Fig.3.(a)Comparison of DHA production between G.oxydans M5/pBBR(open)and

G.oxydans M5AM/GDH(?lled)in a5-L fermentor supplied with100g/L glycerol.(b)

Biotransformation of DHA by G.oxydans M5AM/GDH in a5-L fermentor supplied

with140g/L(?lled)and180g/L(open)glycerol.The pro?les of DHA(circle),

glycerol(triangle)and DO(square)are shown.Each point represents the average

value of two independent experiments.

CDW/h,showing25%loss of productivity after four rounds,which was probably due to cell inactivation by shear forces,DHA damage or the deletion of the essential cofactor(PQQ)for GDH.The stabil-ity of free M5AM/GDH cells was lower than that of immobilized cells which could recover90%of initial activity after?ve cycles of biotransformation in reaction solution containing60g/L glycerol (Wei et al.,2007b).However,M5AM/GDH displayed a much higher conversion rate and productivity at an initial glycerol concentra-tion of100g/L.During the repeated-batch experiments,about 96g/L DHA was accumulated in each round of biotransformation with an increase in reaction time from the?rst to the fourth cycle (Fig.4).In total,385g DHA was formed within34h in the four runs of reaction,giving an average DHA productivity of2.2g/g CDW/h. From an economic viewpoint,the results were of great interest for DHA production by resting cells of G.oxydans M5AM/GDH on an industrial scale.

4.Discussion

We have demonstrated that overexpression of GDH in a strain lacking the gene encoding for ADH led to a signi?cant improve-ment of GDH activity and DHA production.The increased activity led to a2.4-fold increase in productivity compared with M5/pBBR in a batch biotransformation when100g/L glycerol was supplied and shortened the reaction time(Fig.3a).It is also possible that higher levels of GDH increased the tolerance of the strain against DHA inhibition,as suggested by G?tgens et al.(2007).Since in the presence of higher levels of glycerol,a relatively low yield was(Fig.3b),possibly due to inhibition by high concentrations of DHA,further improvements in yield will have to involve changes in fermentation procedures such as those described by Hekmat et al.(2003)who designed a reactor system consisting of a shaking tank and a permeable column harboring immobilized cells.

In addition,more biotransformation cycles may be realized in repeated-batch production of DHA with immobilized M5AM/GDH cells which could maintain consistent oxidative activity more eas-ily than free cells during the oxidation process(Adlercreutz et al., 1985;Wei et al.,2007b).

The current experiments were carried out with pure glycerol.In a commercial setting,most likely biodiesel-derived glycerol would have to be used.This type of glycerol typically contains various contaminants that could cause severe inhibition at high concentra-tions,or even are toxic to the cells(Sabourin-Provost and Hallen-beck,2009).It remains to be determined if our recombinant strain will be able to provide high yields under those conditions.

5.Conclusion

A promising strain for industrial production of DHA was devel-oped by overexpressing the GDH gene in the ADH-negative strain G.oxydans M5AM.Taking advantage of the elevated activity of GDH,the M5AM/GDH strain produced DHA at a high concentration and displayed a substantially increased DHA productivity in a rest-ing cell system.In repetitive batch biotransformation,the cells could be used for several cycles without signi?cant loss of produc-tivity.The recombinant strain thus has potential for industrial pro-duction of DHA.

Acknowledgements

This work was?nancially supported by the National Key Basic Research Development Program of China(‘‘973”Program,No. 2009CB724703),and the National Special Fund for State Key Labo-ratory of Bioreactor Engineering,Grant No.2060204.We also thank professor Yang Susheng(China Agricultural University)for kindly providing the plasmid pBBR1MCS-5.

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