BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

2,3-Butanediol production from starch by engineeredKlebsiella pneumoniae G31-A

Flora Tsvetanova & Penka Petrova & Kaloyan Petrov

Received: 5 November 2013 /Accepted: 14 November 2013 /Published online: 10 December 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract 2,3-Butanediol (2,3-BD) is an organic compound,which is widely used as a fuel and fuel additive and applied inchemical, food, and pharmaceutical industries. Contemporarystrategies for its economic synthesis include the developmentof microbial technologies that use starch as cheap and renew-able feedstock. The present work encompasses the metabolicengineering of the excellent 2,3-BD producer Klebsiellapneumoniae G31. In order to perform direct starch conversioninto 2,3-BD, the amyL gene encoding quite active, liquefyingα-amylase in Bacillus licheniformis was cloned under lacpromoter control in the recombinant K. pneumoniae G31-A.The enhanced extracellular over-expression of amyL led to thehighest extracellular amylase activity (68 U/ml) ever detectedin Klebsiella. The recombinant strain was capable of simulta-neous saccharification and fermentation (SSF) of potato starchto 2,3-BD. In SSF batch process by the use of 200 g/l starch,the amount of total diols produced was 60.9 g/l (53.8 g/l 2,3-BD and 7.1 g/l acetoin), corresponding to 0.31 g/g conversionrate. The presented results are the first to show successfulstarch conversion to 2,3-BD by K. pneumoniae in a one-stepprocess.

Keywords 2,3-Butanediol . Starch .Klebsiella pneumoniae .

Amylase . Bacillus licheniformis

Introduction

2,3-Butanediol (2,3-BD) is a bivalent alcohol with extensiveindustrial uses. The interest in its production increased remark-ably in the last decade because of its application as a liquid fuel,along with its common usage in manufacture of antifreezes,printing inks, perfumes, moistening, and softening agents. 2,3-BD is also a chiral compound, reagent in a number of asym-metric chemical syntheses (Xiu and Zeng 2008; Zeng andSabra 2011). It is platform chemical for valuable derivativessuch as methyl ethyl ketone and 1,3-butadiene (Syu 2001).

The microbial production of 2,3-BD is an attractive alter-native to the more costly chemical synthesis. Several bacterialspecies, belonging to Klebsiella , Enterobacter , Serratia ,Paenibacillus polymyxa , Bacillus licheniformis , Bacillussubtilis , and Bacillus amyloliquefaciens are able to secrete2,3-BD (Ji et al. 2011; Jurchescu et al. 2013). Among them,the species Klebsiella pneumoniae is known as the best forindustrial use because of its more complete fermentation,broad substrate spectrum, and cultural adaptability (Garg andJain 1995; Ma et al. 2009). It is capable of degrading a varietyof hexoses, pentoses, and disaccharides and produces mainlymeso-2,3-BD via mixed-acid pathway that yields several liq-uid by-products: lactic, succinic, and acetic acid and ethanol(Syu 2001). This is the reason that, so far, a number ofengineered Klebsiella strains were manipulated with the pur-pose to redistribute the metabolic flux predominantly to 2,3-BD synthesis (Kumar et al. 2012; Kim et al. 2013; Ji et al.2010, 2013), and at the same time, the attempts to expand thesubstrate utilization properties of 2,3-BD-producing strainsare very limited (Zheng et al. 2008).

In bacterial metabolism, the monosaccharides are first con-verted to pyruvate via a combination of Embden–Mayerhofand pentose phosphate pathways. Then, two molecules ofpyruvate are converted to one molecule of α-acetolactate,subsequently reduced to acetoin. The acetoin is reduced to2,3-BD by a reversible reaction (Ji et al. 2011). Among all

Electronic supplementary material The online version of this article(doi:10.1007/s00253-013-5418-4) contains supplementary material,which is available to authorized users.

F. Tsvetanova :K. Petrov (*)Institute of Chemical Engineering, Bulgarian Academy of Sciences,103, Acad. G. Bonchev str., 1113 Sofia, Bulgariae-mail: kaloian04@yahoo.com

P. PetrovaInstitute ofMicrobiology, Bulgarian Academy of Sciences, 26, Acad.G. Bonchev str., 1113 Sofia, Bulgaria

Appl Microbiol Biotechnol (2014) 98:2441–2451DOI 10.1007/s00253-013-5418-4

carbohydrates, the greatest conversion rates were achieved byglucose fermentation (Ma et al. 2009; Sun et al. 2009), but thehigh cost of the sugar substrates has been identified as a majorfactor affecting the economic viability of this biotechnology.The advancedmicrobial technologies for 2,3-BD synthesis aredevoted to the reduction of expenses by the use of cheaper andrenewable substrates of cellulosic and non-cellulosic origin.Alternative sources are the widely available low-cost cellulos-ic and lignocellulosic agricultural residues, which are subject-ed to hydrolysis and yield sugar mixtures containing glucose,xylose, arabinose, and ribose. Thus, Cao et al. (1997) inves-tigated the potential of corncob hydrolysates, reaching 90 %of the cellulose degraded to glucose. Further, Cheng et al.(2010) achieved successive conversion of the hemicellulosefraction of the plant biomass; Wang et al. (2010) developedcorncob molasses fermentation; Wong et al. (2012) used hy-drolyzed rice straw as feedstock for 2,3-BD production.Renewable substrates are also the carbohydrates extractedfrom plant residues, such as the oligomeric fructans ofJerusalem artichoke (Sun et al. 2009; Li et al. 2010) or thesucrose, glucose, and fructose derived from sugar cane andbagasse (Wong et al. 2012). Industrial waste as bio-diesel by-produced glycerol, whey permeates, or molasses could also beefficiently converted to 2,3-BD (Petrov and Petrova 2010; Jiet al. 2011).

A possible approach to profitable process of 2,3-BD pro-duction is the utilization of starch-containing agriculturalwastes (Perego et al. 2003). Thereby, starch fermentation ispreceded by pretreatment procedures: (1) gelatinization thatbreaks down the intermolecular bonds of starch molecules inthe presence of water and heat, (2) acid hydrolysis, or (3)amylase treatment that degrades the starch to malto-oligosaccharides. The biotechnologies in which the processesof starch saccharification and fermentation are simultaneousare preferred because they are energy saving, avoid the use ofamylase enzymes in industrial scale, and shorten the overallprocess time. Presently, the absence of such developed bio-technology for preparing 2,3-BD from starch suspends the useof this inexpensive substrate.

Several studies showed that the wildK. pneumoniae strainsare able to grow on starch-containing media due to the com-bined action of genes malS , malQ , malZ , and pulA. MalSencodes α-D-1,4-glucan exo-maltohexa-hydrolase, similar tothe periplasmic α-amylase of Escherichia coli (Momma2000) and responsible for maltose and maltodextrines utiliza-tion. The product of the second gene (malQ ) is α-glucanotransferase (CGTase); malZ encodes maltodextringlucosidase, and pulA—an inducible pullulanase.

Up to now, there is only one report describing 2,3-BDsynthesis directly from starch by cloning and expression ofgenes encoding amylase enzymes. Zheng et al. (2008) clonedmalS gene for natural amylase of K. pneumoniae strain KG1and expressed it extracellularly in the same strain. However,

only a slight increase of the amylase activity of the parentalKG1 was achieved, thus suggesting that the natural starch-modifying enzymes of Klebsiella are not able to degrade thestarch sufficiently to assure high 2,3-BD production in onestep. Therefore, simultaneous process of saccharification and2,3-BD synthesis would be possible only by undertaking ofextracellular heterologous expression of a suitable amylasegene in K. pneumoniae .

A prospective source of such genes is the species Bacilluslicheniformis . It is known to produce the most active andstable α-amylase (BLA) that is starch liquefying, i.e., it iscapable to degrade raw starch. In addition, this BLA enzymeretain its activity in a wide pH range, at high temperatures, andeven in the presence of surfactants (Hmidet et al. 2008) andare very suitable for industrial process performance.

In the present study, we report the metabolic engineering ofa strain K. pneumoniae G31, known as an excellent 2,3-BDproducer from glycerol (Petrov and Petrova 2009, 2010).Aiming to enrich its substrate spectrum, we performed thestrain improvement by introduction of heterologous α-amylase derived from the Bulgarian isolate B. licheniformis44 MB82/G. The first successful heterologous expression ofamyL gene in Klebsiella and the first successful direct con-version of starch into 2,3-BD are described in the followingsection.

Materials and methods

Bacterial strains and plasmids

The strains and plasmid constructs used in this work are listedin Table 1. The experiments were carried out with the strain K.pneumoniae subsp. pneumoniae G31, isolated previouslyfrom active slime (Petrova et al. 2009) and deposited in theBulgarian national bank of microorganisms and cell cultures(no. 8650). Bacillus licheniformis strain 44 MB82/G wasobtained from the same culture collection (no. 1542).

The engineered K. pneumoniae G31-A is deposited in theGerman collection of microorganisms and cell cultures inLeibniz Institute (DSMZ 27580).

Media and cultivation conditions

E. coli and K. pneumoniae strains were cultured in Luria–Bertani (LB) medium (Scharlau Chemie S.A., Barcelona,Spain). For LB plates, 1.5 % agar (Oxoid, Thermo FisherScientific, Hampshire, UK) was used. The antibiotics ampi-cillin, kanamycin, tetracycline, erythromycin, and chloram-phenicol (Sigma-Aldrich Chemie GmbH, Steinheim,Germany) were added when needed.

B. licheniformis 44MB82/G was cultured in nutrient brothcontaining (g/l) meat extract—10, peptone—10, and NaCl—

2442 Appl Microbiol Biotechnol (2014) 98:2441–2451

5, supplemented with 0.5 % soluble starch. All strains werecultivated at 37 °C on a rotary shaker. They were maintainedat 4 °C (subcultured every week) or frozen at −80 °C with20 % (w /w) glycerol added.

The basal fermentation medium for 2,3-BD production bythe recombinant strain K. pneumoniae G31-A consisted of (g/l) potato starch (Fluka)—40–300, (NH4)2HPO4—4.9, 3CH3COONa·3H2O—3, KCl—0.4, MgSO4·7H2O—0.2,FeSO4·7H2O—0.02, MnSO4.7H2O—0.01, ZnSO4.7H2O—0.001, and yeast extract—5 (pH 7.0).

Isolation of genomic and plasmid DNA and PCR

Total genomic DNA was isolated from 24-h-old cells of B.licheniformis 44 MB82/G with GeneJET Genomic DNAPurification Kit (Thermo Fisher Scientific Inc., Waltham,MA, USA), following the manufacturer’s recommendations.

Plasmid purifications were either by the maxi-preparativealkaline procedure (Sambrook and Russell 2001) or byGeneJET PlasmidMiniprep Kit (Thermo Fisher Scientific Inc.).

For PCR amplification of amyL gene were used the primersamyF and amyR (Biomers.net GmbH, Ulm, Germany; Table 1)and PCR mixture of GeneAmp® High Fidelity PCR System(Applied Biosystems, CA, USA) in total volume 50 μl, at finalconcentrations of primers 0.5 pmol/μl and template genomicDNA of B. licheniformis 44 MB82/G—2 ng/μl. PCR wasperformed in QB-96 Satellite Gradient thermal cycler (LKBVertriebs GmbH, Vienna, Austria) under the following temper-ature profile: 94 °C for 3.5 min, 35 cycles consisting of 94 °Cfor 1 min, 55 °C for 45 sec, 72 °C for 2 min, followed by finalelongation at 72 °C for 5min. The corresponding PCR productswere visualized in 1 % agarose gel.

Cloning of the amyL gene

The PCR amplified amyL gene was purified using GFX PCRDNA and gel band purification kit (Amersham Biosciences,Uppsala, Sweden) and was cloned in pJET 2.1/blunt vector(2974 bp) following the instructions of CloneJETTM PCRCloning Kit (Thermo Fisher Scientific Inc., Waltham, MA,USA). The construct was designated pJETamyBL and wasused for sequencing of amyL to obtain the gene as a restrictionfragment after Xho I/Xba I digestion (New England Biolabs,MA, USA). The fragment was sub-cloned into vectorpCR®2.1-TOPO® under lac promoter control, givingpCRamyBL.

Bacterial transformation and amylase secretion assay

The strain E. coli DH5α was the cloning host for bothpJETamyBL and pCRamyBL. MAX Efficiency® DH5α™competent cells were purchased from Invitrogen™, LifeTechnologies™ Co. The competent cells of K. pneumoniaeG31 were prepared by TransformAidTM BacterialTransformation Kit (Fermentas, Thermo Fisher Scientific Inc).

Qualitative analysis of the ability of transformants to se-crete amylase was performed at LB-plates, containing 0.5 %starch. After 24 h of cultivation at 37 °C, the colonies wereexposed to iodine vapours. Clear yellow color zonessurrounded the positive clones.

Nucleotide sequence analysis of amyL

The gene amyL was sequenced by Macrogen Inc. (Amsterdam,The Netherlands) using primers pJET2.1-forward and pJET2.1-

Table 1 Strains, plasmids, andprimers used in this study

a The italicized nucleotides repre-sent an introduced KlebsiellaShine–Dalgarno sequence

Strains/plasmids/primers Relevant characteristics Source

Strains

K. pneumoniae G31 2,3-BD producer, Ampr Petrova et al. 2009

K. pneumoniae G31-A 2,3-BD producer, Ampr, Kanr, pCRamyBL This study

E. coli DH5α F-endA1 glnV44 thi-1 recA1 relA1gyrA96 deoR nupG Φ80dlacZΔM15Δ(lacZYA-argF)U169, hsdR17(rK

−mK+), λ–

Invitrogen™

B. licheniformis 44 MB82/G Raw starch degrading, amyL Tonkova 1991

Plasmids

pJET2.1/blunt

pJETamyBL

pCR®2.1-TOPO®

pCRamyBL

Ampr, T7 promoter, rep (MB1)

Ampr, T7 promoter, rep (MB1), amyL

Ampr, Kanr, f1 ori, pUC ori, Plac , lacZα

Ampr, Kanr, f1 ori, pUC ori, Plac , amyL

Thermo Scientific Inc.

This study

Invitrogen™

This study

Primers

amyFa

amyR

pJET2.1-forward

pJET2.1-reverse

5′-gtgtaggat tccatgaaacaacaaaaacggctttac-3′

5′-gattccgttcctatctttgaacataaattgaaacc-3′

5′-cgactcactatagggagagcggc-3′

5′-aagaacatcgattttccatggcag-3′

This study

This study

Thermo Scientific Inc.

Thermo Scientific Inc.

Appl Microbiol Biotechnol (2014) 98:2441–2451 2443

reverse. The sequence analysis was carried out using programsChromas and CAP3 (http://genome.cs.mtu.edu/cap). Thesequence comparison with the GenBank data was byBLASTN and ClustalW programs. The putative proteinstructure of the amylase was analyzed by Expasy program(Swiss Institute of Bioinformatics). The nucleotide sequencedata were deposited in NCBI GeneBank database underaccession number KF261567.

Expression of amyL in K. pneumoniae G31-A

The batch processes were performed using K. pneumoniaeG31-A cells harbouring plasmid pCRamyBL in 500-ml flaskscontaining 100 ml basal fermentation medium, 75 μg/mlkanamycin, and 24-h inoculum culture, grown in the samemedium. The experiments with starch concentration 40 g/lwere performed with 2% inoculum, all others were performedwith 10%. The flasks were incubated on a rotary shaker (NewBrunswick Scientific Co., Edison, NJ, USA) at 37 °C.Agitation was 200 rpm during the batches with 40 g/l starchand 240 rpm when 100, 200, and 300 g/l starch was used.

Separate experiments with addition of variable amounts ofisopropyl β-D-1-thiogalactopyranoside (IPTG), glycine, orCaCl2 (Sigma) were carried out. After withdrawal of samples,the cell-free supernatants were purified through 0.2-μm sterilefilters (Minisart®, Sartorius stedim Biotech, Germany) andanalyzed for amylase activity, residual starch, and total extra-cellular protein content. Mean values and standard deviationsof three independent batches are presented.

Analytical methods

The residual starch content was determined by measuring thelight absorption of the iodine–starch complex color at wave-length 580 nm (Nakamura 1981).

The extracellular amylase activity of cell-free supernatantsamples was estimated. It was detected by measurement of theiodine complexing ability of starch according to the method ofPantschev et al. (1981) at pH 6.0 (0.1 mol/l citrate-phosphatebuffer) and temperature 37 °C. One unit of amylase activitywas defined as the amount of enzyme that hydrolyzes 1 μmolstarch per minute. One unit of specific amylase activity is theamylase activity per milligram total extracellular protein. Theprotein content was estimated by the method of Bradford(1976).

The quantification of the soluble metabolites was carriedout by a high-performance liquid chromatography (HPLC)system. 2,3-BD, acetoin, ethanol, acetic, succinic, and lacticacid content was analyzed by Bio-Rad column Aminex HPX-87H at 65 °C and detected either by refractive index used for2,3-butanediol, acetoin, and ethanol estimation or by UVabsorbance at 210 nm using Knauer variable wavelengthdetector for acid measurement. As a mobile phase, 5 mM

H2SO4 at a flow rate 0.6 ml/min was used. Glucose,maltose, and oligo-sugars were separated by Bio-Rad columnHPX-42A at 85 °C and detected by RI detector (Perkin-Elmer 10). The mobile phase was water with flow rate of0.6 ml/min.

Results

Cloning and sequence analysis of amyL gene of B.licheniformis 44 MB82/G

The amyL gene ofB. licheniformis 44MB82/Gwas amplifiedas a blunt-ended PCR fragment using the primer pair amyFand amyR (Table 1), cloned in the vector pJET2.1/bluntgiving pJETamyBL and sequenced. The gene consisted of1,539 bp, started with ATG codon, and encoded a polypeptideof 512 amino acids (aa) with deduced molecular weight of58.5 kDa. It contains a signal peptide of 29 aa. The protein hasthe conserved domain structure typical for the amylases. Theconserved “DED” triad of catalytic residues (Asp, Glu, andAsp) that performs catalysis are observed at positions 260,290, and 357. The residues 42, 99, 134, 225, 227, 258, 260–264, 290, 292, 293, 319, 356, and 357 form the active centerof the enzyme. The residues Asn and Asp (N, D, D) aresituated at 133, 223, and 229, composing a possible Ca–Na–Ca binding site.

Heterologous expression of amyL in K. pneumoniae G31-A

An important precondition for the choice of expression vectorwas the examination ofK. pneumoniae G31 natural resistanceto antibiotics. It was found that the strain is resistant to allchecked concentrations of ampicillin (50–200 μg/ml), tetra-cycline (10–50 μg/ml), and erythromycin (15–45 μg/ml). Itwas susceptible to high concentrations of chloramphenicol(50 μg/ml) but grew slightly at 25–35 μg/ml. The strain wastotally susceptible only to kanamycin, and this antibioticresistance was chosen as a selection marker.

Thus, the amyL was sub-cloned from pJETamyBL inpCRamyBL (Fig. 1). The recombinants were plated on solidmedium, containing 0.5 % starch, and clear zones of starchdegradation were observed, thus verifying the expres-sion of amyL under lac promoter control by E. coliDH5α. K. pneumoniae G31 was transformed withpCRamyBL isolated from a positive clone of E. coli DH5α.The starch-degrading recombinant K. pneumoniae G31-Ademonstrating the largest halo on the starch–agar mediumwas selected.

The extracellular over-expression of amyL gene by therecombinant K. pneumoniae G31-A was demonstrated bySDS-PAGE of total proteins in cell-free supernatants(Fig. S1).

2444 Appl Microbiol Biotechnol (2014) 98:2441–2451

Metabolite profiles of starch fermentation by recombinant K.pneumoniae G31-A

The amylase activity and the product spectrum of starchfermentation by parental K. pneumoniae G31 and the recom-binant G31-A were investigated in a series of batch experi-ments. The basal fermentation medium was previously opti-mized for 2,3-BD production from glucose. It contains ions ofspecific microelements (Mn2+, Mg2+, Fe2+), acetate, phos-phate, and nitrogen sources required for enhanced 2,3-BDproduction of K. pneumoniae , ensuring minimum by-product formation (Garg and Jain 1995; Syu 2001).

Natural K. pneumoniae G31 was able to degrade 4.9 g/lpotato starch for 48 h but did not display any extracellularamylase activity. There were no amounts of 2,3-BD produced.In contrast, in the absence of any substance enhancing theamylase secretion compounds, the recombinant strain G31-Ahydrolyzed ~35 g/l starch for 48 h and produced 7.3 g/l 2,3-BD.The time profile of degradation of 40 g/l starch and the metab-olites produced by K. pneumoniae G31-A are shown in Fig. 2.

Influence of IPTG addition on the extracellular amylaseactivity and 2,3-BD production from 40 g/l starch by K.pneumoniae G31-A

Since the transcription of amyL was under lac promotercontrol, the influence of the different concentrations of theinductor IPTG was established. Three different final amountsof IPTG (0.1, 1, and 1.5 mM) were added at the beginning ofthe process. The starch degradation, amylase activity, andfermentation profiles of induced and non-induced (control)cultures were compared at Table 2.

The addition of IPTG had a positive effect on starch hy-drolysis by increasing amyL expression in all cases. With therise of the IPTG concentration, the amylase activity increasedalmost twice from 7.95 U/ml (without induction) to 13.44 U/ml (1.5 mM IPTG). However, the corresponding values of theproduced metabolites at the 24th hour showed that the highestIPTG concentration (1.5 mM) has no greater effect on 2,3-BDproduction. Thus, the concentration of 1 mM IPTG giving5.01 g/l 2,3-BD was used in all further experiments.

Influence of Ca2+ addition on the extracellular amylaseactivity and 2,3-BD production from 40 g/l starch by K.pneumoniae G31-A

Ca2+ was added to the medium (containing 1 mM IPTG) asCaCl2·2H2O in concentrations of 0.01, 0.1, and 0.2 g/l.Comparing to the process without calcium, addition of0.01 g/l CaCl2 scarcely affected the amylase activity(16.86 U/ml), diminished the 2,3-BD (4.54 g/l), and slightlyincreased the acetoin, acid, and ethanol production. An in-crease of Ca2+ for 10 and 20 times resulted in extremelyenhanced amylase activity that reached 65–68 U/ml at the24th hour. Unexpectedly, the higher the obtained amylaseactivity, the lower were the concentrations of all fermentationproducts (Table 3). At CaCl2 concentrations 0.1 and 0.2 g/l,only 2.46 and 0.47 g/l of 2,3-BD were produced, respectively.

The estimation of the starch hydrolysis intermediates indi-cates that excessive CaCl2 addition results in exorbitant accu-mulation of Dp 2–Dp 5 oligosaccharides (Dp is degree ofpolymerization). The greatest was the amassment ofmaltopentaose (Dp 5) during the process with 0.2 % CaCl2.In this case 11.2 g/l maltopentaose (Fig. 3a), 5.5 g/lmaltotriose (Fig. 3b), and 1.8 g/l maltose (Fig. 3c) wereaccumulated for 24 h, with a large difference from the fer-mentation without Ca2+, whereby the amount of oligosaccha-rides shorter than Dp 8 was negligible at any moment.Afterwards, slow degradation of the oligosaccharidesoccurred.

Fig. 1 Structure of pCRamyBL. The construct contains amyL gene of B.licheniformis 44 MB82/G cloned in vector pCR®2.1-TOPO® under lacpromoter control

0

5

10

15

20

25

30

35

40

0 8 16 24 32 40 48 56 64 72

Time (h)

Star

ch (

g/l)

0

2

4

6

8

Pro

duct

s (g

/l)

Starch2,3-BDSuccinic acidAcetic acidEthanolAcetoin

Fig. 2 Time course of starch degradation and products formation in batchprocess using the recombinant K. pneumoniae G31-A grown in basalmedium

Appl Microbiol Biotechnol (2014) 98:2441–2451 2445

Influence of glycine addition on the extracellular amylaseactivity and 2,3-BD production from 40 g/l starch by K.pneumoniae G31-A

In order to enhance cell membrane permeability, increasingconcentrations of glycine were added to the cultural mediumat the beginning of the process. The influence of the glycineamount on extracellular amylase activity and 2,3-BD is shownat Table 4. The highest amount of 2,3-BD was produced by theuse of 0.5 % glycine—10.54 g/l (corresponding to 41.5 U/mlamylase and 198 U/mg specific activities). The kinetics ofstarch hydrolysis and major product formation during thisprocess is presented in Fig. 4a. It is evident that 40 g/l starchwas completely degraded until the 19th to the 24th hour,coinciding with the 2,3-BD maximum value. Acetoin appearedin detectable amounts around the 11th hour. The total amount of2,3-BD and acetoin slightly increased until the end of fermen-tation, reaching 12.5 g/l at the 48th hour as the acetoin concen-tration continuously increased on account of 2,3-BD. The by-product formation is presented at Fig. 4b. Their amount alteredin the course of starch fermentation: the lactic and succinic acidincreased to the 24th hour, and then both of them decreased asthe lactic acid was completely converted. The acetic acid wasaccumulated during the first 10 h then decreased, reaching itsminimal value (0.6 g/l, along with 2,3-BD sharp increase), andsteadily increased until the end. Ethanol steadily increasedthroughout fermentation, too. The amylase activity increaseduntil the end of the process (Fig. 4c) as well as the total contentof extracellular protein, possibly caused by cell lysis. The pH ofthe culture declines from 7.0 to 6.3 until the 8th hour and roseagain to 6.5 at the end of the process. These time profiles aremore or less similar for all of the previously describedprocesses.

Conversion of highly concentrated starches into 2,3-BDby the engineered strain K. pneumoniae G31-A

After the process optimization described earlier, the potentialof the engineered strain K. pneumoniae G31-A to convert100, 200, and 300 g/l starch was estimated in a series ofbatches (compared in Table 5). Due to the higher initialconcentration of starch, its hydrolysis occurred slightly slowerin comparison with the previous batches: 100 g/l wascompletely digested after 30 h of fermentation, 200 g/l after90 h, and 300 g/l after 120 h. The highest concentration of 2,3-BD (53.8 g/l) was reached from 200 g/l starch. The finalconcentration of total diols (2,3-BD + acetoin) obtained from200 and 300 g/l starch was equal (60.9–61 g/l total diols).

The kinetics of substrate utilization and the diols produc-tion from 200 g/l starch are shown in Fig. 5a. It is evident thatthe acetoin was the major by-product during this fermentation,reaching 7.13 g/l. Other additional metabolites accumulated atthe end of the process were acetate (6.31 g/l) and ethanol(2.93 g/l) and their share remained low, indicating that thecultivation conditions used favor the diols production. Thelactic and succinic acids were initially formed and then de-graded (Fig. 5b). Amylase activity increased to the 96th hourand then slightly decreased (Fig. 5c). The pH during theprocess dropped to lower values, compared to the fermenta-tion of 40 g/l, and was about pH 5.7 in the period between the48th and the 110th hours.

Discussion

Two metabolic routes of starch utilization are known in wildKlebsiella strains. The first one involves extracellular

Table 2 Influence of isopropyl β-D-1-thiogalactopyranoside on starch conversion by the engineered strain K. pneumoniae G31-A. The amylaseactivity, total extracellular protein content, and concentrations of products formed after 24 h of fermentation are presented

IPTG(mM)

Starchconsumed (g/l)

Amylaseactivity (U/ml)

Protein(mg/ml)

Specificactivity (U/mg)

2,3-BD(g/l)

Acetoin(g/l)

Succinicacid (g/l)

Aceticacid (g/l)

Ethanol(g/l)

0.0 (control) 11.66±1.48 7.95±1.03 0.07±0.00 113.6 4.35±0.18 0.34±0.09 0.83±0.18 0.76±0.30 1.25±0.26

0.1 14.47±0.58 12.48±1.00 0.08±0.00 156.0 4.47±0.11 0.41±0.12 0.88±0.10 0.63±0.16 1.23±0.09

1.0 14.51±1.05 13.30±0.57 0.08±0.00 166.3 5.01±0.05 0.83±0.08 0.83±0.05 0.50±0.10 1.74±0.20

1.5 15.04±0.84 13.44±1.64 0.08±0.00 168.0 4.89±0.13 1.19±0.11 0.71±0.11 0.60±0.13 1.73±0.31

Table 3 Influence of Ca2+ on starch conversion by the engineered strainK. pneumoniae G31-A. The amylase activity, total extracellular protein content,and concentrations of products formed after 24 h of fermentation are presented. All media used were supplemented with 1 mM IPTG

CaCl2·2H2O(g/l)

Starchconsumed (g/l)

Amylaseactivity (U/ml)

Protein(mg/ml)

Specific activity(U/mg protein)

2,3-BD (g/l) Acetoin (g/l) Succinicacid (g/l)

Aceticacid (g/l)

Ethanol (g/l)

0.01 17.84±0.28 16.86±0.82 0.08±0.01 210.8 4.84±0.19 1.16±0.08 1.17±0.10 2.62±0.13 1.50±0.04

0.1 40 65.10±7.55 0.19±0.02 342.6 2.46±0.14 0.54±0.11 0.33±0.06 2.86±0.08 0.65±0.11

0.2 40 68.19±9.62 0.21±0.02 324.7 0.47±0.07 0.23±0.09 0 2.04±0.22 0.25±0.13

2446 Appl Microbiol Biotechnol (2014) 98:2441–2451

degradation of starch by a cell surface-associated pullulanaseand subsequent cleavage of the α-1,4-glycosidic linkages ofthe linear maltodextrins. Through the second pathway, starchis converted extracellularly into cyclodextrins (CDs), whichundergo intracellular linearization by cyclodextrinase (cymH),and the linear maltodextrins enter in the maltose degradationpathway. Considering the cellular localization of the otherstarch-degrading enzymes in Klebsiella , the maltodextrin

glucosidase is cytoplasmic, the maltohexa-hydrolase is peri-plasmic, and the pullulanase is attached to the external mem-brane of the cells. None of these enzymes is extracellular;moreover, none of them cannot accumulate detectable quan-tities of reducing sugars or linear malto-oligosaccharides suit-able for conversion into 2,3-BD (Gawande and Patkar 2001).

Aiming to achieve 2,3-BD production by direct starchconversion, the strategy of heterologous expression was cho-sen. As the most suitable, the amyL gene of B. licheniformiswas determined. Analysis of its nucleotide sequence showed100 % identity with amyL gene of B. licheniformis DSM8785 and amyS gene of B. licheniformis NCIB 8061.Compared to entire genomes, amyL has 100 % homologywith the corresponding gene of B. licheniformisCGMCC3963 and 99 % identity with amyL of B.licheniformis ATCC 14580 genome. Therefore, the selectedamyL gene encodes quite active, liquefying α-amylase in B.licheniformis , capable of attacking raw starch.

The heterologous expression of genes deriving fromGram-positive bacteria encounters many difficulties in Gram-negative hosts. Among them is the choice of promoter andthe effective promoter control, suitable Shine-Dalgarno se-quences, as well as cellular physical barriers against proteinsecretion. Based on the inducible lac promoter (Plac ) of E.coli , the expression of amyL gene by K. pneumoniae G31-Awas expected to be totally dependent on IPTG induction.Surprisingly, the non-induced K. pneumoniae G31-A alsoexpressed the amyL gene at a significant level. The reason isthat the recombinant construct pCRamyBL contains E. colilac promoter, but it does not bear the gene lacI , encoding thecorresponding lac -repressor. In this case, a partial repressionof Plac by the K. pneumoniae chromosomal lac -repressoroccurred. Comparing the amino acid sequences of lac -repres-sors deriving from E. coli and K. pneumoniae , an identitybetween 96 and 98 % was observed. Taking into account thefact that the lac -repressor acts as a tetramer when binding theinverted repeat sequence of the operator DNA, the weakrepression of Plac by K. pneumoniae lac -repressor is mostprobably due to conformation discrepancies.

A common problem facing the extracellular protein expres-sion by Gram-negative bacteria is the cytoplasmic membranebarrier. The obtained high levels of extracellular amylaseactivity suggested that the signal peptide transports the recom-binant amylase to the periplasm. The observation of its de-duced amino acid sequence revealed the presence of N-terminal domain of positively charged amino acids(MKQQKRLYAR), followed by extremely long hydrophobic“h”-region (LLTLLFALIFLLPHSAAAA), known to enhancethe contact between the signal peptide and the cytoplasmicmembrane (Ismail et al. 2011). The extracellular secretion ofthe amylase into the culture medium indicates that the signalpeptide of the enzyme was functional in Klebsiella . SDS-PAGE analysis of the cell-free supernatants showed that the

0

2

4

6

8

10

12

0 0.01 0.1 0.2

Addition of CaCl2.2H2O (g/l)

Mal

tope

ntao

se (

g/l)

0h 6h 24h

0

2

4

6

8

10

12

0 0.01 0.1 0.2

Mal

totr

iose

(g/

l)

0h 6h 24h

0

2

4

6

8

10

12

0 0.01 0.1 0.2

Addition of CaCl2.2H2O (g/l)

Addition of CaCl2.2H2O (g/l)

Mal

tose

(g/

l)

0h 6h 24h

a

b

c

Fig. 3 Effect of increasing Ca2+ concentrations on starch hydrolysisproducts formed in the course of batch fermentations by the recombinantstrain K. pneumoniae G31-A. Time courses of maltopentaose (a ),maltotriose (b), and maltose (c) were presented

Appl Microbiol Biotechnol (2014) 98:2441–2451 2447

enzyme was of the same molecular weight as predicted by thededuced amino acid sequence, namely, 58.5 kDa.

Another obstacle that restrains recombinant protein secre-tion is the outer cell membrane. The heterologous proteins areusually accumulated in the periplasm, and various attempts tofacilitate their secretion have been made. Glycine is known asa commonly used medium supplement for promoting thesecretion of recombinant enzymes into the culture mediumof E. coli (Li et al. 2009), and the same action in Klebsiellawas confirmed for the first time by the results obtained. Theenhanced extracellular secretion of the recombinant enzymesby glycine supplementation might be explained with the in-crease of the cell membrane permeability. Generally, thehigher the glycine amount, the greater was the extracellularamylase activity. However, at glycine concentrations higherthan 0.6 %, the total extracellular protein sharply increaseddue to cell lysis, and the specific amylase activity decreased.

Besides the increased expression levels, higher extracellu-lar amylase activity may be achieved also by stabilization ofthe enzyme in the culture medium. The addition of Ca2+

(which are absent in the basal medium) is considered to benecessary for the stability of Bacillus α-amylases (Hagiharaet al. 2001), and it really was within certain limits. At exces-sive amounts of Ca2+ (0.1–0.2 g/l CaCl2), the activity of theamylase increases, which results in rapid accumulation ofoligo-sugars consisting of two, three, and five glucose units.Dp 5 amassment led to overall retardation of oligosaccharideshydrolysis and, finally, to diminished 2,3-BD production. Theobservation that maltopentaose acts as an amylase enzymeinhibitor is in agreement with Deutch (2002). The maltotrioseand maltose, in turn, hinder DP5 degradation by inhibition ofamylase of competitive and non-competitive type, respec-tively (Al Kazaz et al. 1998).

Regarding the diols productivity, it has to be noted that thepresent study is the first report for successful performance ofsimultaneous saccharification and fermentation (SSF) of starchto 2,3-BD. The engineered K. pneumoniae G31-A is the firststrain capable of one-step conversion of starch into hugeamounts of 2,3-BD and acetoin (Table 5). The diols yieldremained almost the same (0.30–0.31) for starch concentrationsup to 200 g/l. It decreased during the process from 300 g/l.

The value of ~61 g/l total diols, obtained after hydrolysis of200 g/l starch, is 14-fold the yield, obtained from starch byother genetically manipulated Klebsiella strains. For compar-ison, the engineered K. pneumoniae KG1 (pUC18K-amy)formed 3.8 g/l 2,3 BD and was able to degrade 20 g/l starch(Zheng et al. 2008). Furthermore, the high total diols amountobtained by K. pneumoniae G31-A corresponds to a highproductivity (0.51–0.62 g/l/h). This productivity was depen-dent on the initial starch content but in all cases was signifi-cantly higher than those reported for the recombinant K.pneumoniae KG1 (pUC18K-amy)—0.16 g/l/h (Zheng et al.2008). The maximal conversion ratio (0.31 g/g) was alsoTa

ble4

Influenceof

glycineon

starch

conversion

bytheengineered

strainK.pneum

oniaeG31-A

.The

amylaseactiv

ity,totalextracellularp

roteincontent,andquantificationof

accumulated

productsafter

24hof

ferm

entatio

narepresented.Allmediaused

weresupplementedwith

1mM

IPTG

Glycine

(%)

Starch

consum

ed(g/l)

Amylaseactiv

ity(U

/ml)

Protein

(mg/ml)

Specificactiv

ity(U

/mgprotein)

2,3-BD

(g/l)

Acetoin

(g/l)

Lactic

acid

(g/l)

Succinicacid

(g/l)

Acetic

acid

(g/l)

Ethanol

(g/l)

0.1

34.66±1.15

15.63±1.39

0.09

±0.00

173.7

5.80

±0.19

0.58

±0.14

01.54

±0.18

1.01

±0.02

2.07

±0.23

0.3

37.66±0.48

24.82±4.23

0.14

±0.00

177.3

9.08

±0.14

1.17

±0.05

02.15

±0.27

1.05

±0.04

2.14

±0.17

0.5

4041.54±5.48

0.21

±0.02

197.8

10.52±0.17

1.21

±0.14

0.39

±0.21

1.84

±0.28

0.60

±0.26

2.89

±0.34

0.6

4054.55±4.85

0.38

±0.02

143.6

10.16±0.28

0.92

±0.12

01.75

±0.35

0.90

±0.31

2.58

±0.44

0.7

4081.16±7.64

0.52

±0.02

156.1

1.61

±0.43

0.39

±0.26

00.18

±0.16

0.23

±0.10

0.15

±0.07

2448 Appl Microbiol Biotechnol (2014) 98:2441–2451

0

5

10

15

20

25

30

35

40

0 8 16 24 32 40 48

Time (h)

Star

ch (

g/l)

0

2

4

6

8

10

12

14

Ace

toin

; 2,

3-B

D (

g/l)

Starch

2,3-BD

Acetoin

Acetoin + 2,3-BD

0

1

2

3

4

5

0 8 16 24 32 40 48

Time (h)

By-

prod

ucts

(g/

l)

5.5

6

6.5

7

pH

Succinic acid Acetic acid

Ethanol Lactic acid

pH

0

10

20

30

40

50

60

0 8 16 24 32 40 48Time (h)

Am

ylas

e ac

tivi

ty (

U/m

l)

0

0.05

0.1

0.15

0.2

0.25

0.3

Pro

tein

(m

g/m

l)

Amylase activity

Extracellular protein

a

b

c

Fig. 4 Time course of batch fermentation of 40 g/l starch by the recom-binantK. pneumoniae G31-A inmedium supplemented with 1 mM IPTGand 0.5 % (w /v) glycine. Starch degradation and accumulation of 2,3-butanediol and acetoin (a); by-products formation and pH (b); amylaseactivity and total extracellular protein content (c)

Table 5 Conversion of highly concentrated starch by the engineered strain K. pneumoniae G31-A. Product quantifications at the end of the batchfermentations are presented. All media used were supplemented with 1 mM IPTG and 0.5 % glycine

Initial starchconcentration(g/l)

Duration (h) 2,3-BD (g/l) Acetoin (g/l) Diols (g/l) Diols productivity(g/l/h)

Diolsyield (g/g)

Lacticacid (g/l)

Succinicacid (g/l)

Aceticacid (g/l)

Ethanol(g/l)

100 48 25.9±0.3 3.92±0.19 29.8 0.621 0.298 0.55±0.18 2.65±0.12 1.50±0.26 2.03±0.16

200 120 53.8±0.5 7.13±0.22 60.9 0.508 0.305 – – 6.31±0.21 2.93±0.14

300 140 52.0±0.5 9.03±0.17 61.0 0.436 0.203 – – 4.79±0.27 3.15±0.17

0

20

40

60

80

100

120

140

160

180

200

0 24 48 72 96 120

Time (h)

Time (h)

Time (h)

Star

ch (

g/l)

0

10

20

30

40

50

60

70

Ace

toin

; 2,3

-BD

(g/l)

Starch

2,3-BD

Acetoin

Acetoin + 2,3-BD

0

1

2

3

4

5

6

7

0 24 48 72 96 120

By-

prod

ucts

(g/

l)

5.5

6

6.5

7

pH

Succinic acid Acetic acid

Ethanol Lactic acid

pH

0

10

20

30

40

50

60

0 24 48 72 96 120

Am

ylas

e ac

tivi

ty (

U/m

l)

0

0.05

0.1

0.15

0.2

0.25

0.3

Pro

tein

(m

g/m

l)

Amylase activity

Extracellular protein

a

b

c

Fig. 5 Time course of batch fermentation of 200 g/l starch by therecombinant K. pneumoniae G31-A in medium supplemented with1 mM IPTG and 0.5 % (w /v) glycine. Starch degradation and accumu-lation of 2,3-butanediol and acetoin (a); by-product formation and pH(b); amylase activity and total extracellular protein content (c)

Appl Microbiol Biotechnol (2014) 98:2441–2451 2449

higher than the one obtained by the abovementioned strain(0.19). This yield was similar to the diols yields reported forthe SSF processes of conversion of Jerusalem artichoke tubers(Sun et al. 2009) and corn cob cellulose (Cao et al. 1997).However, it has to be underlined that in these studies thesaccharification was carried out by the use of acids and indus-trial enzymes (inulinase, cellulases), pretreatment that highlyincreases the process expenses, while in the present work boththe starch hydrolysis and the fermentation were performed bythe combined enzymatic activities of a single microorganism.

To summarize, the recombinant strain K. pneumoniaeG31-A is an over-producer of extracellular liquefying amylaseenzyme. It is capable of direct starch fermentation and pro-duced ~61 g/l total diols, an amount that had never beenreported before for one-step process of starch conversion.Conversion rate of 0.31 g/g starch and the remarkable pro-ductivity of 0.51–0.62 g/l/h were achieved. Therefore, thepresented results provide new opportunity for economic in-dustrial 2,3-BD biosynthesis development by engagement ofalternative starch materials.

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