The metabolic flux regulation of klebsiella pneumoniae based on quorum sensing system

ABSTRACT Quorum-sensing (QS) systems exist universally in bacteria to regulate multiple biological functions. _Klebsiella pneumoniae_, an industrially important bacterium that produces

bio-based chemicals such as 2,3-butanediol and acetoin, can secrete a furanosyl borate diester (AI-2) as the signalling molecule mediating a QS system, which plays a key regulatory role in

the biosynthesis of secondary metabolites. In this study, the molecular regulation and metabolic functions of a QS system in _K. pneumoniae_ were investigated. The results showed that after

the disruption of AI-2-mediated QS by the knockout of _luxS_, the production of acetoin, ethanol and acetic acid were relatively lower in the _K. pneumoniae_ mutant than in the wild type

bacteria. However, 2,3-butanediol production was increased by 23.8% and reached 54.93 g/L. The observed enhancement may be attributed to the improvement of the catalytic activity of

2,3-butanediol dehydrogenase (BDH) in transforming acetoin to 2,3-butanediol. This possibility is consistent with the RT-PCR-verified increase in the transcriptional level of _budC_, which

encodes BDH. These results also demonstrated that the physiological metabolism of _K. pneumoniae_ was adversely affected by a QS system. This effect was reversed through the addition of

synthetic AI-2. This study provides the basis for a QS-modulated metabolic engineering study of _K. pneumoniae_. SIMILAR CONTENT BEING VIEWED BY OTHERS PYRUVATE-RESPONSIVE GENETIC CIRCUITS

FOR DYNAMIC CONTROL OF CENTRAL METABOLISM Article 07 September 2020 REDESIGNING REGULATORY COMPONENTS OF QUORUM-SENSING SYSTEM FOR DIVERSE METABOLIC CONTROL Article Open access 21 April 2022

AGR QUORUM SENSING INFLUENCES THE WOOD-LJUNGDAHL PATHWAY IN _CLOSTRIDIUM AUTOETHANOGENUM_ Article Open access 10 January 2022 INTRODUCTION A communication system among bacteria was first

discovered in the 1970s while studying the luminescence mechanism of _Photobacterium fischeri_1. This phenomenon was observed in the luminescence organs of marine organisms and was named

quorum sensing (QS)2. In bacterial cells, QS consists of small, diffusible signalling molecules (autoinducers) capable of sensing the density of bacterial cells and subsequently initiating

the coordinated expression of several key genes throughout the entire bacterial community when the autoinducer concentration exceeds a critical threshold in the cells3,4. Recent research has

led to the discovery of some new autoinducers in bacteria, illustrated how these autoinducers are recognized by cognate receptors, revealed new regulatory components embedded in canonical

signalling circuits and identified novel regulatory network designs5. At the same time, QS is also a mechanism for bacterial adaptation to the environment. QS systems exist in multiple

bacterial species3, and the luxR family of proteins in this system play a key role as transcription regulators2. With LuxI protein and signalling molecules, QS systems can modulate a variety

of physiological functions, such as bioluminescence1, symbiosis6, Ti plasmid conjugative transfer7, biofilm formation8, group mobility, pathogenesis9, among others. Notably, the QS system

in _Pseudomonas aeruginosa_ involves two discrete acyl-homoserine lactone (AHL) molecules (OdDHL and BHL) that are generated and sensed by two different signalling systems (LasIR and RhlIR),

which control the production of a diverse variety of virulence factors and biofilm formation, respectively4. Recently, the emergence and worldwide spread of antibiotic-resistant bacteria

have increased the importance of finding therapeutic alternatives to compensate for the reduced effectiveness of antibiotics10. QS systems have also been suggested as promising targets for

developing new anti-infective compounds based on the regulatory function of these systems in the pathogenesis of bacteria11. It is reasonable to consider that targeting the QS system would

put less selective pressure on pathogens, thus avoiding the development of resistant bacteria and combating some of the most hard-to-treat infections that are resistant to powerful

antibiotics11. Fundamental research into the QS mechanism has revealed suprising discoveries4. For example, the signalling molecule _N_-(3-oxododecanoyl)-l-homoserine lactone can cause

inflammation and induce immunomodulatory activity12, which may reveal new drug targets. Currently, the gradual exhaustion of petroleum has revived significant interest in producing bio-based

bulk chemicals, including 2,3-butanediol, from biomass13. The demand and manufacture of 2,3-butanediol are increasing worldwide due to the extensive industrial applications of this

chemical14. For example, 2,3-butanediol can be used as a liquid fuel additive because of its high combustion value (27.198 Jg−1), as an antifreeze agent due to the low freezing point of −60 

°C, as a carrier for pharmaceuticals and as a precursor in the production of methyl ethyl ketone and 1,3-butadiene through dehydration. In addition, 2,3-butanediol is easily dehydrogenated

into acetoin and diacetyl, which can be used in food and cosmetics similar to flavouring agents14,15. _Klebsiella pneumoniae_16, a Gram-negative bacterium, is an industrially important

bacterium that produces bio-based chemicals, including 2,3-butanediol and acetoin17. _K. pneumoniae_ contains AI-1 type QS quenching enzymes, AHL lactonases18. The AHL lactonases in

prokaryotes can be categorized into 2 clusters: _AiiA_ and _AttA_. The lactonase from _K. pneumoniae_ belongs to the _AttA_ cluster and shares only 30% homology with lactonases from other

species18,19. In the submerged fermentation of _K. pneumonia_, because AHL lactonase is present, AHLs cannot reach the threshold concentration in the culture medium, cannot diffuse back into

the cell and cannot be recognized by the receptor protein. Therefore, an AHL-receptor protein complex cannot form, and the AHL-mediated QS system does not work. However, _K. pneumoniae_

contains _metK, pfs_ and _luxS_ genes, which all encode key enzymes in the synthesis of the signalling molecule AI-2, suggesting that _K. pneumoniae_ uses AI-2 as the signalling molecule in

a QS system to regulate group behaviours20,21. In nature, the tight regulation of AI-2 production by _K. pneumoniae_ occurs primarily at the level of _luxS_ transcription in the synthetic

pathway of AI-2, and the maximum AI-2 activity occurs during the late exponential phase, which was determined by our laboratory16. Mutations in _luxS_ induce an increase in the expression of

two lipopolysaccharide (LPS)-synthesis-related genes, _wbbM_ and _wzm_, which affect the biofilm formation of _K. pneumoniae_21. As such, this _LuxS_-dependent signal also plays a main role

in the early stages of biofilm formation by _K. pneumoniae_20. Therefore, it was concluded that AI-2 acts as a regulator of biofilm formation and LPS synthesis in _K. pneumoniae_21.

According to the primary literature, 2,3-butanediol fermentation is dependent on an AHL-dependent QS system in _Serratia_ spp.22. Although 2,3-butanediol production by _K. pneumoniae_ has

also been well studied, it was not known how 2,3-butanediol was regulated by an AI-2-mediated QS system until now. In this study, the molecular regulation and metabolic function of the QS

system in _K. pneumoniae_ was analysed based on previous studies of the cellular metabolic network of _K. pneumoniae_ and transcriptional characteristics of the QS system. Additionally, a

drift in the metabolic flux and changes in the physiological metabolic network of _K. pneumoniae_ were also investigated. The results presented here will lay the foundation for elucidating

the interplay between industrial microbial metabolism and QS systems. RESULTS REGULATION OF THE AI-2-MEDIATED QS SYSTEM IN _K. PNEUMONIAE_ The QS system of _K. pneumoniae_ is

_luxS_-dependent16; therefore, it is possible to regulate QS by inactivating a key gene, _luxS_, through mutagenesis. The gene product of _luxS_ is involved in the synthetic pathway of the

signalling molecule AI-2. Chromosomal _luxS_ was inactivated in each strain using a marker-exchange strategy based on the suicide vector pUTKm as described in the Materials and Methods. The

recombinant plasmid pUTKm-_luxS_ was introduced into _K. pneumoniae_ CICC 10018 competent cells by electrotransformation, followed by screening of the kanamycin-resistant _luxS_ mutants16.

Bacterial growth on the kanamycin plates was evident, suggesting that the _luxS_ gene was knocked out. Clones that were kanamycin-resistant were verified by PCR assays of genomic DNA. An

800-bp (as expected) fragment of the kanamycin-resistance gene was PCR-amplified with primers Kna-1 and Kna-2 (Fig. 1). Additionally, the samples were also verified by commercial sequencing.

The correct recombinant strain containing a kanamycin-resistance gene insertion in a chromosome was picked out (named _K. pneumoniae-6_) and was cultured in LB medium for further

experiments. EFFECTS OF QS QUENCHING ON THE METABOLIC FLUX OF _K. PNEUMONIAE_ As shown in Fig. 2, the production of 2,3-butanediol initially increased and then declined in both _K.

pneumoniae_ and _K. pneumoniae-6_ during fermentation. The production of 2,3-butanediol in _K. pneumoniae_ reached a peak at 12 h, whereas the peak production in _K. pneumoniae-6_ occurred

at 8 h. A possible explanation for this observation is the enhanced bioconversion to 2,3-butanediol under the catalysis of related enzymes in the metabolic flux. During this process, the

production of acetoin, acetic acid and ethanol was relatively lower in _K. pneumoniae-6_ than in _K. pneumoniae_, which may be attributed to the carbon flux shift because there was almost no

change in the catalytic activity of related enzymes involved in the biosynthesis of these compounds (data not shown). Notably, the decreased levels of these 3 compounds in the _luxS_ mutant

could be restored to the levels in the parental strain in the presence of 5 μM synthetic AI-2. Although _K. pneumoniae-6_ grew less well than its parent strain, the defective growth of _K.

pneumoniae-6_ could also be nullified by the addition of 5 μM synthetic AI-2. COMPARISON OF ENZYMATIC ACTIVITY DURING METABOLISM There are two metabolic pathways in bacteria for the

biosynthesis of 2,3-butanediol from α-acetolactic acid. In the first, α-acetolactic acid is converted to acetoin (the key precursor of 2,3-butanediol) by the catalysis of α-acetolactate

decarboxylase (α-ALDC). Next, acetoin is converted to 2,3-butanediol through the catalysis of 2,3-butanediol dehydrogenase (BDH). In the second metabolic pathway, α-acetolactic acid is

oxidized to diacetyl, and diacetyl is converted to acetoin through the catalysis of diacetyl reductase (DR). Subsequently, acetoin is converted to 2,3-butanediol through the catalysis of

BDH. Therefore, the enzymatic activities of BDH, α-ALDC and DR in _K. pneumoniae_ and _K. pneumoniae-6_ were measured at the indicated time points after preparing a crude enzyme solution.

Figure 3 shows that the activities of these 3 enzymes first increased and then declined with time in both strains. The maximal enzymatic activities of BDH, α-ALDC and DR occurred at similar

times in both strains, at 8 h, 12 h and 8 h, respectively. The enzymatic activity of α-ALDC was similar in both strains, whereas the enzymatic activity of BDH and DR was relatively lower in

_K. pneumoniae_. This lower DR activity level decreased the synthesis of acetoin from diacetyl and increased the accumulation of diacetyl in the fermentation broth of _K. pneumoniae_.

Additionally, the activity of α-acetolactate synthase, which catalyses the biosynthesis of α-acetolactic acid from pyruvic acid, was also determined, but there was no significant difference

between these two strains (data not shown). In _K. pneumoniae_, the enzymatic synthesis and metabolic control of 2,3-butanediol and acetoin were tightly regulated by QS. The knockout of

_luxS_ led to the destruction and functional disability of the QS system. As a result, 2,3-butanediol production was improved, and it was confirmed that this improvement was caused by a

change in the expression level of enzymes (BDH and DR) involved in the synthetic pathway of 2,3-butanediol. These results suggest that the knockout of _luxS_ can result in variations in

enzymatic activities and further affect 2,3-butanediol biosynthesis. According to these results, a number of conclusions can be drawn. After quenching the QS system in _K. pneumoniae-6_, the

activity of DR improved significantly, promoting the conversion from diacetyl to acetoin. The increased activity of BDH accelerated the biosynthesis of 2,3-butanediol using acetoin as a

substrate, and therefore, the production of 2,3-butanediol was greatly enhanced. ANALYSIS OF BUDC TRANSCRIPTION The gene _budC_ encodes BDH, which catalyses the reaction of acetoin to

2,3-butanediol. As depicted in Fig. 4, the _budC_ gene was constitutively expressed at the tested time points. Additionally, the number of _budC_ transcripts first increased and then

decreased in _K. pneumoniae-6_, and the maximal transcription levels occurred at 8 h. This result may explain why the maximal enzymatic activity of BDH and the maximal 2,3-butanediol

production took place at 8 h. During the exponential growth phase in _K. pneumoniae-6_, the _budC_ gene expression increased dramatically to a maximal value. However, _budC_ expression

decreased upon entry into the stationary growth phase. Maximal 2,3-butanediol production and _budC_ expression occurred at the same time point (8 h), which suggests that the difference in

2,3-butanediol production by _K. pneumoniae_ and _K. pneumoniae-6_ is due to changes at the transcriptional level of the _budC_ gene during the growth of these two bacteria. DISCUSSION The

chemical butanol is a four-carbon diol that has wide industrial applications for the manufacture of bulk chemicals23. In general, butanol is produced from carbohydrates in submerged

fermentation by _Klebsiella_ spp.24,25,26,27, _Enterobacter_ spp.28,29,30,31, _Bacillus_ spp.32,33,34, and _Clostridia_ spp.35,36,37,38, among others. Since butanol-producing strains were

initially screened from natural environments, the yield, titre and productivity regarding butanol were frequently low, and these strains could not be used in industrial production.

Therefore, the current focus is on finding new strains from natural reservoirs and constructing or modifying strains through mutagenesis, evolutionary engineering and metabolic engineering

strategies to improve their production performance and compliance23. _K. pneumoniae_ and _Klebsiella oxytoca_ are important bio-based chemical-producing bacteria and can ferment glucose

primarily to 2,3-butanediol as a major fermentation end-product with relatively small amounts of acetoin, ethanol and some acids under micro-aerobic conditions because a significant amount

of pyruvate from glycolysis is channelled into the butanediol pathway, through which pyruvate is transformed into butanediol by the catalysis of acetolactate synthase, α-ALDC and BDH in an

orderly fashion17,27. To improve 2,3-butanediol production by _Klebsiella_ spp., _strain_ modification by metabolic engineering has attracted a great deal of attention around the

world17,24,25,26,27. For example, Rathnasingh _et al_. improved the production of 2,3-butanediol in _K. pneumoniae_ by knocking out the genes (_ldhA, adhE and pta-ackA_) involved in the

formation of lactic acid, ethanol and acetic acid; the 2,3-butanediol production thus reached 91 g/L with a yield of 0.45 g per g glucose in batch fermentation27. Guo _et al_. constructed

_K. pneumoniae_ mutants that overexpressed α-ALS, α-ALDC, and AR to improve 2,3-butanediol production. The results revealed that 2,3-butanediol production by the recombinant _K. pneumoniae_

strain (KG-rs) that overexpressed both ALS and AR was 12% higher than that of the parental strain17. Cho _et al_. reported that 2,3-butanediol production reached 115.0 g/L in fed-batch

fermentation with pure glycerol with the construction of a double mutant (_pduC, ldhA) K. oxytoca_ strain to reduce the formation of 1,3-propanediol and lactic acid24. This group also

discovered that if acetoin reductase was overexpressed in _K. oxytoca_, acetoin accumulation was significantly reduced, and the highest titre of 2,3-butanediol (142.5 g/L) was achieved25.

Currently, metabolic engineering strategies at the gene level are commonly used by many scientists to reform certain special production strains. Additionally, there has been growing interest

in developing novel metabolic engineering strategies at the cellular level based on the QS system in bacteria39,40. For example, in bacteria of the _Serratia_ genera, 2,3-butanediol

fermentation has been shown to be affected by an _spl_I-dependent QS system, and the knockout of the _spl_I gene caused a shift towards enhanced acid production. Of course, the biosynthesis

of 2,3-butanediol is not completely shut off by eliminating QS. At the same time, QS also controls the production of extracellular enzymes, including chitinase, nuclease, and protease13,22.

In _Vibrio cholera,_ the production of 2,3-butanediol is regulated by multiple QS systems via the transcriptional activator AphA. Two QS systems use a CAI-1 autoinducer and AI-2 as

signalling molecules, respectively, and act in parallel to trigger a phosphorelay circuit41. In _Aeromonas hydrophila_, 2,3-butanediol fermentation is regulated by AHL-mediated QS because

the disruption of QS by the knockout of _ahyI_, synthesizing C4-HSL, results in medium acidification and blocks the metabolic switch to 2,3-butanediol synthesis13. Furthermore, the

inactivation of the regulatory protein AhyR in QS also suppressed 2,3-butanediol fermentation. Although 2,3-butanediol production by _K. pneumoniae_ has been well studied26,27, the data in

this report represent the first identification of a QS system regulating 2,3-butanediol production. In this study, the effects of QS on 2,3-butanediol formation in _K. pneumoniae_ were

analysed in great detail. _K. pneumoniae_ contains AI-1 type QS quenching enzymes, including AHL lactonase, and possesses _pfs_ and _luxS_ orthologues in its genome; therefore, it can be

inferred that AI-2 is the signalling molecule mediating the QS system40. In the biosynthesis of AI-2, _luxS_ and _pfs_ encode a 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase and

form an operon that utilizes SAH or MTA as a substrate for AI-2 production16. Furthermore, the tight regulation of AI-2 production is largely at the level of _luxS_ transcription.

Therefore, the molecular regulation of the QS system in _K. pneumoniae_ was established by constructing a _luxS_ mutant. Next, changes in the metabolites between _K. pneumoniae-6 (luxS_

mutant) and _K. pneumoniae_ (parental strain) were compared through shaking fermentation with glucose. The results indicated that after quenching the QS system, the production of acetoin,

ethanol and acetic acid was relatively lower in _K. pneumoniae-6_, but the 2,3-butanediol production was increased by 23.8% and reached a maximum level of 54.93 g/L. This increase suggested

that the activation of the 2,3-butanediol pathway is not QS regulated, which is different from _Serratia_ spp.22. The precise mechanism of regulation has yet to be elucidated. This

improvement effect was reversed through the addition of 5 μM synthetic AI-2. At the same time, enzymatic activity analyses revealed that there was no significant difference in α-ALDC

activity between these two strains, whereas the enzymatic activities of BDH and DR were relatively lower in _K. pneumoniae_, in accordance with the transcriptional analysis of _budC._ These

findings should be useful for improving 2,3-butanediol production via QS-based metabolic engineering. Furthermore, this study provides a solid basis for investigating the link between QS and

bacterial physiology. Although the development of butanol-producing microorganisms and fermentation processes has seen remarkable progress, the recovery of butanol from the fermentation

broth still remains a challenging problem under industrial production conditions23. To develop commercially applicable techniques for the recovery of 2,3-butanediol, numerous methods have

been proposed42, _including_ ion exchange, electrodialysis, membrane filtration, pervaporation, reactive extraction and liquid-liquid extraction43,44. However, these methods have inherent

drawbacks that must be overcome, such as generating a large amount of wastewater for resin regeneration, requiring expensive membranes, increasing filtration steps and decreasing the

recovery yield of 2,3-butanediol42. In recent years, many scientists have been developing _in situ_ product recovery techniques, especially for the low concentration of 2,3-butanediol in

fermentation broth. Jeon _et al_. established a four-step recovery process through alcohol precipitation and vacuum distillation, and a recovery yield of 76.2% and a purity of 96.1% were

obtained from fermentation broth containing approximately 90 g/L of 2,3-butanediol produced by the _ldhA_-deficient _K. pneumoniae_42. Xue _et al_. reported a succession of various methods

for the recovery of butanol produced by _Clostridium acetobutylicum_, such as gas stripping37,38, adsorption35, and a vapour stripping-vapour permeation (VSVP) process36. When gas stripping

was applied intermittently in fed-batch fermentation, 195.9 g/L acetone-butanol-ethanol or 150.5 g/L butanol was obtained, and furthermore, energy consumption and water usage was reduced by

at least 90%. Two-stage gas stripping was more effective for producing high-titre butanol, and a highly concentrated product containing 420.3 g/L butanol (532.3 g/L acetone-butanol-ethanol)

can be obtained with this strategy. At the same time, this purification process consumed much less energy. Compared to pervaporation and gas stripping, the VSVP process produced a condensate

containing 212.0–232.0 g/L butanol from a fermentation broth containing ~10 g/L butanol, which suggests that the VSVP process has great potential for efficient butanol recovery. In

acetone-butanol-ethanol fermentation, the _in situ_ product recovery process with activated carbon was also carried out. The results indicated that immobilized-cell fermentation with

adsorption produced 54.6 g/L butanol with a productivity of 0.45 g/L·h, and a condensate containing approximately 167 g/L butanol was obtained after thermal desorption. Furthermore, this

liquid phase _adsorption using_ activated carbon was energy efficient and can be easily applied in butanol fermentation. In the future, integrated downstream processing technologies for

fermentative 2,3-butanediol are especially required regarding yield, purity, and energy consumption. MATERIALS AND METHODS BACTERIAL STRAINS, PLASMIDS, PRIMERS AND REAGENTS The bacterial

strains, plasmids and primers used in this study are listed in Table 1. All _E. coli_ and _K. pneumoniae_ strains were grown in Luria-Bertani (LB) growth medium (0.5% yeast extract, 1%

tryptone, and 1% NaCl) at 37 °C and 30 °C, respectively. Antibiotics were added in the following amounts (per mL) when necessary: 100 μg ampicillin and 50 μg kanamycin for _E. coli_ and _K.

pneumoniae_, respectively. All enzymes, DNA and protein markers, and Kits were from TaKaRa Biotech (Dalian, China). Other chemicals were analytical reagent grade. All the oligonucleotide

primers were synthesized in Bioasia Biotech (Shanghai, China). QUENCHING THE QS SYSTEM IN _K. PNEUMONIAE_ The QS mechanism of _K. pneumoniae_ through the _LuxS_/_AI-2 signalling system_ has

played an important role in understanding the functions of QS systems. Therefore, the regulation of the QS system was determined by constructing a QS _luxS_ knockout mutant strain. A 260-bp

fragment from the _luxS_ gene encoding a key enzyme for AI-2 synthesis was amplified from the genomic DNA of _K. pneumoniae_ CICC 10018 using a PCR technique with the primers luxS-1 and

luxS-2. Commercial sequencing was used to verify these mutants. The _luxS_ fragment and the pUTKm plasmid were double digested with _Kpn_ I and _Sca_ I and then ligated between the _Kpn_ I

and _Sca_ I sites in the plasmids. Successful ligation resulted in generating a marker-exchange plasmid, pUTKm-_luxS_, which was transformed into _E. coli_ cc118 competent cells. The

recombinant pUTKm-_luxS_ cells were selected and confirmed using DNA sequencing and double enzyme digestion. Subsequently, the suicide vector pUTKm-_luxS_ was transformed into _K.

pneumoniae_ CICC 10018 competent cells by electroporation. Mutants were selected on NB medium containing 800 μg/mL ampicillin and 200 μg/mL kanamycin. For each strain with a

kanamycin-resistance gene inserted into the chromosome, the disruption of the locus was confirmed by PCR analysis using the primers Kan-1 and Kan-2 complementary to the KnR cassette. This

mutant was named _K. pneumoniae-6_. GENE EXPRESSION AND METABOLIC FLUX ANALYSIS The _budC_ gene encodes BDH, which is involved in the biosynthetic pathway of 2,3-butanediol. An RT-PCR

analysis was performed with SYBR Green technology to confirm changes in the transcription level of the _budC_ gene. To prepare an external plasmid standard curve, the plasmid pGM-T-_budC_

was constructed. The _budC_ gene was amplified from _K. pneumoniae_ with the primers budC-1 and budC-2 using genomic DNA as a template and was cloned into vector pGM-T to generate the

standard plasmid pGM-T. The purified plasmid pGM-T-_budC_ was serially diluted (1:10) over the appropriate concentration range (usually 105–109). To achieve a reliable standard curve for

each measured parameter, the plasmid was PCR-amplified in five replicates for each standard dilution point over the complete standard curve range. Figure 4A shows the standard curves for the

_budC_ gene, which were used for the determination of _budC_ gene transcription. After preparing the standard, total RNA was isolated from the _K. pneumoniae-6_ sample during growth in the

fermentation medium and was reverse transcribed. The resulting cDNA was directly subjected to real-time PCR with the primers F-budC and R-budC. At the same time, the production of the

primary metabolites (acetic acid, ethanol, acetoin and 2,3-butanediol) by _K. pneumoniae_ was assessed with the corresponding methods listed in the “Analytical methods” section. BATCH

FERMENTATION AT BIOREACTOR SCALE Submerged fermentation experiments were carried out in a bioreactor to investigate the changes in metabolic flux in _K. pneumoniae_. The fermentation medium

was composed of 90 g/L glucose, 13.6 g/L KH2PO4, 5 g/L (NH4)2SO4, 4 g/L MgSO4·7H2O, 4 g/L Citric acid, 15 g/L yeast extract, 4 g/L NaCl, 0.4 g/L CaCl2, 0.08 g/L FeSO4 and 0.3 mL of trace

elements prepared as described. The trace elements consisted of 34.2 g of ZnCl2, 2.7 g of FeCl3·6H2O, 10 g of MnCl2·4H2O, 0.85 g of CuCl2·2H2O, 23.8 g of CoCl2·6H2O, 0.31 g of H3BO3 and 0.25

 g of Na2MoO4·2H2O in 1 L of deionized water. Unless otherwise specified, the submerged cultures of _K. pneumoniae_ for the production of 2,3-butanediol were maintained under the following

culture conditions: temperature, 30 °C; aeration rate, 4 vvm; initial pH, 7.0; and a working volume of 3.5 L. All experiments were performed in triplicate. ANALYTICAL METHODS The primary

components, ethanol, acetic acid, acetoin, and 2,3-butanediol, and other metabolites in the fermentation broth of _K. pneumonia_ were detected using gas chromatography as summarized in the

following steps: (1) An Agilent7890A DB-TPH column was used; (2) the detector temperature was set at 250 °C; (3) the column temperature was set at 100 °C for 2 min; (4) the temperature was

increased to 180 °C at 20 °C/min and maintained for 1 min, then (5) the temperature was increased to 220 °C at 30 °C/min and was maintained for 2 min; finally, (6) the sample was injected in

a volume of 1 μL. DETERMINATION OF ENZYMATIC ACTIVITY * 1 Preparation of crude enzyme Thirty millilitres of bacterial suspension collected at the indicated time points during fermentation

was centrifuged at 8000 rpm for 5 min at 4 °C. The pellet was re-suspended twice with PBS and centrifuged at 8000 rpm for 5 min at 4 °C. Subsequently, 6 mL of buffer was added, and the

mixture was ultrasonicated 99 × for 2s with 5-s intervals. The sonication procedure was repeated three times, followed by centrifugation at 8000 rpm for 20 min at 4 °C. The supernatant was

the crude enzyme. * 2 Determination of α-ALDC activity The amount of enzyme required to convert α-acetolactic acid into 1 μmol of acetoin at 37 °C within a unit interval (min) is defined as

a unit of enzymatic activity. The procedure was as follows: 25 μL of α-acetoxy-α-methyl-ethyl acetate was sufficiently mixed with 750 μL of 1 M NaOH and 750 μL of deionized water and

incubated at room temperature for 20 min. The volume was then adjusted to 10 mL with PBS (pH 6). The pH was adjusted to pH 6 with 0.5 M HCl, and the volume was adjusted to 12.5 mL with PBS

(pH 6). Two hundred microlitres of the final solution was mixed with 200 μL of crude enzyme and 100 μL of PBS (pH 6, containing 0.05% (w/v) Tween 80 and 600 mM NaCl). After sufficient

mixing, the mixture was incubated in a 37 °C water bath for 20 min. Afterwards, 4.5 mL of a chromogenic agent (2.5 g of α-naphthol and 0.25 g of creatine in a volume of 250 mL of 1 M NaOH)

was added to the mixture, which was then incubated for 40 min at room temperature, followed by OD measurement at 522 nm. * 3 Determination of BDH activity The sample solution contained 2330 

μL of PBS buffer (1 mmol of ZnSO4, 20 μL of crude enzyme and 300 μL of 10 mM NAD+). This solution was the same as the control solution. Following the baseline determination, 30 μL of 1 mM

2,3-butanediol solution was added to the sample solution, while 300 μL of deionized water was added to the blank control solution. After mixing, the change in OD was measured at 340 nm. * 4

Determination of DR activity The sample solution contained 2330 μL of PBS buffer, 20 μL of crude enzyme and 300 μL of 10 mM diacetyl solution. To balance the baseline, 300 μL of 10 mM NADH

solution was added to the sample solution, while 300 μL of deionized water was added to the blank control solution. After mixing the solutions, OD measurements were performed at 340 nm.

ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Sun, S. _et al_. The metabolic flux regulation of _Klebsiella pneumoniae_ based on quorum sensing system. _Sci. Rep._ 6, 38725; doi:

10.1038/srep38725 (2016). PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. REFERENCES * Nealson,

K. H., Platt, T. & Hastings, J. W. Cellular control of the synthesis and activity of the bacterial luminescent system. Journal of Bacteriology 104, 313–322 (1970). CAS  PubMed  PubMed

Central  Google Scholar  * Fuqua, W. C., Winans, S. C. & Greenberg, E. P. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. Journal

of Bacteriology 176, 269–275 (1994). Article  CAS  Google Scholar  * Miller, M. B. & Bassler, B. L. Quorum sensing in bacteria. Annual Review of Microbiology 55, 165–199, doi:

10.1146/annurev.micro.55.1.165 (2001). Article  CAS  PubMed  Google Scholar  * Hodgkinson, J. T., Welch, M. & Spring, D. R. Learning the language of bacteria. ACS Chemical Biology 2,

715–717, doi: 10.1021/cb700227k (2007). Article  CAS  PubMed  Google Scholar  * Papenfort, K. & Bassler, B. L. Quorum sensing signal-response systems in Gram-negative bacteria. Nature

Reviews Microbiology 14, 576–588, doi: 10.1038/nrmicro.2016.89 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Verma, S. C. & Miyashiro, T. Quorum sensing in the

squid-_Vibrio_ symbiosis. International Journal of Molecular Sciences 14, 16386–16401, doi: 10.3390/ijms140816386 (2013). Article  CAS  PubMed  PubMed Central  Google Scholar  * Khan, S. R.

& Farrand, S. K. The BlcC (AttM) lactonase of _Agrobacterium tumefaciens_ does not quench the quorum-sensing system that regulates Ti plasmid conjugative transfer. Journal of

Bacteriology 191, 1320–1329, doi: 10.1128/JB.01304-08 (2009). Article  CAS  PubMed  Google Scholar  * McNab, R. et al. LuxS-based signaling in _Streptococcus gordonii_: autoinducer 2

controls carbohydrate metabolism and biofilm formation with _Porphyromonas gingivalis_. Journal of Bacteriology 185, 274–284, doi: 10.1128/JB.185.1.274-284.2003 (2003). Article  CAS  PubMed

  PubMed Central  Google Scholar  * Ohtani, K., Hayashi, H. & Shimizu, T. The _luxS_ gene is involved in cell-cell signalling for toxin production in _Clostridium perfringens_. Molecular

Microbiology 44, 171–179, doi: 10.1046/j.1365-2958.2002.02863.x (2002). Article  CAS  PubMed  Google Scholar  * Rasamiravaka, T. & El Jaziri, M. Quorum-sensing mechanisms and bacterial

response to antibiotics in _P. aeruginosa_. Current Microbiology, doi: 10.1007/s00284-016-1101-1 (2016). * Reuter, K., Steinbach, A. & Helms, V. Interfering with bacterial quorum

sensing. Perspectives in Medicinal Chemistry 8, 1–15, doi: 10.4137/PMC.S13209 (2016). Article  PubMed  PubMed Central  Google Scholar  * Pritchard, D. I. Immune modulation by _Pseudomonas

aeruginosa_ quorum-sensing signal molecules. International Journal of Medical Microbiology 296, 111–116, doi: 10.1016/j.ijmm.2006.01.037 (2006). Article  CAS  PubMed  Google Scholar  * Van

Houdt, R., Aertsen, A. & Michiels, C. W. Quorum-sensing-dependent switch to butanediol fermentation prevents lethal medium acidification in _Aeromonas hydrophila_ AH-1N. Research in

Microbiology 158, 379–385, doi: 10.1016/j.resmic.2006.11.015 (2007). Article  CAS  PubMed  Google Scholar  * Zhang, L. et al. Microbial production of 2,3-butanediol by a surfactant

(serrawettin)-deficient mutant of _Serratia marcescens_ H30. Journal of Industrial Microbiology & Biotechnology 37, 857–862, doi: 10.1007/s10295-010-0733-6 (2010). Article  CAS  Google

Scholar  * Zhang, L. et al. Mechanism of 2,3-butanediol stereoisomers formation in a newly isolated _Serratia_ sp. T241. Scientific Reports 6, 19257, doi: 10.1038/srep19257 (2016). Article 

CAS  ADS  PubMed  PubMed Central  Google Scholar  * Zhu, H., Liu, H. J., Ning, S. J. & Gao, Y. L. A _luxS_-dependent transcript profile of cell-to-cell communication in _Klebsiella

pneumoniae_. Molecular Biosystems 7, 3164–3168, doi: 10.1039/c1mb05314k (2012). Article  CAS  Google Scholar  * Guo, X. W. et al. Enhanced production of 2,3-butanediol by overexpressing

acetolactate synthase and acetoin reductase in _Klebsiella pneumoniae_. Biotechnology and Applied Biochemistry 61, 707–715, doi: 10.1002/bab.1217 (2014). Article  CAS  PubMed  Google Scholar

  * Park, S. Y. et al. AhlD, an _N_-acylhomoserine lactonase in _Arthrobacter_ sp., and predicted homologues in other bacteria. Microbiology 149, 1541–1550, doi: 10.1099/mic.0.26269-0

(2003). Article  CAS  PubMed  Google Scholar  * Carlier, A. et al. The Ti plasmid of _Agrobacterium tumefaciens_ harbors an attM-paralogous gene, aiiB, also encoding _N_-Acyl homoserine

lactonase activity. Applied and Environmental Microbiology 69, 4989–4993, doi: 10.1128/AEM.69.8.4989-4993.2003 (2003). Article  CAS  PubMed  PubMed Central  Google Scholar  * Balestrino, D.,

Haagensen, J. A., Rich, C. & Forestier, C. Characterization of type 2 quorum sensing in _Klebsiella pneumoniae_ and relationship with biofilm formation. Journal of Bacteriology 187,

2870–2880, doi: 10.1128/JB.187.8.2870-2880.2005 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  * De Araujo, C., Balestrino, D., Roth, L., Charbonnel, N. & Forestier, C.

Quorum sensing affects biofilm formation through lipopolysaccharide synthesis in _Klebsiella pneumoniae_. Research in Microbiology 161, 595–603, doi: 10.1016/j.resmic.2010.05.014 (2010).

Article  CAS  PubMed  Google Scholar  * Van Houdt, R., Moons, P., Hueso Buj, M. & Michiels, C. W. _N_-acyl-L-homoserine lactone quorum sensing controls butanediol fermentation in

_Serratia plymuthica_ RVH1 and _Serratia marcescens_ MG1. Journal of Bacteriology 188, 4570–4572, doi: 10.1128/JB.00144-06 (2006). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Xue, C., Zhao, X. Q., Liu, C. G., Chen, L. J. & Bai, F. W. Prospective and development of butanol as an advanced biofuel. Biotechnology Advances 31, 1575–1584, doi:

10.1016/j.biortech.2014.04.047 (2013). Article  CAS  PubMed  Google Scholar  * Cho, S. et al. High production of 2,3-butanediol from biodiesel-derived crude glycerol by metabolically

engineered _Klebsiella oxytoca_ M1. Biotechnology for Biofuels 8, 146, doi: 10.1186/s13068-015-0336-6 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cho, S. et al. Enhanced

2,3-butanediol production by optimizing fermentation conditions and engineering _Klebsiella oxytoca_ M1 through overexpression of acetoin reductase. PLoS One 10, e0138109, doi:

10.1371/journal.pone.0138109 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Kumar, V. et al. Effects of mutation of 2,3-butanediol formation pathway on glycerol metabolism

and 1,3-propanediol production by _Klebsiella pneumoniae_ J2B. Bioresource Technology 214, 432–440, doi: 10.1016/j.biortech.2016.04.032 (2016). Article  CAS  PubMed  Google Scholar  *

Rathnasingh, C., Park, J. M., Kim, D. K., Song, H. & Chang, Y. K. Metabolic engineering of _Klebsiella pneumoniae_ and _in silico_ investigation for enhanced 2,3-butanediol production.

Biotechnology Letters 38, 975–982, doi: 10.1007/s10529-016-2062-y (2016). Article  CAS  PubMed  Google Scholar  * Jung, M. Y., Ng, C. Y., Song, H., Lee, J. & Oh, M. K. Deletion of

lactate dehydrogenase in _Enterobacter aerogenes_ to enhance 2,3-butanediol production. Applied Microbiology and Biotechnology 95, 461–469, doi: 10.1007/s00253-012-3883-9 (2012). Article 

CAS  PubMed  Google Scholar  * Jung, M. Y., Park, B. S., Lee, J. & Oh, M. K. Engineered _Enterobacter aerogenes_ for efficient utilization of sugarcane molasses in 2,3-butanediol

production. Bioresource Technology 139, 21–27, doi: 10.1016/j.biortech.2013.04.003 (2013). Article  CAS  PubMed  Google Scholar  * Li, L. et al. Metabolic engineering of _Enterobacter

cloacae_ for high-yield production of enantiopure (2R,3R)-2,3-butanediol from lignocellulose-derived sugars. Metabolic Engineering 28, 19–27, doi: 10.1016/j.ymben.2014.11.010 (2015). Article

  CAS  PubMed  Google Scholar  * Zhang, C. Y., Peng, X. P., Li, W., Guo, X. W. & Xiao, D. G. Optimization of 2,3-butanediol production by _Enterobacter cloacae_ in simultaneous

saccharification and fermentation of corncob residue. Biotechnology and Applied Biochemistry 61, 501–509, doi: 10.1002/bab.1198 (2014). Article  CAS  PubMed  Google Scholar  * Qiu, Y. et al.

Engineering _Bacillus licheniformis_ for the production of meso-2,3-butanediol. Biotechnology for Biofuels 9, 117, doi: 10.1186/s13068-016-0522-1 (2016). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Fu, J. et al. Metabolic engineering of _Bacillus subtilis_ for chiral pure meso-2,3-butanediol production. Biotechnology for Biofuels 9, 90, doi:

10.1186/s13068-016-0502-5 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Deshmukh, A. N., Nipanikar-Gokhale, P. & Jain, R. Engineering of _Bacillus subtilis_ for the

production of 2,3-butanediol from sugarcane molasses. Applied Biochemistry and Biotechnology 179, 321–331, doi: 10.1007/s12010-016-1996-9 (2016). Article  CAS  PubMed  Google Scholar  * Xue,

C. et al. Butanol production in acetone-butanol-ethanol fermentation with _in situ_ product recovery by adsorption. Bioresource Technology 219, 158–168, doi: 10.1016/j.biortech.2016.07.111

(2016). Article  CAS  PubMed  Google Scholar  * Xue, C. et al. The vital role of citrate buffer in acetone-butanol-ethanol (ABE) fermentation using corn stover and high-efficient product

recovery by vapor stripping-vapor permeation (VSVP) process. Biotechnology for Biofuels 9, 146, doi: 10.1186/s13068-016-0566-2 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Xue, C. et al. Two-stage _in situ_ gas stripping for enhanced butanol fermentation and energy-saving product recovery. Bioresource Technology 135, 396–402, doi:

10.1016/j.biortech.2012.07.062 (2013). Article  CAS  PubMed  Google Scholar  * Xue, C. et al. High-titer n-butanol production by _Clostridium acetobutylicum_ JB200 in fed-batch fermentation

with intermittent gas stripping. Biotechnology and Bioengineering 109, 2746–2756, doi: 10.1002/bit.24563 (2012). Article  CAS  PubMed  Google Scholar  * Goo, E., An, J. H., Kang, Y. &

Hwang, I. Control of bacterial metabolism by quorum sensing. Trends in Microbiology 23, 567–576, doi: 10.1016/j.tim.2015.05.007 (2015). Article  CAS  PubMed  Google Scholar  * Winzer, K.,

Hardie, K. R. & Williams, P. LuxS and autoinducer-2: their contribution to quorum sensing and metabolism in bacteria. Advances in Applied Microbiology 53, 291–396 (2003). Article  CAS 

Google Scholar  * Kovacikova, G., Lin, W. & Skorupski, K. Dual regulation of genes involved in acetoin biosynthesis and motility/biofilm formation by the virulence activator AphA and the

acetate-responsive LysR-type regulator AlsR in _Vibrio cholerae_. Molecular Microbiology 57, 420–433, doi: 10.1111/j.1365-2958.2005.04700.x (2005). Article  CAS  PubMed  Google Scholar  *

Jeon, S. et al. 2,3-Butanediol recovery from fermentation broth by alcohol precipitation and vacuum distillation. Journal of Bioscience and Bioengineering 117, 464–470, doi:

10.1016/j.jbiosc.2013.09.007 (2014). Article  CAS  PubMed  Google Scholar  * Xiu, Z. L. & Zeng, A. P. Present state and perspective of downstream processing of biologically produced

1,3-propanediol and 2,3-butanediol. Applied Microbiology and Biotechnology 78, 917–926, doi: 10.1007/s00253-008-1387-4 (2008). Article  CAS  PubMed  Google Scholar  * Anvari, M.,

Pahlavanzadeh, H., Vasheghani-Farahani, E. & Khayati, G. _In situ_ recovery of 2,3-butanediol from fermentation by liquid-liquid extraction. Journal of Industrial Microbiology &

Biotechnology 36, 313–317, doi: 10.1007/s10295-008-0501-z (2009). Article  CAS  Google Scholar  Download references AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * College of Life Sciences,

Fujian Agriculture and Forestry University, Fuzhou, 350002, P.R. China Shujing Sun, Haiyang Zhang, Shuyi Lu & Chunfen Lai * Centre for Bioengineering and Biotechnology, China University

of Petroleum (East China), Qingdao, 266580, P.R. China Huijun Liu & Hu Zhu * College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, P.R. China Hu Zhu

Authors * Shujing Sun View author publications You can also search for this author inPubMed Google Scholar * Haiyang Zhang View author publications You can also search for this author

inPubMed Google Scholar * Shuyi Lu View author publications You can also search for this author inPubMed Google Scholar * Chunfen Lai View author publications You can also search for this

author inPubMed Google Scholar * Huijun Liu View author publications You can also search for this author inPubMed Google Scholar * Hu Zhu View author publications You can also search for

this author inPubMed Google Scholar CONTRIBUTIONS S.J.S. and H.Z. designed the experiments. S.J.S., H.Y.Z., S.Y.L., C.F.L. and H.J.L. performed the experiments. S.J.S. and H.Z. analysed the

data and prepared the figures and tables. S.J.S. and H.Z. wrote the main manuscript. All the authors reviewed the manuscript. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing financial interests. RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this

article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will

need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Reprints and permissions ABOUT

THIS ARTICLE CITE THIS ARTICLE Sun, S., Zhang, H., Lu, S. _et al._ The metabolic flux regulation of _Klebsiella pneumoniae_ based on quorum sensing system. _Sci Rep_ 6, 38725 (2016).

https://doi.org/10.1038/srep38725 Download citation * Received: 04 August 2016 * Accepted: 11 November 2016 * Published: 07 December 2016 * DOI: https://doi.org/10.1038/srep38725 SHARE THIS

ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard

Provided by the Springer Nature SharedIt content-sharing initiative