Biotechnology Reports 16 (2017) 65–70

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Biotechnology Reports
journal homepage: www.elsevier.com/locate/btre

Enhanced hyluronic acid production in Streptococcus zooepidemicus by

over expressing HasA and molecular weight control with Niscin and
glucose
Alireza Zakeria , Mohammad Javad Rasaeeb,* , Navid Pourzardoshtc
a
Department of Biology Sciences, Faculty of Sciences, Shahid Rajee Teacher Training University, Tehran, Iran
b
Department of Medical Biotechnology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
c
Department of Medical biochemistry, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Article history:
Received 22 August 2016 Hyaluronic acid (HA) is a high molecular weight linear polysaccharide, endowed with unique
Received in revised form 22 February 2017 physiological and biological properties. Given its unique properties, HA have unprecedented applications
Accepted 27 February 2017 in the fields of medicine and cosmetics. The ever growing demand for HA production is the driving force
Available online 2 March 2017 behind the need for finding and developing novel and amenable sources of the HA producers. Microbial
fermentation of Streptococcus zooepidemicus deemed as one the most expeditious and pervasive methods
Keywords: of HA production. Herein, a wild type Streptococcus zooepidemicus, intrinsically expressing high levels of
Streptococcus zooepidemicus HA, was selected and optimized for HA production. HasA gene was amplified and introduced into the wild
Hyaluronic acid
type Streptococcus zooepidemicus, under the control of Nisin promoter. The HasA over-expression
HasA
increased the HA production, while the molecular weight was decreased. In order to compensate for
Glucose
Molecular weight molecular weight loss, the glucose concentration was increased to an optimum amount of 90 g/L. It is
hypostatizes that excess glucose would rectify the distribution of the monomers and each HasA molecule
would be provided with sufficient amount of substrates to lengthen the HA molecules. Arriving at an
improved strain and optimized cultivating condition would pave the way for industrial grade HA
production with high quality and quantity.
© 2017 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Extraction from rooster comb and fermentation of certain

attenuated strains of group A and C Streptococcus are commonly
Hyaluronic acid (HA, also known as hyaluronan) is a high exploited for commercial HA production. However, HA extraction
molecular weight linear polysaccharide. HA is formed from 2000 to from rooster combs is an arduous and costly procedure, suffering
25000 repeating disaccharide units of alternating D-glucuronic from several technical limitations, leading this method to be
acid and N-acetylglucosamine moieties. The monomers of each dwindled away [4]. On the other hands, due to existence of
disaccharide unit are linked by b (1–3) and b (1–4) glycosidic bacterial strains capable of superior HA productivity, fermentation
bonds. The HA molecule is endowed with unique physiological and process becomes more attractive for large-scale production. Non-
biological properties including high water-holding capacity, immunogenic and non-inflammatory properties of the bacterial
viscoelasticity and biocompatibility. Given these properties HA HA renders it as an excellent alternative medical grade HA source.
has a wide range of unprecedented applications in the fields of Moreover, microbial HA production requires simpler downstream
medicine and cosmetics, like osteoarthritis treatment, ophthalmic processing, is devoid of seasonal fluctuations, shows less batch to
surgery, plastic surgery, drug delivery, skin moisturizers and batch variation and reduces the risk of viral contamination. In this
wound healing, to name but a few [1–3]. regard, having regulatory acceptance in both the US and UK, S. equi
subsp. Zooepidemicus is among the most common strains used for
fermentative HA production [5,6]. Various attempts have been
* Corresponding author at: Department of Medical Biotechnology, School of addressed to increase the amount of HA, including conventional
Medical Sciences, Tarbiat, Modares University, Chamran Jalal crossing, Tehran, PO techniques (e.g. optimizing the extraction process, adapting the
Box 14115-331, Iran.
E-mail address: [email protected] (M.J. Rasaee).

http://dx.doi.org/10.1016/j.btre.2017.02.007
2215-017X/© 2017 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
66 A. Zakeri et al. / Biotechnology Reports 16 (2017) 65–70

culture media, and selecting strains with high HA productivity) and electroporation was performed according to the Chen et al. [10]
metabolic engineering methods [7,8]. method with a Gene Pulser device (Eppendorf).
Coming to grips with the metabolic routes involved in HA
synthesis by Streptococci subsp. could play pivotal roles in the 2.3. HasA amplification and sequencing
optimization of its production process. Metabolic engineering
approaches could be expeditiously used to arrive at both better HA Genomic DNA of the wild type S. zooepidemicus was extracted
quality (controlled polymer chain length) and yield of the using standard CTAB method and confirmed on agarose electro-
production process. The Streptococcus equi subsp. zooepidemicus phoresis. Genomic sequences from Streptococcus zooepidemicus
(S. zooepidemicus) operon encodes for five genes involved in HA H70, CY, ATCC35246, 4047 and MGCS were analyzed for their HasA
synthesis, namely HA synthase (hasA), UDP-glucose dehydroge- gene to find the best regions for primer design. The HasA gene was
nase (hasB), UDP-glucose pyrophosphorylase (hasC), a glmU amplified from the extracted genomic DNA template, using
paralog encoding for a dual function enzyme acetyltransferase designed primers (F-HasA: 50 - CCATGGGCAGAACATTAAAAAACCT-
and pyrophosphorylase activity (hasD), and a pgi paralog encoding CATAACTG-30 and R-HasA: 50 -TCTAGATTATAATAATTTTTTACGTG
for phosphoglucoisomerase (hasE) [9,10]. HasA is the key enzyme TTCCCCAGTCAGC-30 ). The NcoI and XbaI restriction sites were
in the production of HA, utilizing two sugar substrates (UDP-GlcA introduced to 50 and 30 end of the PCR product using these primers.
and UDP-GlcNAc) to synthesize HA. The szHasA (hyaluronan The PCR program consisted of 1 cycle of 5 min at 95  C, followed by
synthase of S. zooepidemicus), which is composed of 418 amino 30 cycles of 94  C (30 s), 65  C (30 s), and 72  C (1 min) and one final
acids, harbor’s six membrane domains with the N-terminal and C- extension cycle of 72  C (10 min). The PCR product sizes were
terminal ends inside the cell [11]. confirmed on an agarose gel. To produce blunt-end HasA PCR
Although a broad wealth of Streptococcus zooepidemicus strains product, PCR reactions were done by pfu DNA pol. Then, ligation to
like S. zooepidemicus ATCC 39920 [12], Streptococcus sp. ID9102 blunted pJET 1.2 parental vector was performed at room
[13], S. zooepidemicus ATCC 35426 [14], S. zooepidemicus WSH-24 temperature for 10 min using T4 DNA ligase. Competent Escherichia
[15], S zooepidemicus H23 [16], S. zooepidemicus NJUST01 [17], S. coli DH5a cells were prepared and heat-shock transformation was
equi. ss equi CCUG 22971 (Stahl, S., US patent 2003-0134393 A1, performed on ampicillin selection plates. To confirm the insertion
2003), S. zooepidemicus #104 (Shiseido, Yokohama) [18], S. HasA containing pJET vector was purified and digested with BgLII
zooepidemicus ATCC 35246 HA+ Lac+ Ems (1), S. equi Ferm BP- restriction enzyme, which cuts on both sides of the insert. Gel
879 and S. zooepidemicus BP-878 (Morita, H., and Fuji, M., US patent electrophoresis (1% agarose) was performed to analyze the
5071751, 1991), S. equi FM 100 (Hashimoto, M., Saegusa, H., Chiba, presence of the insert and determine its size. To identify the
S., Kitagawa, H., Miyoshi, T., US patent 4946780, 1990) and S. exact sequence of the HasA gene, it was sequenced using extracted
zooepidemicus MTCC 3523 [19] have been subjected to HA pJET vector by MacroGen Company. The sequencing result was
production studies, exploring novel strains with intrinsic ability submitted as quarry in NCBI nucleotide BLAST tool (http://blast.
of high HA production remains highly valuable to meet the ncbi.nlm.nih.gov/Blast.cgi) to determine the similarities of the
growing HA demand. In the present study, we exerted a HasA sequences in other S. zooepidemicus strains. The search was
combinatorial strategy to achieve the high quality and quantity performed against the nucleotide collection of the BLAST tool,
HA. To this end, a wild type S. zooepidemicus was isolated and restricted to S. zooepidemicus strains.
characterized for high level HA production, while the szHasA
(hyaluronan synthase gene) was introduced into the selected strain 2.4. Construction of recombinant strain
under the control of nisA promoter and the glucose concentration
was optimized for higher HA production. Ultimately, we achieved HasA gene was digested out of pJET plasmid with NcoI and XbaI
an engineered strain capable of over-expressing HasA and restriction enzymes. Then, the DNA fragments were inserted into
increased HA production yield. pNZ8148 expression plasmid, digested with the same restriction
enzymes, by a ligation reaction to construct pNZ8148-szHas
expression plasmid. The pNZ8148 and pNZ8148-szHas plasmids
2. Materials and methods
were then used to transform the E. coli MC1061 as an intermediate
host by heat shock method. The transformants were selected on
2.1. Strain and culture media
TSB plates containing chloramphenicol as selection marker. The
pNZ8148 (as negative control) and pNZ8148-szHas expression
A wild type Streptococcus equi subsp. zooepidemicus (isolated
plasmids subsequently were extracted and electro transformed
and characterized from horse nasal swabs) [20] was utilized as the
into the S. zooepidemicus by electroporation. In order to perform
HA producing strain throughout this study. Stock cultures were
the electroporation we used ice-cold cuvettes of path length of
stored at
80  C in Tryptic soy broth medium (TBS) and 25% (v/v)
0.1 cm, containing 50 ml of washed cells and up to 10 ml of purified
glycerol, while the cultivation was in TSB at 37  C. E.coli MC1061
plasmid DNA. Voltage was set at 1.5 kV, while the resistance was
strain was used as intermediate gene cloning host. Both
set at 200 V and capacitance at 25 microfarads. Immediately
Streptococcus equi subsp. Zooepidemicus and E.coli MC1061
following 5 ms of pulse application, 1.0 ml of cold TSB broth was
strains were cultured in 2.5 mg/ml of chloramphenicol. Moreover,
added to cells, which were then held on ice for 5 min prior to
DH5a strain was used to contain the sequencing vector (pJET).
incubation at 37  C for 2–3 h. The sub-cloning process was assessed
by sequencing of the pNZ8148-szHas expression plasmid,
2.2. Molecular manipulations extracted from the recombinant S. zooepidemicus.

Plasmid DNA isolation, agarose gel electrophoresis, restriction 2.5. HasA enzyme assay
enzyme digestion, DNA ligation and DNA transformation were all
performed by standard procedures [21] or following the specific The recombinant S. zooepidemicus strains containing pNZ8148
recommendations of the manufacturer’s protocol. Plasmid DNA and pNZ8148-szHasA along with the wild type S. zooepidemicus
purification kit was purchased from GeneAll biotechnology and were grown at 37  C to an OD600 of 0.5 and induced by 20 ng/ml of
restriction endonucleases, T4 DNA ligase, PFU polymerase and Taq Nisin. In order to express the HasA gene under the control of Nisin
polymerase were purchased from Thermo Fisher Scientific. The promoter, Nisin (Sigma, USA) was processed and used for
 A. Zakeri et al. / Biotechnology Reports 16 (2017) 65–70 67

expression induction according to the previously described The viscosity of culture broth was measured using a viscometer.
method [22]. The in vitro HasA activity was measured in disrupted Molecular weight of HA was determined by measuring intrinsic
cells containing the membrane-attached HasA. To perform the viscosity [h] using capillary viscometer (Ubbelohde Dilution
enzymatic reaction, cells were harvested in the exponential phase Capillary with 0.63-mm diameter and 5700-mm3 volume). All
of bacterial growth and pelleted by centrifugation at 4000 rpm, measurements were performed at 37  C using 0.15 M NaCl as
4  C for 5 min. The supernatant was discarded, while the pellet was diluent [10]. The intrinsic viscosity [h] was assessed by British
re-suspended in 2 ml of Phosphate- Buffered Saline (PBS, pH7.2, Pharmacopoeia method 2009. The average molecular weight was
Invitrogen). The suspension was disrupted with ultrasonic determined from the intrinsic viscosity using the Mark-Houwink-
processor on ice for 99 cycles with 60W power at 30% duty to Sakurada equation:
release HasA and afterwards 0.5 ml of the obtained crude extract
[h] = 0.0292  MW0.7848
was added into the enzymatic reaction system (0.25 ml UDP-GlcA
(4 mM in PBS, Sigma), 0.25 ml UDP-GlcNAc (4 mM in PBS, Sigma), The HA concentration was measured by carbazole method.
10 ml 1 M MgCl2 and 10 ml 0.1 M DTT). The enzymatic reaction was Assessing the HA concentration, each sample from the bioreactor
commenced by putting the mixed reactants in a 37  C water bath was first incubated with a 10% volume of 5% (w/v) SDS for 10 min to
and incubating for 2 h. The reaction was stopped by 2 min separate the cells and liberate the capsular HA. Then, the culture
immersion into 100  C boiling water, cooling to room temperature broth was centrifuged at 4000 rpm for 30 min. After the cells
and subsequent addition of 1.02 ml of 0.1% SDS to free the HA removal, 2 ml of ethanol was added to 1 ml of the supernatant from
molecules possibly attached on the cell debris. After 10 mins of the culture broth and the solution was then refrigerated at 4  C for
centrifugation of the reaction solution at 4000 rpm, the superna- 24hr to precipitate hyaluronic acid. The precipitate was recovered
tant was ready for the ethanol precipitation and carbazole assay. by centrifugation at 4000 rpm for 30 min and re-precipitated using
The 1 unit/mg activity of the dry cell HA synthase is equivalent to the same procedure. Finally, the pellet was dissolved in distilled
1 mg of HA generated/min/mg of the dry cell. Therefore, one Unit/ water and used for HA concentration analysis by carbazole method.
mg dry cell activity of HasA was calculated as 1 mg HA generated To do so, disodium tetraborate solution (dissolved in sulfuric acid)
per min and per mg dry cell. was added to dissolve HA, and boiled for 15 min. After cooling to
room temperature, carbazole solution was added and heated in
2.6. HasA over expression water bath for 15 min and again cooled to room temperature.
Ultimately, HA concentration was read at 530 nm with D-
Proteins in the crude extract (produced as described in the glucuronic acid as the standard [24]. Moreover, HA purification
previous section) were analyzed by sodium dodecyl sulfate and its molecular weight determination was carried out using the
polyacrylamide gel electrophoresis (SDSPAGE; 10%). Equal procedure employed by Chen et al. at their respective study [10].
amounts of obtained crude extract were employed to perform
standard SDS-PAGE analysis. Bradford method [23] was employed
3. Results
to use equal amounts of the total protein for SDS-PAGE analysis
with bovine serum albumin as the standard.
3.1. HasA gene characterization
2.7. Cultivation
Genomic DNA of the wild type Streptococcus zooepidemicus was
successfully isolated and observed on gel electrophoresis. The PCR
A total of 6 batch fermentations with 2 replicates per strain
reaction using designed primers resulted in a 1254-bp DNA. The
were performed for wild type (WT) strain, WT strain with empty
amplified fragment introduced into the pJET blunt cloning vector
plasmid (pNZ8148) and WT strain with hasA containing plasmid
and the cloning was confirmed by BgLII restriction enzyme. The
(pNZ8148 + HasA) strain. Molecular weight, concentration of HA
sequencing results of the HasA gene indicated a high similarity to
and concentration of biomass were the parameters measured and
other known HasA sequences. The BLAST search results revealed
compared for all three strains. Growth experiments were
that the sequence of the wild type Streptococcus zooepidemicus was
conducted in a 5-l bioreactor (Model KL-5L, Iran Tajhiz Ted Co.
>97% match with the H70, CY, ATCC35246, 4047 and MGCS strains
Ltd, Iran) at a working volume of 1.2 l, and the temperature was
(Table 1).
maintained at 37  C. The reactor was agitated at 300 rpm, and
dissolved oxygen tension (DOT) studies were caried out with air or
3.2. HasA expression and activity assessment
air supplemented with oxygen. The pH automatically was maintain
throughout the experiment at 7 (adding NaOH and 5 M HCl).
The SDSPAGE results demonstrated that the HasA protein
expression in the recombinant strain was enhanced (Fig. 1). The
2.8. Analytical methods
enzyme activity assay recorded up to 1.8 fold of enzyme activity
augmentation for the over expressed HasA (Table 2). Moreover,
Analyzing the effects of HasA over expression on HA produc-
Table 3 reveals that the HA production is significantly increased
tion, a matrix of six (10, 20, 30, 40, 50 and 60 ng/ml) different
due to recombinant HasA activity (Table 3).
concentrations of Nisin along with two different concentrations of
Glucose were used for HA production. Samples were withdrawn at
3.3. Effects of HasA over expression in different expression conditions
regular time intervals and analyzed for Biomass amount, HA
molecular weight and HA concentrations. Cell growth per hour was
The evaluation results of the production concentration of
observed by measuring the optical density of the culture broth at
biomass, HA molecular weight and the concentration of the HA are
530 nm using a UV-spectrophotometer (Shimadzu, UV120-02,
listed in Fig. 2. As the results indicated, at 40 g/L of glucose
Japan). To measure the biomass, the optical density was measured
concentration increasing the Nisin concentration had positive
at 530 nm and converted to biomass using the following equation
effects on HA concentration meanwhile the molecular weight was
[10]:
decreased. However, at 90 g/L of glucose concentration increasing
Biomass (g/l) = A530  0.26  0.01 the Nisin concentration had positive effects on HA concentration
while the molecular weight remained constant.
68 A. Zakeri et al. / Biotechnology Reports 16 (2017) 65–70

Table 1
BLAST search results. The table lists top 7 matches for the sequenced gene along with their scores. From top to down are the best matches with highest scores as compared
with the strain used in this study.

Accession Description Max score Total score Query (%) Max identity (%)
FM204884.1 S.equi subsp. zooepidemicus H70, complete genome 2124 2124 67 97
CP001129.1 S.equi subsp. zooepidemicus MGCS10565, complete genome 2122 2122 67 97
AY173078.1 S. equi subsp. zooepidemicus hyaluronic acid operon, partial sequence 2122 2122 67 97
AF023876.1 S. equisimilis hyaluronan synthase gene, complete cds 2121 2121 67 97
CP006770.1 S.equi subsp. zooepidemicus CY, complete genome 2073 2073 67 97
CP002904.1 S. equi subsp. zooepidemicus ATCC 35246, complete genome 2073 2073 67 97
FM204883.1 S. equi subsp. equi 4047, complete genome 2073 2073 67 97

4. Discussion of previously reported studies (reviewed in [25]), the basal HA

yield over 4.2 g/L prior to any optimization is surprisingly
The ever growing demand for HA production is the driving force compelling for a wild type strain. This extent of metabolic
behind the need for finding and developing novel and amenable adaptation for HA production provides a confounding opportunity
sources of HA producers. Traditional methods of HA production are to achieve highest yields of HA production exerting minimum
not tractable for industrial large scale production, therefore optimization criteria. It has been demonstrated that HAS is
imminently they would be replaced by fermentative production responsible for glycosyl transferase activity required for polymer-
which lacks the concerns associated with animal sourced products. ization of both UDP-GlcNAc and UDP-GlcUA as well as transloca-
Since the first reports of hyaluronic acid isolation from group A tion of hyaluronan across the cell wall of S. zooepidemicus [26]. The
hemolytic streptococci, numerous attempts have been made to coupled nature of HA synthesis and translocation allows the HA
increase the amount of hyaluronan. Optimizing the extraction molecule to be synthesized even in the presence of a large excess of
process, adapting the culture media, selecting strains with high HA-degrading enzyme. Moreover, HasA have a determinant role in
hyaluronan productivity and metabolic engineering are among the the mechanism and amount of HA elongation [27]. Considering the
most common roots of increasing the amount of hyaluronan imperative roles of HasA in the HA production process, extrapo-
(reviewed in [6]). In this regard, we explored a HA producing strain lating better HA yields would be conceivable by increased levels of
and bolstered the production yields both in quantity and quality, HasA activity. Chen et al. cloned and over expressed five genes of
optimizing the culture condition and devising the HasA production the HasA operon in S. zooepidemicus, to manipulate the levels of
rates. UDP-GlcUA and UDP-GlcNAc. They demonstrated that over
S. zooepidemicus is among the most accepted microbial sources expression of the HA synthase gene (HasA) results in a significant
of HA production. Hence, selecting a locally available novel HA increase in HA yield. They also reported that HasA over expression
producing strain of S. zooepidemicus, capable of staggering HA was associated with 47% increase in yield, a significant lowering of
synthesis, would be of great industrial interest. Given the HA yields molecular weight and decrease of microbial growth rate. Thus,
establishing a recombinant strain with controlled HasA over
expression possibility would seems to be rational. In line with this
hypothesis, our results indicate that Nisin mediated induction of
HasA resulted in higher HA yield. However, the higher concen-
trations of Nisin induction reduce the amount of HA production.
This could be a consequence of Nisin toxicity for cells and its
inhibitory effect on bacterial growth [28]. The HasA gene is sub-
cloned under the control of the Nisin promoter of the pNZ8148
plasmid, therefore HasA is the only overexpressed gene. This gene
is responsible for the linkage between N-acetyl glucosamine and
glucuronic acid for HA production which does not play any other
role.
Pummill and Deangelis testified the theory that the relative
strength of the interaction between the catalyst and the precursor
sugars may be a major factor in HA size control via in vitro
confirmation [29]. Later on, Sheng et al. discovered the applicabil-
ity of this theory in their in vivo experiments [30]. They
hypothesized that altered distribution of precursor sugars among
the HasA protein molecules could be the reason behind this
phenomena. They suggested that, HasA is essential, but not
sufficient for high field HA production in heterologous hosts and
the production of UDP-GlcA is the limiting factor in host cell. As an
alternative strategy to enhance UDP-GlcNAc concentration, Chen
et al. suggested feeding the culture with glucosamine. As it was
expected, HasA over expression decreased the HA molecular
weight in our experiment. Our results indicate that HasA over-
expression in 40 g/l of glucose concentration is associated with
increased amount of HA, while MW is decreased. This could be the
consequence of altered distribution of precursor sugars, so that
each HA polysaccharide chain gets less precursor sugars to
Fig. 1. The SDSPAGE results of HasA protein expression in the recombinant strain. lengthen itself by HasA. However, to compensate the MW
Lane 1 and 2 are the recombinant strain, the lane 3 is wild type strain and the lane 4
reduction glucose concentration was contemplated to be
is the protein ladder.
 A. Zakeri et al. / Biotechnology Reports 16 (2017) 65–70 69

Table 2
Activity of HasA enzyme in comparison to wild type strain (p  0.05).

Fold increase in comparison to WT Specific activity mg HA min
1 mg protein
1 Strain

1 0.49  10
3  0.01 WT(S.zooepidemicus)
1.02 0.50  10
3  0.01 RM(pNZ8148)
1.82 0.89  10
3  0.02 RA(pNZ8148 + HasA)

Table 3
The variation in HA concentration comparing wild type and recombinant strains (p  0.05).

Percentage of HA increase in comparison to WT HA concentration (g/l) Strain

0 4.20  0.01 WT(S.zooepidemicus)
0 4.24  0.01 RM (pNZ8148)
43 6.02  0.02 RA(pNZ8148 + HasA)

Fig. 2. The HA fermentation values for different Nisin and glucose concentrations. WT stands for wild type, pNZ8148 stands for WT strain with empty plasmid,
pNZ8148 + HasA stands for WT strain with hasA containing plasmid strain, MW is molecular weight and YX is yield of biomass.

optimized. Additional Glucose would provide the host with weight variation and Nisin plays a more fundamental role. The
sufficient amount of UDP-GlcA, as the limiting factor. Therefore, study conducted by Chen et al. revealed that the increase in MW
suitable distribution of sugar monomers would occur among the could be partially due to the foreign DNA stress exerted by
HasA proteins and MW would increase. Our results revealed that existence of the plasmid within the bacteria. They demonstrated
HasA over-expression along with increased glucose resources up to that the plasmid backbone without any external genes could
90 g/L would lead to increased HA production, while the MW is surprisingly increase the MW. In fact, there are evidences
relatively kept constant in comparison to wild type strain. It should indicating stress in general (plasmid stress, aerobic conditions,
be noted that, the molecular weight of the HA at 40 gr/L glucose changes in pH, and low temperature) is beneficial for high
concentration of the wild type bacteria is 1.7 million Da, while at molecular weight HA production [10]. It is noteworthy to mention
the 90 gr/L glucose concentration the molecular weight of the HA that, the fermentation process is performed by an automatic
molecule reached 1.9 million Da. Therefore, it could not be fermenter which its oxygen sensor was active during the whole
concluded that the molecular weight of HA is constant during process. The oxygen concentration was automatically fine-tuned
glucose fluctuation. On the other hands, Nisin increase does not throughout the process. Due to exerted control, the oxygen
have a tangible effect on molecular weight. Since, aside from the concentration was kept constant; thus the possible influences of
HA production, glucose is involved in energy production processes, dissolved oxygen concentration have been eliminated in the
only 5% of the glucose is consumed in HA production. Therefore, it experiments. In conclusion, it should be noted that optimization of
is anticipated that it does not play a pivotal role in molecular culture media and cultivation conditions along with strain
70 A. Zakeri et al. / Biotechnology Reports 16 (2017) 65–70

improvements are intriguing methods, harnessed for improving Streptococcus sp. ID9102 via a statistical approach, J. Ind. Microbiol.
HA production yield. Performing these improvement on a wild Biotechnol. 36 (2009) 1337–1344.
[14] J.A. Vázquez, M.I. Montemayor, J. Fraguas, M.A. Murado, High production of
type strain which is already capable of high yield HA production hyaluronic and lactic acids by Streptococcus zooepidemicus in fed-batch culture
would circumvent the common obstacles lies ahead of industrial using commercial and marine peptones from fishing by-products, Biochem.
level fermentative HA production. Having an improved strain and Eng. J. 44 (2009) 125–130.
[15] L. Liu, G. Du, J. Chen, M. Wang, J. Sun, Influence of culture modes on the
optimized culture media would pave the way for industrial grade microbial production of hyaluronic acid by Streptococcus zooepidemicus,
HA production with high quality and quantity. Biotechnol. Bioproc. E. 13 (2008) 269–273.
[16] H.-J. Gao, G.-C. Du, J. Chen, Analysis of metabolic fluxes for hyaluronic acid (HA)
production by Streptococcus zooepidemicus, World J. Microbiol. Biotechnol. 22
Conflict of interest (2006) 399–408.
[17] J. Zhang, X. Ding, L. Yang, Z. Kong, A serum-free medium for colony growth and
The authors declare no conflict of interests. hyaluronic acid production by Streptococcus zooepidemicus NJUST01, Appl.
Microbiol. Biotechnol. 72 (2006) 168–172.
[18] S. Hasegawa, M. Nagatsuru, M. Shibutani, S. Yamamoto, S. Hasebe, Productivity
Acknowledgement of concentrated hyaluronic acid using a Maxblend1 fermentor, J. Biosci.
Bioeng. 88 (1999) 68–71.
The authors wish to thank Tarbiat Modares University for [19] K.P. Patil, D.K. Patil, B.L. Chaudhari, S.B. Chincholkar, Production of hyaluronic
acid from Streptococcus zooepidemicus MTCC 3523 and its wound healing
supporting the conduct of this research. The authors declare that activity, J. Biosci. Bioeng. 111 (2011) 286–288.
they have no conflict of interest. [20] A. Jannatabadi, G. Mohammadi, M. Rad, M. Maleki, Molecular identification of
Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus
in nasal swabs samples from horses suffering respiratory infections in Iran, Pak
References J. Biol. Sci. (2008) 468–471.
[21] M.R. Green, J. Sambrook, Molecular Cloning: a Laboratory Manual, Cold Spring
[1] B.F. Chong, L.M. Blank, R. McLaughlin, L.K. Nielsen, Microbial hyaluronic acid Harbor Laboratory Press, New York, 2012.
production, Appl. Microbiol. Biotechnol. 66 (2005) 341–351. [22] Z. Eichenbaum, M.J. Federle, D. Marra, W.M. de Vos, O.P. Kuipers, M.
[2] G. Kogan, L. Soltes, R. Stern, P. Gemeiner, Hyaluronic acid: a natural biopolymer Kleerebezem, et al., Use of the lactococcal nisA promoter to regulate gene
with a broad range of biomedical and industrial applications, Biotechnol. Lett. expression in gram-positive bacteria: comparison of induction level and
29 (2007) 17–25. promoter strength, Appl. Environ. Microbiol. 64 (1998) 2763–2769.
[3] S.-J. Kim, S.-Y. Park, C.-W. Kim, A novel approach to the production of [23] A.M. Crestfield, S. Moore, W.H. Stein, The preparation and enzymatic
hyaluronic acid by Streptococcus zooepidemicus, J. Microbiol. Biotechnol. 16 hydrolysis of reduced and S-carboxymethylated proteins, J. Biol. Chem. 238
(2006) 1849–1855. (1963) 622–627.
[4] E.A. Balazs, Ultrapure hyaluronic acid and the use thereof. United States [24] R. Jaenicke, R. Rudolph, D. Feingold, Dissociation and in vitro reconstitution of
Patent: Biotrics, 1979. bovine liver uridine diphosphoglucose dehydrogenase. The paired subunit
[5] L. Liu, M. Wang, G. Du, J. Chen, Enhanced hyaluronic acid production of nature of the enzyme, Biochemistry 25 (1986) 7283–7287.
Streptococcus zooepidemicus by an intermittent alkaline-stress strategy, Lett. [25] C. Schiraldi, A. LaGatta, M. DeRosa, Biotechnological production and
Appl. Microbiol. 46 (2008) 383–388. application of hyaluronan, in: M. Elnashar (Ed.), Bipolymers, INTECH Open
[6] C.G. Boeriu, J. Springer, F.K. Kooy, L.A. van den Broek, G. Eggink, Production Access Publisher, 2010, pp. 387–412.
methods for hyaluronan, Int. J. Carbohydr. Chem. (2013) 1–14. [26] C. Hubbard, J.T. McNamara, C. Azumaya, M.S. Patel, J. Zimmer, The hyaluronan
[7] B. Holmstrom, J. Ricica, Production of hyaluronic Acid by a streptococcal strain synthase catalyzes the synthesis and membrane translocation of hyaluronan, J.
in batch culture, Appl. Microbiol. 15 (1967) 1409–1413. Mol. Biol. 418 (2012) 21–31.
[8] J.-H. Kim, S.-J. Yoo, D.-K. Oh, Y.-G. Kweon, D.-W. Park, C.-H. Lee, et al., Selection [27] N. Itano, T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, et al., Three
of a Streptococcus equi mutant and optimization of culture conditions for the isoforms of mammalian hyaluronan synthases have distinct enzymatic
production of high molecular weight hyaluronic acid, Enzyme Microb. properties, J. Biol. Chem. 274 (1999) 25085–25092.
Technol. 19 (1996) 440–445. [28] E. Marcellin, W.Y. Chen, L.K. Nielsen, Understanding plasmid effect on
[9] L.M. Blank, P. Hugenholtz, L.K. Nielsen, Evolution of the hyaluronic acid hyaluronic acid molecular weight produced by Streptococcus equi subsp
synthesis (has) operon in Streptococcus zooepidemicus and other pathogenic zooepidemicus, Metab. Eng. 12 (2010) 62–69.
streptococci, J. Mol. Evol. 67 (2008) 13–22. [29] P.E. Pummill, P.L. DeAngelis, Alteration of polysaccharide size distribution of a
[10] W.Y. Chen, E. Marcellin, J. Hung, L.K. Nielsen, Hyaluronan molecular weight is vertebrate hyaluronan synthase by mutation, J. Biol. Chem. 278 (2003) 19808–
controlled by UDP-N-acetylglucosamine concentration in Streptococcus 19814.
zooepidemicus, J. Biol. Chem. 284 (2009) 18007–18014. [30] J.Z. Sheng, P.X. Ling, X.Q. Zhu, X.P. Guo, T.M. Zhang, Y.L. He, et al., Use of
[11] L.J. Chien, C.K. Lee, Hyaluronic acid production by recombinant Lactococcus induction promoters to regulate hyaluronan synthase and UDP-glucose-6-
lactis, Appl. Microbiol. Biotechnol. 77 (2007) 339–346. dehydrogenase of Streptococcus zooepidemicus expression in Lactococcus
[12] S. Jagannath, K. Ramachandran, Influence of competing metabolic processes lactis: a case study of the regulation mechanism of hyaluronic acid polymer, J.
on the molecular weight of hyaluronic acid synthesized by Streptococcus Appl. Microbiol. 107 (2009) 136–144.
zooepidemicus, Biochem. Eng. J. 48 (2010) 148–158.
[13] J.-H. Im, J.-M. Song, J.-H. Kang, D.-J. Kang, Optimization of medium
components for high-molecular-weight hyaluronic acid production by