The Colloquium 12 (2017) pp.

1-3

Production of xylonic acid by recombinant Escherichia coli

Farrah Noor Izzati Zahari, Rosli Md Illias, Zanariah Hashim*

Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor,
Malaysia.

KEYWORDS ABSTRACT
Xylonic acid In this paper, the effect of cultural condition on xylonic acid secretion by
Recombinant E. coli recombinant Escherichia coli (E. coli) was investigated. Then, the
optimized cultural condition was determined to elevate xylonic acid
secretion. To achieve this, analysis on xylonic acid secretion by using
hydroxamate assay and High Performance Liquid Chromatography
(HPLC) was performed. From the experimental works, it was
understood that post induction temperature, inducer concentration,
substrate concentration, medium pH and type of medium have affected
the secretion of xylonic acid.

1. Introduction induction mode, utilization of media additives

(Cheng et al., 2011; Fang et al., 2011).
Over the years, processing of biomass for
The most common strategy engaged in
the production of value-added chemicals has
molecular biology is by changing each variable
become important to both biological and
at a time while holding the others constant,
chemical engineering research (Fitzpatrick et
assuming that each variable is independent to
al., 2010). Xylonic acid, a five-carbon organic
evaluate the influence of such variables on
acid has gained outstanding interest due to its
heterologous protein expression (Larentis et
potential as an important platform chemical. It
al., 2014).
has extensive applications in food,
In this study, parameters affecting xylonic
pharmaceutical and chemical industries.
acid production by recombinant E. coli was
Xylonic acid can be produced by oxidation of
studied.
xylose with microorganism (Cao et al., 2013).
Xylonic acid has been produced by the
2. Experimental procedure
recombinant Escherichia coli, Saccharomyces
cerevisiae and Kluyveromyces lactis (Toivari 2.1 Bacterial strain and chemicals
et al., 2012). Xylonic acid productivity and
The recombinant E. coli strains carrying the
yield by recombinant E. coli are both higher as
genes to express the secretion of xylonic acid
compared to Saccharomyces cerevisiae or
were chosen as the biological hosts in this
Kluyveromyces lactis (Nygård et al., 2011;
research. The strain is grown overnight and 80
Toivari et al., 2010).
% glycerol is prepared. Immobilization
E. coli has native metabolic pathways for
technique was used to express the secretion of
both xylose and xylonic acid, unfortunately it
xylonic acid. Most of the chemicals and
is incapable of converting xylose directly to
reagents that are used in this research are
xylonic acid. Liu et al., (2012) in their studies,
analytical and molecular biology grade,
genetically modified E. coli to produce xylonic
purchased from various companies such as
acid by introducing a xylose dehydrogenase
gene from Caulobacter crescentus. Both Merck, Sigma, Fluka and Calbiochem.
xylose and xylonic acid catabolic pathways
were blocked to prevent the conversion from 2.2 Expression conditions and analytical
xylose or xylonic acid to biomass. methods
Diverse approaches have been studied to Parameters affecting xylonic acid secretion
enhance extracellular production of were determined at five different conditions
recombinant proteins in E. coli. These for each parameter. Xylose and xylonic acid
approaches include improving culture were measured by utilizing High Performance
conditions, such as varying the medium Liquid Chromatography fitted with Bio-Rad
composition, nutritional feeding design, Aminex HPX-87H column (300 x 7.8 mm).

* Corresponding author:
zanariahhashim@utm.my
 The Colloquium 12 (2017) pp.1-3

Xylonic acid concentrations was determined mentioned by Studier (2005), increasing the
by hydroxamate method The conversion of amount of peptone/tryptone or yeast extract
xylonic acid to xylonolactone was done by will lead to higher cell densities thus this in
heating the diluted sample. Then, 1mL turn elevates the secretion of xylonic acid.
hydroxylamine reagent (2M hydroxylamine
HCl in 2 M NaOH) was added to 500 µl of the Acknowledgement
diluted sample. Addition of HCl (650 µl, 3.2
We are grateful to the Ministry of
M) was then done followed with addition of
Education (MOE), Malaysian Genome
500 µl FeCl3 (100 gL-1 FeCl3 in 0.1 M HCl).
Institute (MGI) and Universiti Teknologi
Absorbance was measured at 550 nm.
Malaysia (UTM).
3. Results and discussion
Reference
3.1 Parameters affecting xylonic acid
Cao, Y., Xian, M., Zou, H., & Zhang, H.
synthesis
(2013). Metabolic Engineering of
Post induction temperature is an important Escherichia coli for the Production of
factor in determining the production of xylonic Xylonate. PLoS ONE, 8(7).
acid by immobilized recombinant E. coli. Five Che, R., Fauzi, A., Fatimah, S., Mohd, Z.,
different temperatures were investigated. The Faisal, N., & Illias, R. (2016). Effects of
results indicated that xylonic acid secretion in culture conditions of immobilized
immobilized cells was improved significantly recombinant Escherichia coli on
by increasing the post induction temperature. cyclodextrin glucanotransferase (
According to Hohenblum et al.,(2004), the CGTase ) excretion and cell stability.
metabolic stress generated by heterologous Process Biochemistry, 51(4), 474–483.
gene expression can be slightly relieved by Cheng, J., Wu, D., Chen, S., Chen, J., & Wu, J.
using lower temperatures to perform (2011). High-Level Extracellular
expression. Production of r -Cyclodextrin
Then, immobilized cells were induced Glycosyltransferase with recombinant
using five different concentrations of inducer. Escherichia coli BL21 ( DE3 ), 21,
Highest amount of xylonic acid was obtained 3797–3802.
at an intermediate amount of inducer Fang, S., Li, J., Liu, L., Du, G., & Chen, J.
concentration. If the amount is small, it is not (2011). Bioresource Technology
enough for expression. While high amount of Overproduction of alkaline
inducer concentration reduce xylonic acid polygalacturonate lyase in recombinant
secretion by immobilized recombinant E. coli Escherichia coli by a two-stage glycerol
that was caused by the toxic effect of the feeding approach. Bioresource
inducer and the metabolic burden imposed on Technology, 102(22), 10671–10678.
the cells due to heterologous gene expression Fitzpatrick, M., Champagne, P., Cunningham,
(Larentis et al., 2014). M. F., & Whitney, R. A. (2010).
Substrate concentration plays an important Bioresource Technology A biorefinery
role in determining the secretion of xylonic processing perspective : Treatment of
acid by using immobilized cell. In this lignocellulosic materials for the
research, xylose was used as the substrate. The production of value-added products.
different concentration of substrate seems to Bioresource Technology, 101(23), 8915–
have affected the synthesis of xylonic acid by 8922.
immobilized cell. According to Maier (2009), Hohenblum, H., Gasser, B., Maurer, M., &
cell growth is dependent on the substrate Borth, N. (2004). Effects of Gene Dosage
concentration, when substrate concentration is , Promoters , and Substrates on Unfolded
low, the rate of growth will be low as well. Protein Stress of Recombinant Pichia
The effects of pH on xylonic acid secretion pastoris.
from immobilized recombinant E. coli are Larentis, A. L., Fabiana, J., Quintal, M.,
studied. It was found that an environment that Esteves, S., Vareschini, D. T., Vicente,
is too acidic or too alkaline causes low yield of F.Medeiros, M. A. (2014). Evaluation of
xylonic acid This is because it could impose pre-induction temperature , cell growth at
too much stress on the metabolic activities of induction and IPTG concentration on the
cells, (Che et al., 2015). expression of a leptospiral protein in E .
Next, five different expression media, were coli using shaking flasks and
screened to determine their effect on xylonic microbioreactor, 1–13.
acid production. The production of xylonic
acid was found when the expression medium
consist high amount of yeast and tryptone. As

2
 The Colloquium 12 (2017) pp.1-3

Liu, H., Valdehuesa, K. N. G., Nisola, G. M., Toivari, M. H., Nygard, Y., Penttil, M.,
Ramos, K. R. M., & Chung, W. J. Ruohonen, L., & Wiebe, M. G. (2012).
(2012). High yield production of d- Microbial d-xylonate production.
xylonic acid from d-xylose using Applied Microbiology and
engineered Escherichia coli. Bioresource Biotechnology, 96(1), 1–8.
Technology, 115, 244-248. Toivari, M. H., Ruohonen, L., Richard, P.,
Maier, R. M., Pepper, I. L., & Gerba, C. P. Penttilä, M., & Wiebe, M. G. (2010).
(2009). Environmental microbiology Saccharomyces cerevisiae engineered to
(Vol. 397). Academic press. produce D-xylonate. Applied
Nygård, Y., Toivari, M. H., Penttilä, M. Microbiology and Biotechnology, 88(3),
Ruohonen, L., & Wiebe, M. G. (2011). 751-760.
Bioconversion of d-xylose to d-xylonate
with Kluyveromyces lactis. Metabolic
Engineering, 13(4), 383-391.
Studier, F. W. (2005). Protein production by
auto-induction in high density shaking
cultures. Protein Expr. Purif. 41, 207-
234.