Beruflich Dokumente
Kultur Dokumente
l 43-48
Joumal ofNortheast Agricultural (English Edition)
m
tm
cd
e
Dm
e
r cu
-
M
H
'm aF
,
Lm
4waFMU
-
-
'
V
A
Expression of a Lysine-rich Gene in Bacillus subtilis 168
Li Xue-lin\ Liu Xiao-fd, Gao
t'O
f'Or the lysine standard.
The calibrati'On pl'Ots f'Or lysine were fit t'O linear
equati'Ons 'Of sl'Ope and intercept y =0.0003x+2.3725
(Fig. 4). The c'Orrelati'On c'Oefficient was in the excess
'OfO.99 in case.
The c'Ontent 'Of lysine in the induced pHT43/1ys/
Bacillus subtilis 168 increased by 9.85% c'Ompared
with Bacillus subtilis 168.
Table 1 Lysine content analysis in Bacillus subtilis 168
(geV
1
)
Item Not induced Induced
pHT43/lys/Bacillus
49.03:1:1. 22
pHT43/Bacillus
47.89:1:1.03
subtilis 168
Bacillus subtilis 168
'Of cellular gr'Owth (Fig. 3). A 500 bp amplificati'On Each value is shown as of five individuals. * Significant
product was detected c'Orresp'Onding t'O the expressi'On difference (P<0.05) compared control.
E-mail: xuebaoenglish@neau.edu.cn
Li Xue-lin et al. Expression of a Lysine-rich Gene in Bacil/us subtilis 168
140 I
.!:"" I
I
bo 100
:::L
D
E 60
540
320
10000 20000
.47'
30000 40000 50000
(uy.s)
Fig. 4 Standard curve of Iysine by HPLC
Discussion
The lys gene encoding the lysine-rich protein,
containing 10.8 % of lysine and other essential amino
acids, such as threonine, valine and isoleucine, etc. ,
was the seeds of winged bean. 1n the
present study, lysine-rich gene was c10ned and the
corresponding protein was expressed in Bacillus
subtilis 168. It was an effective method to enhance the
lysine content by transferring alien lysine-rich genes
in cereals. The results in this study were similar to the
previous reports of maize, rice, wheat and lettuce (Li
et al. , 2006; Gao et al. , 2001; Meng et al. , 2001).
L-lysine is L-aspartate as a part
of the aspartate pathway in Bacillus subtilis. The
first step in the common pathway, phosphorylation
of L-aspartate, is catalyzed by aspartate kinase (AK).
Bacillus subtilis has three independently regulated
isozymic aspartate kinases. AKI (encoded by dapG)
is allosterically inhibited by meso-diaminopimelate
AKI1 (encoded by lysC) is inhibited by
L-lysine, and AKII1 (encoded by yclM, also
designated thrD) is regulated by concerted feedback
inhibition by L-lysine and L-threonine (Graves
and Switzer, 1990). Although decreased feedback
inhibition of AK1 and AKII was demonstrated in
Bacillus subtilis, improved L-lysine production was
not demonstrated. Mutations in the 5' untranslated
part of lysC mRNA decreased repression and
increased L-lysine pro-duction in Bacillus subtilis
168, but significant L-lysine production by such
mutants (more than 1 g 0 not been reported.
Unti1 now, no mutation in any AK1II-encoding
genes that leads to increased levels of L-lysine
production in Bacillus species has been described.
Dihydrodipicolinate synthase (DHDPS), encoded
by dapA, has been proved to be another key
for microbial L-lysine production at the end product
of the pathway (Cremer et al. , 1991; Eggeling and
Sahm, 2003; Grundy et al. , 2003). It is suggested that
this enzyme is not feedback inhibited in Bacillus
subtilis. Comparatively, DHDPS from plants is
strongly inhibited by lysine. The last step in the
L-lysine biosynthetic pathway is catalyzed by meso-
DAP decar-boxylase which is encoded by lysA. 1n
Bacil/us subtilis, lysA is repressed by L-lysine (Beli-
tsky, 2002).
Bacillus are gram-positive soil bacteria that
produce and export a variety of hydrolytic
that enable them to utilize complex carbohydrates in
their natural habitats (Huang, 2005). Many genes have
been successful expressed in Bacil/us subtilis, as early
as 1984, Ohmura achieved gene signal
peptide sequence and promoter E. coli
expressed in Bacillus subtilis.
sample of induced culture was detected by SDS-
PAGE and the recombinant protein band had not been
found. It might have been degraded by extra cellular
enzymes secreted by Bacillus subtilis. 1n conc1usion,
lysine content was increased by transferring alien
lysine-rich gene in Bacillus subtilis 168 from 49.32
mgoL-
1
up to 54.18 mgoL-
1
//publish.neau.edu.cn
'48. Agricultural University (English Edition) Vo1. 20 NO.l 2013
sensing by leader RNAs of bacterial lysine biosynthesis genes. Proc
Conclusions
Natl Acad Sci USA, 100: 12057-12062.
He X Y, Tang M Z, Luo Y B, et al. 2009. A 90-day toxicology study of
In this study, lys gene was cloned from winged transgenic lysine-rich maize in rague-Dawley rats. Food
bean and the recombinant plasmid pHT43/1ys was Chem Toxicol, 47(2): 425-32.
constructed, then it was expressed significantly in Wang G, Xiao L. 2005. Cloning, and expression of
Bacillus subtilis 168. Through data analysis, the con- the xylanase gene from a Bacillus subtilis strain BIO in Escherichia
tent oflysine increased by 9.85% 168. coli. Bioresour Technol, 97: 802-808.
ILSI. 2008. Maize with increased lysine (lysine maize-L Y038). In
References nutritional and safety assessments of foods and feeds nutritionally
Anastassiadis S. 2007. L-Lysine fermentation. Recent Patent Bion- studies. Compr Rev Food Sci
technol, 1: 11-24. Food 99-106.
Beauregard M, Hefford M A. 2006. Enhancement of essential amino Jose M, Gasent-Ramirez, Benitez T. 1997. Lysine-overproducing
acid contents in crops by genetic engineering and protein design. mutants of saccharmyces cerevisiae baker's yeast isolated in
Plant Biotechnol J, 4: 561-574. continuous Appl Environ Microbiol, 12: 4800-4806.
Becker J, Klopprogge C, Wittmann C. 2008. Metabolic responses Kircher M, Pfefferle, W. 2001. The fermentative production ofL-lysine
to pyruvate kinase deletion in lysine producing animal feed additive. Chemosphere, 43: 27-31
glutamicum. Microb Cell Fact, 5(13): 7-8. Leuchtenberger W. 1996. Amino acids: technical production and use
R. 2002. of amino acids of the glutamate and Rehm H J, Reed G, Puhler A, et al. Biotechnology: a multi volume
aspartate families, alanine and polyamines. Sonenshein A L, Hoch J A, comprehensive treatise. Products 01 Primary Metabolism. Weinheim,
Losick R. Bacillus subtilis 168 and its closest relatives: from York. pp: 465-502.
cells. ASM Press, Washington, DC. Li X T, Li X, Zhang J W, et al. 2006. Expression and inheritance of
Cremer J, Eggellng L, Sahm H. 1991. Control lysine biosynthesis lysine-rich protein gene in lettuce. Chin J Appl Environ Biol, 12(4):
sequence in Corynebacterium glutamicum as analyzed by over- 472-475
expression of the individual corresponding genes. Appl Environ Meng C M, Chen X Q, Liang R Q, et al. 2004. Expression of lysine-
Microbiol, 57: 1746-1752. rich protein gene and analysis of lysine content in transgenic wheat.
Eggellng L, Sahm H. 2003. New ubiquitous translocators: amino acid Chinese Science Bulletin, 49(19): 2053-2058.
export by Corynebacterium glutamicum and Escherichia coli. Arch Ohnishi J, Mitsuhashi S, Hayashi M, et al. 2002. A novel methodology
Microbiol, 180: 155-160. employing Corynebacterium glutamicum genome information
Frizzi A, Huang S, Gilbertson L A, et al. 2008. Modifying lysine to generate a new L-lysine-producing mutant. Appl Microbiol
biosynthesis and catabolism in com with a single bifunctional Biotechnol, 58(2): 217-223.
expressionlsilencing transgene cassette. Plant Biotechnol J, 6: 13-21. LosickR. 1996. Molecular genetics ofsporulation inBacillus
Galili S, Guenoune D, Wininge S, et al. 2000. Enhanced levels of free subtilis 168. Annu Rev Genet, 30: 297-41.
in transgenic alfalfa (Medicago Sun S S M, Xiong L W, Jing Y X, et al. 1998. Lysine rich protein from
L.) expressing a bacterial feedback-insensitive kinase gene. winged bean: U.S. A. application no, 08/964,722.
Transgenic 9(4): 137-144. Tosaka 0 , shihaya M, Moninaga Y. 1979. Mode of concersion of aspara-
Gao Y F, Jing Y X, Shen S H, et al. 2001. Transfer of lysine-rich to B semialdehyde to L-threonine and L-lysine in B-revibacterium
protein gene and production of fertile transgenic Acta lactofermentum. Agri Biol Chem, 43(2): 265.
Botanica Sinica, 43(5): 506-51 1. Zhu X, Galili G. 2003. Increased lysine synthesis coupled with a
Graves, L M, Switzer R L. 1990. Aspartokinase III, a new in knockout of its catabolism synergistically boosts lysine content
Bacillus subtilis 168. J Bacteriol, 172: 218-223. and also trans regulates the metabolism of other amino acids in
Grundy, F J, Lehman S C, Henkin T M. 2003. L box regulon: lysine Arabidopsis seeds. Plant Cell, 15: 845-853.
E-mail: xuebaoenglish@neau.edu.cn