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\begin{document}

\title{Improvement of Xylose Utilization and L-ornithine Production by
Metabolic Engineering of Corynebacterium glutamicum}



\author[1]{Ge Gao}%
\author[2]{Yan Zhang}%
\author[1]{Ying Zhou}%
\author[3]{Bang-Ce Ye}%
\affil[1]{East China University of Science and Technology}%
\affil[2]{Shihezi University}%
\affil[3]{State Key Laboratory of Bioreactor Engineering}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\selectlanguage{english}
\begin{abstract}
L-ornithine is a basic amino acid, which shows significant value in food
and medicine industries. There is a huge space for L-ornithine
production with strains available for metabolic engineering, and it is
urgent to develop a high-efficiency engineering strain for
industrialization. Here, xylose isomerase and xylulose kinase were
introduced into Corynebacterium glutamicum S9114 to establish xylose
metabolism pathway, and then xylose became a substitute carbon source of
glucose. In addition, the optimization and overexpression of
phosphoenolpyruvate carboxylase and pentose transporter have been
conducted to promote the synthesis of L-ornithine for the first time.
Furthermore, though optimizing the concentration ratio of glucose and
xylose (7:3), adding biotin and thiamine hydrochloride, we arrived at
the highest L-ornithine yield 41.5g/L in shaking flask fermentation so
far. Our results demonstrate that the combination of metabolic
engineering and the optimization of fermentation process can make great
potential for L-ornithine production by lignocellulose hydrolysate.%
\end{abstract}%



\sloppy


\textbf{Improvement of Xylose Utilization and L-ornithine Production by
Metabolic Engineering of \emph{Corynebacterium glutamicum}}

Ge Gao\textsuperscript{1\#}, Yan Zhang\textsuperscript{2\#}, Ying
Zhou\textsuperscript{1}*, Bang-ce Ye\textsuperscript{1,2}*

1: Laboratory of Biosystems and Microanalysis, State Key Laboratory of
Bioreactor Engineering, East China University of Science and Technology,
Shanghai 200237, China; 2: School of Chemistry and Chemical Engineering,
Key Laboratory for Green Processing of Chemical Engineering of Xinjiang
Bingtuan, Shihezi University, Shihezi, 832003, China.

*Corresponding author:\emph{zhouying@ecust.edu.cn}; bcye@ecust.edu.cn;

\section*{ABSTRACT}

{\label{abstract}}

L-ornithine is a basic amino acid, which shows significant value in food
and medicine industries. There is a huge space for L-ornithine
production with strains available for metabolic engineering, and it is
urgent to develop a high-efficiency engineering strain for
industrialization. Here, xylose isomerase and xylulose kinase were
introduced into \emph{Corynebacterium glutamicum} S9114 to establish
xylose metabolism pathway, and then xylose became a substitute carbon
source of glucose. In addition, the optimization and overexpression of
phosphoenolpyruvate carboxylase and pentose transporter have been
conducted to promote the synthesis of L-ornithine for the first time.
Furthermore, though optimizing the concentration ratio of glucose and
xylose (7:3), adding biotin and thiamine hydrochloride, we arrived at
the highest L-ornithine yield 41.5g/L in shaking flask fermentation so
far. Our results demonstrate that the combination of metabolic
engineering and the optimization of fermentation process can make great
potential for L-ornithine production by lignocellulose hydrolysate.

\textbf{Keywords:} L-ornithine; \emph{Corynebacterium glutamicum} ;
Xylose; Metabolic engineering

\section*{INTRODUCTION}

{\label{introduction}}

L-ornithine is non-proteinogenic amino acid, which is an important
intermediate product of the urea cycle pathway. It is a downstream
product of glutamate and a key precursor for the production of
L-citrulline, L-proline and L-arginine. L-ornithine has attracted
special attention for its biological functions (Sivashanmugam, J, V, \&
K, 2017), and is widely used in daily health care and disease treatment
such as the positive effect on the protection and vitality recovery of
human liver and the heart (Butterworth, 2020; Das et al., 2020; Davies
et al., 2009), improving visual function (Sakamoto, Mori, Nakahara,
Morita, \& Ishii, 2015), regulating the secretion of hormones (Matsuo et
al., 2015). Moreover, a recent report demonstrates that L-ornithine as
raw material has a great prospect for the development of new anticancer
drugs (Vargas-Ram\selectlanguage{ngerman}írez et al., 2016).

\emph{Corynebacterium glutamicum} is the most important amino acids
producing strain in the past 40 years, such as glutamate, L-alanine,
L-serine, L-arginine, and L-proline (Gutmann, Hoischen, \& Krämer, 1992;
Jensen, Eberhardt, \& Wendisch, 2015; Jojima, Fujii, Mori, Inui, \&
Yukawa, 2010; Park et al., 2014; X. Zhang et al., 2018).
Although\emph{E. coli} and \emph{Saccharomyces} \emph{cerevisiae} have
ever been engineered to be the producer of L-ornithine (Becker \&
Wittmann, 2015; Lee \& Cho, 2006; Qin et al., 2015), \emph{C.
glutamicum,} which produces large amounts of glutamate, the precursor of
L-ornithine, still to be the preferred dominant strain (Zhang, Yu, Zhou,
\& Ye, 2018). The concentration of intracellular glutamate is an
important driving force for L-ornithine production.

Random mutation together with genetic and metabolic engineering are two
key methods in the breeding process for L-ornithine production
by\emph{C. glutamicum} . However, the random mutation breeding has many
disadvantages, including the complex screening process, low success
rate, high probability of reverse-mutation. Those made directed genetic
metabolic engineering to be the priority option. A series of measures
were taken to increase the L-ornithine production by genetic engineering
modification (Wu, Guo, Zhang, Jiang, \& Ye, 2019). First, focused
primarily on the main L-ornithine synthesis pathway (Hao et al., 2016;
Jensen et al., 2015; Jiang, Zhang, Li, \& Liu, 2013; D. J. Kim, Hwang,
Um, \& Cho, 2015; Q. Shu et al., 2018; Zhang, Ren, Yu, Zhou, \& Ye,
2018; Zhang, Yu, Zhou, Li, \& Ye, 2017); second, the transportation of
L-ornithine amino acids (B. Zhang, L. Q. Ren, et al., 2018); last, based
on glycolysis, acetic acid metabolism, pentose phosphate pathway (S. Y.
Kim, Lee, \& Lee, 2015; Zhang, Yu, Wei, \& Ye, 2018), the tricarboxylic
acid cycle and glucose utilization pathway (Ikeda et al., 2011; Lindner
et al., 2013; Lindner, Seibold, Henrich, Kramer, \& Wendisch, 2011; Xu,
Zhang, Liu, \& Zhang, 2016; Zhang, Gao, Chu, \& Ye, 2019; Zhou, Wang,
Xu, Chen, \& Cai, 2015). In addition to rational modification, adaptive
evolution strategies combined with transcriptional levels analysis
provides another strategy to develop a strain with high performance
(Jiang, Chen, Zhang, \& Liu, 2013). Jensen et al. constructed \emph{C.
glutamicum} ORN6 by knocking out \emph{argF} , \emph{argR}
and\emph{argG} , attenuating the expression of \emph{pgi} and increasing
the copy number of the arginine operon argCJB\textsuperscript{A49V,
M54V}D on the chromosome, the L-ornithine yield was 0.52g/g (Jensen et
al., 2015). Hwang et al. knocked out \emph{ncgl2053} , \emph{ncgl0281}
and\emph{ncgl2582} that encoding NADP\textsuperscript{+}-dependent
oxidoreductase, which resulted in the loss of glucose dehydrogenase
activity and the increasing of 6-phosphate gluconate dehydrogenase
activity. The production of L-ornithine is 66.3\% higher than that of
the starting strain (Hwang \& Cho, 2014). Shu et al. deleted
\emph{proB}and \emph{argF} to block the branch of the L- ornithine
synthesis pathway, mutated \emph{ArgB} and expressed heterologous
\emph{argA} and\emph{argE} to introduce an artificial linear
transacetylation pathway. The production of L-ornithine was 40.4g/L in
5-L bioreactor (Qunfeng Shu et al., 2018). Zhang et al adopted a series
of genetic engineering modifications to achieved the maximum L-
ornithine yield of 43.6g/L in fed batch fermentation by far (Zhang et
al., 2019). Although many strategies have been adapted to increase the
production of L-ornithine, how to build a more efficient industrial
strain with practical applications is still a long way off.

In order to make good use of the most abundant renewable resources and
do not compete with people for food, a large amount of studies have
focused on how to construct an efficient microbial cell factory
utilizing xylose and glucose as mixed carbon sources in the past fifteen
years (Becker, Rohles, \& Wittmann, 2018). Thanks to the weak carbon
catabolite repression, \emph{C. glutamicum} is regarded as a major
industrial force with great potential in recent years (Buschke, Schafer,
Becker, \& Wittmann, 2013; Wendisch et al., 2016). However, due
to\emph{C. glutamicum} lacks of xylose isomerase (XylA), it could not
grow on medium containing xylose as the sole carbon source. Buschke et
al. and Gopinath et al. used the exogenous xylose isomerase (XylA) and
xylulose kinase (XylB) to establish the isomerase pathway in \emph{C.
glutamicum} to realize the utilization of xylose (Buschke, Becker, et
al., 2013; Gopinath, Meiswinkel, Wendisch, \& Nampoothiri, 2011). Five
copies of \emph{xylAB} operon from \emph{E. coli} were integrated
to\emph{C. glutamicum} R chromosome to generate the strain X5C1, which
could consume 40 g/L glucose and 20 g/L xylose in 12h (Sasaki, Jojima,
Inui, \& Yukawa, 2008). In addition, several other strategies were
adopted to improve the xylose utilization, including the introduction of
arabinose transporter (Brusseler et al., 2018; H. Kim et al., 2017),
overexpressing of TAL/TKT in the pentose phosphate pathway (Jo et al.,
2017). Meiswinkel et al. constructed an engineered strain \emph{C.
glutamicum} PUT21 by introducing \emph{xylA} from \emph{X. campestris}
,\emph{xylB} from \emph{C. glutamicum} and \emph{argBAD} operon
from\emph{E. coli} to produce 2.59 g/L of L-ornithine, and the
volumetric ornithine productivity is 43.2 mg/(L\selectlanguage{english}[?]h) (Meiswinkel,
Gopinath, Lindner, Nampoothiri, \& Wendisch, 2013). In addition to
xylose isomerase metabolic pathway, Christian et al. introduced the
\emph{xylXABCD}operon from \emph{Caulobacter crescentus} into \emph{C.
glutamicum}ATCC13032 to establish the Weinberg pathway (Brusseler et
al., 2018). Although the utilization of xylose by \emph{C. glutamicum}
has already been realized, the utilization rate of xylose is still
unsatisfied. More modified strategies are needed to improve the
utilization rate of xylose and the production of L-ornithine., which
opened the door to the efficient utilization of lignocellulose.

In our previous studies, we have successfully constructed the \emph{C.
glutamicum} SO26 with high L-ornithine yield (Zhang et al., 2019; B.
Zhang, L. Q. Ren, et al., 2018). In this study, we attempted to utilize
the most abundant carbon source in lignocellulose hydrolysate - glucose
and xylose. We adopted the approaches of metabolic engineering and
fermentation process control to accelerate the xylose consumption rate
and the yield of L-ornithine. First, a more efficient \emph{xylAB}operon
was screened out from different strains, and the arabinose transporter
araE from \emph{Bacillus subtilis} was knocked into the\emph{iolR} locus
under the promoter P\textsubscript{eftu}. Second, the acetylation of
phosphoenolpyruvate carboxylase (PEPC) was reduced to release the
feedback inhibition of aspartic acid, and a strong constitutive promoter
P\textsubscript{H36} was introduced in the upstream of \emph{pepc} . The
strain after a series of modulations is named \emph{C. glutamicum}
XAB03. Finally, though the optimization of fermentation process, we
found the concentration ratio of glucose and xylose (7:3) and the
coenzyme addition (biotin 0.9 \selectlanguage{greek}μ\selectlanguage{english}M and thiamine-HCl 15 \selectlanguage{greek}μ\selectlanguage{english}M respectively)
can realize the highest L-ornithine yield 41.5g/L in shaking flask
fermentation up to date. All the metabolic engineering process are shown
in Fig.1. Schematic diagram.

\section*{MATERIALS AND METHODS}

{\label{materials-and-methods}}

\section*{Bacterial strains, plasmids and
primers}

{\label{bacterial-strains-plasmids-and-primers}}

The strain SO26 originated from \emph{C. glutamicum} S9114 underwent a
series of modifications (deletion of \emph{argF} , \emph{ncgl1221}
,\emph{argR} , \emph{putP} , \emph{iolR} , and \emph{mscCG2} ;
attenuation of\emph{odhA} , \emph{proB} , \emph{pta} , \emph{cat} , and
\emph{ncgl2228} ; and overexpression of \emph{lysE} , \emph{gdh} ,
\emph{gdh2} ,\emph{cg3035} , \emph{pfkA} , \emph{pyk} , \emph{glt} ,
\emph{tkt} ,\emph{argCJBD} , and \emph{iolT1} ) was used as a starting
strain for further metabolic engineering in this study. \emph{E. coli}
DH5\selectlanguage{greek}α \selectlanguage{english}was used as host for rapid replication of recombinant plasmids.
Xylose isomerase (\emph{xylA} ) and xylulose kinase (\emph{xylB} ) were
amplified from \emph{E. coli} K-12 MG1655 and \emph{X. campestris} . The
arabinose transporter (araE) were derived from \emph{Bacillus subtilis}
. All the strains and plasmids used in this study are presented in Table
1.

\section*{Construction of plasmids and
strains}

{\label{construction-of-plasmids-and-strains}}

The basic DNA manipulation and strain construction was operated
according to the standard molecular cloning manual.\emph{E. coli} DH5\selectlanguage{greek}α
\selectlanguage{english}was used as the host of gene cloning. All the primers used in this study
are presented in Table S1 (Supplementary Information). The suicide
vector pK18\emph{mobsacB} containing the sucrose lethal gene \emph{sacB}
was used to delete or integrate gene on chromosome.

The \emph{xylAB} gene clusters from \emph{E. coli} and \emph{X.
campestris} were amplified by primers EAB-F/EAB-R and XAB-F/XAB-R
respectively, the inserted restriction sites are\emph{Hind}
III/\emph{EcoR} I and \emph{Hind} III/\emph{Sac} I of pXMJ19. The
plasmids pXMJ19, p19P\textsubscript{lac}-EcoAB, and
p19P\textsubscript{lac}-XcaAB were transformed into \emph{C.
glutamicum}SO26 to produce strains \emph{C. glutamicum} pX\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}EAB\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}XAB.
\emph{AraE}from \emph{B. subtilis} with a constitutive
P\emph{\textsubscript{eftu}}promoter were amplified by araE-F/araE-R and
up-eftu-F/araE-eftu-R respectively. The product of fusion PCR was
inserted into the\emph{EcoR} V site of plasmid pk18-^\emph{iolR} by
Gibson assembly, and then generated the recombinant plasmid pk18-
P\textsubscript{eftu}-^iolR::araE and corresponding strain XAB01.

To increase PEPC expression by lowing the acylation of PEPC, a
synthesized strong promoter H36 was inserted in the upstream
of\emph{pepc} gene (Yim, An, Kang, Lee, \& Jeong, 2013), and then the
AAG bases from 1957 bp to 1959 bp were mutated to CGC by overlap PCR,
the primer up-HA-F1 / up-HA-H36-R2 amplified \emph{pepc} upstream
homology arm sequence, primer up-HA-H36-F3 / pepc-H36-R4 amplified H36
promoter, primer mutant-(KR)-F/down-HA-pepc-R8 realizes the base
mutation of K653R, pepc-H36-F5/mutant-(KR)-R amplifies the \emph{pepc}
upstream homology arm sequence, primer down-HA-pepc-F9/down-HA-R10
amplified the homology arm sequence downstream of \emph{pepc} . Finally,
the entire fragment was combined by Gibson assembly under the primer
up-HA-F1/down-HA-R10, resulted the recombinant plasmid
pk18-P\textsubscript{H36}-\emph{pepc} T1. Furthermore, to reduce the
feedback inhibition of aspartic acid on PEPC, the same operation has
been done at 895-897 bp AAC to replace GAT, amplification using
pepc-H36-F5/D299N-R6 and D299N-F7/down-HA-pepc-R8 with
pk18-P\textsubscript{H36}-\emph{pepc} T1 as a template can realize base
mutation of D299N, forming the pk18-P\emph{\textsubscript{H36}}
-\emph{pepc} T2, resulted in the strain\emph{C. glutamicum} XAB03.
Primers pXMJ19-F/pXMJ19-R and M13 fwd/M13 rev are verification primers
for pXMJ19 and pK18\emph{mobsacB} , respectively.

All the recombinant plasmids were constructed in \emph{E. coli} DH5\selectlanguage{greek}α \selectlanguage{english}and
transformed into \emph{C. glutamicum} by electroporation. The mutant
strains were screened through two rounds of homologous recombination,
and further confirmed by colony PCR and sequencing.

\section*{Cultivation medium and
conditions}

{\label{cultivation-medium-and-conditions}}

Luria--Bertani (LB) medium containing NaCl 10 g/L, tryptone 10 g/L and
yeast extract 5 g/L were used for cultivation of \emph{C. glutamicum}and
\emph{E. coli} . Antibiotic was added to the medium for mutants
screening when needed: 50 \selectlanguage{greek}μ\selectlanguage{english}g/mL kanamycin or 30 \selectlanguage{greek}μ\selectlanguage{english}g/mL chloramphenicol
for \emph{E. coli} , 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL kanamycin or 15 \selectlanguage{greek}μ\selectlanguage{english}g/mL chloramphenicol
for\emph{C. glutamicum} .

For the shaking flask fermentation experiment, the correct monoclonal
strains were activated twice on the LB medium for 36 h, and then the
appropriate amount of bacterial seed was inoculated into a 100 mL flask
containing 10 mL of seed solution. The seed medium contains glucose 30
g/L, corn steep liquor 10 g/L, yeast extract 10 g/L,
(NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4} 15 g/L,
MgSO\textsubscript{4} 2.5 g/L, KH\textsubscript{2}PO\textsubscript{4} 1
g/L, K\textsubscript{2}HPO\textsubscript{4} 0.5 g/L,
Na\textsubscript{2}HPO\textsubscript{4}\selectlanguage{ngerman}·12H\textsubscript{2}O 1.26 g/L
and CaCO\textsubscript{3} 10 g/L, the pH was adjusted to 6.7
(Supplementary Information: Figure S1). The monoclonal strains were
incubated at 30°C, 220 rpm for 11 h. Take an appropriate amount of seed
solution and inoculate it in a 250 mL Erlenmeyer flask containing 20 mL
of fermentation medium so that the OD\textsubscript{600} of the initial
fermentation solution is 1 at 600 nm. The fermentation medium contains
xylose 100 g/L or glucose 70 g/L and xylose 30 g/L, yeast extract 6 g/L,
(NH\textsubscript{4})\textsubscript{2}SO\textsubscript{4} 50 g/L,
MgSO\textsubscript{4} 2.5 g/L, KH\textsubscript{2}PO\textsubscript{4} 1
g/L, K\textsubscript{2}HPO\textsubscript{4} 0.5 g/L,
Na\textsubscript{2}HPO\textsubscript{4}·12H\textsubscript{2}O 1.26 g/L,
MnSO\textsubscript{4}·H\textsubscript{2}O 0.02 g/L,
FeSO\textsubscript{4}·7H\textsubscript{2}O 0.02 g/L, biotin 0.9 \selectlanguage{greek}μ\selectlanguage{english}M,
thiamine-HCl 15 \selectlanguage{greek}μ\selectlanguage{english}M and CaCO\textsubscript{3} 10 g/L, while the pH was
adjusted to 6.7. The strains in fermentation medium were incubated at
31.5\selectlanguage{ngerman}°C, 250 rpm for 72 h.

\section*{Analytical methods of cell growth and fermentation
products}

{\label{analytical-methods-of-cell-growth-and-fermentation-products}}

Every 12 hours, three parallel samples (100 \selectlanguage{greek}µ\selectlanguage{english}L) were taken to monitor
the bacteria density, consumption of glucose and xylose, and L-ornithine
production. The OD\textsubscript{600} value was detected to assess the
cell growth by a microplate reader (BioTek Instruments, Winooski, VT,
USA) after adding 0.125 mol/L HCl to dissolve CaCO\textsubscript{3}
(Zhang et al., 2019). The samples were centrifuged to obtain
fermentation supernatant. The glucose concentration was analyzed by
SBA-40E biosensor analyzer (Institute of Biology, Shandong Academy of
Sciences, Jinan, Shandong, China). The level of xylose consumption was
determined by high-performance liquid chromatography (HPLC) (Chen, Zhu,
\& Xia, 2014; Yim et al., 2013). The content of L-ornithine was
determined by ninhydrin colorimetry (B. Zhang, M. Yu, Y. Zhou, et al.,
2018). All experiments had triple parallels, and data are repressed as
mean and standard deviation (SD).

\section*{RESULTS AND DISCUSSION}

{\label{results-and-discussion}}

Comparison with the xylose utilization capacity of xylose isomerases and
xylulose kinases from different sources

{\label{comparison-with-the-xylose-utilization-capacity-of-xylose-isomerases-and-xylulose-kinases-from-different-sources}}

Xylose isomerase (\emph{xylA} ) and xylulose kinase (\emph{xylB} ) exist
in the form of gene clusters. The xylose isomerase metabolic pathway
from different sources established in \emph{C. glutamicum} have
different xylose utilization capacities, this might be the result of the
genetic codon preference between the \emph{xylAB} source strain
and\emph{C. glutamicum} (Meiswinkel et al., 2013). The \emph{xylAB}
genes derived from \emph{E. coli} MG1655 and \emph{X. campestris} were
expressed under IPTG induced vector pXMJ19, in which the
P\textsubscript{tac} is the promoter for gene expression. The resulting
expression plasmids were labeled p19P\textsubscript{lac}-EcoAB and
p19P\textsubscript{tac}-XcaAB. The two plasmids were transformed into
\emph{C. glutamicum} SO26, resulted the \emph{C. glutamicum} EAB and
XAB. Fig.2a showed the growth curve and xylose consumption of strain EAB
and XAB during 72h fermentation. The results indicated that \emph{xylAB}
from \emph{X. campestris} achieved higher xylose consumption (45.1 g/L)
compared with \emph{xylAB} from\emph{E. coli} MG1655 (25.0 g/L). The
growth OD\textsubscript{600}increased from 12.05 (EAB) to 12.95 (XAB).
The L-ornithine concentration in the supernatant of fermentation was
determined (Fig.2b). The L-ornithine production titer of the strain XAB
(21.6 \selectlanguage{ngerman}± 0.19 g/L) is 18.7\% higher than strain EAB (18.2 ± 0.35 g/L),
and the corresponding xylose yield of XAB is 0.48 g/g. The \emph{C.
glutamicum} XAB demonstrated the better capacity of xylose utilization
and L-ornithine synthesis than strain EAB.

Knocking-in the pentose transporter demonstrates the promotion of xylose
utilization and L-ornithine production

{\label{knocking-in-the-pentose-transporter-demonstrates-the-promotion-of-xylose-utilization-and-l-ornithine-production}}

In order to increase the rate of xylose consumption, the pentose
transporter gene (\emph{araE} ) from \emph{Bacillus subtilis} was
integrated into the genome locus of\emph{iolR} with the strong
P\textsubscript{eftu} promoter, to generate the strain termed as XAB01.
The pentose transporter AraE is not only extremely significant for the
arabinose transportation, but also promotes the transportation of xylose
to cells (Mao et al., 2018). As expected, the results showed that strain
XAB01 demonstrated superior xylose consumption and L-ornithine synthesis
than strain XAB (Fig.3a and 3b). strain XAB01 consumed 47.9 g/L xylose
after 72h fermentation, with an average consumption rate of 0.665
g/(L·h). Through the rapid utilization of xylose, the growth and
L-ornithine yield of strain were also improved. The output of
L-ornithine increased by 12.5\% (24.3 ± 0.23g/L) compared with strain
XAB, and the xylose yield is 0.51 g/g.

\section*{Effect of modification of phosphoenolpyruvate carboxylase on
the production of
L-ornithine}

{\label{effect-of-modification-of-phosphoenolpyruvate-carboxylase-on-the-production-of-l-ornithine}}

Phosphoenolpyruvate carboxylase (PEPC) is an enzyme in the glycolysis
pathway of \emph{C. glutamicum} and plays an important role in the
regulation of the TCA cycle (Fig.1). Phosphoenolpyruvate and carbon
dioxide synthesize oxaloacetic acid under the catalysis of PEPC, and
then oxaloacetic acid enters the TCA cycle for further metabolism. The
expression and activity of PEPC affect the synthesis of glutamic acid
in\emph{C. glutamicum} (Nagano-Shoji et al., 2017; Wada et al., 2015).
Lysine at position 653 (K653) is essential for the regulation of PEPC
acetylation. Megumi ed al. found that acetylation of PEPC at K653
decreased enzymatic activity and reduced glutamate production, this
means K653-acetylation regulates PEPC activity negatively. Mutated K653
into arginine can lower the level of acetylation on PEPC, which
correspondingly improve its activity (Nagano-Shoji et al., 2017). In
addition, PEPC will be feedback suppressed by aspartic acid when it is
overexpressed in \emph{C. glutamicum} , mutated aspartic acid at
position 299 of PEPC to asparagine with a similar structure can
effectively reduce the inhibitory effect (Wada et al., 2015). In our
study, we attempt to combine the weakening acetylation by K653R and the
attenuating feedback inhibition by D299N of PEPC to enhance the activity
of this enzyme, leading to promote the glutamate synthesis and the
L-ornithine yield. We selected the more preferred CGC and AAC codons
in\emph{C. glutamicum} S9114 to mutate K653 and D299. At the same time,
the \emph{pepc} gene was overexpressed by adding a strong constitutive
promoter H36 (Yim et al., 2013). Multiple gene fragments are fused into
a complete fragment by PCR overlapping amplification technology. After
two point mutations in \emph{pepc} , strain XAB03 was constructed. The
shaking flask fermentation of strain XAB03 showed that the production of
L-ornithine was 27.1 ± 0.32 g/L, which was 11.5\% higher than that of
strain XAB01 (Fig.4b). Moreover, it has a positive effect on the growth
of \emph{C. glutamicum} (Fig.4a). These results indicate the importance
of PEPC in L-ornithine synthesis, and this modification strategy shows
value for the synthesis of L-glutamate, L-citrulline and L-arginine.

\section*{Addition of biotin and thiamine hydrochloride accelerates the
synthesis of
L-ornithine}

{\label{addition-of-biotin-and-thiamine-hydrochloride-accelerates-the-synthesis-of-l-ornithine}}

In addition to the modification of key enzymes in metabolic pathways,
the addition of key coenzymes is also an important method to promote the
synthesis of products (Y. Cao, Duan, \& Shi, 2014). Biotin and thiamine
hydrochloride are the coenzymes of carboxylase in the metabolic process.
Biotin plays an important role in the metabolism of bacterial proteins,
which could change the content of cell membrane components and
permeability. Different concentrations of biotin affect the
transcription levels of enzymes and promote the synthesis of glutamate
(Y. Cao et al., 2014; Yan Cao, Mpofu, Jian, \& Zuoying, 2012). In order
to further optimize the fermentation process, we attempted to adding the
coenzyme during the L-ornithine production. The concentration of biotin
and thiamine hydrochloride in the fermentation medium was 0.9 \selectlanguage{greek}μ\selectlanguage{english}M and 15
\selectlanguage{greek}μ\selectlanguage{english}M (Hyun Jin, Myung Cho, \& Hoon Park, 2007), and the L-ornithine
production increased to 33.4 g/L compared with no adding coenzyme
fermentation (27.1 g/L) (Fig.5). During the 0-48 h fermentation period
in shake flasks, the strain XAB03 grows with abundant nutrients, the
energy distribution mainly focuses on the growth of the bacteria while
the production of L-ornithine is weak. From 48-72 h, the growth tends to
be stable and the L-ornithine is synthesized rapidly. The results
provide a research direction for the promotion of certain target
products through the addition of coenzymes and some small molecules.

\section*{Effects of a combination of glucose and xylose on
L-ornithine}

{\label{effects-of-a-combination-of-glucose-and-xylose-on-l-ornithine}}

Metabolism of different sugars is an important basement for the
lignocellulose utilization. Glucose and xylose are the most important
six- and five-carbon sugars in the hydrolysis of lignocellulose. We
compared L-ornithine yield in different ratios of glucose and xylose in
the case of the total sugar concentration was constant. On the premise
that the total sugar concentration is 100 g/L, seven groups of different
glucose concentration gradients were chosen (Fig.5). The mixed carbon
sources showed the advantage of L-ornithine yield compared with using
glucose or xylose as the sole carbon source. The results showed that the
maximum L-ornithine production could be obtained (41.5 \selectlanguage{ngerman}± 0.02 g/L) when
glucose is 70 g/L and xylose is 30 g/L (Fig.5 b, c).

Based on all the above conditions, the OD\textsubscript{600} and
L-ornithine production of strain XAB03 could reach to 16.8 ± 0.19 and
41.5 g/L (Fig.6c) respectively after shake flask fermentation for 72h,
which is the highest titer so far to the best of our knowledge (Table
2), 7.8\% higher than Zhang et al. had reported, and increased by 43.4\%
compared with the sole xylose carbon source (Zhang et al., 2019). The
2:1 ratio is close to the ratio of glucose to xylose after
lignocellulose hydrolysis. This result lays a foundation for the
feasibility and superiority of L-ornithine synthesis from lignocellulose
hydrolysate.

\section*{CONCLUSION}

{\label{conclusion}}

In this study, \emph{xylAB} operon was introduced into \emph{C.
glutamicum} SO26 to achieve the consumption of xylose for the production
of L-ornithine. And then, we verified the AraE, the reduction in the
degree of acetylation and the release of feedback inhibition of aspartic
acid of PEPC, the addition of biotin and thiamine hydrochloride, the
resulting strain \emph{C. glutamicum} XAB03 has reached 41.5 g/L shaker
flask output from glucose and xylose. This work also shows the
possibility of making full use of lignocellulose for the synthesis of
L-ornithine and lays the foundation for the further realization of
industrialized strain production.

\section*{FUNDING SOURCES}

{\label{funding-sources}}

This research was supported and funded by the National Key Research and
Development Project (Grant no. 2018YFA0900404), the National Natural
Science Foundation of China (Grant no. 31730004) and the Shanghai
Municipal Science and Technology Commission Project (18JC1410802).

\section*{ACKNOWLEDGMENT}

{\label{acknowledgment}}

We would like to thank Dr. Li-Ming Liu for providing us
with\emph{Corynebacterium glutamine} S9114.

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\textbf{GRAPHICAL ABSTRACT}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig.1. Schematic diagram of the metabolic engineering process of\emph{C.
glutamicum} .

\emph{pepc} encodes phosphoenolpyruvate carboxylase; \emph{pyk} encodes
pyruvate kinase; \emph{xylA} encodes xylose isomerase;
\emph{xylB}encodes xylulose kinase; \emph{aat} encodes aspartate
aminotransferase;\emph{araE} encodes pentose transporter; \emph{argF}
encodes N-ornithine carbamoyl transferase.

Table 1. Strains and plasmids\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
Strains/plasmids & Relevant characteristics & Source\tabularnewline
\midrule
\endhead
\textbf{Strains} & &\tabularnewline
\emph{E. coli} DH5a & Clone host strain & Transgen\tabularnewline
\emph{E. coli} K-12 MG1655 & The source of Eco-\emph{xylAB} genes & Lab
stock\tabularnewline
\emph{X. campestris} & The source of Xca-\emph{xylAB} genes & Lab
stock\tabularnewline
\emph{C. glutamicum} SO26 & Deletion of \emph{argF}, \emph{ncgl1221},
\emph{argR}, \emph{putP}, \emph{iolR}, and \emph{mscCG2}; attenuation of
\emph{odhA}, \emph{proB}, \emph{pta}, \emph{cat}, and \emph{ncgl2228};
and overexpression of \emph{lysE}, \emph{gdh}, \emph{gdh2},
\emph{cg3035}, \emph{pfkA}, \emph{pyk}, \emph{glt}, \emph{tkt}, and
\emph{iolT1} & Lab stock\tabularnewline
\emph{Bacillus subtilis} & The source of pentose transporter \emph{araE}
gene & Lab stock\tabularnewline
\emph{C. glutamicum} pX & \emph{C. glutamicum} SO26 with pXMJ19 & This
study\tabularnewline
\emph{C. glutamicum} EAB & \emph{C. glutamicum} SO26 with
p19P\textsubscript{lac}-EcoAB & This study\tabularnewline
\emph{C. glutamicum} XAB & \emph{C. glutamicum} SO26 with
p19P\textsubscript{lac}-XcaAB & This study\tabularnewline
\emph{C. glutamicum} XAB01 & XAB with the knocking of \emph{araE} gene
into the locus of \emph{iolR} & This study\tabularnewline
\emph{C. glutamicum} XAB02 & XAB01with P\textsubscript{H36} promoter in
the upstream of \emph{pepc} gene and 1957-1959 bp CGC replaces AAG in
\emph{pepc} gene & This study\tabularnewline
\emph{C. glutamicum} XAB03 & XAB02 with 895-897 bp AAC replaces GAT in
\emph{pepc} gene & This study\tabularnewline
\textbf{Plasmids} & &\tabularnewline
pK18mobsacB & The suicide vector containing the \emph{B. subtilis sacB}
gene; kan\textsuperscript{R}, allows for selection of double crossover
in \emph{C. glutamicum} & Lab stock\tabularnewline
pXMJ19 & High copy expression vector,Cm\textsuperscript{R},LacIq
promoter,tac promoter & Lab stock\tabularnewline
p19P\textsubscript{lac}-EcoAB & A derivative of pXMJ19, harboringthe
\emph{xylAB} operon from \emph{E. coli} MG1655 & This
study\tabularnewline
p19P\textsubscript{lac}-XcaAB & A derivative of pXMJ19, harboringthe
\emph{xylAB} operon from \emph{X. campestris} & This
study\tabularnewline
pk18-P\textsubscript{eftu}-^iolR::araE & Using pK18\emph{mobsacB} and
\emph{araE} to connect through the upper and lower homology arms of
^\emph{iolR}, and add P\textsubscript{eftu} promoter in the upstream of
\emph{araE} gene & This study\tabularnewline
pk18-P\textsubscript{H36}-pepcTI & A originated from pK18\emph{mobsacB},
add P\textsubscript{H36} promoter in the upstream of pepc gene and
1957-1959 bp CGC replaces AAG in \emph{pepc} gene & This
study\tabularnewline
pk18-P\textsubscript{H36}-pepcT2 & On the basis of
pk18-P\textsubscript{H36}-pepcTI, harboring 895-897 bp AAC replaces GAT
in \emph{pepc} gene & This study\tabularnewline
\bottomrule
\end{longtable}

Table 2. Synthesis of L-ornithine by \emph{C. glutamicum}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Strains\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
L-ornithine concentration (g\selectlanguage{ngerman}·L\textsuperscript{-1})/yield\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Cultivation\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Sugar source\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
Modulations\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
References\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} SJC8514\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
12.48/ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Shake flask; batch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Glucose\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Overexpression of \emph{ncgl0452} and \emph{argCJBD}mut\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(D. J. Kim et al., 2015)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} SJC8039\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
14/ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Shake flask; batch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Glucose\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Deletion of \emph{argF}, \emph{argR}, and \emph{proB}; Blocking
gluconate biosynthesis\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Hwang \& Cho, 2014)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} YW06\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
51.5/0.240\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Bioreactor; fed-batch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Glucose\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Deletion of \emph{argF}, \emph{argR} and \emph{proB}; Reinforcement of
the PPP pathway flux; The use of a feedback-resistant enzyme\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(S. Y. Kim et al., 2015)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{C. glutamicum} ORN1 (pVWEx1-\emph{araBAD})\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
25.8/0.78\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Shake flask\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Glucose, arabinose\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Deletion of \emph{argF}, \emph{argR}; in-frame deletion of \emph{argF}
and \emph{argR}, auxotrophic for l-arginine\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
(Schneider, Niermann, \& Wendisch, 2011)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} [?]APE6937R42\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
24.1/0.298\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Bioreactor; batch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Glucose\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Deletion of \emph{argF}, \emph{argR}, and \emph{proB}; Adaptive
evolution in presence of L-ornithine\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Jiang, Chen, et al., 2013)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} [?]\emph{APE::rocG}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
14.8/ ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Shake flask; batch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Glucose\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Deletion of \emph{argF}, \emph{proB}, \emph{speE}::Ptac-M-\emph{rocG},
\emph{argR}::Ptac-M-\emph{gapC}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Jiang, Zhang, et al., 2013)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} ORN6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
20.96/0.524\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Shake flask; batch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Glucose\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Deletion of \emph{argF}, \emph{argR}, and \emph{argG}; overexpression of
\emph{argBM}; attenuation of \emph{pgi}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Jensen et al., 2015)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} 1006 \emph{[?]argR-argJ}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
31.6/0.396\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Shake flask; batch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Glucose\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Deletion of \emph{argR}; overexpression of \emph{argJ}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(Hao et al., 2016)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} XAB03\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
41.5/ND\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Shake flask; batch\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Glucose, xylose\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{C. glutamicum} SO26 with \emph{xylAB} from \emph{X. campestris},
expression of \emph{araE}, overexpression of \emph{pepc}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
This study\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig.2. Comparison of the effects of \emph{xylAB} genes from different
strains.

a: Growth curve (solid) and xylose consumption (hollow) curves. b:
L-Ornithine production curves. Strain pX (hollow circular) is a control
strain containing pXMJ19, Strain XAB (hollow square)
overexpresses\emph{xylAB} from \emph{E. coil} , Strain EAB (hollow upper
triangle) overexpresses \emph{xylAB} from \emph{X. campestris} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Fig.3. The promotion of \emph{araE} on xylose and the effect on
L-ornithine production.

a Growth and xylose consumption curves. b L-Ornithine production curves.

Strain XAB01 (blue solid triangle) integrated the \emph{araE} gene
from\emph{B. subtilis} with a strong promoter Peftu, and XAB (black
hollow square) is the control strain.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Fig.4. The PEPC modification reinforces the production of L-ornithine.

a: Growth curves. b: L-Ornithine production curves. XAB01 (hollow
square) integrated the \emph{araE} gene from \emph{B. subtilis} with a
strong promoter Peftu. XAB03 (hollow upper triangle) modified\emph{pepc}
on the basis of XAB01.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Fig.5. The effect of adding biotin and thiamine hydrochloride on
L-ornithine production. a Growth curves for XAB03. b L-Ornithine
production curves for XAB03.

Added (Biotin and thiamine hydrochloride, hollow upper triangle), No
added (hollow square).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Fig.6. The effect of different ratios of glucose and xylose on
L-ornithine production

a: Growth curves for \emph{C. glutamicum} XAB03 at different glucose
concentrations ratios (100\%, blue circle; 90\%, red hollow square;
70\%, green upper triangle; 50\%, purple diamond; 30\%, orange lower
triangle; 10\%, black regular hexagon; 0\%, yellow solid square.). b:
L-Ornithine production curves. c: Optimal shake flask fermentation
experiment of \emph{C. glutamicum} XAB03 in OD\textsubscript{600}(hollow
upper triangle) and L-ornithine production (solid square) in glucose 70
g/L and xylose 30 g/L.

\section*{Supplementary Information}

{\label{supplementary-information}}

Table S1. Primers involved in this article\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Primers & Sequence (5'-3')\tabularnewline
\midrule
\endhead
XAB-F &
ggatcttccagagat\emph{aagctt}aaggagatatacatgagcaacaccgttttcatc\tabularnewline
XAB-R &
caaaacagccaag\textbf{g\emph{agctct}}gatgaatcagccggcgtagag\tabularnewline
EAB-F &
caggaaacagaataatt\emph{aagctt}gggttgacattccgcggcattacctgattatgg\tabularnewline
EAB-R &
caaaacagccaagct\emph{gaattc}ttacgccattaatggcagaagttgc\tabularnewline
up-eftu-F &
ccaccgaagctccgctc\emph{gatatc}cacagggtagctggtagtttgaaaatc\tabularnewline
araE-eftu-R &
ccctcccgaatgttgagtaaagcacttcgtggtggctacgactttc\tabularnewline
araE-F & gaaagtcgtagccaccacgaagtgctttactcaacattcgggaggg\tabularnewline
araE-R &
gaggcgcatatggaattc\emph{gatatc}aacagcccttcccgtagaaagg\tabularnewline
up-HA-F1 &
cctgcaggtcgac\emph{tctaga}gcccttccaaaggaatacttcgagaag\tabularnewline
up-HA-H36-R2 &
ccagatagaggtacccagcttttgaactactttaaacactctttcacattgaggg\tabularnewline
up-HA-H36-F3 &
ccctcaatgtgaaagagtgtttaaagtagttcaaaagctgggtacctctatctgg\tabularnewline
pepc-H36-R4 &
gatgtcatcgcgtagaaaatcagtcatggatcccatgctactcctaccaac\tabularnewline
pepc-H36-F5 &
gttggtaggagtagcatgggatccatgactgattttctacgcgatgacatc\tabularnewline
D299N-R6 & ctcattcatgcg\emph{gtt}cgacaggctcagctcatg\tabularnewline
D299N-F7 & catgagctgagcctgtcg\emph{aac}cgcatgaatgag\tabularnewline
down-HA-pepc-R8 & actacccacccggctggactagccggagttgcgcag\tabularnewline
down-HA-pepc-F9 & ctgcgcaactccggctagtccagccgggtgggtagt\tabularnewline
down-HA-R10 &
tgaccatgattacgaattcagatgtacaccaggctgatgattcc\tabularnewline
mutant-(K-R)-F & gggttgccgta\emph{gcg}agctgagatgatttcg\tabularnewline
mutant-(K-R)-R & cgaaatcatctcagct\emph{cgc}tacggcaaccc\tabularnewline
pXMJ19-F & ttgacaattaatcatcggctcgtataatgtgtg\tabularnewline
pXMJ19-R & ctgatttaatctgtatcaggctgaaaatcttctctc\tabularnewline
M13 fwd & cgttgtaaaacgacggccagt\tabularnewline
M13 rev & acaatttcacacaggaaacagctatgac\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Fig. S1. Effect of different pH on L-ornithine yield

a Growth condition of \emph{C. glutamicum} XAB03 at 72 h under different
pH. b L-Ornithine production of \emph{C. glutamicum} XAB03 at 72 h under
different pH.

\selectlanguage{english}
\FloatBarrier
\end{document}

