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-Ramírez et al., 2016).
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). AlthoughE. coli and Saccharomyces cerevisiae have ever been
engineered to be the producer of L-ornithine (Becker & Wittmann, 2015;
Lee & Cho, 2006; Qin et al., 2015), 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 byC. 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 C.
glutamicum ORN6 by knocking out argF , argR andargG , attenuating the expression of pgi and increasing the
copy number of the arginine operon argCJBA49V, M54VD
on the chromosome, the L-ornithine yield was 0.52g/g (Jensen et al.,
2015). Hwang et al. knocked out ncgl2053 , ncgl0281 andncgl2582 that encoding NADP+-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 proBand argF to block the branch of the L- ornithine synthesis
pathway, mutated ArgB and expressed heterologous argA andargE 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, 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 toC. 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 C.
glutamicum to realize the utilization of xylose (Buschke, Becker, et
al., 2013; Gopinath, Meiswinkel, Wendisch, & Nampoothiri, 2011). Five
copies of xylAB operon from E. coli were integrated toC. 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 C.
glutamicum PUT21 by introducing xylA from X. campestris ,xylB from C. glutamicum and argBAD operon fromE. coli to produce 2.59 g/L of L-ornithine, and the volumetric
ornithine productivity is 43.2 mg/(L⋅h) (Meiswinkel, Gopinath, Lindner,
Nampoothiri, & Wendisch, 2013). In addition to xylose isomerase
metabolic pathway, Christian et al. introduced the xylXABCDoperon from Caulobacter crescentus into C. glutamicumATCC13032 to establish the Weinberg pathway (Brusseler et al., 2018).
Although the utilization of xylose by 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 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 xylABoperon was screened out from different strains, and the arabinose
transporter araE from Bacillus subtilis was knocked into theiolR locus under the promoter Peftu. Second, the
acetylation of phosphoenolpyruvate carboxylase (PEPC) was reduced to
release the feedback inhibition of aspartic acid, and a strong
constitutive promoter PH36 was introduced in the
upstream of pepc . The strain after a series of modulations is
named 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 μM and thiamine-HCl
15 μM 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.