Improvement of Xylose Utilization and L-ornithine Production by Metabolic Engineering of Corynebacterium glutamicum

Background: L-ornithine is a basic amino acid, which shows signicant value in food and medicine industries. Xylose is the most important alternative carbon source of glucose in lignocellulosic hydrolysate. It is urgent to develop a high-eciency cell factory for L-ornithine production with glucose and xylose. Results: In this study, the genes enconding 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 had been conducted to promote the synthesis of L-ornithine for the rst 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 ask fermentation so far. Conclusions: 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.

transportation of L-ornithine amino acids [19] ; glycolysis, acetic acid metabolism and pentose phosphate pathway [24,25] , together with the tricarboxylic acid cycle and glucose utilization pathway [26][27][28][29][30][31] . In addition to rational modi cation, adaptive evolution strategies combined with transcriptional levels analysis provides another strategy to develop a strain with high performance [32] . Jensen et al. constructed C. glutamicum ORN6 by knocking out argF, argR and argG, attenuating the expression of pgi and increasing the copy number of the arginine operon argCJB A49V, M54V D on the chromosome, the Lornithine yield reached 0.52 g/g [12] . Hwang et al. knocked out ncgl2053, ncgl0281 and ncgl2582 that encoding NADP + -dependent oxidoreductase, which resulted in the loss of glucose dehydrogenase activity and the improvement of 6-phosphate gluconate dehydrogenase activity, the production of L-ornithine was 66.3% higher than that of the starting strain [33] . Shu et al.deleted proB and argF to block the branch of the L-ornithine synthesis pathway, mutated ArgB and expressed heterologous argA and argE to introduce an arti cial linear transacetylation pathway, which had increased the production of L-ornithine up to 40.4 g/L in 5-L bioreactor [34] . Zhang et al adopted a series of genetic engineering modi cations to achieved the maximum L-ornithine yield of 43.6 g/L in fed batch fermentation by far [26] . Although many strategies have been adapted to increase the production of L-ornithine, how to build a more e cient industrial strain with practical applications is still a long way off.
In order to take full advantage of abundant renewable resources, many studies have been focused on how to construct an e cient microbial cell factory utilizing xylose and glucose as mixed carbon sources in the past fteen years [35] . Thanks to the weak carbon catabolite repression, C. glutamicum was regarded as a major industrial force with great potential in recent years [36,37] . However, due to lacking of xylose isomerase (XylA), C. glutamicum could not grow in 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 [38,39] . Five copies of xylAB operon from E. coli were integrated to C. glutamicum R chromosome to generate the strain X5C1 which could consume 40 g/L glucose and 20 g/L xylose in 12 hours [40] . In addition, several other strategies were adopted to improve the xylose utilization, including the introduction of arabinose transporter [41,42] , overexpressing of TAL/TKT in the pentose phosphate pathway [43] . Meiswinkel et al. constructed an engineered strain C. glutamicum PUT21 by introducing xylA from X. campestris, xylB from C. glutamicum and argBAD operon from E. coli to produce 2.59 g/L of L-ornithine, and the volumetric ornithine productivity was 43.2 mg/ mg/(L·h) [44] . In addition to xylose isomerase metabolic pathway, Christian et al. introduced the xylXABCD operon from Caulobacter crescentus into C. glutamicum ATCC13032 to establish the Weinberg pathway [42] . Although the utilization of xylose by C. glutamicum has already been realized, the utilization rate of xylose is still unsatis ed. More modi ed strategies are needed to improve the utilization rate of xylose and the production of L-ornithine., which opened the door to the e cient utilization of lignocellulose.
In our previous studies, we have successfully constructed the C. glutamicum SO26 with high L-ornithine yield [19,26] . In this study, we attempted to utilize the most abundant carbon source in lignocellulose hydrolysate -glucose and xylose. The approaches of metabolic engineering and fermentation process control were adopted to accelerate the xylose consumption rate and the yield of L-ornithine. Firstly, a more e cient xylAB operon was screened out from different strains, and the arabinose transporter araE from Bacillus subtilis was knocked into the iolR locus under the promoter P eftu . Secondly, the acetylation of phosphoenolpyruvate carboxylase (PEPC) was reduced to release the feedback inhibition of aspartic acid, and a strong constitutive promoter P H36 was introduced in the upstream of pepc. The strain after a series of modulations was named C. glutamicum XAB03. After the optimization of fermentation process, we have found that the optimal concentration ratio of glucose and xylose(7:3) and the addition of coenzyme (biotin 0.9 µM and thiamine-HCl 15 µM) could reach up to the highest yield of L-ornithine 41.5 g/L in shaking ask fermentation up to date. The metabolic engineering processes had been illustrated in Fig. 1. Schematic diagram.

Results And Discussion
Comparison with the xylose utilization capacity of xylose isomerases and xylulose kinases from different carbon sources Xylose isomerase (xylA) and xylulose kinase (xylB) exist in the form of gene clusters. The xylose isomerase metabolic pathway from different sources established in C. glutamicum have different xylose utilization capacities, this might be the result of the genetic codon preference between the xylAB source strain and C. glutamicum [44] . The xylAB genes derived from E. coli MG1655 and X. campestris were expressed under IPTG induced vector pXMJ19, in which the P tac is the promoter for gene expression. The resulting expression plasmids were labeled p19P lac -EcoAB and p19P tac -XcaAB. The two plasmids were transformed into C. glutamicum SO26 to obtain the C. glutamicum EAB and XAB. Figure 2a showed the growth curve and xylose consumption of strain EAB and XAB during 72 hours fermentation. The results indicated that xylAB from X. campestris achieved higher xylose consumption (45.1 g/L) in comparison with xylAB from E. coli MG1655 (25.0 g/L). The growth OD 600 value increased from 12.05 (EAB) to 12.95 (XAB). The L-ornithine concentration in the supernatant of fermentation was determined as shown in Fig. 2b. The L-ornithine production titer of the strain XAB (21.6 ± 0.19 g/L) was 18.7% higher than strain EAB (18.2 ± 0.35 g/L), and the corresponding xylose yield of XAB was 0.48 g/g. The C. glutamicum XAB had been performed 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 In order to increase the rate of xylose consumption, the pentose transporter gene (araE) from Bacillus subtilis was integrated into the genome locus of iolR with the strong P eftu promoter, to generate the strain termed as XAB01. The pentose transporter AraE is not only extremely signi cant for the arabinose transportation, but also promotes the transportation of xylose to cells [45] . 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 72 h fermentation, with an average consumption rate of 0.665 g/(L·h). Through the rapid utilization of xylose, the growth and Lornithine yield of strain were also improved. The output of L-ornithine increased by 12.5% (24.3 ± 0.23 g/L) compared with strain XAB, and the xylose yield was 0.51 g/g.
Effect of modi cation of phosphoenolpyruvate carboxylase on the production of L-ornithine Phosphoenolpyruvate carboxylase (PEPC) is an enzyme in the glycolysis pathway of C. glutamicum and plays an important role in the regulation of the TCA cycle. 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 affects the synthesis of glutamic acid in C.
glutamicum [46,47] . Lysine at position 653 (K653) is essential for the regulation of PEPC acetylation. Megumi ed al. found that acetylation of PEPC at K653 could decrease enzymatic activity and glutamate production, which refer to the K653-acetylation could regulate PEPC activity negatively. Mutated K653 into arginine could decrease the level of acetylation on PEPC, which correspondingly improve its activity [47] . In addition, PEPC would be feedback suppressed by aspartic acid when it was overexpressed in C. glutamicum. The inhibitory effect could be effectively reduced when the aspartic acid at position 299 was mutated to asparagine with a similar structure [46] . In our study, we attempted 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. The preferred CGC and AAC codons in C. glutamicum S9114 were chosen to mutate K653 and D299. At the same time, the pepc gene was overexpressed by adding a strong constitutive promoter H36 [48] . Multiple fragments had been fused into a complete fragment by PCR overlapping ampli cation technology. After two-point mutations in pepc, strain XAB03 was constructed. The results of shaking ask fermentation of strain XAB03 have 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, this strain had a positive effect on the growth of C. glutamicum (Fig. 4a). These results indicated the importance of PEPC in L-ornithine synthesis, and the modi cation strategies were available and effective for the synthesis of L-glutamate, L-citrulline and L-arginine.

Addition of biotin and thiamine hydrochloride accelerates the synthesis of L-ornithine
In addition to the modi cation of key enzymes in metabolic pathways, the addition of key coenzymes is also an important method to promote the synthesis of products [49] . 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 [49,50] . In order to further optimize the fermentation process, we attempted to adding the coenzyme during the L-ornithine production. The optimal concentration of biotin and thiamine hydrochloride in the fermentation medium was 0.9 µM and 15 µM [51] , 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 fermentation period from 0-48 hours in shake asks, the strain XAB03 had been grown with abundant nutrients, the energy distribution mainly distributed on the growth of the bacteria while the production of L-ornithine was weak. From 48-72 hours, the growth was tended to be stable and the L-ornithine was synthesized rapidly. The results provided a research direction for the promotion of certain target products through the addition of coenzymes and some small molecules.
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 ve-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 was 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 ± 0.02 g/L) when glucose was 70 g/L and xylose was 30 g/L (Fig. 5b, c).
Based on all the above conditions, the OD 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 ask fermentation for 72 hours, which was the highest titer so far to the best of our knowledge (  [26] . The 2:1 ratio was 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. Overexpression of ncgl0452 and argCJBDmut [22] C. glutamicum SJC8039 14/ND Shake ask; batch

Conclusions
In this study, xylAB operon was introduced into C. glutamicum SO26 to achieve the consumption of xylose for the production of L-ornithine. And then, we veri ed 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 C. glutamicum XAB03 has reached 41.5 g/L shaker ask 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.

Bacterial strains, plasmids and primers
The strain SO26 originated from C. glutamicum S9114 with a series of modi cations (deletion of argF, ncgl1221, argR, putP, iolR, and mscCG2; attenuation of odhA, proB, pta, cat, and ncgl2228; and overexpression of lysE, gdh, gdh2, cg3035, pfkA, pyk, glt, tkt, argCJBD, and iolT1) was used as a starting strain for further metabolic engineering in this study. E. coli DH5α was used as host for rapid replication of recombinant plasmids. Xylose isomerase (xylA) and xylulose kinase (xylB) were ampli ed from E. coli K-12 MG1655 and X. campestris, respectively. The arabinose transporter (araE) were derived from Bacillus subtilis. All the strains and plasmids used in this study were presented in Table 1. On the basis of pk18-P H36 -pepcTI, harboring 895-897 bp AAC replaces GAT in pepc gene This study

Construction of plasmids and strains
The basic DNA manipulation and strain construction were operated according to the standard molecular cloning manual. All the primers used in this study were presented in Table S1 (Supplementary Information). The suicide vector pK18mobsacB containing the sucrose lethal gene sacB was used to delete or integrate gene on genome.
The xylAB gene clusters from E. coli and X. campestris were ampli ed by primers EAB-F/EAB-R and XAB-F/XAB-R respectively, the inserted restriction sites were HindIII/EcoRI and HindIII/SacI of pXMJ19. The plasmids pXMJ19, p19P lac -EcoAB, and p19P lac -XcaAB were transformed into C. glutamicum SO26 to produce strains C. glutamicum pX EAB XAB. AraE from B. subtilis with a constitutive P eftu promoter were ampli ed by primers araE-F/araE-R and up-eftu-F/araE-eftu-R respectively. The products of fusion PCR were inserted into the EcoRV site of plasmid pk18-△iolR by Gibson assembly, and then generated the recombinant plasmid pk18-P eftu -△iolR::araE and corresponding strain XAB01.
All the recombinant plasmids were constructed in E. coli DH5α and transformed into C. glutamicum by electroporation. The mutant strains were screened through two rounds of homologous recombination, and further con rmed by colony PCR and sequencing.

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 C. glutamicum and E. coli strains. Antibiotic was added to the medium for mutants screening when needed: 50 µg/mL kanamycin or 30 µg/mL chloramphenicol for E. coli, 10 µg/mL kanamycin or 15 µg/mL chloramphenicol for C. glutamicum.
For the shaking ask fermentation experiment, the correct monoclonal strains were activated twice on the LB medium for 36 hours, and then the appropriate amount of bacterial seed was inoculated into a 100 mL ask 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 4 ) 2 SO 4  Three parallel samples (100 µL) were taken to monitor the bacteria density, consumption of glucose and xylose, and L-ornithine production for every 12 hours interval. The OD 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 3 [26] . The samples were centrifuged to obtain fermentation supernatant for subsequent analysis. 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) [48,52] . The content of L-ornithine was determined by ninhydrin colorimetry [16] . All experiments had triple parallels, and data had been presented as mean and standard deviation (SD).

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information les. Authors' contributions G.G., Y.Z., acquisition, analysis, and interpretation of data, and manuscript preparation. These two authors contributed to the work equally and should be regarded as co-rst authors. Y.Z., B.-C.Y., study conception and design, data analysis, and nal approval of the manuscript. All authors read and approved the nal manuscript. Figure 1 Schematic diagram of the metabolic engineering process of C. glutamicum. pepc encodes phosphoenolpyruvate carboxylase; pyk encodes pyruvate kinase; xylA encodes xylose isomerase; xylB encodes xylulose kinase; aat encodes aspartate aminotransferase; araE encodes pentose transporter; argF encodes N-ornithine carbamoyl transferase.

Figure 2
Comparison of the effects of 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 xylAB from E. coil, Strain EAB (hollow upper triangle) overexpresses xylAB from X. campestris.

Figure 3
The promotion of 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 araE gene from B. subtilis with a strong promoter Peftu, and XAB (black hollow square) is the control strain.

Figure 4
The PEPC modi cation reinforces the production of L-ornithine. a: Growth curves. b: L-Ornithine production curves. XAB01 (blue solid triangle) integrated the araE gene from B. subtilis with a strong promoter Peftu. XAB03 (hollow upper triangle) modi ed pepc on the basis of XAB01.

Figure 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).

Figure 6
The effect of different ratios of glucose and xylose on L-ornithine production a: Growth curves for C.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1.pdf