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Adaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum… Lee, Jungseok; Saddler, Jack N; Um, Youngsoon; Woo, Han M Jan 22, 2016

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Lee et al. Microb Cell Fact  (2016) 15:20 DOI 10.1186/s12934-016-0420-zRESEARCHAdaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum that co-utilizes cellobiose and xyloseJungseok Lee1, Jack N. Saddler2, Youngsoon Um1,3 and Han Min Woo1,3,4*Abstract Background: An efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels. The industrially important bacterium Corynebacterium glutamicum has been studied to broaden substrate utilizations for lignocellulose-derived sugars. However, C. glutami-cum ATCC 13032 is incapable of PTS-dependent utilization of cellobiose because it has missing genes annotated to β-glucosidases (bG) and cellobiose-specific PTS permease.Results: We have engineered and evolved a cellobiose-negative and xylose-negative C. glutamicum that utilizes cel-lobiose as sole carbon and co-ferments cellobiose and xylose. NGS-genomic and DNA microarray-transcriptomic anal-ysis revealed the multiple genetic mutations for the evolved cellobiose-utilizing strains. As a result, a consortium of mutated transporters and metabolic and auxiliary proteins was responsible for the efficient cellobiose uptake. Evolved and engineered strains expressing an intracellular bG showed a better rate of growth rate on cellobiose as sole carbon source than did other bG-secreting or bG-displaying C. glutamicum strains under aerobic culture. Our strain was also capable of co-fermenting cellobiose and xylose without a biphasic growth, although additional pentose transporter expression did not enhance the xylose uptake rate. We subsequently assessed the strains for simultaneous saccharifi-cation and fermentation of cellulosic substrates derived from Canadian Ponderosa Pine.Conclusions: The combinatorial strategies of metabolic engineering and adaptive evolution enabled to construct C. glutamicum strains that were able to co-ferment cellobiose and xylose. This work could be useful in development of recombinant C. glutamicum strains for efficient lignocellulosic-biomass conversion to produce value-added chemicals and fuels.Keywords: Corynebacterium glutamicum, Cellobiose and xylose, Cofermentation, Intracellular β-glucosidase, Adaptive evolution© 2016 Lee et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.BackgroundAdvances in metabolic engineering and synthetic biology have opened-up opportunities for us to engineer microbial hosts to produce a range of industrially-relevant chemi-cals and fuels [1, 2]. In addition, oligonucleotide-mediated or CRISPR-CAS9-mediated genome editing technolo-gies have been used to accelerated genomic evolution and enhance development of new strains [3, 4]. More efficient utilization of hexose and pentose derived sugars from lignocellulosic biomass (cellulose:  ~48  %, xylan:  ~22  %, lignin:  ~25  %) [5] is advantageous to achieve economi-cally-attractive bioprocesse for improving titers, produc-tivities, and yields of value-added chemicals.An industrial amino acid producer, Corynebacterium glutamicum [6] showed a broad range of sugar utilization Open AccessMicrobial Cell Factories*Correspondence:  hmwoo@kist.re.kr 1 Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Hwarangro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of KoreaFull list of author information is available at the end of the articlePage 2 of 16Lee et al. Microb Cell Fact  (2016) 15:20 such as hexose (i.g. glucose and gluconate) and disaccha-ride (i.g. maltose and sucrose) but some pentose (arab-inose but xylose) [7]. Since there are great potentials of C. glutamicum as a microbial cell factory to produce other commercially relevant chemicals and fuels [8–10] from renewable lignocellulosic biomass, efficient utilization of cellulosic sugars is an inevitable goal. C. glutamicum has been successfully engineered for cell growth and pro-duction of biochemical using pentose sugars via either the heterologous xylose-isomerase pathway [11, 12] or Weimberg pathway [13]. For cellobiose utilization in C. glutamicum, recent genome sequencing of C. glutami-cum R strain showed possible gene clusters that encode for functional EII permeases of PTS (BglF1 and BglF2), and for functional phospho-β-glucosidases (BglG1 and BglG2) [14]. Thus, an adaptive strain of C. glutamicum R strain grown in minimal medium with 0.2 % cellobiose and glucose has revealed a single-substitution mutation BglF(V217A or V317  M) for cellobiose utilization [14, 15]. The C. glutamicum R-CEL strain has been shown to utilize cellobiose, glucose and xylose simultaneously, but only possible under anaerobic conditions [16].However, C. glutamicum ATCC 13032 is incapable of PTS-dependent utilization of methyl β-D-glucoside and cellobiose because it does not have any genes annotated to β-glucosidases (bG). In addition, no genes encoding for cellobiose-specific PTS permease were annotated [17]. To metabolize cellobiose, C. glutamicum ATCC 13032 must have an enzyme that cleaves the β-(1 →  4)-glyco-sidic linkage of cellobiose. Thus, bG-displaying or secret-ing C. glutamicum strains have been developed and exhibited the complete consumption of 20  g/L cellobi-ose in 4 days with l-lysine production [18]. The cellobi-ose utilization was quite slow, compared to the glucose consumption of current C. glutamicum strains. Thus, optimizing gene expression and maximizing the activ-ity of bG was necessary for better production of l-lysine and other chemicals. Recently, Saccharomyces cerevisiae (a cellobiose- and xylose-negative yeast strain) was engi-neered for cellobiose utilization by expressing cellodex-trin transporter (CDT-1) and intracellular bG along with a xylose-consuming pathway in order to resolve carbon catabolite repression by glucose in hydrolysates [19]. This engineering has enabled simultaneous utilization of cel-lobiose and xylose as a model hydrolysate, and increased the productivity of ethanol.Like S. cerevisiae, C. glutamicum ATCC 13032 is not able to utilize either cellobiose or xylose as sole carbon source. First, we performed metabolic engineering of C. glutamicum for cellobiose utilization by expressing a cellodextrin transporter and an intracellular bG (Fig.  1) and evolved the strains for the efficient cellobiose utiliza-tion. Subsequently, NGS-genomic and DNA microarray transcriptomic analysis were performed to characterize the evolved strains. Next, we have introduced the xylose-isomerase pathway [11, 12, 20] to the cellobiose-utilizing C. glutamicum strain for co-utilization of cellobiose and xylose. Our cellobiose-utilizing engineered strains were used to ferment the cellobiose and glucose derived from Canadian Ponderosa Pine in simultaneous saccharifica-tion and fermentation (SSF). Canadian Ponderosa Pine was used as a model lignocellulosic biomass.Results and discussionUtilization of cellobiose in C. glutamicum using metabolic engineering and adaptive evolutionTo test whether heterologous expressions of either CDT-1 transporter and bG or bG alone allow utiliza-tion of cellobiose in C. glutamicum, the N. crassa cdt-1 and gh1-1 gene were codon-optimized and introduced into a CoryneBrick vector [11], pBbEB1c (Table  1). We attempted to grow Cg-Cello01 strain containing both the cdt-1 and gh1-1 genes, and Cg-Cello02 containing the gh1-1 gene in CgXII minimal medium containing 2  % (w/v) cellobiose as sole carbon source. No growth of Cg-Cello01 was observed for 4  d. Surprisingly, the cultures of Cg-Cello01 strain exhibited the growth only after day 16 (Fig.  2). As soon as we observed the maximal cell growth of each strain (corresponding to the growth in presence of 2  % glucose), the adapted strain was trans-ferred to fresh CgXII minimal medium containing 2  % (w/v) cellobiose for 48  h. After the first transfer, the growth and residual sugars in the culture medium were determined for each cell culture. However, no cell growth was observed for Cg-pBbEB1c as a control. During the adaptive evolution of Cg-Cello01 strain by three serial transfers, cellobiose was gradually consumed during cell cultures (Fig.  2b). Interestingly, glucose in the medium was detected up to 12 g/L during the evolution and then glucose derived from cellobiose was consumed after cel-lobiose was completely depleted. In the last evolutionary round (third serial transfer), Cg-Cello01(evo) showed the complete cellobiose consumption in 12  h and glucose (5 g/L) was minimally secreted.On the other hand, fewer adaptations of Cg-Cello02 were performed to obtain Cg-Cello02(evo). Cg-Cello02 strains exhibited the growth after day 11. Only twice serial transfers were performed to obtain the desired phenotype, of which the strain completely consumed its cellobiose in 12  h. No glucose derived from cellobi-ose was secreted during the adaptive evolution rounds (Fig.  2c). Finally, we confirmed no phenotypic changes (cell growth and cellobiose consumption) shown over more than twenty’s serial transfers into fresh medium (not shown) after Cg-Cello01(evo) and Cg-Cello02(evo) were obtained. As a result, the patterns of cell growth and Page 3 of 16Lee et al. Microb Cell Fact  (2016) 15:20 2 % (w/v) sugar consumption of Cg-Cello01(evo) and Cg-Cello01(evo) were almost identical to those of the wild-type [11], regardless of cellobiose or glucose. Finally, we obtained Cg-Cello01(evo) and Cg-Cello02(evo) strains as cellobiose-utilizing C. glutamicum ATCC 13032 deriva-tive strains. Cg-Cello01(evo) strain is the fastest cellobi-ose-utilizing strain known under aerobic conditions.Cellobiose-utilizing C. glutamicum requires activities of bG and glucokinase (Glk) for the catabolism of cellobi-ose in the cytosol. Thus, we checked whether the bG and Glk activities of the Cg-Cello01(evo) and Cg-Cello02(evo) strains were altered (Fig.  3). As a result, the control (Cg-pBbEB1c; the wild-type carrying the empty vec-tor) showed no bG activity (both cell extract and super-natant). On the other hand, levels of the bG activities in the cell extracts of Cg-Cello01(evo) and Cg-Cello02(evo) grown on 2  % cellobiose were measured at 0.2  ±  0.01 and 0.17 ± 0.002 U/mg, respectively (Fig. 3). Significantly low or none of the bG activities were seen in the cell-free supernatants from Cg-Cello01(evo) or Cg-Cello02(evo) cultures. Also, the bG activity levels were quite similar to a bG activity from wild-type expressing GH-1-1 alone (initial Cg-Cello02) was grown on 2  % glucose, the bG activity (0.17  ±  0.01). Thus, these results indicated that the adaptive evolution did not alter the intracellu-lar expression of heterologous bG. Also, no exogenous expression of bG was occurred due to possible genetic mutations. In addition, we measured Glk activity over the culture interval until the carbon sources were depleted. The Glk activity of Cg-Cello01(evo) and Cg-Cello02(evo) was not significantly different the Glk activity from Cg-pBbEB1c. Therefore, having bG activity in the cytosol of C. glutamicum is one of key steps to utilization of cello-biose, but increasing or high bG activity is not necessary for better cellobiose utilization.Characterization of the evolved cellobiose‑positive  C. glutamicum strainsThrough metabolic engineering and adaptive evolution of C. glutamicum, we obtained the cellobiose-utilizing strains, Cg-Cello01(evo) and Cg-Cello02(evo). Since C. glutamicum wild-type does not have any genes annotated Fig. 1 Scheme of reconstruction of cellobiose-utilizing and xylose-utilizing pathway in C. glutamicum ATCC 13032. C. glutamicum wild-type is not able to utilize cellobiose and xylose as sole carbon source (left). No genes for xylose isomerase, cellobiose transporters, and β-glucosidase are annotated (shown as no arrow). Through metabolic engineering and adaptive evolution of C. glutamicum strains (right), the cells were able to utilize cellobiose and xylose. Extracellular cellobiose was transported and intracellular β-glucosidase encoded by the gh1-1 gene hydrolyzed intracel-lular cellobiose to glucose, which further was metabolized by glucokinase (Glk) into glycolysis. Xylose metabolic pathway consists of heterologous xylose isomerase (xylA from E. coli) and additional xylulose kinase (xylB from E. coli). Transporter of Gxf1 (Candida intermedia) and Sut1 (Pichia stipitis), respectively was introduced as a xylose transporterPage 4 of 16Lee et al. Microb Cell Fact  (2016) 15:20 cellobiose transporter, we investigated if the evolved cells have either functional CDT-1 transporters or altered transporters for the uptake of cellobiose.First, plasmids used for metabolic engineering were isolated to characterize genetic mutations occurred during the adaptive evolution. When we compared the original sequence of plasmids, pBbEB1c-CT-bG and pBbEB1c-bG, in-frame deletion and a point mutation were found in the region of replication of origins (Addi-tional file 1: Figure S1), but no mutations were found on the sequence of the gh1-1 gene. Interestingly, the cdt-1 gene that was present on the pBbEB1c-CT-bG in the Cg-Cello01 strain was missing, which was confirmed by DNA sequencing. This could be due to intra-molecular recombination that occurs during the adaptive evolu-tion at the identical and ribosomal binding synthetic DNA sequences of the cdt-1 and gh1-1 genes. Also, the gel images of the cdt-1 gene by colony PCR were shown for the loss of cdt-1 gene during the evolutionary process of the Cg-Cello01 to Cg-Cello01(evo) strains (Additional file 1: Figure S2). This result indicated that heterologous expression of CDT-1 was not suitable for the cellobiose uptake in C. glutamicum. Also, we found that there were no mutations found in the sequence of the gh1-1 gene encoding for bG on the plasmids although a substitu-tion mutation of BglF (V217A or V317 M) was found on the adaptive C. glutamicum R strain [15]. Thus, the plas-mid sequencing results confirmed that the intracellular expression of heterologous bG was sufficient to utilize cellobiose as sole carbon source in Cg-Cello01(evo) and Cg-Cello02(evo) strains (Fig. 3).To characterize the genetic basis of cellobiose-utiliz-ing C. glutamicum, next-generation sequencing (NGS) analysis was applied to fully-evolved Cg-Cello01(evo) and Cg-Cello02(evo) strains, compared with the refer-ence genome sequence of C. glutamicum ATCC 13032 (Additional file 1: Table S1 and Table S2). As a result, in the genome sequence of Cg-Cello01(evo) strain thirty-six genes were mutated with thirty-one single-nucle-otide variants including missense (15 variants) and Table 1 Bacteria strains and plasmids used in this studyStrain or plasmid Relevant characteristics Source or referenceStrains E. coli HIT-DH5α F−(80d lacZ M15) (lacZYA-argF) U169 hsdR17(r− m+) recA1 endA1 relA1 deoR96 [38] C. glutamicum ATCC 13032 C. glutamicum wild-type strain (ATCC 13032) ATCC Cg-pBbEB1c C. glutamicum wild-type harboring pBbEB1c, Cmr CoryneBrick empty vector [39] Cg-EcXylAB C. glutamicum wild-type harboring pBbEB1c-EcXylA(cg.co) -EcXylB(cg.co), Cmr [11] Cg-Cello01 C. glutamicum wild-type harboring pBbEB1c-CT-bG, Cmr This study Cg-Cello02 C. glutamicum wild-type harboring pBbEB1c-bG, Cmr This study Cg-Cello01(evo) Cellobiose-adapted Cg-Cello01 strain, Cmr This study Cg-Cello02(evo) Cellobiose-adapted Cg-Cello02 strain, Cmr This study Cg-Evo1 Cellobiose-adapted Cg-Cello01 strain after plasmid curing (plasmid-free), cellobiose−, Cms This study Cg-Evo2 Cellobiose-adapted Cg-Cello02 strain after plasmid curing (no plasmids), cellobiose−, Cms This study Cg-Cello03 Cg-Evo1 harboring pBbEB1c-bG, Cmr This study Cg-Cello04 Cg-Evo2 harboring pBbEB1c-bG, Cmr This study Cg-Cello03-Xyl01 Cg-Cello03 harboring pBbEB1c-bG-XIK, Cmr This study Cg-Cello03-Xyl02 Cg-Cello03 harboring pBbEB1c-bG-XIK-XTg, Cmr This study Cg-Cello03-Xyl03 Cg-Cello03 harboring pBbEB1c-bG-XIK-XTs, Cmr This study Cg-Cello04-Xyl01 Cg-Cello04 harboring pBbEB1c-bG-XIK, Cmr This study Cg-Cello04-Xyl02 Cg-Cello04 harboring pBbEB1c-bG-XIK-XTg, Cmr This study Cg-Cello04-Xyl03 Cg-Cello04 harboring pBbEB1c-bG-XIK-XTs, Cmr This studyPlasmids pBbEB1c ColE1 (Ec), pBL1 (Cg), Cmr, Ptrc, BglBrick sites, CoryneBrick vector [39] pBbEB1c-CT-bG pBbEB1c derivative containing each codon-optimized N. crassa cdt1 and gh1-1 genes This study pBbEB1c-bG pBbEB1c derivative containing each codon-optimized N. crassa gh1-1 gene This study pBbEB1c-bG-XIK pBbEB1c derivative containing each codon-optimized N. crassa gh1-1 gene and E. coli xylA and  xylB genesThis study pBbEB1c-bG-XIK-XTg pBbEB1c derivative containing each codon-optimized N. crassa gh1-1 gene and E. coli xylA and  xylB genes and C. intermedia gxf1 geneThis study pBbEB1c-bG-XIK-XTs pBbEB1c derivative containing each codon-optimized N. crassa gh1-1 gene and E. coli xylA and  xylB genes and P. stipitis sut1 geneThis studyPage 5 of 16Lee et al. Microb Cell Fact  (2016) 15:20 silent mutations (five variants) in the coding regions, two multi-nucleotide variants, one insert and two dele-tions (Additional file 1: Table S1). Cg-Cello02(evo) strain exhibiting shorter adaption were shown for more muta-tions occurred. Three hundred single-nucleotide variants including missense (123 variants), nonsense (six vari-ants), silent mutations (98 variants) in the coding regions were identified along with 41 insertions and 28 deletions in the nucleotide sequence. Yet, the reasons for the high number of mutations for the evolved strains were unclear Fig. 2 Adaptive evolution of engineered C. glutamicum strains. a Scheme of metabolic engineering and adaptive evolution of Cg-Cello01 and Cg-Cello02 strains were described. CoryneBrick vectors containing the cdt-1 and/or gh1-1 gene(s) were introduced into C. glutamicum wild-type, in which no growth and consumption of cellobiose were observed (b and c; the first column). Growth of Cg-Cello01 and Cg-Cello02 strain was not appeared initially. However, the maximal cell growths of Cg-Cello01 and Cg-Cello02 were observed after 16 d (b; the second column) and after 11 d (c; the second column), respectively. Subsequently, serial cell transfers were performed for adaptive evolutions of Cg-Cello01 and Cg-Cello02 in 48 h (b and c). Finally, Cg-Cello01(evo) and Cg-Cello02(evo) were obtained since growth and cellobiose consumption were unchanged. Growth at OD600, cel-lobiose (g/L) and glucose (g/L) were shown in a symbol of circle (black), square (blue) and triangle (red), respectively. Data represents mean values of at least three cultivations. (N.A.) not availablePage 6 of 16Lee et al. Microb Cell Fact  (2016) 15:20 since the complete genome sequences of cellobiose-utilizing C. glutamicum R-CEL and CEL2 strains were not available [14, 15]. Specifically, multiple genetic vari-ants were found in genes encoding for putative proteins, phage integrase, ATPase component of ABC-type trans-port system (Cg2184), GTPase, tranposase (Cg2461), and intergentic regions. Thus, we searched the identi-cal genetic variants in between Cg-Cello01(evo) and Cg-Cello02(evo) strains. Finally, 10 identical genetic vari-ants were identified (Table 2). Three genes encoding for membrane proteins (ABC-type transporter, RibX, LysE-type transloactor) were shown for the missense muta-tions, which could be responsible membrane proteins for the cellobiose uptake. In addition, two genes (wzz and fruR) involved in sugar metabolism were mutated, chang-ing its amino acid sequences (Glu363Asp and Gly75Val), respectively. MiaB (tRNA methylthiotransferase (MiaB), Maltose-bninding protein (AmyE), and Benoate 1,2-diox-genase (BenA) were also confirmed to be mutated. Unlike evolved C. glutamicum R strains, C. glutamicum ATCC1304 required multiple mutations for the cellobi-ose uptake in a concert of altered membrane proteins, metabolic and regulatory proteins, translational pro-cessing, and auxiliary proteins. The mechanism of those muteins for cellobiose utilization remains unclear.Next, to characterize the genome-wide gene expres-sion of the Cg-Cello01(evo) and Cg-Cello02(evo) strains utilizing cellobiose, we performed a DNA microar-ray-transcriptomic analysis to investigate whether the gene-expression levels of sugar transporters, or other membrane proteins, were differentially altered in the evolved strains during cellobiose consumption. We ana-lyzed three groups: (1) gene expression of the evolved strains grown on glucose with a control (Cg-pBbEB1c) on glucose, (2) gene expression of the evolved strains grown on cellobiose with a control (Cg-pBbEB1c) on glucose, and (3) gene expression of the evolved strains grown on cellobiose with evolved strains on glucose. Those with gene expression that was 2-fold up- and 0.5-fold down-regulated in Group 2 and Group 3 were selected for fur-ther analysis (Fig. 4 and Additional file 1: Figure S3 and Table S3). Among 32 and 66 differentially altered genes for Cg-Cello01(evo) and Cg-Cello02(evo), respectively, the gene expressions of four and fourteen membrane proteins (respectively) were changed significantly. Most of them are hypothetically annotated, according to the National Center for Biotechnology Information (NCBI) (accession no. NC003450).In a comparison of transcriptomic analysis with next-generation sequencing-analysis, the mRNA expression of the regulon of FruR (the ptsH, ptsF, pfkB1, ptsI gene encoding for EII components of the PTS system) were not altered although the transcriptional FruR has been known to attenuate the induction of EII components of the PTS system in C. glutamicum R [21]. Thus, mutated FurR in the evolved strain may be not functional as a transcriptional regulator. However, the mRNA expression of a DeoR-type transcriptional regulator (FruR, Cg2118) was highly up-regulated (5-fold) in the Cg-Cello02(evo) strain in presence of cellobiose than glucose (Group 3). Also, the mRNA expression of benoate 1,2-dioxgenase (BenA) was only down-regulated (0.3-fold) in the Cg-Cello02(evo) strain in presence of cellobiose than glucose Fig. 3 Measurement of β-glucosidase (bG) and glucokinase (Glk) activities in C. glutamicum wild-type or evolved strains. The Cg-pBbEB1c (black) and Cg-Cello02 (grey) were cultivated in CgXII medium with 2 % (w/v) glucose. The evolved Cg-Cello01(evo) (red) and Cg-Cello02(evo) (blue) strains were cultivated in CgXII medium with 2 % (w/v) cellobiose. The cell extract (E) and cell-free superna-tant (S) were used for the measurement of the bG activities (U/mg protein) when the strains were cultivated for 24 h (upper panel). The cell extracts from the strains grown for 6, 12, or 24 h were used for the measurement of the Glk activities (mU/mg protein) (lower panel). Data represents mean values of at least three cultivations and error bars represent standard deviations. (N.D.) not detectedPage 7 of 16Lee et al. Microb Cell Fact  (2016) 15:20 (Group 3), in which mRNA expression is repressed by global transcriptional regulator GlxR in sugar metabo-lism [22].Since we observed the overexpressed hypothetical membrane proteins from the gene expression profiling of the cellobiose-utilizing strains (Fig. 4), we looked into the fatty acid profiles of cellular membranes of the wild-type strain, Cg-Cello01(evo), and Cg-Cello02(evo). Com-pared to the fatty acid profiles of the wild-type, lower palmitic acids (C16:0) and higher unsaturated steric acids (C18:1w9c) were measured in the lipids of the cellobiose-utilizing strains (Cg-Cello01(evo) and Cg-Cello01(evo)) (Table  3). This altered lipid profile of C. glutamicum was also shown for the wild-type growing on sodium oleate, and for mutants [23]. Moreover, cellobiose utili-zation influenced the fatty acid profiles of a recombinant Rhodococcus opacus PD630 expressing bG, which accu-mulated fatty acids from cellobiose [24]. Multiple muta-tions metabolic and regulatory proteins, translational processing, and auxiliary proteins, intergenic regions in evolved C. glutamicum could be responsible for altered lipid profiles.Based on the NSG- and transcriptomic analysis, altered ABC-type transporters/hypothetical membrane proteins and sugar metabolism were responsible for efficient cel-lobiose utilization in C. glutamicum. However, it was difficult to pinpoint which single transporter is mainly designated for the cellobiose uptake. Rather, multiple gene mutations could be required for the efficient cello-biose uptake in C. glutamicum. Those mutated genes in common could be good targets for further engineering of C. glutamicum wild-type to explorer cellobiose uptake Table 2 List of common mutations of C. glutamicum Cg-Cello01(evo) and Cg-Cello02(evo) strainsFull list of all mutations of C. glutamicum Cg-Cello01(evo) and Cg-Cello01(evo) strains were described in the Additional file 1: Table S1 and S2a The mRNA expression of the cg2118 gene was highly up-regulatedb The mRNA expression of the cg2637 gene was highly down-regulated. See the details in the textReference  positionGene name Type Reference nucleotideAllele  nucleotideCoding region changeAmino acid changeAnnotation32227 cg0045 MISSENSE A T 551A > T Asn184Ile Probable ABC transport protein, membrane com-ponent364912 cg0414 MISSENSE A C 1089A > C Glu363Asp Wzz, cell surface polysaccharide biosynthesis/chain length determi-nant protein1689677 cg1796 MISSENSE C T 103G > A Glu35Lys RibX, putative mem-brane protein-C. ammoniagenes RibX homolog2041951 cg2118a MISSENSE G T 224G > T Gly75Val FruR, transcriptional regulator of sugar metabolism, DeoR family2058943 cg2135 MISSENSE A G 490T > C Ser164Pro MiaB, tRNA methyl-thiotransferase2296630 cg2380 Deletion C – 270delG Gly90 fs Hypothetical protein Cg23802331324 cg2412 MISSENSE C T 154C > T Pro52Ser Hypothetical protein Cg24122545730 cg2637b MISSENSE G A 436G > A Asp146Asn BenA, benzoate 1,2-dioxygenase alpha subunit (aromatic ring hydroxylation dioxygenase A)2607484 cg2705 MISSENSE G A 407C > T Ala136Val AmyE, maltose-binding protein precursor2826260 cg2941 MISSENSE T C 577A > G Ile193Val LysE type translo-catorPage 8 of 16Lee et al. Microb Cell Fact  (2016) 15:20 and corresponding sugar metabolism or protein struc-tures. Thus, comprehensive next-generation sequencing-analysis could be required to analyze the evolving and evolved strains to investigate the most critical mutations for the cellobiose utilization.Reconstruction of cellobiose‑positive chassis of the adaptive evolved C. glutamicum strainsInverse engineering the C. glutamicum wild-type is nec-essary to construct rational microbial cells for cellobiose utilization. However, lack of multiple genome editing technology such as RNA-guided CRISPR-CAS9 [4] or MAGE [3] system of C. glutamicum has led to limitation of inverse engineering of C. glutamicum in this study. Thus, we decided to re-construct a cellobiose-positive chassis in which all multiple genetic changes were already reflected, for further engineering. We obtained plasmid-free strains by curing of plasmids in Cg-Cello01(evo) and Cg-Cello02(evo), resulting in Cg-Evo1 and Cg-Evo2 (Table 1).After the cultivation of Cg-Evo1 and Cg-Evo2 using 2 % cellobiose as sole carbon source, we confirmed that Cg-Evo1 and Cg-Evo2 did not grow at all. Thus, pBbEB1c-bG plasmid was introduced to Cg-Evo1 and Cg-Evo2, yielding Cg-Cello03 and Cg-Cello04 strains (Fig.  5). Cg-Cello03 and Cg-Cello04 strains showed complete growth and consumption of cellobiose in the CgXII medium contain-ing 2 % (w/v) cellobiose, without any adaptations or pre-culture with cellobiose (Fig. 5). This result supports that the Cg-Evo1 and Cg-Evo2 have already multiple genetic changes from its parental strain for the efficient cellobi-ose uptake.When we compared the profiles of growth and cellobi-ose consumption, the Cg-Cello03 strain showed almost identical patterns of growth and cellobiose consumption Fig. 4 Heat map of altered gene expressions of C. glutamicum strains with cellobiose or glucose. Evolved C. glutamicum strains [Cg-Cello01(evo) and Cg-Cello02(evo)] grown on 2 % (w/v) cellobiose were tested with either a control (Cg-pBbEB1c) or Cg-Cello01(evo) and Cg-Cello02(evo) grown 2 % (w/v) glucose. The mRNA expression changed with 2-fold up- and 0.5-fold down-regulated were selected in the evolved strains with cellobiose over glucose (the third columns) as well as a control (the second columns). The mRNA ratios are averages from at least duplicated experiments. Heat maps generated by MeV (MutiExperiment Viewer ver. 4.8) showed differential gene expression of significantly changed genes. Up-regulated signals relative to the mean were colored in red. Down-regulated were colored in green (scale bar, log 2 of mRNA ratio). The criterion used for selection of RNA ratios was a signal-to-noise ratio of >3 for either Cy5 fluorescence. For the significantly changed genes, P < 0.05 as determined by a one-way ANOVA. The ID numbers of C. glutamicum were given at the last column, of which data were described in the Additional file 1: Table S1▸Page 9 of 16Lee et al. Microb Cell Fact  (2016) 15:20 as its parental strain, Cg-Cello01(evo). Glucose derived from cellobiose was secreted into the medium while both Cg-Cello01(evo) and Cg-Cello03 strains consumed the cellobiose. In the case of Cg-Cello04 strain, of which the parental strain is the Cg-Cello02(evo) strain, the rates of cell growth and cellobiose consumption were slightly retarded but no glucose was detected as for the paren-tal strain. The reason for slower cellobiose consumption remains unclear.Instead of inverse engineering, we successfully con-structed cellobiose-positive C. glutamicum chassis strain that utilize cellobiose as sole carbon source under the conditions of aerobic culture by expressing intracellular bG alone. Moreover, the strains expressing intracellular bG exhibited better cellobiose utilization than any other strain either secreting bG into the medium, or display-ing bG on the cell surface [18] in terms of the cellobiose consumption rate under aerobic conditions. Therefore, we obtained cellobiose-positive C. glutamicum chassis strains to perform metabolic engineering with cellobiose as sole carbon source.Co‑utilization of cellobiose and xylose in C. glutamicum through metabolic engineeringUsing the cellobiose-positive C. glutamicum chas-sis strains, we focused on co-utilization of xylose and cellobiose in C. glutamicum via direct cellobiose uptake and intracellular hydrolysis of cellobiose. As a result, Cg-Cello03-Xyl01 and Cg-Cello04-Xyl01 strains were able to co-utilize cellobiose and xylose under aerobic conditions (Fig.  6). Compared to the xylose consumption by Cg-EcXylAB in the presence of glucose, the xylose consump-tion rates of the engineered strains were improved in the presence of cellobiose, instead of glucose.As shown in the culture of Cg-EcXylAB strain with glu-cose and xylose, a biphasic growth of Cg-Cello03-Xyl01 strain was observed after 12 h when cellobiose was com-pletely consumed. However, a biphasic growth behavior was not shown by Cg-Cello04-Xyl01 strain because the cellobiose was slowly consumed before 12  h, and cel-lobiose and xylose were almost simultaneously utilized and depleted in the culture between 12 and 24 h (Fig. 6). Moreover, Cg-Cello04-Xyl01 strain reached higher opti-cal density at first, compared to the Cg-Cello03-Xyl01 strain. To further improve the engineered cellobiose- and xylose-utilizing strains, we introduced genes coding for sugar transporters (Gxf1 and Sut1) into Cg-Cello03-Xyl01 and Cg-Cello04-Xyl01 strains, respectively. But, the engi-neered strains with additional heterologous transporters did not show significant improvement of xylose utiliza-tion or co-utilization under the conditions of aerobic cul-ture (Fig. 7).Simultaneous consumption of cellobiose and xylose in engineered S. cerevisiae has resolved carbon catabo-lite repression and significantly increased ethanol pro-ductivity through co-fermentation [19]. Engineering of cellobiose-positive C. glutamicum also enabled the co-consumption of cellobiose and xylose (Figs. 6 and 7). However, compared to the cellobiose-utilizing S. cer-evisiae, our cellobiose-positive C. glutamicum strains showed much faster cellobiose-consumption rates dur-ing aerobic culture. On the other hand, their xylose consumption rate was not much increased during co-fer-mentation. Expression of a pentose-specific transporter did not increase the rate, either. Therefore, we concluded that inefficient xylose utilization by C. glutamicum was another bottleneck for the co-fermentation of cellobiose and xylose although we did optimize the codon-usage of the corresponding Escherichia coli gene sequence [11]. Exploring an alternative xylose utilization pathway in C. glutamicum is necessary to provide faster xylose uptake [13, 20]. This strategy could then be further applied for co-fermentation of xylose and cellobiose.Hydrolysis of Canadian biomass and efficient SSF by engineered C. glutamicumTaking advantage of the capability for cellobiose utili-zation by two engineered C. glutamicum strains (Cg-Cello03 and in Cg-Cello04), we applied the strains for Table 3 Fatty acid profiles of  the cellobiose-utilizing C. glutamicum strains and the wild-typeThe Cg-Cello01(evo) and Cg-Cello02(evo) strains were cultivated with 2 % cellobiose as sole carbon source. The Cg-pBbEB1c stain was cultivated with 2 % glucose as sole carbon source. The fatty acid profiles of the Cg-pBbEB1c were almost identical to the profiles of the C. glutamicum wild-type [23]. Analysis of fatty acid and fatty acid methyl ester were followed by the standard protocol of Sherlock® Microbial Identification System (MIS) of MIcrobial IDentification Inc. (MIDI)Data represents mean values of duplicated cultivations* Equivalent chain lengths (ECL) value = 18.846Fatty acid  composition (%)Cg‑pBbEB1c Cg‑Cello01(evo) Cg‑Cello02(evo)C10:0 0.15 0.08 0.08C12:0 0.25 – –C12:0 3OH – 0.06 0.05C14:0 1.33 0.43 0.39C16:1 w9c 0.84 0.51 0.46C16:0 41.04 35.89 33.28C16:0 3OH 0.31 0.69 0.34C18:1 w9c 54.24 59.97 63.4C18:0 0.47 0.44 0.41C18:0 10-methyl 1.27 1.54 1.26C19:1 iso I 0.09 0.33 0.33C19:1 w6c/unknown fatty acids*– 0.06 –Page 10 of 16Lee et al. Microb Cell Fact  (2016) 15:20 efficient SSF of Canadian cellulosic hydrolysates. Before fermentation by C. glutamicum, we hydrolyzed either 1 % (w/v) Avicel® PH-101, or 1 % (w/v) dissolving pulp (DP), with cellulase (Celluclast 1.5  L) under the same culture conditions (except for the cell type). As a result, equal amounts of cellobiose and glucose were detected for both cellulosic substrates (Fig.  8; upper panels), and similar conversion yields of total sugar (16.7 % and 16.15 %) were obtained for Avicel and DP, respectively. Also, enzymatic hydrolysis terminated after 12  h. There were not many differences between Avicel and DP as a cellulosic sub-strate for enzymatic hydrolysis with Celluclast 1.5 L.Based on the enzymatic hydrolysis, we investigated whether the engineered strains (Cg-Cello03 and in Cg-Cello04) were able to utilize cellulosic substrates during SSF. Thus, we cultivated 1  % (w/v) cellulosic substrate of either Avicel or DP as sole carbon source for the evolved C. glutamicum strains with cellulase. No lag phase was shown by either culture (Fig. 8; lower panels). Compared to a control (Cg-pBbEB1c; the wild-type with empty vector), the Cello03 and Cg-Cello04 strains showed faster growth, and reached nearly double growth at the end. When total amount of sugars was quantified independently, the total amount of sugars consumed in the Cg-Cello03 and Cg-Cello04 cultures were higher than that of a control. Moreo-ver, we measured cellobiose and glucose in the supernatant, with the result that no cellobiose and glucose were detected during the culture of Cello03 and Cg-Cello04. Therefore, the engineered Cg-Cello03 and in Cg-Cello04 strains were able to grow by simultaneously utilizing cellobiose and glucose from cellulosic hydrolysates although low conversion yields of cellulosic substrates limit further cell growth during SSF. Along with improvements of the enzymatic hydrolysis, the simultaneous saccharification and co-fermentation (SSCF) of pretreated plant biomass (hexose and pentose) could be accomplished using engineered C. glutamicum strains (Cg-Cello03-Xyl01 and Cg-Cello04-Xyl01).ConclusionsAdaptive evolution of the microbial host to acquire desired environmental phenotypes was quite diffi-cult unless growth-associated evolutions [25, 26]. In this study, integrated metabolic engineering and adap-tive evolution allowed us to develop a cellobiose- and Fig. 5 A comparison of cell growth and cellobiose consumption of evolved C. glutamicum strains. The evolved C. glutamicum strains was compared with reconstructed cellobiose-positive chassis of C. glutamicum strains. By plasmid-curing and re-transformation of pBbEB1c-bG plasmid, Cg-Cello03 (upper panels; open symbols) and Cg-Cello04 (lower panels; open symbols) strains as reconstructed cellobiose-positive chassis were obtained from Cg-Cello01(evo) (upper panels; solid symbols) and Cg-Cello02(evo) (lower panels; solid symbols) strains, respectively. The strains were cultivated in CgXII medium with 2 % (w/v) cellobiose as sole carbon source after the pre-cultivation in BHIS medium. Growth (left panels; black circle), cellobiose (right panels; blue square), glucose (right panels; red triangle) were shown. Data represents mean values of at least three cultivationsPage 11 of 16Lee et al. Microb Cell Fact  (2016) 15:20 xylose-negative C. glutamicum strain that co-utilize cel-lobiose and xylose using. For further studies, we envi-sion development of recombinant C. glutamicum strains based on the chassis strain, for efficient lignocellulosic-biomass conversion to create valuable products such as l-glutamate or l-lysine.Fig. 6 Co-consumption of cellobiose and xylose of engineered cellobiose-positive chassis of C. glutamicum strains. Two different cellobiose-neg-ative strains (Cg-pBbEB1c and Cg-EcXylAB) and two different cellobiose-positive strains (Cg-Cello03-Xyl01 and Cg-Cello04-Xyl01) co-expressing XylA and XylB were tested. The cellobiose-negative strains were cultivated in CgXII medium with a mixture of 2 % (w/v) glucose and 1 % (w/v) xylose. On the other hand, the cellobiose-positive strains were cultivated in CgXII medium with a mixture of 2 % (w/v) cellobiose and 1 % (w/v) xylose. Growth (left panels; black circle), cellobiose (right panels; blue square), glucose (right panels; black triangle), xylose (right panels; red circle) were shown. Data represents mean values of at least three cultivations and error bars represent standard deviationsPage 12 of 16Lee et al. Microb Cell Fact  (2016) 15:20 MethodsBacterial strains, plasmids and culture conditionsAll bacterial strains and plasmids used or constructed in this work are listed in Table 1. E. coli strains were grown in LB medium (containing per liter: 10  g tryptone, 5  g yeast extract, and 5  g NaCl) at 37  °C and 200  rpm. C. glutamicum ATCC 13032 and its derivatives were culti-vated in BHIS medium (containing per liter: 37  g brain heart infusion, 91 g sorbitol) [27] at 30  °C and 200 rpm overnight and then incubated aerobically in CgXII medium (50 in 250  mL baffled Erlenmeyer flasks) [27] containing 2 % (w/v) cellobiose or a mixture of 2 % (w/v) cellobiose and 1  % (w/v) xylose supplemented with 25  μg/mL chloramphenicol at 30  °C on a rotary shaker at 200  rpm. All chemicals used in this study were pur-chased from Sigma-Aldrich (St. Louis, Mo). 0.5  mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for induction.Fig. 7 Additional sugar transporters for co-consumption of cellobiose and xylose. Two different cellobiose-positive strains [Cg-Cello03-Xyl01 (top left panel) and Cg-Cello04-Xyl01 (top right panel)] co-expressing XylA and XylB were further engineered with additional sugar transporters such as a Gxf1 (Candia intermedia) [29] and Sut1 (Pichia stipites) [30], yielding Cg-Cello03-Xyl02 and Cg-Cello03-Xyl03 (left panels) and Cg-Cello04-Xyl02 and Cg-Cello04-Xyl03 (right panels), respectively. The cellobiose-positive and xylose-positive strains were cultivated in CgXII medium with a mixture of 2 % (w/v) cellobiose and 1 % (w/v) xylose. Cellobiose (blue square), xylose (red circle) were shown. Data represents mean values of at least three cultiva-tions and error bars represent standard deviationsPage 13 of 16Lee et al. Microb Cell Fact  (2016) 15:20 Construction of plasmids and recombinant C. glutamicum strainsThe cdt-1 (NCU00801) and gh1-1 (NCU00130) genes from Neurospora crassa [19] encoding for a cellodex-trin transporter and a bG, respectively, were chosen for constructing a synthetic pathway in C. glutamicum. Each target gene was synthesized (Genscript, USA) with codon-optimization (Gene Designer 2.0 software [28]) for C. glutamicum and was assembled using a standard Bgl-Brick cloning method, where the target gene is inserted at the EcoRI and XhoI sites of the CoryneBrick plasmid pBbEB1c [11]. Thus, the synthesized gh1-1 gene was inserted, resulting pBbEB1c-bG. Subsequently, the syn-thesized cdt-1 gene was placed in front of the gh1-1 gene, resulting pBbEB1c-CT-bG. For utilization of xylose, the codon-optimized xylA and xylB genes from E. coli [11] were, subsequently, inserted to pBbEB1c-bG, resulting pBbEB1c-bG-XIK. In addition, the gxf1 (Candia interme-dia) [29] and sut1 (Pichia stipites) [30] gene encoding for sugar-transporter were codon-optimized and inserted to pBbEB1c-bG-XIK, yielding pBbEB1c-bG-XIK-XTg and pBbEB1c-bG-XIK-XTs, respectively. Cloned DNA frag-ments were correctly verified by DNA sequencing.The resulting plasmids were introduced into C. glu-tamicum by electroporation, and strain validation was performed by colony PCR [27]. The resulting strains are listed in Table 1.Adaptive Evolution of recombinant C. glutamicum strainsCg-Cello01 and Cg-Cello02 strains were cultivated in CgXII minimal medium containing 2 % (w/v) cellobiose as sole carbon source. After the maximal cell growth of Cg-Cello01 and Cg-Cello02 were observed in 16 and 11  days, respectively, the cells were transferred to the Fig. 8 Profiles of conversion of Avicel® PH-101 or DP by C. glutamicum strains. Celluclast 1.5 L (Sigma; Cat C2730) [75 filter paper unit (FPU)/g-glucan] was used as the cellulolytic enzymes for saccharification of Avicel® PH-101 (left panels) or DP (right panels). For cellulolytic hydrolysis (upper panels), Avicel (1 % [w/v]) or DP (1 % [w/v]) were hydrolyzed at 30 °C and cellobiose (blue bar) and glucose (red bar) were measured. For SSF (lower panels), Cg-pBbEB1c (black square), Cg-Cello03 (blue triangle) and Cg-Cello04 (red circle) were cultivated with either Avicel (1 % [w/v]) or DP (1 % [w/v]) as sole carbon source in the presence of Celluclast 1.5 L and optical densities at 600 nm were measured after the sedimentation of the residual sub-strate (lower panels; lines and symbols with left Y-axis). For the measurement of the residual substrate (g/L), each residual substrate was measured at 0 and 24 h from the SSF cultures (lower panels; bars with right Y-axis). During SSF, no cellobiose and glucose were detected in the supernatant from the cultures. Data represents mean values of at least three cultivations and error bars represent standard deviationsPage 14 of 16Lee et al. Microb Cell Fact  (2016) 15:20 fresh CgXII minimal medium containing 2  % (w/v) cel-lobiose, starting OD600 of 1 (Fig.  2a). Subsequently, the cells were transferred to the same fresh medium after every 48  h. Each culture of the cell was analyzed using HPLC to investigate the changes of the profiles of cello-biose utilization. The cell transfers were conducted until the rates of growth and cellobiose consumption were not changed, yielding Cg-Cello01(evo) and Cg-Cello02(evo) strains. Cg-Cello01(evo) and Cg-Cello02(evo) strains were further analyzed using DNA microarray. Plasmids from Cg-Cello01(evo) and Cg-Cello02(evo) strains were isolated and their DNA sequences were identified using partial overlapping primer-walking. In addition, plas-mid-free strains were obtained by curing of plasmids in C. glutamicum as follows: after the electroporation of Cg-Cello01(evo) and Cg-Cello02(evo) strains, nonselec-tive BHIS medium was inoculated at 30  °C. Each single colony was streaked onto BHIS agar plates either with or without chloramphenicol (25  μg/mL), yielding plasmid-free (Cms) Cg-Evo1 and Cg-Evo2 strains, respectively. For co-utilization of cellobiose and xylose, the xylA (encod-ing for xylose isomerase) and xylB (encoding for xylulose kinase) genes was introduced into cellobiose-utilizing Cg-Cello03 and Cg-Cello04 strains, yielding Cg-Cello03-Xyl01 and Cg-Cello04-Xyl01 strains.HPLC analysis for glucose, xylose, and cellobiose quantificationFor the measurement of the concentrations of glucose, xylose and cellobiose, culture supernatant was passed through a syringe filter (pore size of 0.2 μm) after centrif-ugation at 10,000g for 10 min. The concentrations of glu-cose and xylose were determined by high-performance liquid chromatography (HPLC system Agilent 1260, Waldbronn, Germany) equipped with a refractive index detector (RID) and an Aminex HPX-87 H Ion Exclusion Column (300  mm by 7.8  mm, Bio-Rad, Hercules, CA, USA) under the following conditions: sample volume of 20 μL, mobile phase of 5 mM H2SO4, flow rate of 0.6 mL/min, and column temperature of 65 °C.Enzymatic measurement of β‑glucosidase and glucokinase activityRecombinant strains were cultivated in CgXII medium containing 2  % (w/v) cellobiose but 2  % (w/v) glucose was used for the control (Cg-pBbEB1c). After incubation at 30 °C for 24 h, bG activities in the cell-free extracts or in the culture supernatants, respectively, were quanti-tatively measured in a 1  mL mixture containing 590  μL 500  mM potassium phosphate buffer (pH 7.0), 10  μL 500 mM MgCl2, 200 μL sample, 200 μL p-nitrophenyl-β-d-glucopyranoside (pNPG) as a substrate at 410 nm [16] (U; μmol of pNPG reduced min−1). For the determination of Glk activity (U/L) [31], the Glk activity in cell-free extracts was determined at 25  °C by measuring of the formation of NADPH at 340  nm in a coupled reaction containing 100 mM potassium phosphate buffer (pH 7.0), 20 mM glucose, 2 mM ATP, 25 mM MgCl2, 2 mM NADP and 2 U glucose-6-phosphate dehydrogenase (U; μmol of NADP reduced min−1).NGS‑based genomic DNA sequencing analysisGenomic DNAs of Cg-Cello01(evo) and Cg-Cello02(evo) were isolated from a single colony’s culture and purified using Wizard Genomic DNA purification kit (Promega, Cat.No. A1125). The genomes of Cg-Cello01(evo) and Cg-Cello02(evo) strains were sequenced using the Illu-mina Miseq 300 bp paired-end system (Illumina, Inc, San Diego, CA, USA) at ChunLab, Inc. (Seoul, South Korea). We obtained 5,753,368 reads of the genome to reach a 428.63-fold depth of coverage on Cg-Cello01(evo) and Cg-Cello02(evo). The re-sequencing data were annotated by RNAmmer 1.2, tRNA scan-SE 1.21, Rapid Annota-tion using Subsystem Technology (RAST), Pipeline, and CLgenomics program (ChunLab, Inc). The detail proce-dures were described in the previous study [32].Transcriptomic analysisTotal RNA from Cg-Cello01(evo) and Cg-Cello02(evo) were sampled in the exponential phase. For Transcrip-tome analysis, extraction of total RNA and prepara-tion of cDNA was followed by previous methods [33]. The cDNA probes were cleaned up using Microcon YM-30 column (Millipore, Bedford, MA) and then fol-lowed by Cy5-dye (Amersham Pharmacia, Uppsala, Sweden). The Cy5-labelled cDNA probes were cleaned up using the QIAquick PCR Purification Kit (Qiagen). Dried Cy5-labelled cDNA probes were re-suspended in hybridization buffer. Hybridization to a microarray slide (Corynebacterium_glutamicum 3  ×  20  K) (Mycroar-ray.com, Ann Arbor, MI), staining, and scanning of the probe array were performed according to the manufac-turer’s instructions. Hybridization image on the slide was scanned by Axon 4000B (Axon Instrument, Union City, CA). The analysis of the microarray data was per-formed using GenePix Pro 6.0 (Axon Instruments). The averages of the normalized ratios were calculated by dividing the average normalized signal channel intensity by the average normalized control channel intensity. All measurements were performed on duplicated techni-cal replicates. Two independent samples were analyzed; their measurements were averaged for further analy-sis. The microarray data were deposited at the NCBI Gene Expression Omnibus, GEO under accession no. GSE65076 and at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65076.Page 15 of 16Lee et al. Microb Cell Fact  (2016) 15:20 Fatty acids and lipid analysisFatty acid methyl esters were prepared as described previously [23, 34], and identified by gas chromatog-raphy with model 5898A microbial identification sys-tem (Microbial ID). Trimethylsilylated derivatives of fatty acids and methyl esters were analyzed by high-temperature gas chromatography on an HP 5790A gas chromatograph (Hewlett Packard), equipped with a flame-ionization detector on a 12  m high throughput screening (HTS) column with hydrogen gas as the carrier. Derivatives were identified by comparing their retention times to those of standards and by gas chromatography mass spectrometry analysis on a KRATOS MS50 spec-trometer (ion source temperature set to 200 °C and ioni-zation energy to 70  eV), respectively. For the analysis, colonies of Cg-Cello01(evo) and Cg-Cello02(evo) strains were obtained on CgXII agar plate containing 2 % cello-biose and colonies of Cg-pBbEB1c stain was obtained on CgXII agar plate containing 2 % glucose as.Cellulolytic hydrolysis of Avicel® PH‑101 and Canadian biomass and SSF by C. glutamicumAvicel® PH-101 (Sigma), microcrystalline cellulose and dissolving pulp (DP, pure cellulosic substrate less than 0.5 % lignin, less than 3 % xylan) [35] from Canadian Pon-derosa Pine were used as substrate for cellulolytic hydrol-ysis and SSF by the cellobiose-utilizing C. glutamicum strains. Each cellulolytic hydrolysis and SSF was carried out in CgXII medium (pH 7.0) with 1  % (w/v) Avicel® PH-101 or 1  % (w/v) dissolvping pulp at 30  °C and 200  rpm. Celluclast 1.5  L (Sigma; Cat C2730) [75 filter paper unit (FPU)/g-glucan] was used as the cellulolytic enzymes for saccharification of Avicel® PH-101 or DP. Cellulase actitivity of the Celluclast 1.5 L was determined by the standard filter paper assay with the 3,5-dinitrosali-cylic acid (DNS) method [36]. One unit of cellulose activ-ity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per mint at pH 4.8 and 50 °C. The enzyme activity of Celluclast 1.5  L was measured to be 28  FPU/mL. A colorimetric method based on the phenol–sulfuric acid reaction was used to determine the amount of residual substrate during SSF by quantifying total sugars [37].Availability of supporting dataThe data set supporting the results of this article is availa-ble at NCBI GEO repository, [GSE65076 and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65076].Additional fileAdditional file 1. Additional data and analysis for the evolved C. glutamicum strains.AbbreviationsbG: β-glucosidase; SSF: simultaneous saccharification and fermentation; HPLC: high-performance liquid chromatography; RID: refractive index detector; NGS: next-generation sequencing; GEO: gene expression omnibus; Glk: glucokinase; DP: dissolving pulp; SSCF: simultaneous saccharification and co-fermentation.Authors’ contributionsJL performed and analyzed all experiments and helped in drafted the manu-script. JNS and YU assisted in analysis and helped in drafting the manuscript. HMW analyzed all experiments and coordinated the study and drafted the manuscript. All authors read and approved the final manuscript.Author details1 Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Hwarangro 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea. 2 Department of Wood Science, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. 3 Department of Clean Energy and Chemical Engineer-ing, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea. 4 Green School (Gradu-ate School of Energy and Environment), Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea. AcknowledgementsAuthors appreciate to Dr. Changsoo Kim at UBC-KIST on-site laboratory for valuable comments and also thank to Dr. Jinguang Hu at UBC for sending DP (Canadian biomass). This work was financially supported from the R&D Convergence Program of NST (National Research Council of Science and Tech-nology) of Republic of Korea and KIST (Korea Institute of Science and Technol-ogy) (2E25402) and Korea CCS R&D Center (KCRC) (no. 2014M1A8A1049277) and the National Research Foundation of Korea grant-funded by the Korean Government (Ministry of Science, ICT and Future Planning) (2015, University-Institute Cooperation program).Competing interestsThe authors declare that they have no competing interests.Received: 9 January 2016   Accepted: 11 January 2016References 1. Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, Del Cardayre SB, Keasling JD. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature. 2010;463:559–62. 2. Peralta-Yahya PP, Zhang F, Del Cardayre SB, Keasling JD. Microbial engi-neering for the production of advanced biofuels. Nature. 2012;488:320–8. 3. 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