Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli

Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli

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Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli Suk Min Kim a,1, Bae Young Choi b,1, Young Shin Ryu a,1, Sung Hun Jung b, Jung Min Park b, Goo-Hee Kim b, Sung Kuk Lee a,b,n a b

School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 16 February 2015 Received in revised form 20 May 2015 Accepted 21 May 2015

After glucose, xylose is the most abundant sugar in lignocellulosic carbon sources. However, wild-type Escherichia coli is unable to simultaneously utilize both sugars due to carbon catabolite repression (CCR). In this paper, we describe GX50, an engineered strain capable of utilizing glucose and xylose simultaneously. This strain was obtained by evolving a mutant from which araC has been deleted, and in which genes required for pentose metabolism are constitutively expressed. The strain acquired four additional mutations during adaptive evolution, including intergenic mutations in the 50 -flanking region of xylA and pyrE, and missense mutations in araE (S91I) and ybjG (D99G). In contrast to wild type E. coli, GX50 rapidly converts xylose to xylitol even if glucose is available. Notably, the strain grows well when cultured on glucose, unlike some well-known CCR-insensitive mutants defective in the glucose phosphotransferase system. Our work will advance efforts to design a metabolically efficient platform strain for potential use in producing chemicals from lignocellulose. & 2015 International Metabolic Engineering Society. Published by Elsevier Inc.

Keywords: Lignocellulose Xylose Glucose Metabolic engineering Escherichia coli

1. Introduction Xylose is the most abundant sugar in lignocellulosic biomass after glucose. However, wild-type Escherichia coli does not simultaneously utilize both sugars due to carbon catabolite repression (CCR), specifically of genes related to xylose transport and catabolism. As a result, E. coli consumes glucose and xylose sequentially. Thus, E. coli is not an ideal host strain to produce chemicals from lignocellulosic biomass (Gorke and Stulke, 2008) because of diauxic growth (Deutscher, 2008), which lengthens fermentation time. Nevertheless, E. coli remains attractive as host strain, because it is capable of naturally metabolizing both glucose and xylose, grows rapidly in inexpensive media, and can be genetically manipulated easily (Cirino et al., 2006; Nakamura and Whited, 2003; Su et al., 2013). The key is to develop strains that metabolize both sugars at the same time.

Abbreviations: CAP, catabolite activator protein; cAMP, cyclic adenosine monophosphate; CCR, carbon catabolite repression; EFB-FH, empty palm fruit bunch fiber hydrolysate; IPTG, isopropyl β-D-1-thiogalactopyranoside; PPP, pentose phosphate pathway; PTS, phosphoenolpyruvate carbohydrate phosphotransferase system; TCA, tricarboxylic acid n Corresponding author at: School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea. Fax: þ82 52 217 2509. E-mail address: [email protected] (S.K. Lee). 1 These authors contributed equally to this work.

In E. coli, CCR is induced by glucose (Deutscher, 2008), which is preferentially transported by the glucose transporter EIIBCGlc (encoded by ptsG), and delivered to the phosphoenolpyruvate carbohydrate phosphotransferase system (Fig. 1). This event dephosphorylates EIIAGlc (encoded by crr), reduces intracellular cAMP, and blocks the complex between cAMP and catabolite activator protein. As a result, expression of enzymes that catabolize other sugars is suppressed. Additionally, arabinose transcriptional regulator (AraC) imposes a second layer of CCR. AraC activates the arabinose-catabolism genes araAB, araE, and araFGH, and suppresses the xylose-catabolism genes xylAB and xylFGH by inhibiting XylR, the xylose transcriptional activator. Consequently, arabinose is also preferentially consumed over xylose (Desai and Rao, 2010). Mutation of regulatory genes (Eppler et al., 2002; Warrens et al., 1997; Yao et al., 2011) and ptsG (Nichols et al., 2001), as well as deletion of mgsA (Gawand et al., 2013; Yomano et al., 2009), relieves CCR, and improves co-utilization of glucose and xylose. However, these mutations impair growth by reducing glucose uptake. Additionally, it is unclear whether CCR remains at high sugar concentrations. In this study, a strain that simultaneously consumes glucose and xylose was obtained by genetic engineering and adaptive evolution. Importantly, the strain, termed GX50, has no obvious growth defect even in the presence of arabinose. Finally, we also demonstrate that GX50 effectively produces xylitol from empty palm fruit bunch-fiber hydrolysate.

http://dx.doi.org/10.1016/j.ymben.2015.05.002 1096-7176/& 2015 International Metabolic Engineering Society. Published by Elsevier Inc.

Please cite this article as: Kim, S.M., et al., Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.002i

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Fig. 1. Carbon catabolite repression in E. coli and promoter replacement of genes in pentose metabolism. Central pathways related to sugar repression are shown, where  represents regulated genes. The structure of relevant genes in wild-type and GX50 E. coli is also depicted. araA, arabinose isomerase; araB, ribulokinase; araC, arabinose transcription factor; araE, arabinose-proton symporter; araFGH, arabinose ABC transporters; crp, cAMP receptor protein; ptsG, glucose PTS permease; xylA, xylose isomerase; xylB, xylulokinase; xylE, xylose-proton symporter; xylFGH, xylose ABC transporters; xylR, xylose transcription factor; xyl1, xylose reductase from Candida boidinii; cAMP, cyclic adenosine monophosphate; PPP, pentose phosphate pathway; TCA, tricarboxylic acid; *PTS, phosphoenolpyruvate carbohydrate phosphotransferase system.

Table 1 Strains and plasmids. Strains and plasmids

Genotype and description

Source

Strains E. coli MG1655 MG1655ΔptsG MG1655-pyrEup JARA1 AXcp GX50 GX50-inactived araES91I GX50ΔptsG GX50-pyrE MG1655ΔxylAB GXcp50ΔxylAB MGZ GXZ AXcpM AXM-1a AXM-2a AXM-2b AXcp-xF GX50-xF

Wild type MG1655 with ΔptsG::FRT MG1655 with pyrE::pyrEup MG1655 with PCP25-araB, PCP6-araF, ΔaraC: FRT JARA1 with PCP6-araE, PCP25-xylA, PCP6-xylF AXcp adapted for 50 days in arabinose and xylose minimal media GX50 with araES91I::FRT (at 100–440 bases in coding sequence) GX50 with ΔptsG::FRT GX50 with pyrEup::pyrE MG1655 with ΔxylA::FRT, ΔxylB::FRT GXcp50 with ΔxylA::FRT, ΔxylB::FRT MG1655ΔxylAB with pXR GX50ΔxylAB with pXR Genome integrated pRED-2 in AXcp AXcpM with xylAup AXcpM with xylAup, araES91I AXcpM with xylAup, ybjGD99G AXcp with xylA::xylA-FLAG tag GX50 with xylA::xylA-FLAG tag

Blattner et al. (1997) Baba et al. (2006) In this study Park et al. (2012) In this study In this study In this study In this study In this study In this study In this study In this study In this study In this study In this study In this study In this study In this study In this study

Plasmids pSIM5 pCP20 pKD13 pBbB6a pXR pRED-2

pSC101-ts ori, carrying λ-RED recombinase expression controlled by temperature, CmR Yeast FLP recombinase gene and replication controlled by temperature, AmR, CmR Template for kanamycin cassette flanked by FRT sites. KmR BBR1 ori, carrying LlacO-1 promoter and GFP, AmR pBbB6a with Δgfp::xyl1 (Candida boidinii, xylose reductase), AmR pSIM5 carrying mutS fragment, R6Kori, CmR

Datta et al. (2006) Cherepanov and Wackernagel (1995) Datsenko and Wanner (2000) Lee et al. (2011) In this study Ryu et al. (2014)

2. Materials and methods 2.1. Bacterial strains and plasmids Bacterial strains and plasmids are listed in Table 1. Wild-type E. coli MG1655 was used as parental strain.

Engineered mutations were introduced using the λ RED recombination system with pSIM5 (Datta et al., 2006) and pCP20 (Cherepanov and Wackernagel, 1995). Using splice overlapextension PCR (Datsenko and Wanner, 2000; Jensen and Hammer, 1998; Warrens et al., 1997), the synthetic constitutive promoter CP6 or CP25 (Jensen and Hammer, 1998) was spliced

Please cite this article as: Kim, S.M., et al., Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.002i

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with a kanamycin resistance (KmR) cassette flanked by a Flp recognition target (FRT). To generate the AXcp strain, this construct was used to replace native promoters in genes involved in pentose catabolism (araBAD and xylAB) and transport (araFGH, araE, and xylFGH), in a strain from which araC had been deleted (Fig. 1). Subsequently, AXcp was adapted in minimal media containing 2 g/L arabinose and 2 g/L xylose. At OD600 1.0, cells were sub-cultured by 100  dilution into fresh media to select for a faster growing subpopulation (Balderas-Hernandez et al., 2011). After 50 days, a strain efficiently growing in xylose medium was obtained and designated GX50. Mutations acquired during adaptive evolution were identified by sequencing and introduced into the unadapted strain AXcp through genome editing, as described (Ryu et al., 2014; Wang et al., 2009). Briefly, the plasmid pRED-2 was integrated by homologous recombination into the chromosomal mutS locus (Ryu et al., 2014). The resulting strain, designated AXcpM, was grown in LB media until OD600 0.5, whereupon it was heatshocked at 42 1C for 15 min to induce the λ RED system. Cells were harvested, rendered electrocompetent, and transformed with 0.5 mM oligos (Table S1). Around 4–10 cycles of mutagenesis were required to achieve the desired mutation. The xylose reductase gene (xyl1) was amplified by PCR using primers XR_F and XR_R (Table S1) from Candida boidinii strain KCTC 17776, which was obtained from Korean Collection for Type Culture. The PCR product was digested with NdeI and BamHI and cloned into the pBbB6a plasmid to create pXR, which was used to generate a xylitol-producing strain.

2.2. Batch culture A single colony growing on LB plates was inoculated into 5 mL liquid LB and grown at 37 1C for 8 h. Cells were then diluted 1:100 in 50 mL defined M9 media (BioShop Canada Inc., Burlington, Canada) supplemented with 2 mM MgSO4, 0.1 mM CaCl2, 2 g/L CaCO3, 2.5 g/L glucose, and 2.5 g/L xylose. After 10 h, these cultures were diluted 1:10 into 50 mL fresh M9 media supplemented with magnesium and calcium as described, along with glucose or xylose at the appropriate concentration. Batch cultures were grown in 250 mL flasks at 37 1C and 200 rpm.

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2.4. Analysis of cell growth and sugar metabolism Cell density was monitored by absorbance at 600 nm (OD600) using a Biochrom Libra S22 spectrophotometer. Residual sugar was measured by HPLC. Briefly, 1 mL culture media was collected and centrifuged for 30 min. The supernatant was heated at 80 1C for 1 h, and centrifuged a second time for 30 min at 16,500g. The final sample was diluted 10–20 fold, and 20 mL aliquots were injected into an HPX-87P column (Bio-Rad) at 0.6 mL/min and 80 1C. HPLC-grade water was used as mobile phase, and analysis was performed on a Shimadzu HPLC station equipped with a refractive index detector (Shimadzu) and a SIL-20A auto-sampler (Shimadzu). 2.5. Genome sequencing Genomic DNA was isolated from GX50 using GeneAll DNA isolation kit (GeneAll Biotechnology Co., Korea), and sequenced by Next Generation Sequencing (Macrogen Inc., Korea) on an Illumina HiSeq 2000 platform. The parental strain MG1655 (Table 1) (NC00913.2) was used as reference for genome assembly. Mutated sequences were amplified by PCR and verified by DNA sequencing.

3. Results 3.1. An E. coli strain without preference for glucose We obtained an E. coli strain that consumes xylose and glucose simultaneously by deleting araC, constitutively expressing arabinose and xylose operons, and adaptation in minimal media containing arabinose and xylose. The strain, termed GX50, grows faster than wild-type E. coli MG1655 in xylose minimal media (Fig. 2). However, strains that metabolize arabinose and glucose at the same time were not found. In contrast, the strain AXcp has a severe growth defect over 16 h in the presence of 4 g/L xylose (Fig. 2). Because GX50 was derived from AXcp by adaptive evolution, this finding indicates that further genetic alterations acquired during adaptation successfully rescued the growth defect and enhanced xylose metabolism. Further, this result suggests that evolutionary engineering is a powerful approach to improve or acquire strains with desired traits. 3.2. Mutations acquired during adaptive evolution

2.3. Fed-batch fermentation in mini-bioreactors Cells grown in LB broth were inoculated 1:100 into 50 mL M9 media supplemented with 2 mM MgSO4, 0.1 mM CaCl2, 10 mg/mL ampicillin, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), 4 g/L glucose, and 1 g/L xylose. Cells were harvested after 8 h and resuspended in 70 mL fresh M9 media supplemented with MgSO4, CaCl2, ampicillin, and IPTG as described, along with 8 g/L glucose and 2 g/L xylose. Cells were grown in a homemade mini-bioreactor (Fig. S1). Fed-batch fermentation was carried out at 37 1C and 700 rpm. pH was maintained at 7.0 by feeding 3 M NaOH. At intervals of 6 h, 3.5 mL of a mixture of 160 g/L glucose and 40 g/L xylose was added. The total amount of sugar used was 3.5 g. Fermentation of lignocellulosic hydrolysate was performed as described above, except that pre-cultures were supplemented with 3.12 g/L glucose and 1.88 g/L xylose. During fermentation, 4.46 mL concentrated fiber hydrolysate containing  97.8 g/L glucose and 59.4 g/L xylose was added every 5 h. The total amount of sugar used was 3.5 g. Fiber hydrolysate was obtained from Gendocs (Daejeon, Korea) (Kim et al., 2013), concentrated with a freeze-dryer, and sterilized through a 0.22 mm filter.

The GX50 genome was sequenced and compared with AXcp. As summarized in Table 2, GX50 acquired four notable mutations during adaptive evolution. These include deletion and replacement in non-

Fig. 2. Cell growth of E. coli strains with xylose as the sole carbon source. Filled circles (●), wild-type E. coli MG1655; open inverted triangles (∇), engineered AXcp; open squares (□), GX50. Error bars are standard deviations of three independent experiments.

Please cite this article as: Kim, S.M., et al., Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.002i

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Table 2 Mutations acquired by GX50 during adaptive evolution. Gene function

Nucleotide (positiona)

Codon

Protein change

Coding sequences Arabinose-proton symporter araES91I ybjGD99G Undecaprenyl pyrophosphatase

Carbohydrate metabolism Glycan biosynthesis and metabolism

g272t (2979933) a296g (882316)

AGC-ATC GAC-GGC

S91I D99G

Non-coding sequences pyrEup pyrE attenuator xylAup 50 -Untranslated region

Pyrimidine metabolism Carbohydrate metabolism

Δ1bp of c (3813833) g(þ6)t (CP25b)

Mutation

Gene product

– –

Nucleotide and amino acid changes are numbered according to the respective gene or protein. a b

Reference sequence: NC_000913.2 from NCBI. From the transcription start site of the synthetic promoter CP25 (Jensen and Hammer, 1998).

coding regions upstream of pyrE and xylA (pyrEup and xylAup), and the point mutations S91I and D99G in araE and ybjG, respectively. These mutations indicate that the molecular basis of glucose and xylose coutilization in GX50 is completely different from that in other strains without CCR (Chiang et al., 2013; Gawand et al., 2013; HernándezMontalvo et al., 2001; Khankal et al., 2009; Nichols et al., 2001; Yao et al., 2011; Yomano et al., 2009). xylA encodes a xylose isomerase, the first enzyme required to catabolize xylose, while pyrE encodes orotate phosphoribosyltransferase, an enzyme involved in pyrimidine biosynthesis. The mutation xylAup is a guanine to thymine substitution at position þ6 of the synthetic CP25 promoter (Jensen and Hammer, 1998), which in GX50 is inserted upstream of xylA. On the other hand, the pyrEup mutation is a deletion of a cytosine within the attenuator structure 39–60 bases upstream of the start codon of pyrE (Fig. S2). These mutations, while noncoding, may alter expression of the corresponding genes. On the other hand, araE encodes a symporter that transports arabinose and protons simultaneously into the cytoplasm. YbjG, a predicted protein, has putative undecaprenyl-diphosphatase activity, and is reported to confer bacitracin resistance when overexpressed (Ghachi et al., 2005). araES91I is a point mutation at the edge of the cytoplasmic loop between transmembrane helices (Stoner and Schleif, 1983), while ybjGD99G is a missense mutation in the middle of a predicted cytoplasmic loop between transmembrane helices (UniProt database [http://www.uniprot.org/]). These mutations may directly affect protein structure and activity. 3.3. Effect of acquired mutations on sugar metabolism To verify that acquired mutations produce phenotypic changes, these mutations were reintroduced combinatorially into the unadapted strain AXcpM, which is AXcp expressing the λ RED recombination system. Like AXcp, AXcpM is incapable of simultaneously consuming glucose and xylose (Fig. 3A). The xylAup mutation effectively enhanced xylose metabolism in AXcpM (Fig. 3B), and a combination of araES91I with xylAup increased glucose uptake (Fig. 3E). Indeed, the strain with both mutations simultaneously utilized glucose and xylose with similar kinetics as GX50 (Fig. 3D), suggesting that araES91I positively affects glucose metabolism. In addition, deletion of the mutated AraE in GX50 did not significantly affect glucose uptake and metabolism of both sugars (Fig. 3F), indicating that the both point mutation araES91I and araE deletion may have the same phenotype, that supports glucose metabolism. However, combination of ybjGD99G with xylAup resulted in no further changes in sugar metabolism (Fig. 3B), indicating that a mutation in ybjG is not required to generate the GX50 phenotype. Interestingly, reversal of pyrEup back to wild type slightly impeded the growth of GX50 in minimal media (Fig. S3A). Further, we found

that pyrEup enhanced glucose consumption in the parental strain MG1655 (Fig. S3B). A previous study reported that pyrEup helps to relieve pyrimidine starvation (Jensen, 1993). We propose that this mutation helps GX50 co-utilize glucose and xylose by increasing cell growth. xylAup is located in the 50 untranslated region, suggesting that the mutation may change expression of the XylA enzyme. Thus, we measured of expression of XylA-FLAG in the AXcp and GX50 genetic background (Fig. S4). Indeed, XylA is more abundantly expressed in GX50 than in the unadapted strain AXcp. Taken together, data indicate that improved xylose metabolism in GX50 is mainly due to abundant expression of xylose isomerase as a result of mutations in the 50 untranslated region. Nevertheless, additional studies are required to elucidate the mechanistic details by which these mutations induce changes in xylose metabolism.

3.4. Characterization of GX50 The unadapted strain AXcp grows negligibly on xylose, while GX50 grew faster than wild type (Fig. 2). Additionally, GX50 metabolizes both glucose and xylose simultaneously when provided in equal concentrations (Fig. 4A). In contrast, the wild-type parental strain MG1655 has the typical diauxic growth pattern where xylose is consumed only after glucose is depleted (Fig. 4B). To determine whether the GX50 phenotype is caused by inactivation of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS), ptsG was deleted from G50 and MG1655 (Fig. 4C and D). Interestingly, GX50 metabolizes glucose and xylose more efficiently than ptsG-deficient MG1655, a well-known strain without CCR. In any case, both ptsG-deficient mutants consumed glucose less efficiently, highlighting the importance of the phosphotransferase system. This result further indicates that the glucose transporter EIIBCGlc, which is encoded by ptsG, is active in GX50 (Fig. S5A), and is unmutated based on genome sequencing (Table 2). Thus, GX50 metabolizes glucose and xylose without cost to glucose uptake, unlike ptsG-deficient mutants that have been described (Flores et al., 1996; Hernández-Montalvo et al., 2001; Nichols et al., 2001). Hence, GX50 may efficiently metabolize lignocellulosic materials that contain high amounts of glucose. To evaluate suitability in fermenting various lignocellulosic materials, it is important to characterize sugar metabolism at different ratios of glucose and xylose because of the potential heterogeneity and/or diversity of the lignocellulose feed in industrial operation. Thus, we characterized GX50 in media containing 1:1, 2:1, 3:1, and 4:1 ratio of glucose to xylose (Fig. 5). In all cases, GX50 metabolized both sugars simultaneously, highlighting its versatility and potential ability to process various lignocellulosic materials.

Please cite this article as: Kim, S.M., et al., Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.002i

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Fig. 3. Consumption of glucose and xylose in AXcpM (A), AXcpM with xylAup (B), AXcpM with xylAup and ybjGD99G (C), GX50 (D), AXcpM with xylAup and araES91I (E), and GX50 without AraE (F). Fermentation was carried out in batches. Closed circles (●), glucose; closed inverted triangles (▼), xylose; open squares (□), optical density at 600 nm. Error bars are standard deviations of six independent experiments.

Fig. 4. Utilization of glucose and xylose (5 g/L) by GX50 (A), parental strain MG1655 (B) and ptsG-deficient GX50 (C) and MG1655 (D). Fermentation was carried out in batches. Closed circles (●), glucose; closed inverted triangles (▼), xylose; open squares (□), optical density at 600 nm. Error bars are standard deviations of four independent experiments.

3.5. Xylitol production by fed-batch fermentation To demonstrate potential application, we used GX50 to produce xylitol from mixtures of glucose and xylose. For this purpose, the xylose-catabolism genes xylAB were deleted from GX50 and MG1655. A copy of C. boidinii xylose reductase, which converts xylose directly into xylitol (Cirino et al., 2006), was transformed into deletion strains to obtain the strains GXZ and MGZ, respectively (Table 1). Fig. 6A shows negligible xylitol production in MGZ, whereas Fig. 6B shows significantly improved xylitol production in GXZ, with yield of 0.993 g xylitol per g xylose consumed. These results indicate that GXZ uptakes glucose and xylose simultaneously, and produces xylitol from xylose in the presence of glucose.

We also attempted to produce xylitol from empty palm fruit bunch fiber hydrolysate (Fig. 6C and D). MGZ produced 1 g/L of xylitol, which may be attributed to intermittent expression of xylose transporters due to suboptimal timing of substrate delivery. In contrast, GXZ produced 4.8 g/L xylitol within 25 h. These results highlight the ability of GX50 to process complex lignocellulosic biomass.

4. Discussion We constructed GX50, an E. coli strain that simultaneously utilizes glucose and xylose, the most abundant sugars in lignocellulose. GX50 was derived by replacement of pentose operon

Please cite this article as: Kim, S.M., et al., Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.002i

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Fig. 5. Sugar utilization in GX50 at different ratios of glucose and xylose. GX50 was grown at glucose to xylose ratio 1:1 (A), 2:1 (B), 3:1 (C), and 4:1 (D). Closed circles (●), glucose; closed inverted triangles (▼), xylose; open squares (□), optical density at 600 nm. Error bars are standard deviations of four independent experiments.

Fig. 6. Xylitol production from mixtures of glucose and xylose in E. coli MGZ (A) and GXZ (B), and from empty palm fruit bunch fiber hydrolysate (C and D). Strains were grown in fed batches. Closed blue circles ( ), glucose; closed red triangles ( ), xylose; green squares ( ), xylitol; open black diamonds ( ), optical density at 600 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

promoters with synthetic constitutive promoters, deletion of araC, and adaptive evolution. These strategies were designed to eliminate carbon catabolite repression of xylose metabolism by glucose and arabinose. Indeed, results suggest that constitutive expression of genes that are otherwise regulated by sugars maybe an effective strategy to eliminate CCR. As noted, the glucose transporter encoded by ptsG is intact and active in GX50. Accordingly, one of the most significant characteristics of GX50 is robust growth in mixtures of glucose and xylose. This can be attributed to the specific deregulation of catabolite-

repressed genes, but not of the phosphotransferase system or of global regulatory genes that control growth. This is in contrast to previous approaches, in which modulation of crp, ptsG, or crr changed global regulation, decreased glucose uptake, significantly slowed growth, and suppressed xylose metabolism via arabinose (Hernández-Montalvo et al., 2003, 2001; Hollands et al., 2007). Thus, we anticipate that, on the basis of robust growth, GX50 will be useful in industrial-scale processes. It is also important that arabinose should not suppress xylose metabolism, since most lignocellulosic materials

Please cite this article as: Kim, S.M., et al., Simultaneous utilization of glucose and xylose via novel mechanisms in engineered Escherichia coli. Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.05.002i

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contain some arabinose (Parisutham et al., 2014). In GX50, xylose utilization is not affected by arabinose because of AraC is not present (Fig. S5B). Thus, GX50 may be more suitable than other arabinose-sensitive strains to process actual biomass. Indeed, a GX50-derived strain generated xylitol from fiber hydrolysate. Key mutations acquired by GX50 during adaptation include xylAup and araES91I. xylAup is a point mutation in a non-coding region upstream of xylA that increases expression of xylose isomerase and thereby boosts xylose utilization. This indicates that, in GX50, constitutive expression of xylA is required to eliminate catabolite repression by glucose, while the xylAup mutation is essential to increase xylose metabolism. On the other hand, araES91I enhances glucose uptake. The AraE arabinose-proton symporter transports xylose promiscuously, but not glucose, when it is constitutively expressed (Hasona et al., 2004). Thus, it is possible that araES91I enhances glucose metabolism by decreasing xylose uptake. However, further studies are required to elucidate the exact mechanism underlying this effect. Nevertheless, we propose that an overall balance between sugar metabolic rates is important for efficient co-metabolism of glucose and xylose. Another notable mutation acquired by GX50 is pyrEup, a mutation in the pyrE attenuator that enhances the growth of wild-type E. coli in glucose. Mutations upstream of pyrE are frequently found at or near the attenuator, and allow adaptation to relieve pyrimidine starvation (Conrad et al., 2009; Jensen, 1993). Thus, pyrEup is likely to destabilize the attenuator loop, probably resulting in increased pyrE gene expression. The effect of the last mutation, ybjGD99G, remains to be clarified. These two mutations seem to be less important for co-metabolism than the others. Taken together, we conclude that simultaneous utilization of glucose and xylose in GX50 is likely due to the combined effect of four individual mutations, in addition to AraC deletion and constitutive expression of the pentose operon. Although the AXcp strain constitutively expresses pentosemetabolism genes under the synthetic promoters CP6 and CP25, the strain did not grow in xylose media, and an additional mutation in CP25 was required. This indicates that constitutive expression might not always be sufficient, and that additional optimization steps maybe required to engineer new metabolic pathways. Based on this and many other studies (Nduko et al., 2013; Utrilla et al., 2012; Zhou et al., 2012), one such step is evolutionary engineering. Genome sequencing is another, it being a powerful tool to characterize desirable phenotypes and provide a template for reverse engineering. Cost-effective, industrial-scale production of useful chemicals from renewable feedstock such as lignocellulose requires suitable and efficient host strains, which may have to be engineered (Akinterinwa and Cirino, 2009; Ren et al., 2010; Wu et al., 2015). Such engineering may require a combination of several strategies, as we have done. Indeed, the GX50 strained obtained by this approach has great potential for processing lignocellulosic biomass containing glucose and xylose. Moreover, a multipronged approach may also help solve sequential utilization of other sugars in lignocellulose.

5. Conclusions We constructed GX50, a strain that consumes xylose and glucose simultaneously, using genetic engineering and evolutionary adaptation. The mechanism underlying this metabolic change maybe different than has been reported for other engineered strains. Thus, GX50 is an alternative to these strains, with its own set of advantages. For instance, GXZ, a GX50-derived strain, produces xylitol efficiently, and will be useful in industrial-scale production. Thus, GX50 can serve as a platform to engineer strains that completely metabolize xylose and glucose into other useful biochemicals.

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Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) through grants funded by the Ministry of Science, ICT & Future Planning (MSIP) (NRF-2009-C1AAA001-20090093491 and 2011-0031948), and by a grant from the NextGeneration Bio Green 21 Program (SSAC, PJ0099058), Rural Development Administration, Republic of Korea. We thank Gendocs (Daejeon, Korea) for providing empty palm fruit bunch fiber hydrolysate.

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