Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose–xylose mixtures
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- Ji, X., Nie, Z., Huang, H. et al. Appl Microbiol Biotechnol (2011) 89: 1119. doi:10.1007/s00253-010-2940-5
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Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.
Keywords2,3-ButanediolKlebsiella oxytocaGlucoseXyloseCarbon catabolite repressionMutant cAMP receptor protein
As fossil fuel supplies are becoming increasingly scarce, the bio-refinery systems that integrate biomass conversion processes and equipment to produce fuels, power, and chemicals from annually renewable resources, are at the stage of development worldwide (Li et al. 2010; Ragauskas et al. 2006). Lignocellulose derived from agricultural residues is a kind of potential low-cost feedstock for fermentative production of different types of chemicals (van Haveren et al. 2008). Hydrolysis of lignocellulose yields a mixture of sugars containing mainly glucose and xylose (Sheehan and Himmel 1999), thus it is important that the applied microorganism could utilize both substrates to maximize carbon conversion to a product. Klebsiella species belong to such organisms, and they are able to produce 2,3-butanediol (2,3-BD) from both glucose and xylose (Cheng et al. 2010; Kosaric et al. 1990; Wang et al. 2010). Recently, microbial production of 2,3-BD has attracted worldwide interest due to the wide industrial application of 2,3-BD (Ji et al. 2009a; Syu 2001; Celińska and Grajek 2009).
In our previous study, a 2,3-BD hyper-producing Klebsiella oxytoca strain was obtained by altering the mixed acid–butanediol fermentation pathway (Ji et al. 2008; 2010). And, an industrial medium containing urea as a sole nitrogen source, low levels of corn steep liquor and mineral salts as nutrition factors to obtain high 2,3-BD production through co-fermentation of glucose and xylose was developed (Ji et al. 2009b). However, when glucose and xylose were present at the same time, xylose consumption generally did not commence until glucose was depleted, i.e., sugars were sequentially consumed resulting in several exponential growth phases that were separated by intermediate lag phases, developing so-called diauxic growth (Ji et al. 2009b). This phenomenon is universally displayed among the other enteric bacteria such as Klebsiella pneumoniae and Escherichia coli (Gosset 2005; Wang et al. 2010), and it hinders the possibility of utilizing the lignocellulose hydrolysates as carbon sources for efficient chemical production. Moreover, the utilization of xylose in mixed sugar fermentation was usually delayed and was slower than fermentation of pure xylose; furthermore, it was often incomplete in some cases (Bothast et al. 1994; Moniruzzaman et al. 1996). This would be disadvantageous for the fermentative production process using the sugar mixtures derived from lignocellulose hydrolysates as substrates.
The described above trait was attributed to carbon catabolite repression (CCR) or glucose effect (Görke and Stülke 2008; Jojima et al. 2010). CCR has been studied extensively in enteric bacteria with respect to the ability of glucose to block induction of the genes for other substrate utilizations, such as xylose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and cyclic adenosine monophosphate (cAMP; Postma et al. 1993; Roseman and Meadow 1990). In the enteric bacteria, when glucose is absent from the medium, the IIA component of the glucose-specific PTS (EIIAGlc) is mainly in its phosphorylated state, bound to an enzyme adenylate cyclase (EC 22.214.171.124) and activating its cAMP biosynthetic capacity, which leads to increase in cAMP concentration. The cAMP further binds to a cAMP receptor protein (CRP) and causes the induction of carbon catabolite repressed genes. However, if the glucose is present in the culture medium, EIIAGlc protein is mainly in its nonphosphorylated state, bound to various non-PTS permeases and inhibiting uptake of non-PTS sugars, which is called “inducer exclusion” (Gosset 2005). This response is mediated mainly by the CRP whose active form is a homodimer requiring cAMP for functioning (Saier and Ramseier 1996). In the present study, a mutant CRP, which does not require cAMP for functional dimerization, was characterized and overexpressed in a 2,3-BD producing K. oxytoca strain to eliminate the CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD.
Materials and methods
Strains and plasmids construction
Bacterial strains, plasmids, and primers used in this study
Strain, plasmid or primer
Genotype, properties, or sequence
Source or reference
K. oxytoca ME-XJ-8
Apr, parent strain
Ji et al. (2010)
K. oxytoca ME-CRPin
Apr, Cmr, ME-XJ-8/pDK7-crp(in)
E. coli DH5α
supE44 ∆lacU169 (φ80 lacZ ∆M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1
Apr, crp(in) PCR product from E. coli ET25 in pUC18 (3,813 bp)
Expressing vector, Cmr (4,800 bp)
Kleiner et al. (1988)
Apr (2,692 bp)
Apr, pMD18-T Simple derivative carrying a 1,109-bp DNA fragment containing the crp(in) gene
Cmr, pDK7 derivative, where a 1,109-bp DNA fragment containing the crp(in) gene was inserted
5′-TTAGGATCCCTCCACTGCGTCAATTTTC-3′ (BamH I)
5′-TAAGTCGACCAGGAACGAGGGAGAAGAG-3′ (Sal I)
Medium and culture methods
Luria–Bertani (LB) medium was used for the seed cultures of E. coli, K. oxytoca, and their derivatives. When K. oxytoca was subjected to electroporation, EDTA was added to the LB medium to a final concentration of 0.7 mM (Zhu et al. 2009). After the electroporation, the cells were regenerated in SOC medium (Joseph and David 2001). When necessary, ampicillin (60 μg/mL) and/or chloromycetin (12 μg/mL) were added to the medium as selection pressure.
For a seed culture preparation, a full loop of K. oxytoca from a fresh slant was inoculated into an Erlenmeyer flask (500-mL) containing 100 mL of fresh seed medium and cultivated on a rotary shaker at 200 rpm for 24 h. Seed culture (5%, v/v) was then inoculated into the glucose–xylose co-fermentation medium in which the glucose and xylose were set at three ratios (w/w, 2:1, 1:1, 1:2), representing what was present in different sources of lignocellulose hydrolysates. The composition of the fermentation medium besides the sugars is provided in our previous study (Ji et al. 2009b). Batch fermentation was carried out in a 3-L stirred fermenter (BioFlo 100; New Brunswick Scientific Co., NJ, USA) with a working volume of 2 L. All cultivations were carried out at 37 °C with the aeration rate and agitation speed at 1.0 vvm and 200 rpm, respectively. pH was controlled at 6.5 automatically by adding 3 M NaOH (for the engineered K. oxytoca strain, when OD600 reached about 0.7, 1 mmol/L isopropyl beta-d-thiogalactoside (IPTG) was added to induce expression of the recombinant protein).
Biomass was determined by measuring turbidity at 600 nm with appropriate dilution using a UV–visible spectrophotometer (Lambda-25, Perkin-Elmer, USA). Dry cell weight (DCW) was estimated from the absorbance, and 1 unit of optical density was determined to be equivalent to 0.35 g DCW per liter. The substrates (glucose and xylose) and the metabolites (2,3-BD and acetoin) were analyzed by high pressure liquid chromatography as indicated in our previous study (Ji et al. 2008; Yan et al. 2009).
Characterization of the crp(in) gene
The BLAST results of crp(in) gene and its predicted amino acid sequences in NCBI
GenBank accession number
E. coli ET25
E. coli K12
Riley et al. (2006)
K. oxytoca M5al
Li et al. (2002)
K. aerogenes KC1043
Osuna and Bender (1991)
K. pneumoniae MGH 78578
E. coli ET25
E. coli K12
Riley et al. (2006)
K. oxytoca M5al
Li et al. (2002)
K. aerogenes KC1043
Osuna and Bender (1991)
K. pneumoniae MGH 78578
Altered pattern of glucose and xylose utilization in the engineered K. oxytoca
Comparison of the metabolic characteristics of K. oxytoca ME-XJ-8 and its recombinant K. oxytoca ME-CRPin in different glucose–xylose mixtures
Composition of sugar mixture (g/L)
Sugar consumption time (h)
Glucose + Xylose
2,3-Butanediol production by engineered K. oxytoca cultured in glucose–xylose mixtures
The use of lignocellulose hydrolysates offers a means to increase the economics of 2,3-BD production beyond current starch-based production. However, the step utilization of mixed sugars caused by the regulatory mechanism of CCR delays the fermentation of lignocellulose hydrolysates as raw materials. To some extent, the CCR is a prevailing obstacle that must be overcome for efficient utilization of the lignocellulosic biomass (Jojima et al. 2010). Many previous efforts at relieving the CCR and eliminating diauxie have focused on mutations of the PTS (Dien et al. 2002; Hernández-Montalvo et al. 2001; Li et al. 2007; Lindsay et al. 1995; Nichols et al. 2001). Due to inactivation of ptsG gene encoding the major glucose transporter IICBGlc involved in PTS, the strains lacked the molecular machinery required for the CCR. Many of the obtained mutants did co-utilize glucose and xylose simultaneously, but this came at the expense of glucose utilization rates. To recover growth deficits in the PTS mutant strain, increased expression of a secondary glucose transporter and/or glucokinase was needed (Gosset 2005; Hernández-Montalvo et al. 2003; Snoep et al. 1994; Wang et al. 2005).
In the present study, a simple metabolic engineering strategy was developed to relieve the CCR in K. oxytoca for simultaneous consumption of glucose and xylose to produce 2,3-BD. A mutant CRP protein (CRP(in)) which is independent of cAMP was characterized and overexpressed in a 2,3-BD producing K. oxytoca. The recombinant turned to be able to consume glucose and xylose simultaneously to produce 2,3-BD in glucose–xylose mixtures. Moreover, the operating time was reduced and the 2,3-BD productivity was increased for the recombinant compared with the parent strain. To the best of our knowledge, this is the first report showing that elimination of the CCR in K. oxytoca has a prominent role in simultaneous consumption of glucose and xylose for 2,3-BD production.
The above response might be attributed by the following reasons. In the enteric bacteria, the use of sugar is transcriptionally regulated by CRP (Görke and Stülke 2008; Jojima et al. 2010). The active form of CRP is a homodimer requiring cAMP for functioning. In the present study, we have shown that the mutations of CRP(in) were located within the region of the protein known to be involved in functional dimerization with cAMP (Harman et al. 1986), and thus, conferred a cAMP-independent phenotype. Therefore, in the recombinant, the overexpressed CRP(in) did not require cAMP for functional dimerization and played the regulatory role in the absence of cAMP. Thus, it could replace the native CRP, and facilitate xylose uptake from mixtures of glucose and xylose. The CRP(in) phenotype should promote xylose uptake in the presence of glucose by activating the native xylose transporters and/or by activating other CRP-controlled promiscuous transporters capable of xylose uptake. Furthermore, in order to make the process economically feasible, the promoter of the plasmid used in present study should be exchanged, some other stress-induced (such as temperature-induced) promoters could be applied. In addition, the lactose could be used instead of IPTG to induce the CRP(in) expression for industrial production.
In the present work, it was shown that an engineered K. oxytoca harboring the CRP(in) phenotype could metabolize glucose and xylose mixtures simultaneously, which would be beneficial for efficient mixed substrates utilization. Thus, this recombinant has the potential for producing 2,3-BD directly from the inexpensive lignocellulose hydrolysates. As a consequence of the co-metabolism of glucose and xylose, the total sugars could be consumed faster, leading to higher growth rate and 2,3-BD productivity by the recombinant compared to the parent strain. This is another example showing the advantage of metabolic engineering to improve the microbial physiological functionality. The idea developed in this paper could be applied to the other similar industrial microorganisms to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass.
We are grateful to Prof. Mike J. Merrick, John Innes Centre, UK, and Prof. Lonnie O. Ingram, University of Florida, USA, for providing the pDK7 and pLOI 3244 plasmids. This work was financially supported by the National Natural Science Foundation of China (Nos. 20606018 and 21006049), the Key Program of National Natural Science Foundation of China (No. 20936002), the National Basic Research Program of China (Nos. 2007CB707805, 2009CB724700 and 2011CB200906), and the Program for New Century Excellent Talents in University from the Ministry of Education of China (No. NCET-09-0157). X.-J. Ji was supported by the Innovation Fund for Doctoral Dissertation of Nanjing University of Technology (No. BSCX200808), the China Postdoctoral Science Foundation Funded Project (No. 20100471328) and the Jiangsu Planned Projects for Postdoctoral Research Funds of China (No. 1001015 C).