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Engineering Escherichia coli for an efficient aerobic fermentation...

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Engineering Escherichia coli for an efficient aerobic fermentation...

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     Journal of Biotechnology 144 (2009) 58?C63

     Contents lists available at ScienceDirect

     Journal of Biotechnology

     journal homepage: www.elsevier.com/locate/jbiotec

     Engineering Escherichia coli for an ef?cient aerobic fermentation platform

     Zhen Kang a , Yanping Geng a , Yong zhen Xia a , Junhua Kang a , Qingsheng Qi a,b,?

     a b

     State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People??s Republic of China National Glycoengineering Research Center, Shandong University, Jinan 250100, People??s Republic of China

     a r t i c l e

     i n f o

     a b s t r a c t

     Acetate, as a major by-product, was excreted by Escherichia coli when aerobic fermentation runs at high growth rates. In order to reduce the acetate secretion during the fermentation fundamentally, a list of genes related to acetate accumulation in E. coli was selected and knocked out. Physiological characterization of each mutant demonstrated that the growth and metabolites accumulation properties of these mutations exhibited signi?cant change upon pathway engineering. The ?nal engineered E. coli QZ1110 with ptsG, poxB, pta and iclR gene mutations was con?rmed to accumulate 270% more biomass with 90% less acetate secretion than that of wild type E. coli in LB medium supplied with 1% glucose. Polyhydroxybutyrate biosynthesis experiment showed that the acetate reduction of the engineered strain in minimal medium also reduced 90% while the PHB accumulation increased almost 100% compare to wild type E. coli. ? 2009 Elsevier B.V. All rights reserved.

     Article history: Received 3 February 2009 Received in revised form 20 April 2009 Accepted 13 June 2009 Keywords: Acetate Escherichia coli Metabolic engineering PHB

     1. Introduction Escherichia coli is the most well known microorganism for biosynthetic pathway engineering in synthetic biology (Keasling, 2008). It has also been widely applied in industrial biotechnology for proteins and biochemicals production. Aerobic high cell density cultivation of E. coli was frequently used to arrive at high biomass yields and high metabolite/protein concentrations (De Mey et al., 2007). However, E. coli excretes acetate as a major by-product

    of its aerobic metabolism and acetate production represents a diversion of carbon that might otherwise have generated biomass or protein product (Andersen and von Meyenburg, 1980; March et al., 2002). Meanwhile, acetate was known to reduce the rate of RNA, DNA, protein and lipid synthesis (Cherrington et al., 1990), particularly those involved in the E. coli transcription?ªtranslation machinery, the general stress response and regulation. Additionally, acetate interferes with methionine biosynthesis, which causes the inhibitor homocysteine to accumulate (Roe et al., 2002). It is commonly observed that E. coli excretes 10?C30% of carbon ?ux from glucose to acetate in glucose-containing media even when the culture is fully aerated. Acetate is produced under an imbalance between the glycolytic and the TCA cycle ?uxes (Farmer and Liao, 1997; Majewski and

     Corresponding author at: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People??s Republic of China. Tel.: +86 531 88365628; fax: +86 531 88365628. E-mail address: qiqingsheng@sdu.edu.cn (Q. Qi). 0168-1656/$ ?C see front matter ? 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2009.06.021

     Domach, 1990; Wong et al., 2008). To solve this problem, many methods have been developed through controlling the glucose concentration in the fed-batch fermentation process (Kleman et al., 1991a,b; Paalme et al., 1990; Shiloach et al., 1996). Although most of these approaches have reduced acetate production, they undermine maximum growth and production capacity, or lead to undesirable pyruvate accumulation (Diaz-Ricci et al., 1991). Thus, a number of attempts with metabolic engineering to reduce carbon ?ow to acetate-producing pathways have been tried (Aristidou et al., 1994; Chou et al., 1994; Delgado and Liao, 1997; Farmer and Liao, 1997; Hosono et al., 1995; San et al., 1994). In E. coli, the two major aerobically active acetate-producing pathways are phosphotransacetylase/acetate kinase (Pta?CAckA) and pyruvate oxidase (PoxB). In the former pathway, acetate is produced from acetyl coenzyme A via acetyl phosphate by the Pta?CAckA; while in the later pathway, PoxB converts pyruvate, ubiquinone and H2 O to acetate, ubiquinol and CO2 (De Mey et al., 2007). Dittrich et al. recently con?rmed that the PoxB pathway is more active in E. coli during the late exponential and stationary phases, whereas the Pta?CAckA pathway is more active in the exponential stage of the cell growth (Dittrich et al., 2005). However, most of these genetic modi?cations were only cloning or deleting one or two genes. In the present study, based on the analysis of available information (March et al., 2002; Farmer and Liao, 1997; Dittrich et al., 2005; De Mey et al., 2007), a list of genes ptsG, poxB, pta and iclR in E. coli related to acetate secretion were selected and knocked out. The growth and physiological properties of these mutants were characterized. As an example, the production of

polyhydroxybutyrate was performed.

     Z. Kang et al. / Journal of Biotechnology 144 (2009) 58?C63 Table 1 Strains and plasmids used in this study. Strains and plasmids Strains E. coli MG1655 E. coli DH5 Genotype Reference or source

     59

     rph?1 fnr F?C 80 lacZ M15 recA endA1 (lacZYA-argF) U169 deoR gyrA96 thi-I hsdR17 supE44 relAI MG1655( ptsG) MG1655( ptsG poxB) MG1655( ptsG poxB pta) MG1655( ptsG poxB pta iclR) MG1655 harboring plasmid pHBS01 QZ1110 harboring plasmid pHBS01 oriR6K , CmR , rgnB(Ter) oriR6K , kmR , rgnB(Ter) araBp-gam-bet-exo, bla(ApR ), repA101(ts), oriR101 ApR ,CmR , FLP recombinance spcR , plac , low copy number pBluescript SK? , phbCAB operon from R. eutropha SpcR , PrpoS , low copy number SpcR , PrpoS , phbCAB operon

     Lab stock Invitrogen

     LR1010 QZ1011 QZ1100 QZ1110 QZ2000 QZ2001 Plasmids pKD3 pKD4 pKD46 pCP20 pCL1920 pBHR68 pSCP pHBS01

     Li et al. (2007) This study This study This study This study This study

     CGSC (Datsenko and Wanner, 2000) CGSC (Datsenko and Wanner, 2000) CGSC (Datsenko and Wanner, 2000) CGSC (Datsenko and Wanner, 2000) Lerner and Inouye (1990) Spiekermann et al. (1999) This study This study

     pHBS01 was transformed into E. coli MG1655 and E. coli QZ1110. Transformants were designated E. coli QZ2000 and E. coli QZ2001, respectively. E. coli mutants were created using the one-step inactivation method with some modi?cation (Datsenko and Wanner, 2000). This method includes following steps: ampli?cation of the resistance gene with polymerase chain reaction (PCR) using pKD4 or pKD3 as template. PCR products ?anked by FRT (FLP recognition target) sites and homologous sequences to the gene of interest were puri?ed and transformed into the cells by electroporation (Bio-Rad, Gene Pulser). The knocking out of the target gene in the cells which carried with plasmid pKD46 that expresses Red recombinase was happened by recombining the PCR product into the chromosome. Transformants were selected with

    antibiotic-resistance plate. The kanamycin or chloramphenicol cassette was removed with the helper plasmid pCP20 that expresses FLP before next mutation. All other genetic operations were performed according to the protocols provided by the manufactures. 2.2. Medium and growth conditions Cultivation of E. coli was performed in LB medium (10 g L?1 tryptone, 5 g L?1 yeast extract, and 10 g L?1 NaCl, pH 7.2). Antibiotic, such as chloramphenicol (25 g mL?1 ), kanamycin (25 g mL?1 ), Spectinomycin (25 g mL?1 ) or ampicillin (100 g mL?1 ), was added when necessary. For characterization of the engineered E. coli, 1% (w/v) glucose was added. Flask cultivations were carried out in 300 mL Erlenmeyer ?ask supplied with 50 mL LB medium at 37 ? C at an agitation

    of 250 rpm. pH controlled cultivation was performed in 1 L bioreactor using 2 M NaOH as neutralization agent. M9 mineral salts medium (Sambrook et al., 1989) was used to characterize the performance of the engineered E. coli. Glucose as the sole carbon source was added at the indicated amount. PHB fermentation was performed in 5 L fermentor. The inlet air?ow was 0.6 L min?1 while the dissolved oxygen was maintained above 50% saturation throughout the experiment by changing the stirrer speed. 2.3. Analytical methods Bacterial growth was monitored by measuring the optical density (OD) at 600 nm, the culture was diluted to the linear range with 0.15N NaCl. For analyzing the substrate and the extracellular metabolite concentrations, 1 mL of culture was centrifuged at 12,000 ?Á g for 5 min

     2. Materials and methods 2.1. Genetic methods E. coli strains, plasmids and oligonucleotides used in this study were summarized in Tables 1 and 2. Molecular cloning and manipulation of plasmids were done with E. coli DH5 . Plasmid pSCP derived from pCL1920, in which lac promoter was replaced by a stress-induced promoter (Kang et al., 2008). After ampli?cation and double enzyme BamHI and ApaI digestion, the puri?ed fragments ApaI-spc-pSC101ori-BamHI and BamHISIR-ApaI were linked by T4 ligase and the new plasmid was named as pSCP. The phbCAB operon which encodes PHB synthase (PhbC), -ketothiolase (PhbA) and acetoacetyl-CoA reductase (PhbB), respectively, was ampli?ed from plasmid pBHR68 by PCR and subcloned to pSCP to generate plasmid pHBS01. Plasmid

     Table 2 Oligonucleotides used in this study. Primer pKD-poxB F pKD-poxB R poxB test F poxB test R pKD-pta F pKD-pta R Pta test F Pta test R pKD-iclR F pKD-iclR R iclR test F iclR test R pSCP F pSCP R SIR F SIR R Sequence

     5

    -AAACTTGTTACCGTTATCACATTCAGGAGATGGAGAACCGTGTAGGCTGGAGCTGCTTC-3 5 -CATGGCATGTCCTTATTATGACGGGAAATGCCACCCTTTATGGGAATTAGCCATGGTCC-3 5 -TCCCCCTCCGTCAGATGA-3 5 -GGTATCACTGCGTAAATCAA-3 5

    -GTAACCCGCCAAATCGGCGGTAACGAAAGAGGATAAACCGTGTAGGCTGGAGCTGCTTC-3 5 -TCAGATATCCGCAGCGCAAAGCTGCGGATGATGACGAGAATGGGAATTAGCCATGGTCC-3 5 -TCAGCTGGCGGTGCTGTTT-3 5 -ACCGGAAATAGTGATTATTTCCGG-3 5

    -ATGAAAATGATTTCCACGATACAGAAAAAAGAGACTGTCGTGTAGGCTGGAGCTGCTTC-3 5 -TATGATGGGCAGAATATTGCCTCTGCCCGCCAGAAAAAGATGGGAATTAGCCATGGTCC-3 5 -TAAAAGCGACCACCACG-3 5 -GCGATTAACAGACACCCT-3 5

    -TTTGGATCCCGACAGTAAGACGGGTAAGCCTGTTGATGAT-3 5

    -ACTGGGCCCGAGCTCCTTGAACGAATTGTTAGACATTATTTG-3 5

    -AAAGGATCCGCCTGCACAAAATTCCACCGTTGCTG-3 5

    -TTTGGGCCCCCCCTCGAGGTCGACGGTAT-3

     Annotation: the underlined bases meant enzyme site.

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     al., 2005). The pH was measured using a glass electrode. PHB was quantitatively analyzed with GC (Li et al., 2007). 3. Results 3.1. Construction of acetate-reducing E. coli strains In E. coli, the phosphoenolpyruvate?Ccarbohydrate phosphotransferase system (PTS) played an important role in metabolic utilization of glucose and carbon catabolite repression (Gorke and Stulke, 2008). Comparing the growth of E. coli ptsG mutant (E. coli LR1010) with wild type E. coli in LB medium, we found that E. coli LR1010 exhibited a faster growth and more biomass accumulation (Fig. 1). When both E. coli strains were grown in LB glucose medium, biomass accumulation of E. coli LR1010 was doubled as that of wild type E. coli (Fig. 2A). Meanwhile, the glucose consuming rate of the ptsG mutant at the initial growth phase was getting slow in LB glucose medium (Fig. 2C). The direct effect of slow glucose consuming rate of the ptsG mutant was the less acetate secretion (Fig. 2B). Previous studies also demonstrated that inactivation of ptsG may reduce glucose consumption rate and decrease transformation of PEP to pyruvate, causing a reduced acetyl-CoA accumulation (Chou et al., 1994; Li et al., 2007). However, signi?cant amount of acetate was still secreted into the medium during the fermentation in the ptsG mutant (Fig. 2B). To further reduce the acetate secretion, both poxB and pta genes in the ptsG mutant were selected and knocked out sequentially to block the direct acetate production pathways. Furthermore, iclR (encoding an isocitrate lyase regulator) gene was knocked out to activate the glyoxylate bypass. The modi?ed metabolic pathway

     Fig. 1. Growth curve of each strain in LB medium.

     and the supernatant was then ?ltered through a 0.22 m syringe ?lter for HPLC analysis. The HPLC system (Shimadzu-10A Systems, Shimadzu, Columbia, MD) was equipped with a cation-exchange column (Waters), a UV detector (SPD-10A) and a differential refractive index (RI) detector (RI-10A). A 0.8 mL min?1 mobile phase using 0.1% formate solution was applied to the column. The column was operated at 45 ? C. Standards of glucose, acetate were prepared for both the RI detector and UV detector, and calibration curves were created. Glucose and acetate were measured by the RI detector and pyruvate was measured by the UV detector at 210 nm (Lin et

     Fig. 2. Growth characterization of each strain in LB glucose medium: (A) optical density at 600 nm; (B) acetate excretion; (C) glucose consumption; (D) culture pH.

     Z. Kang et al. / Journal of Biotechnology 144 (2009) 58?C63

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     coli QZ1100 and E. coli QZ1110 accumulated the acetate only during the middle stage of the cultivation. The ?nal acetate concentrations in glucose medium of E. coli MG1655, E. coli LR1010, E. coli QZ1011,

    E. coli QZ1100 and E. coli QZ1110 were 4.24, 3.33, 2.74, 0.81 and 0.53 g L?1 , respectively. The total acetate secretiondecreased more than 90% in E. coli QZ1110 comparing with that of the wild type E. coli. Calculation results of total acetate secretion in wild type E. coli grown in LB glucose medium indicated that most glucose was used to produce acetate (1 g L?1 acetate accumulation consume 1.5 g L?1 glucose). In wild type E. coli cultures, the culture medium pH decreased to 4.2 after only 8 h cultivation, the point that E. coli has began to lysis (Fig. 2D). Thus, the wild type E. coli grew even worse in LB glucose medium than in LB medium (Figs. 1 and 2A). The slow glucose consuming rate of the mutants at the initial growth stage caused the low acetate secretion (Fig. 2C). Reduction of acetate secretion indicated the high biomass accumulation. The ?nal OD600 of E. coli MG1655, E. coli LR1010, E. coli QZ1011, E. coli QZ1100 and E. coli QZ1110 in LB glucose medium was 4.5, 10.2, 10.7, 15.6 and 16.8, respectively (Fig. 2A). 3.3. Characterization of engineered E. coli strains under controlled condition The glucose consumption rate, as well as the acetate secretion rate of the mutants, was much faster at pH controlled conditions than that at non-controlled conditions (Fig. 4B and C). The maximum acetate secretion of E. coli MG1655, E. coli LR1010, E. coli QZ1011, E. coli QZ1100 and E. coli QZ1110 were appeared at the middle stage of cultivation and were 7.0, 5.6, 5.1, 0.7 and 0.52 g L?1 , respectively, which is even more than that secreted at non-controlled conditions (Fig. 4B). In the wild type E. coli, 7.0 g L?1 acetate secretion indicated that almost all glucose supplied in the medium was used to produce acetate. However, the secreted acetate began to be re-used by E. coli after the middle stage of cultivation (normally when glucose was consumed up); the re-utilization of acetate by E. coli did not cause the cell biomass to increase. Interestingly, disruption of ptsG caused the co-consumption of glucose and acetate at the late exponential growth phase before glucose was completely consumed up (Fig. 4B and C); this is more obvious in case of E. coli QZ1100 and E. coli QZ1110. In E. coli QZ1110, the re-consumption of acetate was started after 14 h cultivation, when there is still 4 g L?1 glucose and only 0.5 g L?1 acetate in the medium. This indicated that the re-consumption of acetate in this strain was neither due to the glucose content nor the acetate content in the medium. The ?nal OD600 of E. coli MG1655, E. coli LR1010, E. coli QZ1011, E. coli QZ1100 and E. coli QZ1110 was 7.1, 13.4, 14.1, 15.8 and 16.0, respectively (Fig. 4A). 3.4. Application example of the engineered E. coli In order to investigate the performance of the engineered strain, E. coli QZ1110 was studied with respect to its growth under M9 minimal medium and its PHB biosynthesis capability under low copy number plasmid (Fig. 5). The result showed that the OD600 of engineered E. coli increased 76% while the acetate secretion reduced

    95% compare to wild type E. coli when cultivated in M9 medium. PHB accumulation experiment was performed in 5 L fermentor. The results showed that the ?nal cell concentration and PHB content of E. coli QZ2001 obtained were 9.63 g L?1 , 28.92 wt%,

     Fig. 3. Metabolic pathway of central carbon source in reconstructed E. coli strains. PEP: phosphoenolpyruvate. Bar indicated the knockout site.

     of central carbon source under aerobic condition was shown in Fig. 3. 3.2. Characterization of engineered E. coli strains The mutants obtained by sequential knocking out of ptsG, poxB, pta and iclR genes exhibited different growth phenomena in LB medium (Fig. 1). Inactivation of ptsG increased the growth rate and biomass accumulation clearly. Inactivation of pta also increased the biomass accumulation but resulted a comparative long growth lag phase, which revealed that pta gene played an important role in energy production with substrate level phosphorylation at early exponential phase. PoxB and IclR were found to contribute comparatively less to the growth rate and cell biomass accumulation although their effect was also positive. E. coli QZ1110, which possesses an iclR mutation, showed a relative faster growth rate but similar biomass when compared with E. coli QZ1100, which further proved the results previous (Farmer and Liao, 1997). Acetate secretion was evidently found under excess glucose conditions (Fig. 2B). E. coli MG1655, E. coli LR1010 and E. coli QZ1011 accumulated a large amount of acetate throughout the cultivation with highest accumulation rate at the initial growth phase, while E.

     Table 3 Batch fermentation results of E. coli QZ2000 and QZ2001. Stains E. coli QZ2000 E. coli QZ2001 Cell dry weight (g L?1 ) 9.04 ?À 0.1 9.63 ?À 0.1

     Acetate secretion (g L?1 ) 11.44 ?À 0.2 1.41 ?À 0.1

     PHB content (wt%) 14.91 ?À 0.12 28.92 ?À 0.14

     Glucose consumption (g L?1 ) 36.5 ?À 0.5 35.0 ?À 0.5

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     Fig. 4. Growth characterization of each strain in LB glucose medium with pH control: (A) optical density at 600 nm; (B) acetate excretion; (C) glucose consumption.

     respectively. While the ?nal cell concentration and PHB content of E. coli QZ2000 obtained were 9.04 g L?1 , 14.91 wt%, respectively (Table 3). The maximum acetate secretion of E. coli QZ2000 was 11.44 g L?1 , while acetate concentration of E. coli QZ2001 was only 1.41 g L?1 . 4. Discussion In this study, we analyzed the main metabolic properties of enteric bacteria, combined the available information and constructed a series of acetate reducing E. coli strains with sequential ptsG, poxB, pta and iclR mutations. The acetate secretion was moni-

     Fig. 5. Flask cultivation results in M9 medium with 2% glucose.

     tored throughout the fermentation at both non-pH controlled and pH controlled conditions. In the past few years, many researchers have tried a wide variety of strategies to reduce acetate accumulation to increase the cell biomass in E. coli high cell density fermentations. These strategies are situated at two levels: the bioprocess level and the genetic level (De Mey et al., 2007). Because the methods used in bioprocess level undermined maximum growth and production capacity and complex to operate, genetic approaches to minimize acetate formation attracted more attention (Chou et al., 1994; Eiteman and Altman, 2006; March et al., 2002). Among genetic methods which has obvious effect, pta and poxB mutation directly blocked the acetate accumulation pathway from glycolysis which catalyzes about 72% of carbon supply in enteric bacteria, but these mutants caused the imbalanced cell growth (Yang et al., 1999); over-expression of ppc and glyoxylate increase may reduce the acetate production by diverting the central glycolytic pathway intermediates, PEP and acetyl-CoA to TCA cycle or glyoxylate bypass, but the acetate reduction was not monitored throughout the fermentation. It is proved that acetate excretion is physiologically due to a metabolic over?ow mechanism, caused by an imbalance between the rapid uptake of glucose and its conversion into biomass and products, diverting acetyl-CoA from the TCA-cycle toward acetate (Akesson et al., 1999). Controlled glucose uptake and glycolytic pathway may reduce the acetate secretion and increase the cell biomass. Wong et al. recently constructed an engineered E. coli strain GJT001 with reduced acetate accumulation and increased protein production by only disabling the phosphoenolpyruvate: sugar phosphotransferase system. Our experiment result demonstrated that E. coli ptsG mutant resulted in 25?C30% reduction in acetate yield. Meanwhile, inactivation of ptsG also partially released

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     the catabolite repression and resulted the co-consumption of glucose and acetate at the middle stage of cultivation before glucose was completely consumed (Fig. 4). This phenomenon was also useful when fermentation process was performed in a mixed substrate culture. PoxB and pta mutations reduced about 10% and 50% acetate accumulation, respectively. However, only inactivation of these two genes caused the over?ux of glycolytic pathway, resulting pyruvate accumulation and imbalanced cell growth (Dittrich et al., 2005). Our experiment data also support this conclusion (data not shown). Inactivation of iclR enabled the operation of glyoxylate pathway which is normally repressed in the presence of glucose and condenses another acetyl-CoA to form malate. The continuous consumption of an acetyl-CoA by glyoxylate

    pathway may release the metabolic burden caused by pta and poxB knockout. Experiment resulted demonstrated that E. coli QZ1110 grew faster than E. coli QZ1100, which indicated at least the partial recovery of the imbalanced growth of E. coli QZ1110 (Fig. 4A). Meanwhile, activation of the glyoxylate bypass responds to the availability of a wide variety of carbon sources and culture conditions (Gui et al., 1996). The ?nal constructed E. coli strain QZ1110 was con?rmed to have a similar performance in minimal medium as that in complex medium. The PHB accumulation of the engineered E. coli in M9 medium increased 100% than that of wild type E. coli. Since the Pta?CAckA pathway and PoxB also involved in the anaerobic growth (Abdel-Hamid et al., 2001; Chang et al., 1999), this system can also useful under anaerobic conditions. Acknowledgements We appreciate the plasmids pKD3, pKD4, pKD46 and pCP20 provided by Dr. Wanner and the CGSC, the Coli Genetic Stock Center, Yale University. This work was ?nancially supported by research grants from the National High-Tech Research and Development Plan of China (2006AA02Z218), the National Basic Research Program of China (2007CB707803) and a grant from the National Natural Science Foundation of China (30870022). References

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