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Construction of an efficient Escherichia coli whole-cell biocatalyst for d -mannitol production
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e4, 2014 www.elsevier.com/locate/jbiosc
Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production Shamlan M.S. Reshamwala,1, * Sandip K. Pagar,1 Vishal S. Velhal,1 Vijay M. Maranholakar,1 Vishal G. Talangkar,1 and Arvind M. Lali1, 2 DBT-ICT-Centre for Energy Biosciences, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai 400019, Maharashtra, India1 and Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai 400019, Maharashtra, India2 Received 28 February 2014; accepted 4 May 2014 Available online xxx
Mannitol is a six carbon sugar alcohol that finds applications in the pharmaceutical and food industries. A novel Escherichia coli strain capable of converting D-glucose to D-mannitol has been constructed, wherein native mannitol-1-phosphate dehydrogenase (MtlD) and codon-optimized Eimeria tenella mannitol-1-phosphatase (M1Pase) have been overexpressed. Codon-optimized Pseudomonas stutzeri phosphite dehydrogenase (PtxD) was overexpressed for cofactor (NADH) regeneration with the concomitant oxidation of phosphite to phosphate. Whole-cell biotransformation using resting cells in a medium containing D-glucose and equimolar sodium phosphite resulted in D-mannitol yield of 87 mol%. Thus, production of an industrially relevant biochemical without using complex media components and elaborate process control mechanisms has been demonstrated. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: D-Mannitol production; Whole-cell biocatalyst; Cofactor regeneration; Biotransformation; Resting cells]
Mannitol is a six carbon sugar alcohol that is used in the pharmaceutical, food, medicine and chemical industries (1,2). The global market for mannitol is estimated to be 13.6 million kg year1 (3). The exudate of the manna tree, Fraxinus ornus, was the commercial source of mannitol till the 1920s (4). Currently, mannitol is produced at an industrial scale by catalytic reduction of a mixture of glucose and fructose, leading to the formation of mannitol and sorbitol, which are then separated by selective crystallization (5,6). Low yields and cost associated with the chemical production of mannitol has generated interest in possible selective fermentative production of mannitol from glucose or fructose. Lactic acid bacteria, yeasts and fungi known to naturally produce mannitol have been used and manipulated to achieve high titres of mannitol (3,7). However, many of the fermentative processes developed and reported lead to co-production of byproducts like acetic acid and lactic acid (8,9) and require complex media components (10,11). Using Leuconostoc mesenteroides, yields upto 93e97% from fructose have been reported at a 100 L pilot scale (12). The process, however, requires a significant amount of glucose to be co-fed, leading to a mannitol yield of 61e62% on the basis of sugar consumed (von Weymarn, N., Ph.D. thesis, Helsinki University of Technology, Helsinki, 2002). Recently, the marine cyanobacterium Synechococcus sp. PCC 7002 has been genetically modified to photosynthetically produce mannitol from carbon dioxide as the sole carbon source (13). The * Corresponding author. Tel.: þ91 22 33611111, þ91 22 33612222; fax: þ91 22 33611020. E-mail address: [email protected] (S.M.S. Reshamwala).
highest productivity was obtained using a glycogen synthasedeficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol l1 and a production rate of 0.15 g mannitol l1 day1. Metabolic engineering of Escherichia coli has been reported to convert fructose to mannitol. Kaup et al. (14) expressed the Leuconostoc pseudomesenteroides mdh gene, coding for mannitol dehydrogenase, to reduce fructose to mannitol. The glucose facilitator protein (GLF) of Zymomonas mobilis was expressed to allow fructose to be taken up by the cells without simultaneous phosphorylation, and the fdh gene from Mycobacterium vaccae N10, coding for formate dehydrogenase, was co-expressed to provide a source of reducing equivalents. The authors obtained a yield of 84 mol% by carrying out biotransformations conducted under pH control by formic acid addition (14). Similar work has been carried out using Corynebacterium glutamicum (15) and Bacillus megaterium (16). To allow production of mannitol using glucose as substrate, Kaup et al. (17) supplemented recombinant E. coli expressing mdh, glf and fdh with extracellular glucose isomerase, leading to a yield of 80% on glucose. On the other hand, expression of xylA, coding for xylose isomerase, which can convert glucose to fructose under nonphysiological conditions (18), resulted in a mannitol yield of 42% from glucose (17). The present report describes our attempt at construction of a recombinant E. coli strain that can efficiently convert glucose to mannitol without addition of extracellular enzymes. This has been achieved by diverting carbon flux from fructose-6-phosphate towards mannitol biosynthesis, resulting in a mannitol yield of 87 mol% from glucose.
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.05.004
Please cite this article in press as: Reshamwala, S. M. S., et al., Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.004
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RESHAMWALA ET AL.
J. BIOSCI. BIOENG.,
TABLE 1. Plasmids used in this study. Plasmid
Size (kb)
Plasmid
Description
pSR-M1
w4.8
pSR-M1
pSR-M2
w5.9
pSR-M2
pSR-M3
w6.9
pSR-M3
Codon-optimized E. tenella M1Pase CDS cloned under T5 promoter in plasmid pJexpress401; KanR E. coli mtlD cloned in plasmid pSR-M1 downstream of M1Pase Codon-optimized P. stutzeri ptxD cloned in plasmid pSR-M2 downstream of mtlD
MATERIALS AND METHODS Bacterial strains and growth conditions E. coli DH5a (19) was used for cloning and E. coli BL21(DE3) (20) for gene expression and whole-cell biotransformation. Bacteria were routinely cultivated in Luria-Bertani medium (Himedia, Mumbai, India) or M9 minimal medium supplemented with 1% glucose at 37 C. Plasmids used in this work are listed in Table 1. When required, kanamycin and ampicillin were used at a concentration of 50 mg ml1 and 100 mg ml1, respectively. For induction of gene expression, isopropyl-b-D-1thiogalactopyranoside (IPTG) was added to achieve a final concentration of 1 mM. Growth was monitored by measuring optical density at 600 nm (OD600). Cloning and plasmid construction All DNA techniques were carried out using standard methods (21). Codon-optimization and synthesis of genes were outsourced to DNA2.0 (Menlo Park, CA, USA). The codon-optimized Eimeria tenella mannitol-1-phosphatase (M1Pase) coding sequence (22) was synthesized and cloned in plasmid pJexpress401 by DNA2.0 to obtain plasmid pSR-M1. The E. coli mannitol-1-phosphate dehydrogenase gene mtlD was amplified by polymerase chain reaction from E. coli MG1655 genomic DNA using primers mtlD-Fw (50 -GGGAATTCCATATGAAAGCATTACATTTTGGC) and mtlD-Rv (50 -CCCAAGCTTTTATTGCATTGCTTTATAAGCG) and cloned between the NdeI and XhoI restriction sites of plasmid pET43.1b. The mtlD sequence was verified by sequencing (Scigenom Labs, Kerala, India) and sub-cloned into the blunted XhoI recognition site w10 bp downstream of the M1Pase coding sequence in plasmid pSR-M1 to obtain plasmid pSR-M2. The codon-optimized Pseudomonas stutzeri phosphite dehydrogenase gene ptxD (23), synthesized and cloned in plasmid pJexpress404 by DNA2.0, was excised along with the ribosome binding sequence by digesting pJexpress404 with XbaI and XhoI, blunted and cloned into the SmaI recognition site w35 bp downstream of the mtlD gene in plasmid pSR-M2 to obtain plasmid pSR-M3. All constructs were verified for correct orientation of the genes by restriction digestion. The synthetic mannitol operon thus constructed is depicted in Fig. 1. Mannitol production from glucose To assess mannitol production, E. coli BL21(DE3) transformed with plasmids pSR-M1, pSR-M2 or pSR-M3 was grown in M9 medium supplemented with 55 mM glucose at 37 C in a rotary shaker set at 180 rpm. IPTG was added when the culture reached a cell density of OD600 0.5, and incubation was continued for a further 18 h under the same conditions. When required, sodium phosphite was added at the time of IPTG addition. Whole-cell mediated biotransformation was carried out using cultures grown overnight in M9 medium supplemented with 55 mM glucose and diluted 4:100 in the same medium and grown till cell density of OD600 0.5 was attained. IPTG was then added and the culture allowed to grow for a further 12 h, after which the cells were pelleted, washed with M9 salts and resuspended in 55 mM glucose or 55 mM glucose supplemented with equimolar concentration of sodium phosphite. The resuspended cells were incubated at 37 C in a rotary shaker set at 180 rpm and samples drawn periodically to quantify metabolites. HPLC analysis Cells were pelleted by centrifugation at 10,000 g for 10 min and the supernatant was stored at 20 C till analysis. Glucose and mannitol in the supernatant were quantified using a Bio-Rad Aminex HPX-87H column maintained at 50 C coupled to an RI detector. The eluent was 5 mM sulphuric acid, and flow was 0.6 ml min1.
RESULTS Assembling the mannitol biosynthesis pathway in E. coli A synthetic pathway was assembled in E. coli to divert carbon flow
T5 promoter
lac O
RBS
M1Pase
FIG. 2. Synthetic pathway cloned in E. coli leading to mannitol production from glucose. Bold arrows indicate overexpression.
towards mannitol production, comprising of two enzymes, the native mannitol-1-phosphate dehydrogenase (MtlD) and a mannitol-1-phosphatase (M1Pase) from the protozoan parasite E. tenella (22). MtlD reduces fructose-6-phosphate to mannitol-1phosphate, while M1Pase dephosphorylates mannitol-1phosphate to mannitol, which is transported across the membrane and released into the medium (Fig. 2). E. coli can metabolize mannitol as a source of carbon, but cannot biosynthesize this sugar alcohol as it lacks mannitol-1-phosphatase. Expression of MtlD in E. coli leads to toxicity (24), which may be because of the accumulation of mannitol-1-phosphate, the product of the reaction catalysed by mannitol-1-phosphate dehydrogenase. Therefore, this enzyme was expressed by cloning it in plasmid pSRM1 downstream of M1Pase, leading to the construction of plasmid pSR-M2, ensuring that mannitol-1-phosphate is readily converted to mannitol by the action of mannitol-1-phosphatase. Reduction of fructose-6-phosphate is accompanied by the oxidation of NADH (25). In order to regenerate NADH, PtxD, a phosphite dehydrogenase from P. stutzeri, was selected for its ability to catalyse the nearly irreversible oxidation of phosphite to phosphate, with the concomitant reduction of NADþ to NADH (23). ptxD was cloned downstream of the mtlD gene in plasmid pSR-M2 to obtain plasmid pSR-M3. All genes in pSR-M3 are transcribed from a single T5 promoter to form the synthetic mannitol biosynthesis operon (Fig. 1). Mannitol production by recombinant E. coli Mannitol production by E. coli harbouring the pSR-M1, pSR-M2 and pSR-M3 plasmids was quantified by growing the cells in M9 minimal medium supplemented with glucose. Expression of M1Pase alone led to mannitol production of 21 mol%, indicating that the level of MtlD in the cell was enough to divert carbon flow towards mannitol. Overexpression of MtlD along with M1Pase increased mannitol production to 28 mol% (Table 2). Regeneration of NADH facilitated by overexpression of PtxD along with M1Pase and MtlD resulted in an increase in mannitol production to 43 mol% in the presence of equimolar phosphite (Table 2). This demonstrated that carbon flux towards mannitol
mtlD
RBS
ptxD
term
FIG. 1. Schematic diagram of the synthetic mannitol operon cloned in plasmid pSR-M3. M1Pase, codon-optimized E. tenella mannitol-1-phosphatase; mtlD, E. coli mannitol-1-phosphate dehydrogenase; ptxD, codon-optimized P. stutzeri phosphite dehydrogenase.
Please cite this article in press as: Reshamwala, S. M. S., et al., Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.004
VOL. xx, 2014
MANNITOL PRODUCTION BY RECOMBINANT E. COLI
TABLE 2. Mannitol formation by E. coli transformed with different plasmids. Strain
M9 medium supplemented with
BL21(DE3) BL21(DE3)/pSR-M1 BL21(DE3)/pSR-M2 BL21(DE3)/pSR-M3 BL21(DE3)/pSR-M3 BL21(DE3)/pSR-M3
Mannitol yield (mol%)
55 mM glucose 55 mM glucose 55 mM glucose 55 mM glucose 55 mM glucose þ 28 mM sodium phosphite 55 mM glucose þ 55 mM sodium phosphite
e 21.8 28.83 30.2 35.25 43.74
3.42 1.93 0.62 2.42 1.96
biosynthesis could indeed be efficiently diverted by providing a mechanism for NADH regeneration. Whole-cell biotransformation for mannitol biosynthesis from glucose To increase the yield of mannitol, a whole-cell biotransformation approach was adopted. E. coli BL21(DE3) transformed with plasmid pSR-M3 was induced to produce the enzymes of the synthetic mannitol pathway, and w25 g l1 cells (wet cell weight basis) were suspended in an aqueous solution of 55 mM glucose and equimolar concentration of phosphite. The yield of mannitol obtained using this approach was 87 mol% (Fig. 3). As no other metabolites were detected in the supernatant, we conclude that E. coli BL21(DE3)/pSR-M3 is an efficient biocatalyst for production of mannitol from glucose. DISCUSSION Mannitol production from fructose using biological systems has been described in the literature. Though a number of studies have focused on the use of lactic acid bacteria for mannitol production (26), glucose has to be coutilized with fructose for production of NADH (27), leading to reduced mannitol yields on the basis of total sugar consumed. The industrial workhorse E. coli has been reported to produce mannitol from fructose with a yield of 84 mol% (14). However, the construct was not stable over a long term, possibly due to cofactor loss, intracellular pH changes and accumulation of mannitol in the cells (28). Moreover, fructose being a costlier substrate compared to glucose, Kaup et al. (17) tried to enzymatically isomerize glucose to fructose using glucose isomerase, and also tried to express an isomerase to allow intracellular conversion of glucose to fructose. The latter approach led to lower yields (42 mol%), while the former process may not be feasible at large scales due to the cost of enzyme. In the present study, mannitol production from glucose was demonstrated using a recombinant E. coli strain that channels carbon from glucose to mannitol via the glycolytic intermediate
Glucose, Mannitol (mM)
60 50 40 30
Glc (mM) Man (mM)
20 10 0 0
4
8
12 Time (h)
16
20
24
FIG. 3. Whole-cell mediated biotransformation of glucose to mannitol using resting E. coli BL21(DE3) cells transformed with plasmid pSR-M3 (see Materials and methods for details). The time course of mannitol formation was determined by averaging values of two independent biotransformations.
3
fructose-6-phosphate (Fig. 2). Overexpressed mannitol-1-phosphate dehydrogenase catalyses the reduction of fructose-6-phosphate to mannitol-1-phosphate. Though the reverse reaction may be operative in the cell when E. coli utilizes mannitol as a carbon source, MtlD has a 20-fold higher affinity for NADH than for NADþ (25). Dephosphorylation of mannitol-1-phosphate to mannitol by M1Pase completes the pathway. Expression of formate dehydrogenase (FDH) from the yeast Candida boidinii has been frequently used for NADH regeneration (29e32). FDH catalyses the NADþ-dependent oxidation of formate to carbon dioxide, and in the process regenerates NADH. The equimolar formation of sodium hydroxide during FDH-catalysed oxidation of sodium formate makes the medium alkaline, necessitating pH control (14). In the present work, a phosphite dehydrogenase from P. stutzeri was overexpressed for NADH cofactor regeneration. This enzyme catalyses the NADþ-dependent oxidation of phosphite to phosphate. Phosphate is an innocuous element present in biological systems, and along with providing a physiological environment, obviates the need for a buffer reaction system. Using resting cells expressing the synthetic mannitol pathway for whole-cell biotransformation of glucose to mannitol, a molar yield of 87% was achieved without the use of complex media components and elaborate process control mechanisms. The present study demonstrates the feasibility of this simple yet effective approach for bioproduction of industrially relevant metabolites from glucose, and may potentially be extended to other sugars derived from lignocellulosic agricultural residues. Increased yields and higher productivity may be achieved by deleting pathways for carbon dissimilation that compete with end-product biosynthesis and by using optimized high cell density biotransformation, approaches that we are actively pursuing. References 1. Soetaert, W., Buchholz, K., and Vandamme, E. J.: Production of D-mannitol and D-lactic acid by fermentation with Leuconostoc mesenteroides, Agro Food Ind. Hi-Tech., 6, 41e44 (1995). 2. Wisselink, H. W., Weusthuis, R. A., Eggink, G., Hugenholtz, J., and Grobben, G. J.: Mannitol production by lactic acid bacteria: a review, Int. Dairy J., 12, 151e161 (2002). 3. Saha, B. C. and Racine, F. M.: Biotechnological production of mannitol and its applications, Appl. Microbiol. Biotechnol., 89, 879e891 (2011). 4. Soetaert, W.: Production of mannitol with Leuconostoc mesenteroides, Med. Fac. Landbouww. Rijksuniv. Gent, 55, 1549e1552 (1990). 5. Makkee, M., Kieboom, A. P. G., and Van Bekkum, H.: Production methods of D-mannitol, Starch/Stärke, 37, 136e141 (1985). 6. Soetaert, W., Vanhooren, P. T., and Vandamme, E. J.: The production of mannitol by fermentation, pp. 261e275, in: Bucke, C. (Ed.), Methods in biotechnology, vol. 10. Humana Press, Totowa (1999). 7. Kiviharju, K. and Nyyssölä, A.: Contributions of biotechnology to the production of mannitol, Recent Pat. Biotechnol., 2, 73e78 (2008). 8. Erten, H.: Metabolism of fructose as an electron acceptor by Leuconostoc mesenteroides, Process Biochem., 33, 735e739 (1998). 9. Gaspar, P., Neves, A. R., Ramos, A., Gasson, M. J., Shearman, C. A., and Santos, H.: Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system, Appl. Environ. Microbiol., 70, 1466e1474 (2004). 10. Saha, B. C.: A low-cost medium for mannitol production by Lactobacillus intermedius NRRL B-3693, Appl. Microbiol. Biotechnol., 72, 676e680 (2006). 11. Saha, B. C. and Racine, F. M.: Effects of pH and corn steep liquor variability on mannitol production by Lactobacillus intermedius NRRL B-3693, Appl. Microbiol. Biotechnol., 87, 553e560 (2010). 12. von Weymarn, F. N., Kiviharju, K. J., Jääskeläinen, S. T., and Leisola, M. S.: Scale-up of a new bacterial mannitol production process, Biotechnol. Prog., 19, 815e821 (2003). 13. Jacobsen, J. H. and Frigaard, N. U.: Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium, Metab. Eng., 21, 60e70 (2014). 14. Kaup, B., Bringer-Meyer, S., and Sahm, H.: Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a wholecell biotransformation, Appl. Microbiol. Biotechnol., 64, 333e339 (2004). 15. Bäumchen, C. and Bringer-Meyer, S.: Expression of glfZ.m. increases D-mannitol formation in whole cell biotransformation with resting cells of Corynebacterium glutamicum, Appl. Microbiol. Biotechnol., 76, 545e552 (2007).
Please cite this article in press as: Reshamwala, S. M. S., et al., Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.004
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16. Bäumchen, C., Roth, A. H., Biedendieck, R., Malten, M., Follmann, M., Sahm, H., Bringer-Meyer, S., and Jahn, D.: D-Mannitol production by resting state whole cell biotransformation of D-fructose by heterologous mannitol and formate dehydrogenase gene expression in Bacillus megaterium, Biotechnol. J., 2, 1408e1416 (2007). 17. Kaup, B., Bringer-Meyer, S., and Sahm, H.: D-mannitol formation from Dglucose in a whole-cell biotransformation with recombinant Escherichia coli, Appl. Microbiol. Biotechnol., 69, 397e403 (2005). 18. Wovcha, M. G., Steuerwald, D. L., and Brooks, K. E.: Amplification of D-xylose and D-glucose isomerase activities in Escherichia coli by gene cloning, Appl. Environ. Microbiol., 45, 1402e1404 (1983). 19. Hanahan, D.: Studies on the transformation of Escherichia coli with plasmids, J. Mol. Biol., 166, 557e580 (1983). 20. Studier, F. W. and Moffatt, B. A.: Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol., 189, 113e130 (1986). 21. Sambrook, J. and Russell, D. W.: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001). 22. Liberator, P., Anderson, J., Feiglin, M., Sardana, M., Griffin, P., Schmatz, D., and Myers, R. W.: Molecular cloning and functional expression of mannitol-1phosphatase from the apicomplexan parasite Eimeria tenella, J. Biol. Chem., 273, 4237e4244 (1998). 23. Costas, A. M., White, A. K., and Metcalf, W. W.: Purification and characterization of a novel phosphorus-oxidizing enzyme from Pseudomonas stutzeri WM88, J. Biol. Chem., 276, 17429e17436 (2001). 24. Kitagawa, M., Ara, T., Arifuzzaman, M., Ioka-Nakamichi, T., Inamoto, E., Toyonaga, H., and Mori, H.: Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research, DNA Res., 12, 291e299 (2005).
J. BIOSCI. BIOENG., 25. Teschner, W., Serre, M. C., and Garel, J. R.: Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli, Biochimie, 72, 33e40 (1990). 26. Akinterinwa, O., Khankal, R., and Cirino, P. C.: Metabolic engineering for bioproduction of sugar alcohols, Curr. Opin. Biotechnol., 19, 461e467 (2008). 27. von Weymarn, N., Hujanen, M., and Leisola, M.: Production of D-mannitol by heterofermentative lactic acid bacteria, Process Biochem., 37, 1207e1213 (2002). 28. Heuser, F., Marin, K., Kaup, B., Bringer, S., and Sahm, H.: Improving Dmannitol productivity of Escherichia coli: impact of NAD, CO2 and expression of a putative sugar permease from Leuconostoc pseudomesenteroides, Metab. Eng., 11, 178e183 (2009). 29. Berríos-Rivera, S. J., Bennett, G. N., and San, K. Y.: Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NADþ-dependent formate dehydrogenase, Metab. Eng., 4, 217e229 (2002). 30. Kratzer, R., Pukl, M., Egger, S., and Nidetzky, B.: Whole-cell bioreduction of aromatic alpha-keto esters using Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase co-expressed in Escherichia coli, Microb. Cell Fact., 7, 37 (2008). 31. Mädje, K., Schmölzer, K., Nidetzky, B., and Kratzer, R.: Host cell and expression engineering for development of an E. coli ketoreductase catalyst: enhancement of formate dehydrogenase activity for regeneration of NADH, Microb. Cell Fact., 11, 7 (2012). 32. Zhang, Y., Huang, Z., Du, C., Li, Y., and Cao, Z.: Introduction of an NADH regeneration system into Klebsiella oxytoca leads to an enhanced oxidative and reductive metabolism of glycerol, Metab. Eng., 11, 101e106 (2009).
Please cite this article in press as: Reshamwala, S. M. S., et al., Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.004
Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production Shamlan M.S. Reshamwala,1, * Sandip K. Pagar,1 Vishal S. Velhal,1 Vijay M. Maranholakar,1 Vishal G. Talangkar,1 and Arvind M. Lali1, 2 DBT-ICT-Centre for Energy Biosciences, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai 400019, Maharashtra, India1 and Department of Chemical Engineering, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (East), Mumbai 400019, Maharashtra, India2 Received 28 February 2014; accepted 4 May 2014 Available online xxx
Mannitol is a six carbon sugar alcohol that finds applications in the pharmaceutical and food industries. A novel Escherichia coli strain capable of converting D-glucose to D-mannitol has been constructed, wherein native mannitol-1-phosphate dehydrogenase (MtlD) and codon-optimized Eimeria tenella mannitol-1-phosphatase (M1Pase) have been overexpressed. Codon-optimized Pseudomonas stutzeri phosphite dehydrogenase (PtxD) was overexpressed for cofactor (NADH) regeneration with the concomitant oxidation of phosphite to phosphate. Whole-cell biotransformation using resting cells in a medium containing D-glucose and equimolar sodium phosphite resulted in D-mannitol yield of 87 mol%. Thus, production of an industrially relevant biochemical without using complex media components and elaborate process control mechanisms has been demonstrated. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: D-Mannitol production; Whole-cell biocatalyst; Cofactor regeneration; Biotransformation; Resting cells]
Mannitol is a six carbon sugar alcohol that is used in the pharmaceutical, food, medicine and chemical industries (1,2). The global market for mannitol is estimated to be 13.6 million kg year1 (3). The exudate of the manna tree, Fraxinus ornus, was the commercial source of mannitol till the 1920s (4). Currently, mannitol is produced at an industrial scale by catalytic reduction of a mixture of glucose and fructose, leading to the formation of mannitol and sorbitol, which are then separated by selective crystallization (5,6). Low yields and cost associated with the chemical production of mannitol has generated interest in possible selective fermentative production of mannitol from glucose or fructose. Lactic acid bacteria, yeasts and fungi known to naturally produce mannitol have been used and manipulated to achieve high titres of mannitol (3,7). However, many of the fermentative processes developed and reported lead to co-production of byproducts like acetic acid and lactic acid (8,9) and require complex media components (10,11). Using Leuconostoc mesenteroides, yields upto 93e97% from fructose have been reported at a 100 L pilot scale (12). The process, however, requires a significant amount of glucose to be co-fed, leading to a mannitol yield of 61e62% on the basis of sugar consumed (von Weymarn, N., Ph.D. thesis, Helsinki University of Technology, Helsinki, 2002). Recently, the marine cyanobacterium Synechococcus sp. PCC 7002 has been genetically modified to photosynthetically produce mannitol from carbon dioxide as the sole carbon source (13). The * Corresponding author. Tel.: þ91 22 33611111, þ91 22 33612222; fax: þ91 22 33611020. E-mail address: [email protected] (S.M.S. Reshamwala).
highest productivity was obtained using a glycogen synthasedeficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol l1 and a production rate of 0.15 g mannitol l1 day1. Metabolic engineering of Escherichia coli has been reported to convert fructose to mannitol. Kaup et al. (14) expressed the Leuconostoc pseudomesenteroides mdh gene, coding for mannitol dehydrogenase, to reduce fructose to mannitol. The glucose facilitator protein (GLF) of Zymomonas mobilis was expressed to allow fructose to be taken up by the cells without simultaneous phosphorylation, and the fdh gene from Mycobacterium vaccae N10, coding for formate dehydrogenase, was co-expressed to provide a source of reducing equivalents. The authors obtained a yield of 84 mol% by carrying out biotransformations conducted under pH control by formic acid addition (14). Similar work has been carried out using Corynebacterium glutamicum (15) and Bacillus megaterium (16). To allow production of mannitol using glucose as substrate, Kaup et al. (17) supplemented recombinant E. coli expressing mdh, glf and fdh with extracellular glucose isomerase, leading to a yield of 80% on glucose. On the other hand, expression of xylA, coding for xylose isomerase, which can convert glucose to fructose under nonphysiological conditions (18), resulted in a mannitol yield of 42% from glucose (17). The present report describes our attempt at construction of a recombinant E. coli strain that can efficiently convert glucose to mannitol without addition of extracellular enzymes. This has been achieved by diverting carbon flux from fructose-6-phosphate towards mannitol biosynthesis, resulting in a mannitol yield of 87 mol% from glucose.
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.05.004
Please cite this article in press as: Reshamwala, S. M. S., et al., Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.004
2
RESHAMWALA ET AL.
J. BIOSCI. BIOENG.,
TABLE 1. Plasmids used in this study. Plasmid
Size (kb)
Plasmid
Description
pSR-M1
w4.8
pSR-M1
pSR-M2
w5.9
pSR-M2
pSR-M3
w6.9
pSR-M3
Codon-optimized E. tenella M1Pase CDS cloned under T5 promoter in plasmid pJexpress401; KanR E. coli mtlD cloned in plasmid pSR-M1 downstream of M1Pase Codon-optimized P. stutzeri ptxD cloned in plasmid pSR-M2 downstream of mtlD
MATERIALS AND METHODS Bacterial strains and growth conditions E. coli DH5a (19) was used for cloning and E. coli BL21(DE3) (20) for gene expression and whole-cell biotransformation. Bacteria were routinely cultivated in Luria-Bertani medium (Himedia, Mumbai, India) or M9 minimal medium supplemented with 1% glucose at 37 C. Plasmids used in this work are listed in Table 1. When required, kanamycin and ampicillin were used at a concentration of 50 mg ml1 and 100 mg ml1, respectively. For induction of gene expression, isopropyl-b-D-1thiogalactopyranoside (IPTG) was added to achieve a final concentration of 1 mM. Growth was monitored by measuring optical density at 600 nm (OD600). Cloning and plasmid construction All DNA techniques were carried out using standard methods (21). Codon-optimization and synthesis of genes were outsourced to DNA2.0 (Menlo Park, CA, USA). The codon-optimized Eimeria tenella mannitol-1-phosphatase (M1Pase) coding sequence (22) was synthesized and cloned in plasmid pJexpress401 by DNA2.0 to obtain plasmid pSR-M1. The E. coli mannitol-1-phosphate dehydrogenase gene mtlD was amplified by polymerase chain reaction from E. coli MG1655 genomic DNA using primers mtlD-Fw (50 -GGGAATTCCATATGAAAGCATTACATTTTGGC) and mtlD-Rv (50 -CCCAAGCTTTTATTGCATTGCTTTATAAGCG) and cloned between the NdeI and XhoI restriction sites of plasmid pET43.1b. The mtlD sequence was verified by sequencing (Scigenom Labs, Kerala, India) and sub-cloned into the blunted XhoI recognition site w10 bp downstream of the M1Pase coding sequence in plasmid pSR-M1 to obtain plasmid pSR-M2. The codon-optimized Pseudomonas stutzeri phosphite dehydrogenase gene ptxD (23), synthesized and cloned in plasmid pJexpress404 by DNA2.0, was excised along with the ribosome binding sequence by digesting pJexpress404 with XbaI and XhoI, blunted and cloned into the SmaI recognition site w35 bp downstream of the mtlD gene in plasmid pSR-M2 to obtain plasmid pSR-M3. All constructs were verified for correct orientation of the genes by restriction digestion. The synthetic mannitol operon thus constructed is depicted in Fig. 1. Mannitol production from glucose To assess mannitol production, E. coli BL21(DE3) transformed with plasmids pSR-M1, pSR-M2 or pSR-M3 was grown in M9 medium supplemented with 55 mM glucose at 37 C in a rotary shaker set at 180 rpm. IPTG was added when the culture reached a cell density of OD600 0.5, and incubation was continued for a further 18 h under the same conditions. When required, sodium phosphite was added at the time of IPTG addition. Whole-cell mediated biotransformation was carried out using cultures grown overnight in M9 medium supplemented with 55 mM glucose and diluted 4:100 in the same medium and grown till cell density of OD600 0.5 was attained. IPTG was then added and the culture allowed to grow for a further 12 h, after which the cells were pelleted, washed with M9 salts and resuspended in 55 mM glucose or 55 mM glucose supplemented with equimolar concentration of sodium phosphite. The resuspended cells were incubated at 37 C in a rotary shaker set at 180 rpm and samples drawn periodically to quantify metabolites. HPLC analysis Cells were pelleted by centrifugation at 10,000 g for 10 min and the supernatant was stored at 20 C till analysis. Glucose and mannitol in the supernatant were quantified using a Bio-Rad Aminex HPX-87H column maintained at 50 C coupled to an RI detector. The eluent was 5 mM sulphuric acid, and flow was 0.6 ml min1.
RESULTS Assembling the mannitol biosynthesis pathway in E. coli A synthetic pathway was assembled in E. coli to divert carbon flow
T5 promoter
lac O
RBS
M1Pase
FIG. 2. Synthetic pathway cloned in E. coli leading to mannitol production from glucose. Bold arrows indicate overexpression.
towards mannitol production, comprising of two enzymes, the native mannitol-1-phosphate dehydrogenase (MtlD) and a mannitol-1-phosphatase (M1Pase) from the protozoan parasite E. tenella (22). MtlD reduces fructose-6-phosphate to mannitol-1phosphate, while M1Pase dephosphorylates mannitol-1phosphate to mannitol, which is transported across the membrane and released into the medium (Fig. 2). E. coli can metabolize mannitol as a source of carbon, but cannot biosynthesize this sugar alcohol as it lacks mannitol-1-phosphatase. Expression of MtlD in E. coli leads to toxicity (24), which may be because of the accumulation of mannitol-1-phosphate, the product of the reaction catalysed by mannitol-1-phosphate dehydrogenase. Therefore, this enzyme was expressed by cloning it in plasmid pSRM1 downstream of M1Pase, leading to the construction of plasmid pSR-M2, ensuring that mannitol-1-phosphate is readily converted to mannitol by the action of mannitol-1-phosphatase. Reduction of fructose-6-phosphate is accompanied by the oxidation of NADH (25). In order to regenerate NADH, PtxD, a phosphite dehydrogenase from P. stutzeri, was selected for its ability to catalyse the nearly irreversible oxidation of phosphite to phosphate, with the concomitant reduction of NADþ to NADH (23). ptxD was cloned downstream of the mtlD gene in plasmid pSR-M2 to obtain plasmid pSR-M3. All genes in pSR-M3 are transcribed from a single T5 promoter to form the synthetic mannitol biosynthesis operon (Fig. 1). Mannitol production by recombinant E. coli Mannitol production by E. coli harbouring the pSR-M1, pSR-M2 and pSR-M3 plasmids was quantified by growing the cells in M9 minimal medium supplemented with glucose. Expression of M1Pase alone led to mannitol production of 21 mol%, indicating that the level of MtlD in the cell was enough to divert carbon flow towards mannitol. Overexpression of MtlD along with M1Pase increased mannitol production to 28 mol% (Table 2). Regeneration of NADH facilitated by overexpression of PtxD along with M1Pase and MtlD resulted in an increase in mannitol production to 43 mol% in the presence of equimolar phosphite (Table 2). This demonstrated that carbon flux towards mannitol
mtlD
RBS
ptxD
term
FIG. 1. Schematic diagram of the synthetic mannitol operon cloned in plasmid pSR-M3. M1Pase, codon-optimized E. tenella mannitol-1-phosphatase; mtlD, E. coli mannitol-1-phosphate dehydrogenase; ptxD, codon-optimized P. stutzeri phosphite dehydrogenase.
Please cite this article in press as: Reshamwala, S. M. S., et al., Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.004
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TABLE 2. Mannitol formation by E. coli transformed with different plasmids. Strain
M9 medium supplemented with
BL21(DE3) BL21(DE3)/pSR-M1 BL21(DE3)/pSR-M2 BL21(DE3)/pSR-M3 BL21(DE3)/pSR-M3 BL21(DE3)/pSR-M3
Mannitol yield (mol%)
55 mM glucose 55 mM glucose 55 mM glucose 55 mM glucose 55 mM glucose þ 28 mM sodium phosphite 55 mM glucose þ 55 mM sodium phosphite
e 21.8 28.83 30.2 35.25 43.74
3.42 1.93 0.62 2.42 1.96
biosynthesis could indeed be efficiently diverted by providing a mechanism for NADH regeneration. Whole-cell biotransformation for mannitol biosynthesis from glucose To increase the yield of mannitol, a whole-cell biotransformation approach was adopted. E. coli BL21(DE3) transformed with plasmid pSR-M3 was induced to produce the enzymes of the synthetic mannitol pathway, and w25 g l1 cells (wet cell weight basis) were suspended in an aqueous solution of 55 mM glucose and equimolar concentration of phosphite. The yield of mannitol obtained using this approach was 87 mol% (Fig. 3). As no other metabolites were detected in the supernatant, we conclude that E. coli BL21(DE3)/pSR-M3 is an efficient biocatalyst for production of mannitol from glucose. DISCUSSION Mannitol production from fructose using biological systems has been described in the literature. Though a number of studies have focused on the use of lactic acid bacteria for mannitol production (26), glucose has to be coutilized with fructose for production of NADH (27), leading to reduced mannitol yields on the basis of total sugar consumed. The industrial workhorse E. coli has been reported to produce mannitol from fructose with a yield of 84 mol% (14). However, the construct was not stable over a long term, possibly due to cofactor loss, intracellular pH changes and accumulation of mannitol in the cells (28). Moreover, fructose being a costlier substrate compared to glucose, Kaup et al. (17) tried to enzymatically isomerize glucose to fructose using glucose isomerase, and also tried to express an isomerase to allow intracellular conversion of glucose to fructose. The latter approach led to lower yields (42 mol%), while the former process may not be feasible at large scales due to the cost of enzyme. In the present study, mannitol production from glucose was demonstrated using a recombinant E. coli strain that channels carbon from glucose to mannitol via the glycolytic intermediate
Glucose, Mannitol (mM)
60 50 40 30
Glc (mM) Man (mM)
20 10 0 0
4
8
12 Time (h)
16
20
24
FIG. 3. Whole-cell mediated biotransformation of glucose to mannitol using resting E. coli BL21(DE3) cells transformed with plasmid pSR-M3 (see Materials and methods for details). The time course of mannitol formation was determined by averaging values of two independent biotransformations.
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fructose-6-phosphate (Fig. 2). Overexpressed mannitol-1-phosphate dehydrogenase catalyses the reduction of fructose-6-phosphate to mannitol-1-phosphate. Though the reverse reaction may be operative in the cell when E. coli utilizes mannitol as a carbon source, MtlD has a 20-fold higher affinity for NADH than for NADþ (25). Dephosphorylation of mannitol-1-phosphate to mannitol by M1Pase completes the pathway. Expression of formate dehydrogenase (FDH) from the yeast Candida boidinii has been frequently used for NADH regeneration (29e32). FDH catalyses the NADþ-dependent oxidation of formate to carbon dioxide, and in the process regenerates NADH. The equimolar formation of sodium hydroxide during FDH-catalysed oxidation of sodium formate makes the medium alkaline, necessitating pH control (14). In the present work, a phosphite dehydrogenase from P. stutzeri was overexpressed for NADH cofactor regeneration. This enzyme catalyses the NADþ-dependent oxidation of phosphite to phosphate. Phosphate is an innocuous element present in biological systems, and along with providing a physiological environment, obviates the need for a buffer reaction system. Using resting cells expressing the synthetic mannitol pathway for whole-cell biotransformation of glucose to mannitol, a molar yield of 87% was achieved without the use of complex media components and elaborate process control mechanisms. The present study demonstrates the feasibility of this simple yet effective approach for bioproduction of industrially relevant metabolites from glucose, and may potentially be extended to other sugars derived from lignocellulosic agricultural residues. Increased yields and higher productivity may be achieved by deleting pathways for carbon dissimilation that compete with end-product biosynthesis and by using optimized high cell density biotransformation, approaches that we are actively pursuing. References 1. Soetaert, W., Buchholz, K., and Vandamme, E. J.: Production of D-mannitol and D-lactic acid by fermentation with Leuconostoc mesenteroides, Agro Food Ind. Hi-Tech., 6, 41e44 (1995). 2. Wisselink, H. W., Weusthuis, R. A., Eggink, G., Hugenholtz, J., and Grobben, G. J.: Mannitol production by lactic acid bacteria: a review, Int. Dairy J., 12, 151e161 (2002). 3. Saha, B. C. and Racine, F. M.: Biotechnological production of mannitol and its applications, Appl. Microbiol. Biotechnol., 89, 879e891 (2011). 4. Soetaert, W.: Production of mannitol with Leuconostoc mesenteroides, Med. Fac. Landbouww. Rijksuniv. Gent, 55, 1549e1552 (1990). 5. Makkee, M., Kieboom, A. P. G., and Van Bekkum, H.: Production methods of D-mannitol, Starch/Stärke, 37, 136e141 (1985). 6. Soetaert, W., Vanhooren, P. T., and Vandamme, E. J.: The production of mannitol by fermentation, pp. 261e275, in: Bucke, C. (Ed.), Methods in biotechnology, vol. 10. Humana Press, Totowa (1999). 7. Kiviharju, K. and Nyyssölä, A.: Contributions of biotechnology to the production of mannitol, Recent Pat. Biotechnol., 2, 73e78 (2008). 8. Erten, H.: Metabolism of fructose as an electron acceptor by Leuconostoc mesenteroides, Process Biochem., 33, 735e739 (1998). 9. Gaspar, P., Neves, A. R., Ramos, A., Gasson, M. J., Shearman, C. A., and Santos, H.: Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system, Appl. Environ. Microbiol., 70, 1466e1474 (2004). 10. Saha, B. C.: A low-cost medium for mannitol production by Lactobacillus intermedius NRRL B-3693, Appl. Microbiol. Biotechnol., 72, 676e680 (2006). 11. Saha, B. C. and Racine, F. M.: Effects of pH and corn steep liquor variability on mannitol production by Lactobacillus intermedius NRRL B-3693, Appl. Microbiol. Biotechnol., 87, 553e560 (2010). 12. von Weymarn, F. N., Kiviharju, K. J., Jääskeläinen, S. T., and Leisola, M. S.: Scale-up of a new bacterial mannitol production process, Biotechnol. Prog., 19, 815e821 (2003). 13. Jacobsen, J. H. and Frigaard, N. U.: Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium, Metab. Eng., 21, 60e70 (2014). 14. Kaup, B., Bringer-Meyer, S., and Sahm, H.: Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a wholecell biotransformation, Appl. Microbiol. Biotechnol., 64, 333e339 (2004). 15. Bäumchen, C. and Bringer-Meyer, S.: Expression of glfZ.m. increases D-mannitol formation in whole cell biotransformation with resting cells of Corynebacterium glutamicum, Appl. Microbiol. Biotechnol., 76, 545e552 (2007).
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Please cite this article in press as: Reshamwala, S. M. S., et al., Construction of an efficient Escherichia coli whole-cell biocatalyst for D-mannitol production, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.05.004