Abstract
The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).
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References
Alexandrov A, Vignali M, LaCount DJ, Quartley E, de Vries C, De Rosa D, Babulski J, Mitchell SF, Schoenfeld LW, Fields S (2004) A facile method for high-throughput co-expression of protein pairs. Mol Cell Proteomics 3(9):934–938. doi:10.1074/mcp.T400008-MCP200
Burda E, Hummel W, Gröger H (2008) Modular chemoenzymatic one-pot syntheses in aqueous media: combination of a palladium-catalyzed cross-coupling with an asymmetric biotransformation. Angew Chem Int Ed 47(49):9551–9554. doi:10.1002/anie.200801341
Chandrayan SK, Wu C-H, McTernan PM, Adams MW (2015) High yield purification of a tagged cytoplasmic [NiFe]-hydrogenase and a catalytically-active nickel-free intermediate form. Protein Expr Purif 107:90–94. doi:10.1016/j.pep.2014.10.018
Curran KA, Karim AS, Gupta A, Alper HS (2013) Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metab Eng 19:88–97. doi:10.1016/j.ymben.2013.07.001
Fernández-Lucas J (2015) Multienzymatic synthesis of nucleic acid derivatives: a general perspective. Appl Microbiol Biotechnol 99(11):4615–4627. doi:10.1007/s00253-015-6642-x
Fisch KM (2013) Biosynthesis of natural products by microbial iterative hybrid PKS–NRPS. RSC Adv 3(40):18228–18247. doi:10.1039/C3RA42661K
Glanemann C, Loos A, Gorret N, Willis L, O’Brien X, Lessard P, Sinskey A (2003) Disparity between changes in mRNA abundance and enzyme activity in Corynebacterium glutamicum: implications for DNA microarray analysis. Appl Microbiol Biotechnol 61(1):61–68 doi:10.1007/s00253-002-1191-5
Higgins MK, Demir M, Tate CG (2003) Calnexin co-expression and the use of weaker promoters increase the expression of correctly assembled shaker potassium channel in insect cells. Biochim Biophys Acta Biomem 1610(1):124–132. doi:10.1016/S0005-2736(02)00715-0
Jiang W, Fang B (2016) Construction of a tunable multi-enzyme-coordinate expression system for biosynthesis of chiral drug intermediates. Sci Rep 6:30462. doi:10.1038/srep30462
Johnston K, Clements A, Venkataramani RN, Trievel RC, Marmorstein R (2000) Coexpression of proteins in bacteria using T7-based expression plasmids: expression of heteromeric cell-cycle and transcriptional regulatory complexes. Protein Expr Purif 20(3):435–443. doi:10.1006/prep.2000.1313
Jones KL, Kim S-W, Keasling J (2000) Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria. Metab Eng 2(4):328–338. doi:10.1006/mben.2000.0161
Kealey JT, Liu L, Santi DV, Betlach MC, Barr PJ (1998) Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl Acad Sci U S A 95(2):505–509. doi:10.1006/prep.2000.1313
Kim KJ, Kim HE, Lee KH, Han W, Yi MJ, Jeong J, Oh BH (2004) Two-promoter vector is highly efficient for overproduction of protein complexes. Protein Sci 13(6):1698–1703. doi:10.1110/ps.04644504
Kim S, Lee SB (2006) Rare codon clusters at 5′-end influence heterologous expression of archaeal gene in Escherichia coli. Protein Expr Purif 50(1):49–57. doi:10.1016/j.pep.2006.07.014
Lopez-Gallego F, Schmidt-Dannert C (2010) Multi-enzymatic synthesis. Curr Opin Chem Biol 14(2):174–183. doi:10.1016/j.cbpa.2009.11.023
Lu W, Ye L, Xu H, Xie W, Gu J, Yu H (2014) Enhanced production of coenzyme Q10 by self-regulating the engineered MEP pathway in Rhodobacter sphaeroides. Biotechnol Bioeng 111(4):761–769. doi:10.1002/bit.25130
Lv X, Xu H, Yu H (2013) Significantly enhanced production of isoprene by ordered coexpression of genes dxs, dxr, and idi in Escherichia coli. Appl Microbiol Biotechnol 97(6):2357–2365. doi:10.1007/s00253-012-4485-2
Makrides SC (1996) Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol Mol Biol Rev 60(3):512–538
Mantis J, Tague BW (2000) Comparing the utility of β-glucuronidase and green fluorescent protein for detection of weak promoter activity in Arabidopsis thaliana. Plant Mol Biol Rep 18(4):319–330. doi:10.1007/BF02825059
Myung S, Rollin J, You C, Sun F, Chandrayan S, Adams MW, Zhang Y-HP (2014) In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose. Metab Eng 24:70–77. doi:10.1016/j.ymben.2014.05.006
Naoumkina MA, Modolo LV, Huhman DV, Urbanczyk-Wochniak E, Tang Y, Sumner LW, Dixon RA (2010) Genomic and coexpression analyses predict multiple genes involved in triterpene saponin biosynthesis in Medicago truncatula. Plant Cell 22(3):850–866. doi:10.1105/tpc.109.073270
Neumann D, Woods A, Carling D, Wallimann T, Schlattner U (2003) Mammalian AMP-activated protein kinase: functional, heterotrimeric complexes by co-expression of subunits in Escherichia coli. Protein Expr Purif 30(2):230–237. doi:10.1016/S1046-5928(03)00126-8
Ninh PH, Honda K, Sakai T, Okano K, Ohtake H (2015) Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering. Biotechnol Bioeng 112(1):189–196. doi:10.1002/bit.25338
Nogueira T, Springer M (2000) Post-transcriptional control by global regulators of gene expression in bacteria. Curr Opin Microbiol 3(2):154–158. doi:10.1016/S1369-5274(00)00068-0
Opgenorth PH, Korman TP, Bowie JU (2016) A synthetic biochemistry module for production of bio-based chemicals from glucose. Nat Chem Biol:393–395 doi: 10.1038/nchembio.2062
Otte KB, Kittelberger J, Kirtz M, Nestl BM, Hauer B (2014) Whole-cell one-pot biosynthesis of azelaic acid. ChemCatChem 6(4):1003–1009. doi:10.1002/cctc.201300787
Rollin JA, Tam TK, Zhang Y-HP (2013) New biotechnology paradigm: cell-free biosystems for biomanufacturing. Green Chem 15(7):1708–1719. doi:10.1039/C3GC40625C
Romier C, Ben Jelloul M, Albeck S, Buchwald G, Busso D, Celie PH, Christodoulou E, De Marco V, Van Gerwen S, Knipscheer P (2006) Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies. Acta Crystallogr, Sect D: Biol Crystallogr 62(10):1232–1242. doi:10.1107/S0907444906031003
Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27(10):946–950. doi:10.1038/nbt.1568
Santacoloma PA, Gr S, Gernaey KV, Woodley JM (2010) Multienzyme-catalyzed processes: next-generation biocatalysis. Org Proc Res Dev 15(1):203–212. doi:10.1021/op1002159
Sørensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115(2):113–128. doi:10.1016/j.jbiotec.2004.08.004
Sun J, Hopkins RC, Jenney FE Jr, McTernan PM, Adams MW (2010) Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production. PLoS One 5(5):e10526. doi:10.1371/journal.pone.0010526
Tan S (2001) A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli. Protein Expr Purif 21(1):224–234. doi:10.1006/prep.2000.1363
Taniguchi Y, Choi PJ, Li G-W, Chen H, Babu M, Hearn J, Emili A, Xie XS (2010) Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329(5991):533–538. doi:10.1126/science.1188308
Tolia NH, Joshua-Tor L (2006) Strategies for protein coexpression in Escherichia coli. Nat Meth 3(1):55–64. doi:10.1038/nmeth0106-55
Vind J, Sørensen MA, Rasmussen MD, Pedersen S (1993) Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes: expression from reporter genes does not always reflect functional mRNA levels. J Mol Biol 231(3):678–688. doi:10.1006/jmbi.1993.1319
Wakagi T, Oshima T, Imamura H, Matsuzawa H (1998) Cloning of the gene for inorganic pyrophosphatase from a thermoacidophilic archaeon, Sulfolobus sp. strain 7, and overproduction of the enzyme by coexpression of tRNA for arginine rare codon. Biosci Biotechnol Biochem 62(12):2408–2414. doi:10.1271/bbb.62.2408
Wang Y, Huang W, Sathitsuksanoh N, Zhu Z, Zhang Y-HP (2011) Biohydrogenation from biomass sugar mediated by in vitro synthetic enzymatic pathways. Chem Biol 18(3):372–380. doi:10.1016/j.chembiol.2010.12.019
Wenzel SC, Gross F, Zhang Y, Fu J, Stewart AF, Müller R (2005) Heterologous expression of a myxobacterial natural products assembly line in Pseudomonads via red/ET recombineering. Chem Biol 12(3):349–356. doi:10.1016/j.chembiol.2004.12.012
Woodyer R, Simurdiak M, van der Donk WA, Zhao H (2005) Heterologous expression, purification, and characterization of a highly active xylose reductase from Neurospora crassa. Appl Environ Microbiol 71(3):1642–1647. doi:10.1128/AEM.71.3.1642-1647.2005
Wu S, Chen Y, Xu Y, Li A, Xu Q, Glieder A, Li Z (2014) Enantioselective trans-dihydroxylation of aryl olefins by cascade biocatalysis with recombinant Escherichia coli coexpressing monooxygenase and epoxide hydrolase. ACS Catal 4(2):409–420. doi:10.1021/cs400992z
You C, Zhang X-Z, Zhang Y-HP (2012) Simple cloning via direct transformation of PCR product (DNA multimer) to Escherichia coli and Bacillus subtilis. Appl Environ Microbiol 78(5):1593–1595. doi:10.1128/AEM.07105-11
Zha W, Rubin-Pitel SB, Shao Z, Zhao H (2009) Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering. Metab Eng 11(3):192–198. doi:10.1016/j.ymben.2009.01.005
Zhang Y-HP (2015) Production of biofuels and biochemicals by in vitro synthetic biosystems: opportunities and challenges. Biotechnol Adv 33(7):1467–1483. doi:10.1016/j.biotechadv.2014.10.009
Zhang Y-HP, Lynd LR (2005) Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum. J Bacteriol 187(1):99–106. doi:10.1128/JB.187.1.99-106.2005
Zhong C, Wei P, Zhang Y-HP (2016) Enhancing functional expression of codon-optimized heterologous enzymes in Escherichia coli BL21(DE3) by selective introduction of synonymous rare codons. Biotechnol Bioeng. doi:10.1002/bit.26238
Zhou W, You C, Ma H, Ma Y, Zhang Y-HP (2016) One-pot biosynthesis of high-concentration α-glucose 1-phosphate from starch by sequential addition of three hyperthermophilic enzymes. J Agr Food Chem 64:1777–1783. doi:10.1021/acs.jafc.5b05648
Zhu Y, Li H, Liu P, Yang J, Zhang X, Sun Y (2015) Construction of allitol synthesis pathway by multi-enzyme coexpression in Escherichia coli and its application in allitol production. J Ind Microbiol Biotechnol 42(5):661–669. doi:10.1007/s10295-014-1578-1
Zhu Z, Sun F, Zhang X, Zhang Y-HP (2012) Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenase. Biosens Bioelectron 36(1):110–115. doi:10.1016/j.bios.2012.04.001
Zhu Z, Tam TK, Sun F, You C, Zhang Y-HP (2014) A high-energy-density sugar biobattery based on a synthetic enzymatic pathway. Nat Commun 5:3026. doi:10.1038/ncomms4026
Acknowledgements
This project was carried out with the support of the Biological System Engineering Department, Virginia Polytechnic Institute and State University, Virginia. It was mainly funded by the DOE EERE award (DE-EE0006968) and partially supported by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, US Department of Agriculture.
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Chen, H., Huang, R. & Zhang, YH.P. Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3). Appl Microbiol Biotechnol 101, 4481–4493 (2017). https://doi.org/10.1007/s00253-017-8206-8
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DOI: https://doi.org/10.1007/s00253-017-8206-8