Abstract
The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200 U l-1 ADH and 370 U l-1 G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700 U l-1 ADH and 170 U l-1 GDH activity. However, the crude extract of both strains reduced 40 mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99 % in 60 min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH–LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP+, makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74 g l−1 day−1 or 406 g kg−1 dry cell weight (dcw) day−1 isolated 1-(R)-phenylethanol with an ee of 100 % and a TTN of 206 mol product per mole NADP+. In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols.
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References
Alvaro-Benito M, Fernández-Lobato M, Baronian K, Kunze G (2012) Assessment of Schwanniomyces occidentalis as a host for protein production using the wide-range Xplor2 expression platform. Appl Microbiol Biotechnol 97:4443–4456
Anwar A, Qader SAU, Raiz A, Iqbal S, Azhar A (2009) Calcium alginate: a support material for immobilization of proteases from newly isolated strain of Bacillus subtilis KIBGE-HAS. Appl Sci J 7:1281–1286
Aragozzini F, Maconi E, Craveri R (1968) Stereoselective reduction of non-cyclic carbonyl compounds by some eumycetes. Appl Microbiol Biotechnol 24:175–177
Böer E, Bode R, Mock HP, Piontek M, Kunze G (2009a) Atan1p-an extracellular tannase from the dimorphic yeast Arxula adeninivorans: molecular cloning of the ATAN1 gene and characterization of the recombinant enzyme. Yeast 26:323–337
Böer E, Piontek M, Kunze G (2009b) Xplor 2 - an optimized transformation/expression system for recombinant protein production in the yeast Arxula adeninivorans. Appl Microbiol Biotechnol 84:583–594
Borzęcka W, Lavandera I, Gotor V (2013) Synthesis of enantiopure fluorohydrins using alcohol dehydrogenases at high substrate concentrations. J Org Chem 78:7312–7317
Chemler JA, Fowler ZL, McHugh KP, Koffas MAG (2010) Improving NADPH availability for natural product biosynthesis in Escherichia coli by metabolic engineering. Metab Eng 12:96–104
Cheng C, Ma JH (1996) Enantioselective synthesis of S-(−)-1-phenylethanol in Candida utilis semi-fed-batch cultures. Process Biochem 31:119–124
Csuk R, Glanzer B (1991) Baker’s yeast mediated transformations in organic chemistry. Chem Rev 91:49–97
Daußmann T, Rosen TC, Dünkelmann P (2006) Oxidoreductases and hydroxynitrilase lyases: complementary enzymatic technologies for chiral alcohols. Eng Life Sci 6:125–129
Giersberg M, Degelmann A, Bode R, Piontek M, Kunze G (2012) Production of a thermostable alcohol dehydrogenase from Rhodococcus ruber in three different yeast species using the Xplor®2 transformation/expression platform. J Ind Microbiol Biotechnol 39:1385–1396
Hildebrand F, Lütz S (2006) Immobilisation of alcohol dehydrogenase from Lactobacillus brevis and its application in a plug-flow reactor. Tetr Asym 17:3219–3225
Hildebrandt P, Musidlowska A, Bornscheuer UT, Altenbuchner J (2002) Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106. Appl Microbiol Biotechnol 59:483–487
Hummel W (1990) Reduction of acetophenone to R(+)-phenylethanol by a new alcohol dehydrogenase from Lactobacillus kefir. Appl Microbiol Biotechnol 34:15–19
Hummel W (1997) New alcohol dehydrogenases for the synthesis of chiral compounds. Adv Biochem Eng Biotechnol 58:145–184
Hummel W, Riebel B (1997) Alkohol-Dehydrogenase und deren Verwendung zur enzymatischen Herstellung chiraler Hydroxyverbindungen, EP 0796914 A2
Isotani K, Kurokawa J, Itoh N (2012) Production of (R)-3-quinuclidinol by E. coli biocatalysts possessing NADH-dependent 3-quinuclidinone reductase (QNR or bacC) from Microbacterium luteolum and Leifsonia alcohol dehydrogenase (LSADH). Int J Mol Sci 13:13542–13553
Jobanputra AH, Karode BA, Chincholkar SB (2011) Calcium alginate as supporting material for the immobilization of rifamycin oxidase from Chryseobacterium species. Biotechnol Bioinf Bioeng 4:529–535
Kizaki N, Yasohara Y, Hasegawa J, Wada M, Kataoka M, Shimizu S (2001) Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes. Appl Microbiol Biotechnol 55:590–595
Klabunde J, Kunze G, Gellissen G, Hollenberg CP (2003) Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element. FEMS Yeast Res 4:185–193
Kunze G, Kunze I (1994) Characterization of Arxula adeninivorans strains from different habitats. Antonie Van Leeuwenhoek 65:29–34
Leuchs S, Greiner L (2011) Alcohol dehydrogenase from Lactobacillus brevis: a versatile robust catalyst for enantioselective transformations. Chem Biochem Eng Q 25:267–281
Li L, Wang Y, Zhang L, Ma C, Wang A, Tao F, Xu P (2012) Biocatalytic production of (2S,3S)-2,3-butanediol from diacetyl using whole cells of engineered Escherichia coli. Bioresour Technol 115:111–116
Machielsen R, Looger LL, Raedts J, Dijkhuizen S, Hummel W, Hennemann HG, Daussmann T, van der Oost J (2009) Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design. Eng Life Sci 9:38–44
Meese CO (1986) (S)-(−)-und (R)-(+)-1-(Pentafluorphenyl)ethanol. Liebigs Ann Chem 1986:2004–2007
Meyer HP (2010) Sustainability and biotechnology. Organ Proc Res Develop 15:180–188
Miozzari GF, Niederberger P, Hütter R (1978) Permeabilisation of microorganisms by Triton X-100. Anal Biochem 90:220–233
Rauter M, Schwarz M, Becker K, Baronian K, Bode R, Kunze G, Vorbrodt H-M (2013) Synthesis of benzyl ß-D-galactopyranoside by transgalactosylation using a β-galactosidase produced by the over expression of the Kluyveromyces lactis LAC4 gene in Arxula adeninivorans. J Mol Catal B: Enzym 97:319–327
Rauter M, Kasprzak J, Becker K, Baronian K, Bode R, Kunze G, Vorbrodt HM (2014a) ADH from Rhodococcus ruber expressed in Arxula adeninivorans for the synthesis of 1-(S)-phenylethanol. J Mol Catal B: Enzym 104:8–16
Rauter M, Kasprzak J, Becker K, Baronian K, Bode R, Kunze G, Vorbrodt HM (2014b) Reusability of ADH and GDH producing Arxula adeninivorans cells and cell extract for the production of 1-(S)-phenylethanol. J Mol Catal B: Enzym 108:72–76
Riebel B (1996) Biochemische und molekularbiologische Charakterisierung neuer mikrobieller NAD(P)-abhängiger Alkoholdehydrogenasen. PhD thesis, Heinrich-Heine-University Düsseldorf, Germany
Rissom S, Beliczey J, Giffels G, Kragl U, Wandrey C (1999) Asymmetric reduction of acetophenone in membrane reactors: comparison of oxazaborolodine and alcohol dehydrogenase catalyzed processes. Tetrahedron Asymmet 10:923–928
Rose MD, Winston F, Hieter P (1990) Methods in yeast genetics. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Rosen TC, Daußmann T, Stohrer J (2004) Bioreduction forms optically active 3-hydroxyesters. Spec Chem 39–40
Steinborn G, Gellissen G, Kunze G (2007) A novel vector element providing multicopy vector integration in Arxula adeninivorans. FEMS Yeast Res 7:1197–1205
Talekar S, Chavare S (2012) Optimization of immobilization of α-amylase in alginate gel and its comparative biochemical studies with free α-amylase. Rec Res Sci Technol 4:01–05
Tanaka A, Ohnishi N, Fukui S (1967) Studies on the formation of vitamins and their function in hydrocarbon fermentation. Production of vitamins and their function in hydrocarbon medium. J Ferment Technol 45:617–623
Wartmann T, Böer E, Pico AH, Sieber H, Bartelsen O, Gellissen G, Kunze G (2003) High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans. FEMS Yeast Res 2:363–369
Weckbecker A (2005) Entwicklung von Ganzzellkatalysatoren zur Synthese von chiralen Alkoholen. PhD thesis, Heinrich-Heine University Düsseldorf, Germany
Weckbecker A, Hummel W (2004) Improved synthesis of chiral alcohols with Escherichia coli cells co-expressing pyridine nucleotide transhydrogenase, NADP+-dependent alcohol dehydrogenase and NAD+-dependent formate dehydrogenase. Biotechnol Lett 26:1739–1744
Weckbecker A, Hummel W (2005a) Glucose dehydrogenase for the regeneration of NADPH and NADH, microbial enzymes and biotransformations. Methods Biotechnol 17:225–238
Weckbecker A, Hummel W (2005b) Glucose dehydrogenase for the regeneration of NADPH and NADH. In: Barredo JL (eds) Microbial enzymes and biotransformations. Humana, Totowa, NJ, p 225–238
Wohlgemuth R (2011) Molecular and engineering perspectives of the biocatalysis interface to chemical synthesis. Chem Biochem Eng Q 25:125–134
Wolberg M, Hummel W, Wandrey C, Müller M (2000) Highly regio- and enantioselective reduction of 3,5-dioxocarboxylates. Angew Chem 112:4476–4478
Wolberg M, Hummel W, Müller M (2001) Biocatalytic reduction of β, δ-diketo esters: a highly stereoselective approach to all four stereoisomers of a chlorinated β, δ-dihydroxy hexanoate. Chemistry 7:4562–4571
Wolberg M, Filho MV, Bode S, Geilenkirchen P, Feldmann R, Liese A, Hummel W, Müller M (2008) Chemoenzymatic synthesis of the chiral side-chain of statins: application of an alcohol dehydrogenase catalysed ketone reduction on a large scale. Bioprocess Biosyst Eng 31:183–191
Zhang J, Witholt B, Lia Z (2006) Bioreduction with efficient recycling of NADPH by coupled permeabilised microorganisms. Adv Synth Catal 348:429–433
Zhang W, O’Connor K, Wang DIC, Li Z (2009) Bioreduction with efficient recycling of NADPH by coupled permeabilised microorganisms. Appl Environ Microbiol 75:687–694
Zhao H, van der Donk WA (2003) Regeneration of cofactors for use in biocatalysis. Curr Opin Biotechnol 14:583–589
Zhou S, Zhang SC, Lai DY, Zhang SL, Chen ZM (2013) Biocatalytic characterization of a short-chain alcohol dehydrogenase with broad substrate specificity from thermophilic Carboxydothermus hydrogenoformans. Biotechnol Lett 35:359–365
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The researchers thank Ina Lemke and David Sjaba for their support. The research work was supported by grant from the BMWi (Grant No. KF2131613MD2 BMWi).
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Rauter, M., Prokoph, A., Kasprzak, J. et al. Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol. Appl Microbiol Biotechnol 99, 4723–4733 (2015). https://doi.org/10.1007/s00253-014-6297-z
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DOI: https://doi.org/10.1007/s00253-014-6297-z