Sains Malaysiana 51(10)(2022): 3163-3170

http://doi.org/10.17576/jsm-2022-5110-04

Expression of Furfural Reductase Improved Furfural Tolerance in Antarctic Bacterium Pseudomonas extremaustralis

(Pengekspresan Furfural Reduktase Meningkatkan Ketoleranan Furfural dalam Bakteria AntartikaPseudomonas extremaustralis)

AHMAD BAZLI RAMZI^1,*, MATTHLESSA MATTHEW MINGGU¹, UMMUL SYAFIQAH RUSLAN¹, MOHAMAD HAZWAN FIKRI KHAIRI¹& PEER MOHAMED ABDUL^2,3

¹Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Research Centre for Sustainable Process Technology, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

³Program of Chemical Engineering, Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

Received: 14 December 2021/Accepted: 15 June 2022

Abstract

Whole-cell biocatalysis using Antarctic bacteria is presently hampered by a lack of genetic information, limited gene tools and critically, a poor range of cultivation conditions. In this work, biological engineering strategy was employed for developing Pseudomonas extremaustralis, a metabolically-versatile and biopolymer-producing Antarctic bacterium, as a new whole-cell biocatalytic host. For this purpose, gene cloning and plasmid construction were carried out for overexpression of furfural reductase (FucO), an industrially-important enzyme for degradation of toxic furfural compound commonly found in lignocellulosic biorefinery. FucO gene from Escherichia coli BL21 was cloned in pJM105 plasmid and transformed into competent cells of P. extremaustralis to generate a biologically-engineered pFucO strain. For functional characterization of the enzyme, furfural reductase activity was assayed, where the P. extremaustralis pFucO strain exhibited increased furfural reductase activity of about 15.6 U/mg, an 18.8-fold higher than empty plasmid-carrying control pJM105 strain (0.83 U/mg). Furfural detoxification activity using whole cells was also determined by which the overexpression of FucO led to increased tolerance and cell growth with an OD₆₀₀ value of 5.3 as compared to the control pJM105 strain that was inhibited with 10 mM furfural during 48-hour cultivation. Therefore, the findings obtained in this study successfully demonstrated the development of P. extremaustralis as biocatalytic host for the production of recombinant furfural reductase. The bioengineering would serve as a modular biotechnological platform for polar strain and bioproduct development tailored towards industrial biotechnology applications.

Keywords: Antarctic bacteria; biological engineering; furfural reductase; Pseudomonas extremaustralis; whole-cell biocatalysis 

Abstrak

Penggunaan bakteria Antartika sebagai biopemangkin keseluruhan sel buat masa ini adalah sangat terbatas akibat kurangnya maklumat genetik, alatan genetik yang terhad dan ketidakserasian keadaan pengkulturan. Dalam kajian ini, strategi kejuruteraan biologi digunakan untuk membangunkan Pseudomonas extremaustralis, sejenis bakteria yang berasal dari Antartika yang mempunyai metabolisme versatil, berupaya menghasilkan biopolimer dan sesuai digunakan sebagai perumah baru biopemangkin keseluruhan sel. Untuk mencapai tujuan ini, pengklonan gen dan pembinaan plasmid telah dilakukan bagi penghasilan furfural reduktase (FucO), sejenis enzim yang penting dalam penguraian sebatian toksik furfural yang kerap ditemui dalam proses penapisan sisa lignoselulosa. Gen FucO daripada Escherichia coli BL21 diklonkan ke dalam plasmid pJM105 dan dimasukkan ke dalam sel kompeten P. extremaustralis untuk menghasilkan strain terjurutera biologi pFucO. Untuk pencirian enzim dan kefungsian bakteria terubah suai biologi ini, aktiviti furfural reduktase telah diasai dengan strain P. extremaustralis pFucO menghasilkan sebanyak 15.6 U/mg, iaitu 18.8 kali ganda lebih banyak daripada sel yang mengandungi plasmid kawalan pJM105 (0.83 U/mg). Aktiviti penguraian furfural menggunakan seluruh sel juga dilakukan yang mana pengaruhan enzim furfural reduktase berjaya meningkatkan pertumbuhan sel oleh strain pFucO yang menunjukkan OD₆₀₀ sebanyak 5.3 selepas 48 jam dikultur berbanding strain kawalan pJM105 yang direncatkan apabila diuji dengan 10 mM furfural. Penemuan kajian ini membuktikan P. extremaustralis terjurutera biologi ini berjaya dibangunkan sebagai perumah biopemangkin untuk penghasilan furfural reduktase rekombinan. Strategi kejuruteraan biologi ini akan menjadi pelantar berasaskan bioteknologi untuk pembangunan strain dan penghasilan bioproduk kutub untuk aplikasi bioteknologi industri.

Kata kunci: Bakteria Antartika; biopemangkin seluruh sel; furfural reduktase; kejuruteraan biologi; Pseudomonas extremaustralis

REFERENCES

Abdul, P.M., Md. Jahim, J., Harun, S., Markom, M., Hassan, O., Mohamad, A.W. & Asis, A.J. 2013. Biohydrogen production from pentose-rich oil palm empty fruit bunch molasses: A first trial. International Journal of Hydrogen Energy 38(35): 15693-15699.

Ali, M.S.M., Ganasen, M., Rahman, R.N.Z.R.A., Chor, A.L.T., Salleh, A.B. & Basri, M. 2013. Cold-adapted RTX lipase from Antarctic Pseudomonas sp. strain AMS8: Isolation, molecular modeling and heterologous expression. The Protein Journal 32(4): 317-325.

Dietrich, D., Illman, B. & Crooks, C. 2013. Differential sensitivity of polyhydroxyalkanoate producing bacteria to fermentation inhibitors and comparison of polyhydroxybutyrate production from Burkholderia cepacia and Pseudomonas pseudoflava. BMC Research Notes 6: 219.

Fendrihan, S. & Negoiţa, T.G. 2017. Psychrophilic microorganisms as important source for biotechnological processes. Adaptation of Microbial Life to Environmental Extremes: Novel Research Results and Application. 2nd ed., edited by Stan-Lotter, H. & Fendrihan, S. Springer.

Franden, M.A., Pienkos, P.T. & Zhang, M. 2009. Development of a high-throughput method to evaluate the impact of inhibitory compounds from lignocellulosic hydrolysates on the growth of Zymomonas mobilis. Journal of Biotechnology 144(4): 259-267.

Guarnieri, M.T., Ann Franden, M., Johnson, C.W. & Beckham, G.T. 2017. Conversion and assimilation of furfural and 5-(hydroxymethyl)furfural by Pseudomonas putida KT2440. Metabolic Engineering Communications 8(4): 22-28.

Hashim, N.H.F., Bharudin, I., Nguong, D.L.S., Higa, S., Bakar, F.D.A., Nathan, S., Rabu, A., Kawahara, H., Illias, R.M., Najimudin, N., Mahadi, N.M. & Murad, A.M.A. 2013. Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12. Extremophiles 17(1): 63-73.

Heer, D. & Sauer, U. 2008. Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain. Microbial Biotechnology 1(6): 497-506.

Jiang, W., Wang, S., Wang, Y. & Fang, B. 2016. Key enzymes catalyzing glycerol to 1,3-propanediol. Biotechnology for Biofuels 9: 57.

Markley, A.L., Begemann, M.B., Clarke, R.E., Gordon, G.C. & Pfleger, B.F. 2015. Synthetic biology toolbox for controlling gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002. ACS Synthetic Biology 4: 595-603.

Martínez-Rosales, C., Fullana, N., Musto, H. & Castro-Sowinski, S. 2012. Antarctic DNA moving forward: Genomic plasticity and biotechnological potential. FEMS Microbiology Letter 331(1): 1-9.

Marx, C.J. & Lidstrom, M.E. 2001. Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147: 2065-2075.

Piette, F., D’Amico, S., Mazzucchelli, G., Danchin, A., LePrince, P. & Feller, G. 2011. Life in the cold: A proteomic study of cold-repressed proteins in the antarctic bacterium Pseudoalteromonas haloplanktis TAC125. Applied and Environmental Microbiology 77(11): 3881-3883.

Raiger Iustman, L.J., Tribelli, P.M., Ibarra, J.G., Catone, M.V., Venero, E.S.C. & Lopez, N.I. 2015. Genome sequence analysis of Pseudomonas extremaustralis provides new insights into environmental adaptability and extreme conditions resistance. Extremophiles 19(1): 207-220.

Raj, K., Partow, S., Correia, K., Khusnutdinova, A.N., Yakunin, A.F. & Mahadevan, R. 2018. Biocatalytic production of adipic acid from glucose using engineered Saccharomyces cerevisiae. Metabolic Engineering Communications 6: 28-32.

Ramzi, A.B., Ku Bahaudin, K.N.A., Baharum, S.N., Che Me, M.L., Goh, H.H., Hassan, M. & Mohd Noor, N. 2018. Rapid assembly of yeast expression cassettes for phenylpropanoid biosynthesis in Saccharomyces cerevisiae. Sains Malaysiana 47(12): 2969-2974.

Ramzi, A.B., Hyeon, J.E., Kim, S.W., Park, C. & Han, O.S. 2015. 5-aminolevulinic acid production in engineered Corynebacterium glutamicum via C5 biosynthesis pathway. Enzyme and Microbial Technology 81: 1-7.

Sarmiento, F., Peralta, R. & Blamey, J.M. 2015. Cold and hot extremozymes: Industrial relevance and current trends. Frontiers in Bioengineering and Biotechnology 3: 148.

Tomás, A.F., Karagöz, P., Karakashev, D. & Angelidaki, I. 2013. Extreme thermophilic ethanol production from rapeseed straw: Using the newly isolated Thermoanaerobacter pentosaceus and combining it with Saccharomyces cerevisiae in a two-step process. Biotechnology and Bioengineering 110(6): 1574-1582.

Tribelli, P.M., Lujan, A.M., Pardo, A., Ibarra, J.G., Do Porto, D.F., Smania, A. & Lopez, N.I. 2019. Core regulon of the global anaerobic regulator Anr targets central metabolism functions in Pseudomonas species. Scientific Reports 9: 9065.

Tribelli, P.M., Rossi, L., Ricardi, M.M., Gomez-Lozano, M., Molin, S., Raiger lustman, L.J. & Lopez, N.I. 2018. Microaerophilic alkane degradation in Pseudomonas extremaustralis: A transcriptomic and physiological approach. Journal of Industrial and Microbiology and Biotechnology 45(1): 15-23.

Tribelli, P.M., Venero, E.C.S., Ricardi, M.M., Gomez-Lozano, M., Raiger lustman, L.J., Molin, S. & Lopez, N.I. 2015. Novel essential role of ethanol oxidation genes at low temperature revealed by transcriptome analysis in the antarctic bacterium Pseudomonas extremaustralis. PLoS ONE 10(12): e0145353.

Tribelli, P.M., Di Martino, C., López, N.I. & Raiger Iustman, L.J. 2012. Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis. Biodegradation 23: 645-651.

Wang, Q., Mueller, A.P., Leong, C.R., Matsumoto, K., Taguchi, S. & Nomura, C.T. 2010. Quick and efficient method for genetic transformation of biopolymer-producing bacteria. Journal of Chemical Technology and Biotechnology 85(6): 775-778.

Wang, X., Yomano, L.P., Lee, J.Y., York, S.W., Zheng, H., Mullinnix, Shanmugam, K.T. & Ingram, L.O. 2013. Engineering furfural tolerance in Escherichia coli improves the fermentation of lignocellulosic sugars into renewable chemicals. Proceedings of the National Academy of Sciences of the United States of America 110(10): 4021-4026.

Wang, X., Miller, E.N., Yomano, L.P., Zhang, X., Shanmugam, K.T. & Ingram, L.O. 2011. Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in Escherichia coli strains engineered for the production of ethanol and lactate. Applied and Environmental Microbiology 77: 5132-5140.

^*Corresponding author; email: bazliramzi@ukm.edu.my