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

Diserahkan: 14 Disember 2021/Diterima: 15 Jun 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

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^*Pengarang untuk surat-menyurat; email: bazliramzi@ukm.edu.my