Sains Malaysiana 51(1)(2022): 95-105

http://doi.org/10.17576/jsm-2022-5101-08

Engineering Lactococcus lactis as a Cell Factory for the Production of Limonene

(Kejuruteraan Lactococcus lactis sebagai Kilang Sel untuk Penghasilan Limonena)

NURUL ‘AISHAH SHAILI¹, ADELENE AI-LIAN SONG^2,3*, SARAH OTHMAN¹, LIONEL LIAN AUN IN⁴, JANNA ONG-ABDULLAH¹ & RAHA ABDUL RAHIM^1,3

¹Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

²Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

³Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

⁴Department of Biotechnology, Faculty of Applied Sciences, UCSI University, KL Campus, 56000 Kuala Lumpur, Federal Territory, Malaysia

Received: 25 December 2020/Accepted: 25 May 2021

ABSTRACT

Limonene is a plant monoterpene which contributes significantly to the scent of most essential oils due to its pleasant fragrance. The compound had been reported to have anti-cancer properties against several types of cancer including colorectal cancer. However, the production of this compound in nature is limited because it is produced as a secondary metabolite. To overcome these challenges, Lactococcus lactis was developed as a heterologous host for the production of limonene. A synthesized limonene synthase (LS) from Mentha spicata (mint) was cloned into L. lactis NZ9000. Western blot analysis using mouse IgG His-Tag monoclonal antibody showed successful LS expression by L. lactis at the size of ~55 kDa. GC-MS analysis results showed that limonene production was optimum after 24 h of induction (~8.0 ppm). Metabolic engineering was attempted to enhance the limonene production by overexpression of lactococcal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and mevalonate kinase (mvk) genes in the bacterial host. The recombinant L. lactis carrying pNZ:LSMM plasmid successfully enhanced the limonene production to two-fold (~15.1 ppm) after 24 h of induction. The outcomes of this study show the potential of L. lactis to produce plant proteins and bioactive compounds production, which prospectively leads to an oral delivery system for anti-cancer compounds.

Keywords: Isoprenoid; lactic acid bacteria; metabolic engineering; monoterpene

ABSTRAK

Limonena adalah monoterpena yang menyumbang secara signifikan kepada aroma bagi kebanyakan minyak pati kerana harumannya yang menyenangkan. Bahan ini dilaporkan mempunyai sifat anti-kanser terhadap beberapa jenis kanser termasuk kanser kolorektum. Walau bagaimanapun, penghasilan bahan ini adalah terhad kerana ia dihasilkan sebagai metabolit sekunder. Untuk mengatasi cabaran ini, Lactococcus lactis telah dibangunkan sebagai hos heterologus untuk penghasilan limonena. Gen limonena sintes (LS) yang disintesis daripada Mentha spicata (pudina) telah diklon ke dalam L. lactis NZ9000. Analisis pemblotan Western menggunakan antibodi monoklon IgG His-Tag tikus menunjukkan protein LS berjaya diekspreskan dan mempunyai berat molekul ~ 55 kDa. Keputusan analisis GC-MS menunjukkan bahawa pengeluaran limonena adalah optimum selepas 24 jam induksi (~8.0 ppm). Kejuruteraan metabolik dilakukan untuk meningkatkan penghasilan limonena dengan memasukkan gen 3-hidroksi-3-metilglutaril koenzim A reduktase (HMGR) dan mvk ke dalam hos bakteria. L. lactis rekombinan yang mengandungi plasmid pNZ:LSMM hanya berjaya meningkatkan pengeluaran limonena kepada dua kali ganda (~15.1 ppm) selepas 24 jam induksi. Hasil kajian ini menunjukkan potensi L. lactis untuk penghasilan protein tumbuhan dan sebatian bioaktif, secara prospektif membawa kepada sistem penghantaran oral untuk sebatian anti-kanser.

Kata kunci: Bakteria asid laktik; isoprenoid; kejuruteraan metabolik; monoterpena

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*Corresponding author; email: adelene@upm.edu.my