Engineering Lactococcus lactis as cell factory for the production of plant limonene
Limonene and perillyl alcohol (POH) are plant monoterpenes which give a significant contribution to the aroma of most essential oils due to its pleasant fragrance. Both compounds are synthesized via two pathways known as the mevalonate or 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway by meta...
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| Format: | Thesis |
| Language: | English |
| Published: |
2019
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/92859/1/FBSB%202021%201%20-%20IR.1.pdf |
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| Summary: | Limonene and perillyl alcohol (POH) are plant monoterpenes which give a
significant contribution to the aroma of most essential oils due to its pleasant
fragrance. Both compounds are synthesized via two pathways known as the
mevalonate or 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway by
metabolizing geranyl pyrophosphate (GPP) substrate. These compounds have
been studied to exhibit an anti-cancer effect against several types of cancer
including colorectal cancer. However, isoprenoid metabolic engineering work
has been mostly studied in Escherichia coli. To date there is no report on
limonene and POH production in Lactococcus lactis . In this study, L. lactis was
developed as a heterologous host for the production of limonene using the
recombinant L. lactis as an oral delivery for anti-cancer compounds against
colorectal cancer in the future. Limonene synthase (LS) and cytochrome P450
alkane hydroxylase (ahpGHI ) genes were cloned and expressed in L. lactis
NZ9000 host. Western blot analysis using mouse IgG His-Tag monoclonal
antibody revealed a successful LS expression by L. lactis with the size of ~56
kDa. The expression ahpGHI of gene by L. lactis was not detected by Western
blot despite induction time up to 48 h. According to RT-PCR analysis, the ahpGHI
gene was transcribed only after 24 h post induction, although protein
expression of ahpGHI gene remained unresolved. GC-MS analysis result
showed that limonene production was optimum after 24 h post induction.
However, production of limonene was still low (~4.0 ppm) and did not reach
the LD50 value required to inhibit the proliferation of cancer cells. Therefore
metabolic engineering was attempted to increase the production of limonene
by introducing the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)
and mvk genes in the bacterial host. There are two types of HMGR that was used which are mvaA gene from L. lactis and HMGI from baker's yeast. The constructs containing mvaA and mvk genes from L. lactis was described as pNZ : LSMM plasmid, wheras the one from baker's yeast written as pNZ:LS: HMGI plasmid. The recombinant L. lactis carrying pNZ:LSMM plasmid
successfully enhanced the limonene production over three folds (4.9-15.1
ppm) after 24 h of induction. Trypan blue exclusion test was conducted to
investigate the viability of SW480 colorectal cancer cell after treatment with
recombinant L. lactis producing limonene. From the result it showed that the
cell viability was decreased in dose-dependent manner with the highest
inhibition effect observed at 29.2% after 4 h of treatment at MOI 1500:1. The
outcomes of this study shed light on the potential of food grade L. lactis for plant proteins and bioactive compounds production which prospectively leads to a delivery system for anti-cancer compounds. |
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