Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis.
Candida tropicalis
L-limonene
Metabolic engineering
Perillic acid
Synthetic biology
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
27 Aug 2024
27 Aug 2024
Historique:
received:
08
12
2023
accepted:
08
08
2024
revised:
04
08
2024
medline:
27
8
2024
pubmed:
27
8
2024
entrez:
27
8
2024
Statut:
epublish
Résumé
Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid's precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis. KEY POINTS: • Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis. • Perillic acid was first synthesized de novo in engineered microorganisms. • The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.
Identifiants
pubmed: 39190181
doi: 10.1007/s00253-024-13279-z
pii: 10.1007/s00253-024-13279-z
doi:
Substances chimiques
Limonene
9MC3I34447
perillic acid
7694-45-3
Monoterpenes
0
pinene cyclase I
EC 5.5.-
Intramolecular Lyases
EC 5.5.-
Intramolecular Transferases
EC 5.4.-
Terpenes
0
Cyclohexenes
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
447Subventions
Organisme : National Natural Science Foundation of China
ID : 32271533
Informations de copyright
© 2024. The Author(s).
Références
Bhataya A, Schmidt-Dannert C, Lee PC (2009) Metabolic engineering of Pichia pastoris X-33 for lycopene production. Process Biochem 44:1095–1102
doi: 10.1016/j.procbio.2009.05.012
Bicas JL, Dionísio AP, Pastore GM (2009) Bio-oxidation of terpenes: an approach for the flavor industry. Chem Rev 109:4518–4531
doi: 10.1021/cr800190y
pubmed: 19645444
Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95:4126–4133
doi: 10.1073/pnas.95.8.4126
pubmed: 9539701
pmcid: 22453
Da Fonseca CO, Simão M, Lins IR, Caetano RO, Futuro D, Quirico-Santos T (2011) Efficacy of monoterpene perillyl alcohol upon survival rate of patients with recurrent glioblastoma. J Cancer Res Clin Oncol 137:287–293
doi: 10.1007/s00432-010-0873-0
pubmed: 20401670
Davies FK, Work VH, Beliaev AS, Posewitz MC (2014) Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp. PCC 7002. Front Bioeng Biotechnol 2:21
doi: 10.3389/fbioe.2014.00021
pubmed: 25152894
pmcid: 4126464
DeLoache WC, Russ ZN, Dueber JE (2016) Towards repurposing the yeast peroxisomefor compartmentalizing heterologous metabolic pathways. Nat Commun 7:11152
doi: 10.1038/ncomms11152
pubmed: 27025684
pmcid: 5476825
Duetz WA, Bouwmeester H, Van Beilen JB, Witholt B (2003) Biotransformation of limonene by bacteria, fungi, yeasts, and plants. Appl Microbiol Biotechnol 61:269–277
doi: 10.1007/s00253-003-1221-y
pubmed: 12743755
Ferrara MA, Almeida DS, Siani AC, Lucchetti L, Lacerda PSB, Freitas A, Tappin MRR, Bon EPS (2013) Bioconversion of R-(+)-limonene to perillic acid by the yeast Yarrowia lipolytica. Braz J Microbiol 44:1075–1080
doi: 10.1590/S1517-83822014005000008
pubmed: 24688495
Fickers P, Benetti PH, Wache Y, Marty A, Mauersberger S, Smit MS, Nicaud JM (2005) Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications. FEMS Yeast Res 5:527–543
doi: 10.1016/j.femsyr.2004.09.004
pubmed: 15780653
Gellissen G, Kunze G, Gaillardin C, Cregg JM, Berardi E, Veenhuis M, Van der Klei I (2005) New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica – a comparison. FEMS Yeast Res 5:1079–1096
doi: 10.1016/j.femsyr.2005.06.004
pubmed: 16144775
Grabinska K, Palamarczyk G (2002) Dolichol biosynthesis in the yeast Saccharomyces cerevisiae: an insight into the regulatory role of farnesyl diphosphate synthase. FEMS Yeast Res 2:259–265
pubmed: 12702274
Hara A, Arie M, Kanai T, Matsui T, Matsuda H, Furuhashi K, Ueda M, Tanaka A (2001) Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene. Arch Microbiol 176:364–369
doi: 10.1007/s002030100338
pubmed: 11702078
Hu FF, Liu JD, Du GC, Hua ZZ, Zhou JW, Chen J (2012) Key cytomembrane ABC transporters of Saccharomyces cerevisiae fail to improve the tolerance to D-limonene. Biotechnol Lett 34:1505–1509
doi: 10.1007/s10529-012-0931-6
pubmed: 22526424
Ignea C, Pontini M, Maffei ME, Makris AM, Kampranis SC (2014) Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase. ACS Synth Biol 3:298–306
doi: 10.1021/sb400115e
pubmed: 24847684
Jiang G, Yao M, Wang Y, Zhou L, Song TQ, Liu H, Xiao WH, Yuan YJ (2017) Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae. Metab Eng 41:57–66
doi: 10.1016/j.ymben.2017.03.005
pubmed: 28359705
Liu S, Zhang M, Ren Y, Jin G, Tao Y, Lyu L, Zhao ZK, Yang X (2021) Engineering Rhodosporidium toruloides for limonene production. Biotechnol Biofuels 14:241
doi: 10.1186/s13068-021-02094-7
pubmed: 34920742
pmcid: 8684234
Liu JD, Zhu YB, Du GC, Zhou JW, Chen J (2023) Exogenous ergosterol protects Saccharomyces cerevisiae from D-limonene stress. J Appl Microbiol 114:482–491
doi: 10.1111/jam.12046
Mars AE, Gorissen JPL, van den Beld I, Eggink G (2001) Bioconversion of limonene to increased concentrations of perillic acid by Pseudomonas putida GS1 in a fed-batch reactor. Appl Microbiol Biotechnol 56:101–107
doi: 10.1007/s002530100625
pubmed: 11499915
Mirata MA, Heerd D, Schrader J (2009) Integrated bioprocess for the oxidation of limonene to perillic acid with Pseudomonas putida DSM 12264. Process Biochem 44:764–771
doi: 10.1016/j.procbio.2009.03.013
Pang Y, Zhao Y, Li S, Zhao Y, Yu A (2019) Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil. Biotechnol Biofuels 12:241
doi: 10.1186/s13068-019-1580-y
pubmed: 31624503
pmcid: 6781337
Parveen M, Hasan MK, Takahashi J, Murata Y, Kitagawa E, Kodama O, Iwahashi H (2004) Response of Saccharomyces cerevisiae to a monoterpene: evaluation of antifungal potential by DNA microarray analysis. J Antimicrob Chemother 54:46–55
doi: 10.1093/jac/dkh245
pubmed: 15201226
Sun C, Theodoropoulos C, Scrutton NS (2019) Techno-economic assessment of microbial limonene production. Bioresour Technol 300:122666
doi: 10.1016/j.biortech.2019.122666
pubmed: 31901556
Szkopinska A, Plochocka D (2005) Farnesyl diphosphate synthase: regulation of product specificity. Acta Biochim Pol 52:45–55
doi: 10.18388/abp.2005_3485
pubmed: 15827605
Turner G, Gershenzon J, Nielson EE, Froehlich JE, Croteau R (1999) Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localized to leucoplasts of oil gland secretory cells. Plant Physiol 120:879–886
doi: 10.1104/pp.120.3.879
pubmed: 10398724
pmcid: 59327
Van Beilen JB, Holtackers R, Lüscher D, Bauer U, Witholt B, Duetz WA (2005) Biocatalytic production of perillyl alcohol from limonene by using a novel Mycobacterium sp. cytochrome P450 alkane hydroxylase expressed in Pseudomonas putida. Appl Environ Microbiol 71:1737–1744
doi: 10.1128/AEM.71.4.1737-1744.2005
pubmed: 15811996
pmcid: 1082528
Wang J, Peng J, Fan H, Xiu X, Xue L, Wang L, Su J, Yang X, Wang R (2018) Development of mazF-based markerless genome editing system and metabolic pathway engineering in Candida tropicalis for producing long-chain dicarboxylic acids. J Ind Microbiol Biotechnol 45:971–981
doi: 10.1007/s10295-018-2074-9
pubmed: 30187242
Wang L, Qi AD, Liu JY, Shen Y, Wang JS (2023) Comparative metabolic analysis of the adaptive Candida tropicalis to furfural stress response. Chem Eng Sci 267:118348
doi: 10.1016/j.ces.2022.118348
Wei CL, Zhang LH, Shen W, Xia YY, Chen XZ, Yang HQ (2024) Enhancement of squalene synthesis in Candida tropicalis via combinatorial metabolic engineering strategies to rebuild pathways. Biochem Eng J 208:109348
doi: 10.1016/j.bej.2024.109348
Willrodt C, Halan B, Karthaus L, Rehdorf J, Julsing MK, Buehler K, Schmid A (2017) Continuous multistep synthesis of perillic acid from limonene by catalytic biofilms under segmented flow. Biotechnol Bioeng 114:281–290
doi: 10.1002/bit.26071
pubmed: 27530691
Wu J, Cheng S, Cao J, Qao J, Zhao GR (2019) Systematic optimization of limonene production in engineered Escherichia coli. J Agric Food Chem 67:7087–7097
doi: 10.1021/acs.jafc.9b01427
pubmed: 31199132
Yeruva L, Pierre KJ, Elegbede A, Wang RC, Carper SW (2007) Perillyl alcohol and perillic acid induced cell cycle and apoptosis in non small cell lung cancer. Cancer Lett 257:216–226
doi: 10.1016/j.canlet.2007.07.020
pubmed: 17888568
Zhang LH, Chen XZ, Chen Z, Wang ZZ, Jiang S, Li L, Pötter M, Shen W, Fan Y (2016) Development of an efficient genetic manipulation strategy for sequential gene disruption and expression of different heterologous GFP genes in Candida tropicalis. Appl Microbiol Biotechnol 100:9567–9580
doi: 10.1007/s00253-016-7762-7
pubmed: 27522195
Zhang C, Li M, Zhao GR, Lu W (2020) Harnessing yeast peroxisomes and cytosol acetyl-CoA for sesquiterpene α-humulene production. Agric Food Chem 68:1382–1389
doi: 10.1021/acs.jafc.9b07290
Zhang X, Liu X, Meng Y, Zhang L, Qiao J, Zhao GR (2021) Combinatorial engineering of Saccharomyces cerevisiae for improving limonene production. Biochem Eng J 176:108155
doi: 10.1016/j.bej.2021.108155
Zhang LH, Yang HQ, Xia YY, Shen W, Liu LM, Li Q, Chen XZ (2022) Engineering the oleaginous yeast Candida tropicalis for α-humulene overproduction. Biotechnol Biofuels Bioprod 15:59
doi: 10.1186/s13068-022-02160-8
pubmed: 35619177
pmcid: 9137083
Zhao J, Bao X, Chen L, Yu S, Jin H (2016) Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 100:4561–4571
doi: 10.1007/s00253-016-7375-1
pubmed: 26883346
Zhao D, Gao QD, Zheng XC, Liu SS, Qi QS, Wang X, Yang XB (2023) Optimization of fermentation conditions for elevating limonene production with engineered Rhodosporidium toruloides. Fermentation 9:431
doi: 10.3390/fermentation9050431