Protocols for the Production and Analysis of Isoprenoids in Bacteria and Yeast

Vickers, Claudia E.; Bongers, Mareike; Bydder, Sarah; Chrysanthopoulos, Panagiotis K.; Hodson, Mark P.

doi:10.1007/8623_2015_107

Cited by 13 publications

(9 citation statements)

References 17 publications

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“…Limonene, a highly volatile monoterpene, can be relatively easy to be captured by overlaying the microbial fermentation system with n -dodecane [30, 34, 35]. Although n -dodecane is a known carbon source for Y. lipolytica , it did not contribute significantly to the growth of our engineered strains in the rich medium that we used for cultivation (Additional file 1: Fig.…”

Section: Resultsmentioning

confidence: 99%

Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil

Pang

Zhao

et al. 2019

Biotechnol Biofuels

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Background Limonene is an important biologically active natural product widely used in the food, cosmetic, nutraceutical and pharmaceutical industries. However, the low abundance of limonene in plants renders their isolation from plant sources non-economically viable. Therefore, engineering microbes into microbial factories for producing limonene is fast becoming an attractive alternative approach that can overcome the aforementioned bottleneck to meet the needs of industries and make limonene production more sustainable and environmentally friendly. Results In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was successfully engineered to produce both d-limonene and l-limonene by introducing the heterologous d-limonene synthase from Citrus limon and l-limonene synthase from Mentha spicata, respectively. However, only 0.124 mg/L d-limonene and 0.126 mg/L l-limonene were produced. To improve the limonene production by the engineered yeast Y. lipolytica strain, ten genes involved in the mevalonate-dependent isoprenoid pathway were overexpressed individually to investigate their effects on limonene titer. Hydroxymethylglutaryl-CoA reductase (HMGR) was found to be the key rate-limiting enzyme in the mevalonate (MVA) pathway for the improving limonene synthesis in Y. lipolytica. Through the overexpression of HMGR gene, the titers of d-limonene and l-limonene were increased to 0.256 mg/L and 0.316 mg/L, respectively. Subsequently, the fermentation conditions were optimized to maximize limonene production by the engineered Y. lipolytica strains from glucose, and the final titers of d-limonene and l-limonene were improved to 2.369 mg/L and 2.471 mg/L, respectively. Furthermore, fed-batch fermentation of the engineered strains Po1g KdHR and Po1g KlHR was used to enhance limonene production in shake flasks and the titers achieved for d-limonene and l-limonene were 11.705 mg/L (0.443 mg/g) and 11.088 mg/L (0.385 mg/g), respectively. Finally, the potential of using waste cooking oil as a carbon source for limonene biosynthesis from the engineered Y. lipolytica strains was investigated. We showed that d-limonene and l-limonene were successfully produced at the respective titers of 2.514 mg/L and 2.723 mg/L under the optimal cultivation condition, where 70% of waste cooking oil was added as the carbon source, representing a 20-fold increase in limonene titer compared to that before strain and fermentation optimization. Conclusions This study represents the first report on the development of a new and efficient process to convert waste cooking oil into d-limonene and l-limonene by exploiting metabolically engineered Y. lipolytica strains for fermentation. The results obtained in this study lay the foundation for more future applications of Y. lipolytica in converting waste cooking oil into various industrially valuable products.

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Section: Resultsmentioning

confidence: 99%

Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil

Pang

Zhao

et al. 2019

Biotechnol Biofuels

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“…Nerolidol-producing strains were evaluated through two-phase flask cultivation using dodecane as a non-toxic organic extractant phase [ 76 ]. Synthetic minimal (SM) medium containing 6.7 g L −1 YNB (Sigma-Aldrich #Y0626; pH 6.0) with 20 g L −1 glucose as the carbon source was used (SM-glucose).…”

Section: Methodsmentioning

confidence: 99%

“…Pre-cultured cells were collected by centrifugation and re-suspend in fresh media before initiating two-phase cultivations. Two-phase flask cultivation was initiated by inoculating pre-cultured cells to OD 600 = 0.2 in 25 mL MES-buffered SM 20 g L −1 glucose medium (or 23 mL MES-buffered SM 20 g L −1 sucrose medium when cultures were not sampled for RNA extraction) in 250 mL flasks with solvent-resistant screw-caps [ 76 ]; 2 mL dodecane was added to extract nerolidol. Flask cultivation was performed at 30°C and 200 rpm.…”

Section: Methodsmentioning

confidence: 99%

Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast

Peng

Plan

Carpenter

et al. 2017

Biotechnol Biofuels

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BackgroundAssembly of heterologous metabolic pathways is commonly required to generate microbial cell factories for industrial production of both commodity chemicals (including biofuels) and high-value chemicals. Promoter-mediated transcriptional regulation coordinates the expression of the individual components of these heterologous pathways. Expression patterns vary during culture as conditions change, and this can influence yeast physiology and productivity in both positive and negative ways. Well-characterized strategies are required for matching transcriptional regulation with desired output across changing culture conditions.ResultsHere, constitutive and inducible regulatory mechanisms were examined to optimize synthetic isoprenoid metabolic pathway modules for production of trans-nerolidol, an acyclic sesquiterpene alcohol, in yeast. The choice of regulatory system significantly affected physiological features (growth and productivity) over batch cultivation. Use of constitutive promoters resulted in poor growth during the exponential phase. Delaying expression of the assembled metabolic modules using the copper-inducible CUP1 promoter resulted in a 1.6-fold increase in the exponential-phase growth rate and a twofold increase in productivity in the post-exponential phase. However, repeated use of the CUP1 promoter in multiple expression cassettes resulted in genetic instability. A diauxie-inducible expression system, based on an engineered GAL regulatory circuit and a set of four different GAL promoters, was characterized and employed to assemble nerolidol synthetic metabolic modules. Nerolidol production was further improved by 60% to 392 mg L−1 using this approach. Various carbon source systems were investigated in batch/fed-batch cultivation to regulate induction through the GAL system; final nerolidol titres of 4–5.5 g L−1 were achieved, depending on the conditions.ConclusionDirect comparison of different transcriptional regulatory mechanisms clearly demonstrated that coupling the output strength to the fermentation stage is important to optimize the growth fitness and overall productivities of engineered cells in industrially relevant processes. Applying different well-characterized promoters with the same induction behaviour mitigates against the risks of homologous sequence-mediated genetic instability. Using these approaches, we significantly improved sesquiterpene production in yeast.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0728-x) contains supplementary material, which is available to authorized users.

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“…Two-phase flask cultivation was performed as described previously (Peng et al, 2017a;Peng et al, 2017b;Vickers et al, 2015b). Synthetic minimal (SM) medium (containing 6.7 g L -1 yeast nitrogen base, YNB, Sigma-Aldrich #Y0626; pH 6.0) with 20 g L -1 glucose as the carbon source was used (SM-glucose).…”

Section: Two-phase Flask Cultivationmentioning

confidence: 99%

Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae

Peng

Nielsen

Kampranis

et al. 2018

Metabolic Engineering

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Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene/sesquiterpene (linalool/nerolidol) synthase, AcNES1, was used as a reporter of intracellular GPP and FPP production. Transcription of the synthetic pathway was controlled by either constitutive or diauxie-inducible promoters. A combination of degron K3K15 and the ERG1 promoter increased linalool titre by 27-fold to 11 mg L in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L in the strain with diauxie-inducible promoter constructs. The sesquiterpene nerolidol remained the major product in both strains. The same strategies were applied to construct a limonene-producing strain, which produced 76 mg L in batch cultivation. The FPPS regulation method developed here successfully redirected metabolic flux toward monoterpene production. Examination of growth defects in various strains suggested that the intracellular FPP concentration had a significant effect on growth rate. Further strategies are required to balance intracellular production of FPP and GPP so as to maximise monoterpene production without impacting on cellular growth.

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Protocols for the Production and Analysis of Isoprenoids in Bacteria and Yeast

Cited by 13 publications

References 17 publications

Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil

Engineering the oleaginous yeast Yarrowia lipolytica to produce limonene from waste cooking oil

Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast

Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae

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