Team:Toulouse INSA-UPS/Design

Overview

We developed a modular platform to produce the odorous molecules composing the violet smell, the emblematic flower of Toulouse.

In order to adopt a sustainable and environmentally friendly approach, we designed a synthetic microbial consortium consisting of a yeast (Saccharomyces cerevisiae) growing with a sucrose-secreting cyanobacterium (Synechococcus elongatus UTEX 2973 CscB+).

The yeast was engineered to produce violet terpenes providing flowery scent: α-ionone, β-ionone, dihydro-β-ionone and linalool.

We engineered the cyanobacterium to produce the violet leaf aldehyde with a green scent: nona-2,6-dienal and nona-2,6-dienol.

The proportions of each molecule is planned to be modulated by a regulation system, in order to generate custom-made mixtures of these odorant molecules.

Here is described the rationale behind the design of the parts of our system as well as an overview of our experimental design.

Module 1 : Engineering Saccharomyces cerevisiae for the production of the violet terpenes

Terpenoids, a highly diverse and industrially relevant family of natural molecules

Terpenes constitute one of the largest and diverse families of natural molecules with over 80,000 different chemical structures. They are classified into hemiterpenoids (C5), monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20), triterpenoids (C30) and tetraterpenoids (C40). Very diverse in structure and origins, terpenoids have highly interesting properties for various applications, such as pharmaceuticals, flavors and fragrances, biofuels and fuel additives, or in agriculture (Vickers et al. 2017; Moser and Pichler 2019; Chen et al. 2020; Zhang and Hong 2020) (Figure 1).

Figure 1: Exemples of isoprenoid (terpenoid) products with (real or potential) industrial applications. These include pharmaceuticals, nutraceuticals, flavours, fragrances, dyes, agricultural and industrial chemicals, chemical feedstocks, and fuels/fuel additives (Vickers et al. 2017)

Although many of these molecules are still extracted from their natural source - mainly plants - this approach generally suffers from the weak concentrations of the desired compounds, poor extraction yields and seasonal and geographical variations (Moser and Pichler 2019; Chen et al. 2020; Zhang and Hong 2020). With recent advances in synthetic biology and metabolic engineering, microbial terpenoid synthesis is being viewed as an attractive alternative to chemistry for more sustainable and natural industrial production (Cataldo et al. 2016; Vickers et al. 2017; Moser and Pichler 2019; Chen et al. 2020; Zhang and Hong 2020).

Yeast as a host for de novo synthesis of terpenoids

Among numerous available chassis, Saccharomyces cerevisiae is one of the best-established host for the production of industrially relevant titers, yields and rates of terpenoid compounds. (Vickers et al. 2017; Moser and Pichler 2019; Chen et al. 2020; Zhang and Hong 2020).

Advantages of yeast include ease of handling and genetic manipulation, in-depth physiological characterization, rapid growth, relatively high flux of the native terpenoid pathway and substantial knowledge on the engineering of this pathway, as well as adaptability to industrial bioprocess conditions (Vickers et al. 2017; Moser and Pichler 2019; Chen et al. 2020; Zhang and Hong 2020).

Consequently, we chose to use this organism for the production of the violet terpenes: α-ionone, β-ionone, dihydro-β-ionone and linalool.

As shown in Figure 2, all terpenoids are built from C5 blocks: isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). IPP and DMAPP are naturally produced either through the mevalonate (MVA) pathway in eukaryotes or the methylerythritol-phosphate (MEP) pathway in prokaryotes and plant plastids (Vickers et al. 2017; Moser and Pichler 2019; Chen et al. 2020; Zhang and Hong 2020).

As illustrated in the center of Figure 2, the C5 precursors IPP and DMAPP can be condensed sequentially to give geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), or geranylgeranyl diphosphate (GGPP, C20) which constitute a pool of precursors for terpenoid biosynthesis.

Figure 2: Overview of precursor production for terpenoid biosynthesis starting with the mevalonate (MVA) pathway (most eukaryotes) or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway (bacteria and plant chloroplasts) and examples for terpenoid classes derived from these prenyl diphosphate precursors. Ionones can be produced starting from geranylgeranyl diphosphate (GGPP) while linalool can be produced from geranyl diphosphate (GPP) (Moser and Pichler 2019)

Strain choice and metabolic pathway for the production

We have four molecules to produce in the yeast, most importantly ionones plus linalool. Since these molecules do not naturally occur in S. cerevisiae, we need to identify the enzymes to create new synthetic pathways. These metabolic pathways are presented in this part and summarized in Figure 3.

Figure 3: Native and heterologous pathways for the production of violet α-ionone, β-ionone, dihydro-β-ionone, and linalool terpenes in Saccharomyces cerevisiae. In red are the modifications of the LycoYeast strain: different genes were either overexpressed or deleted to increase the metabolic flux to lycopene and hinder some competing pathways (for more information, see our Strain plasmids and primers page). In green are the genetic modifications that we introduced during our iGEM project.

α-Ionone and β-Ionone

Ionones belong to the family of C13 apocarotenoids. They are produced in certain plants from lycopene in two steps. Lycopene is first converted to a carotene, which is then cleaved by a carotenoid cleavage dioxygenase (CCD) to produce two ionone molecules. α-ionone is produced by the cleavage ε-carotene while β-ionone comes from β-carotene (Cataldo et al. 2016; Zhang 2018; Chen et al. 2019; López et al. 2020).

For the α-ionone production, because of the previous demonstration of its successful expression in S. cerevisiae, the lycopene ε-cyclase (LcyE) from Latuca sativa (C. Zhang et al. 2018; Chen et al. 2019) was used to convert lycopene into ε-carotene. For β-ionone production, for the same reason as above, the lycopene β-cyclase (CrtY) from Pantoea ananatis (C. Zhang et al. 2018; Werner et al. 2019; López et al. 2020) was used to convert lycopene into β-carotene.

The next enzymatic step involving the CCD enzyme has previously been shown to be rate limiting (Chen et al. 2019; Werner et al. 2019). To overcome this limitation, a rational enzyme engineering strategy of the most performant CCD for α-ionone production in E. coli was carried out recently (Chen et al. 2019). In this work, the CCD1 from Osmanthus fragrans (OfCCD1) was subjected to site-directed mutagenesis. While this approach allowed the isolation of mutants with improved catalytic properties, a significant proportion of ε-carotene and lycopene were still not converted. The authors hypothesized that it might be caused by the poor accessibility of the OfCCD1 enzyme, which is cytoplasmic, to its substrate which usually is trapped inside the membrane. To address this, the engineered OfCCD1 was fused to the membrane-bound LcyE. This approach aims to facilitate substrate channeling and at the same time, the CCD1 enzyme would be now positioned close to the membrane. Altogether, these modifications allowed a >2.5-fold enhancement of α-ionone in E. coli (Chen et al. 2019).

Dihydro-β-Ionones

Dihydro-β-ionone is obtained from β-ionone by action of the enoate reductase DBR1 from Artemisia annua (X. Zhang et al. 2018).

Linalool

Linalool is a monoterpenoid (C10) derived from GPP (Amiri et al. 2016).The production of linalool can be catalyzed by a linalool synthase (LIS) from geranyl diphosphate (GPP). We chose the LIS gene from Lavandula angustifolia which has already been used to produce linalool in yeast (Amiri et al. 2016).

Chassis

To complete the chosen production in the short time available for our iGEM project, we took advantage of a previously engineered lycopene producing yeast strain, kindly provided by Dr. Sara Castaño-Cerezo from the Molecular and Metabolic Engineering team of the Toulouse Biotechnology Institute (TBI). Lycopene is also a tetraterpenoid (C40) and is the precursor of both ε-carotene and β-carotene (Zhang 2018; Rabeharindranto et al. 2019; López et al. 2020). This strain is based on the laboratory strain CEN-PK2-1C and is named yGPP034 ipENZ011 ipENZ078 (hereafter LycoYeast for simplicity). It produces a quantity of lycopene of the order of 8 mg/gCDW (unpublished data) thanks to the modifications indicated in red in Figure 3.

Construction design for the production of the violet terpenes

Inspired by the molecular engineering work on the CCD enzymes carried out by Chen et al. (2019) and since the problematics addressed by the authors for α-ionone also exist for β-ionone, we chose to design two fusion enzymes. First, for the production of α-ionone in S. cerevisiae, we fused the engineered OfCCD1 to the C-terminal end of LCYe using a (GGGS)₄ linker, in an identical attempt to Chen et al. (2019) (except that the construction was codon-optimized for S. cerevisiae and not E. coli) (see Figure 4.a). The same approach was applied for β-ionone with some slight modifications. In this case, we have chosen the CCD1 from Petunia hybrida (PhCCD1) because it was demonstrated in a previous work that the PhCCD1 should be preferred in this case due to its higher selectivity for β-carotene (C. Zhang et al. 2018) (see Figure 4.b).

In addition to the fusion enzyme between CrtY and PhCCD1, another design was made as a backup plan in the case of β-ionone. For this strategy, the genes of CrtY and PhCCD1 were separated. To achieve substrate channeling and to increase the accessibility of PhCCD1 to its substrate, this enzyme was in this case addressed to the membrane using a fyn destination peptide. In addition to a single amino-acid mutation (K164L), this strategy has been shown to increase up to 4-fold the β-ionone production compared to the native enzyme in yeast (Werner et al. 2019; López et al. 2020) (see Figure 4.c).

Figure 4: Summary of the enzymatic strategies for the biosynthesis of α-ionone and β-ionone, the central fragrance compounds of the violet odour. A) LcyE -OfCCD1 fusion to produce α-ionone (see plasmid pViolette in Figure 5). B) CrtY-PhCCD1 fusion to produce β-ionone (see plasmid pFramboise-fused in Figure 6.a). C) Membrane addressing of an engineered version of PhCCD1 with a fyn peptide to produce β-ionone (see plasmid pFramboise-notfused in Figure 6.b).

All our genetic constructions to engineer S. cerevisiae were inserted into the integrative vectors available in the EasyClone Yeast Toolkit (Stovicek et al. 2015). Genomic integration should allow stable gene expression in our strains. For each construction, since the goal is to modulate the proportions of each molecule, the expression of the key enzymes was placed under the control of inducible promoters.

pVIOLETTE for α-ionone

Three different plasmids were designed for the production of α- and β-ionone. First, since α-ionone is the characteristic molecule of the violet odour, we chose to name the plasmid pVIOLETTE and placed the expression of the key LcyE-OfCCD1 fusion enzyme under the control of a strong galactose-inducible promoter, P_GAL1 (Adams 1972).

Figure 5: pVIOLETTE. The integrative locus used is XII-4. The selective marker is NsrR. The gene coding for LcyE comes from Latuca sativa and the one coding for the OfCCD1 comes from Osmanthus fragrans. The enzymes are fused with a long glycine-serine linker (LGS linker). We use the galactose inducible promoter PGAL1 and a TVPS13 terminator.

What’s in the registry ?

There are already several parts on the registry for Gal1 promoter, coming from S. cerevisiae strains. The closest sequence of our Gal1 promoter is referenced as BBa_J63006, but ours is 107 base pairs shorter. The gene coding for nourseothricin resistance is also already present on the registry, with the affiliate code BBa_K3629011. Nonetheless, this part is just a design with a codon optimization for expression into Yarrowia lipolytica. We therefore codon optimized it for an expression into S. cerevisiae.

How will we test it ?

α-ionone will be detected by GC-MS. This molecule can also be detected by the smell of the culture. Its precursor, ε-carotene can be analyzed using a C18 RP HPLC column with a mixture of acetonitrile:methanol:isopropyl (85:10:5 v/v) as eluant.

pFRAMBOISE for β-ionone

For β-ionone, the two constructions were named pFRAMBOISE since the odor of this molecule is usually associated with raspberry. The first one carrying the enzymatic fusion was named pFRAMBOISE-fused while the one with the two genes separated was named pFRAMBOISE-notfused. Since β-ionone also contributes significantly to the violet scent, we chose to place its expression under the control of the finely regulated promoter P_TetO7, controlled by the rtTA activator present in the construction, responding to the addition of doxycycline (Garí et al., 1997).

Figure 6: pFRAMBOISE. The integrative locus used is X-3. The selective marker is NeoR. The genes coding for CrtY comes from Pantoea ananatis and the one coding for the PhCCD1 comes from Petunia hybrida. A) pFRAMBOISE-fused. Fusion enzyme CrtY-PhCCD1 whose expression is controlled by the doxyciclin inducible promoter P_TetO7 and a TVPS13 terminator. B) pFRAMBOISE-notfused. The two key enzymes are separated. The expression of CrtY is controlled by the doxyciclin inducible promoter P_TetO7 and membrane-adressed-PhCCD1is expressed constitutively.

What’s in the registry ?

The P_TetO7, for use in S. cerevisiae, is already present on the registry, referenced as BBa_K2601000. Nevertheless, the sequence of our P_TetO7 is coming from the vector described by Tominaga et al. (2021). There are also already two parts on the registry for the TEF1 promoter (BBa_K2117000 and BBa_K2117005) but they are coming from Y. lipolytica. Our TEF1 promoter is coming from S. cerevisiae, and therefore will be more relevant for a gene expression in our yeast.

How will we test it ?

β-ionone will be detected by GC-MS. This molecule can also be detected by the smell of the culture. Its precursor, β-carotene can be analyzed using a C18 RP HPLC column with a mixture of acetonitrile:methanol:isopropyl (85:10:5 v/v) as eluant.

pFLEUR for Linalool & Dihydro-β-ionone

Finally, in a practical consideration regarding their size, the genes coding for the LIS and DBR1 were placed on the same construction which was named pFLEUR (linalool and dihydro-β-ionone are floral notes) which is presented in Figure 7. DBR1 expression was placed under the control of the estradiol-inducible promoter PZ3eV and linalool synthase under the control of the copper-inducible promoter P_CUP1 (Butt et al. 1984; Liu et al. 2020).

Figure 7: pFLEUR. The integrative locus used is XII-1. The selective marker used is HygR. The genes coding for LIS and DBR1 come respectively from Lavandula angustifolia and Artemisia annua. The expression of DBR1 is controlled by the copper-inducible promoter P_CUP1 while the expression of LIS is controlled by a β-estradiol inducible promoter PZ3eV.

What’s in the registry ?

There is already BBa_K3052002 on the registry for linalool synthase. This part is codon optimized for translation in E. coli, but ours is codon optimized for translation in S. cerevisiae We take advantage of the referenced promoter CUP1 on the registry for the design of pFLEUR (BBa_K1969005).

How will we test it ?

Linalool and dihydro-β-ionone can also be detected by GC-MS.

Module 2 : Engineering S. elongatus for the sustainable production of violet green scents

As explained in our project Description, we imagined a synthetic microbial consortium between our modified terpene-producing yeast strain and an already engineered sucrose-secreting cyanobacterium (S. elongatus) which provides a carbon source to the yeast. To complete the violet fragrance we aim to produce, we also chose to further engineer our cyanobacterium to make it produce the violet leaf aldehydes. These molecules are secondary in the violet fragrance but could allow to get as close as possible to a perfect violet scent. We describe here the different elements that led us to these choices as well as the engineering approach for the cyanobacteria.

Synechococcus elongatus UTEX 2973, an attractive chassis for sustainable bioproduction

Cyanobacteria are oxygenic photosynthetic prokaryotes which efficiently fix CO₂ as a carbon source while powering their metabolic processes by absorbing sunlight, the most abundant form of renewable energy (Li et al. 2018; Santos-Merino et al. 2019; Wang et al. 2020). These organisms can be grown on non-arable lands and developed from saltwater or wastewater. Thus, their cultivation does not compete with food production (Nozzi et al. 2013; Santos-Merino et al. 2019). Compared to other phototrophs like plants or eukaryotic microalgae, cyanobacteria are relatively fast to grow and easy to genetically engineer (Yu et al. 2015; Ungerer et al. 2018). Such carbon-neutral chassis show therefore a great potential as bioproduction platforms for industrial biotechnology (Yu et al. 2015; Li et al. 2018; Ungerer et al. 2018; Santos-Merino et al. 2019; Wang et al. 2020).

However, despite the advantages of using cyanobacteria in biotechnological settings, some limitations have prevented these organisms from becoming a classic microbial platform. Compared to model heterotrophs such as E. coli or S. cerevisiae, cyanobacteria are generally slow growers. The set of characterized genetic parts and synthetic biology tools available is also much narrower (Li et al. 2018; Santos-Merino et al. 2019; Wang et al. 2020). Finally, several technical challenges remain to be addressed, particularly to achieve the scaling-up of cyanobacteria-based biotechnology processes in an economically efficient way (Lin et al. 2020; Jodlbauer et al. 2021).

Synechococcus elongatus UTEX 2973 (hereafter Syn UTEX 2973), a freshwater cyanobacterium strain isolated in 2015, grows photo-autotrophically with one of the fastest rates reported for any photosynthetic microbe, with generation times as low as 1.5 hours which is equivalent to heterotrophic organisms such as certain yeasts (Yu et al. 2015; Ungerer et al. 2018). Efficient genetic engineering of this strain can be achieved through triparental conjugation with E. coli and a growing number of molecular tools and parts are being developed and characterized by the scientific community (Yu et al. 2015; Li et al. 2018; Ungerer et al. 2018). Examples include Cyanogate, a standardized modular cloning (MoClo) toolkit for cyanobacteria (Vasudevan et al. 2019), or CRISPR-Cas interference technology (Knoot et al. 2020).

Figure 8: Electron micrographs of Synechococcus elongatus UTEX 2973. Labels are carboxysomes (C) and thylakoid membranes (T) (adaptated from Yu et al. 2015).

Sucrose secretion & Coculture with S. cerevisiae

Industrial biotechnology provides alternative solutions to reduce the dependency on fossil fuel. Current biotechnological processes involving heterotrophic organisms rely on sugar inputs derived from agriculture, often competing with the food and feed market. As a consequence, the cost of the carbon source in commercial fermentations represents up to 50% of the overall operating cost (Ducat et al. 2012; Ortiz-Marquez et al. 2013). The choice of sugar feedstock therefore remains a challenge to achieve a sustainable and cost-effective bioproduction (Ducat et al. 2012; Lin et al. 2020).

Many cyanobacterial species are known to accumulate sucrose to resist osmotic stress. Several strain engineering studies have been carried out in the last few years to further enhance the sucrose production in Synechococcus sp. (Ducat et al. 2012; Lin et al. 2020; Zhang et al. 2020). In particular, the sucrose/H+ symporter CscB, from E. coli, was recently introduced in UTEX 2973 under the control of the IPTG-inducible promoter PlacUV5 leading to sucrose production with a rate of 1.9 g.L⁻¹.day⁻¹ in salt stress conditions, reaching titers as high as 8 g.L⁻¹ (Lin et al. 2020). The authors also overexpressed the sucrose-phosphate syntase (sps) and sucrose-phosphate phosphatase (spp) genes from Synechocystis sp. PCC 6803 resulting in a sucrose secretion with a rate of 1.4 g.L⁻¹.day⁻¹ without additional NaCl (Lin et al. 2020).

Figure 9: Engineering sucrose secretion in Syn UTEX 2973 (adaptated from Lin et al. 2020) A) Metabolic pathway for sucrose secretion in Syn UTEX 2973 B) Genetic construction inserted in the genome of the cscB strains developed by Lin et al. 2020 which secrete sucrose upon salt stress and IPTG induction. The additional overexpression of the spp and sps genes allows sucrose production without need of salt stress.

The recovery of sugars from the culture media being expensive and inefficient, an alternative strategy is to develop a “one pot” synthetic microbial consortium between the sucrose secreting cyanobacteria and another heterotrophic organism (Hays et al. 2017). This modular approach has been successfully applied for many organisms, including E. coli, B. subtilis, P. putida as well as various yeast species, and further used to produce CO₂-derived high value products such as bioplastics, organic acids or recombinant proteins (Hays et al. 2017; Li et al. 2017; Löwe et al. 2017; Zhang et al. 2020).

Engineering microbial communities holds a great potential for synthetic biology applications. Advantages include enhanced robustness, reduced metabolic burden due to division of labor, as well as exchange of resources and communication between the organisms (McCarty and Ledesma-Amaro 2019). To ensure the sustainability of our system, we therefore decided to establish a co-culture between the engineered terpene-producing yeast strain and the sucrose secreting Syn UTEX 2973 strains (kindly provided to us by Dr. Michelle Liberton from the Pakrasi Lab). The sucrose and O2 produced by the cyanobacteria are used by the yeast, which produces CO₂ as it grows, supplying additional carbon source to the cyanobacteria module. This way, the two organisms interact in a mutually beneficial way, leading to sustainable production of our CO₂-based violet fragrance ingredient.

Figure 10: ELIXIO, a synthetic microbial consortium for sustainable violet fragrances. Sucrose secreting Syn UTEX 2973 provides a carbon source for the yeast. The production of molecules of interest is shared between the two organisms by taking advantage of their physiological specificities.

How will we test it ?

The sucrose secreted by our cyanobacterium strains can be detected in the culture supernatant using HPLC with a K+ Column and mQ water as mobile phase. After demonstrating that our yeast can grow in the coculture medium with exogenous sucrose, our goal would be to demonstrate that the yeast can grow in the same reactor as the cyanobacterium using as a sole carbon source the sucrose secreted by these strains.

Violet leaf aldehydes production through the Lipoxygenase pathway

In addition to ensuring the sustainability of our violet ingredient through sucrose secretion, S. elongatus is also capable of producing unsaturated fatty acids, such as linoleic acid or α-linolenic acid, in conditions of high light and CO₂ enrichment (Silva et al. 2014). This feature is particularly interesting for our project since these molecules are the precursors of the “violet leaf aldehydes” we could be interested in: (2E,6Z)-nonadienal and (2E,6Z)-nonadienol. The production of these “green leaf aldehydes” occurs naturally in plants through the lipoxygenase pathway. In this pathway, fatty acids are oxygenated by a lipoxygenase (LOX) to form corresponding hydroperoxides, which are then subjected to the action of a hydroperoxide lyase (HPL) (Hassan et al. 2015; Vincenti et al. 2019) (see Figure 11).

Figure 11: Biosynthesis of violet leaf volatile compounds following the LOX pathway. 9-LOX oxygenates α-linolenic acid into 9-HPOT. 9-HPLs act on 9-hydroperoxides to form C9 aldehydes. Conversion of aldehyde form to alcohol form can be catalyzed by an alcohol dehydrogenase (ADH)

S. cerevisiae does not produce α-linolenic acid naturally (Yazawa et al. 2009) and hydroperoxides have besides been shown to exhibit toxicity in this yeast (Buchhaupt et al. 2012). To avoid the metabolic burden resulting from the additional modifications required to produce polyunsaturated fatty acids in S. cerevisiae, we decided to engineer Syn UTEX 2973 to produce these aldehydes by introducing the LOX and HPL genes in its genome.

To do so, we designed pCONCOMBRE, a plasmid based on a pAM4951 backbone. This plasmid is designed for chromosomal integration into S. elongatus Neutral Site I (NSI) which should result in a stable strain with medium copy number of the inserts, since S. elongatus contains multiple copies of chromosomes (Yu et al. 2015). The backbone also contains an E. coli pBR322 origin of replication as well as a conjugation origin of transfer oriT allowing mobilization of the plasmid for triparental conjugation . We chose to use codon optimized versions of the LOX from Nicotiana benthamiana (Nb-9-LOX) in association with the HPL from Cucumis melo (Cm-HPL), which specificity and biotechnological potential have already been described (Huang and Schwab 2011).

Figure 12: pCONCOMBRE. The integrative locus used is Neutral site I (NSI). The selective marker is SpecR. The genes coding for the LOX and HPL come respectively from Nicotiana benthamiana and Cucumis melo. The expression of these two genes is controlled at the transcriptional level by LacI (IPTG induction) and at the transcriptional level by the theophylline-inducible riboswitch theoE*.

As for the yeast productions, we wanted to control the production of the violet leaf aldehydes. We therefore used induction systems to control the expression of the LOX and HPL genes. We chose the theophylline-inducible riboswitch theoE* in association with a P_trc promoter. This device ensures a perfect linear relationship between inducer concentrations and induced intensities up to 190-fold in Syn UTEX 2973 (Nakahira et al. 2013; Li et al. 2018).

The sucrose secreting Syn UTEX 2973 strains expresses the CscB transporter and the sps and spp genes under the control of LacI repressible promoters. lacI was therefore also introduced into these strains to ensure that there is no leakage of these promoters in the absence of IPTG. The two expression cassettes of the LOX and HPL genes involve the P_trc promoter, which is also a LacI repressible promoter. To ensure that there was enough LacI to prevent the leakage of all these promoters, we added an extra copy of lacI. This was done in accordance with Li et al. (2020) who demonstrated that the amount of LacI repressor was already insufficient to control the expression of the three genes involved in sucrose secretion (Lin et al. 2020).

Thus, as summarized in Figure 13, our key genes Nb-9-LOX and Cm-HPL were designed to be tightly controlled, both at the transcriptional level (LacI repressor deactivated with IPTG) and at the translational level (theophylline-inducible riboswitch).

Figure 13: Summary of the regulation strategy for both the LOX and HPL genes. The transcription of the gene of interest is induced by IPTG. Translation initiation is dependent on the presence of theophylline.

What’s in the registry ?

The LacI basic part is present on the iGEM registry and is classified with the code BBa_C0012.

How will we test it ?

(2E,6Z)-nonadienal and (2E,6Z)-nonadienol can also be detected by GC-MS.

References

Adams BG. 1972. Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. J Bacteriol. 111(2):308–315. doi:10.1128/jb.111.2.308-315.1972.

Amiri P, Shahpiri A, Asadollahi MA, Momenbeik F, Partow S. 2016. Metabolic engineering of Saccharomyces cerevisiae for linalool production. Biotechnol Lett. 38(3):503–508. doi:10.1007/s10529-015-2000-4.

Buchhaupt M, Guder JC, Etschmann MMW, Schrader J. 2012. Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase. Appl Microbiol Biotechnol. 93(1):159–168. doi:10.1007/s00253-011-3482-1.

Butt TR, Sternberg EJ, Gorman JA, Clark P, Hamer D, Rosenberg M, Crooke ST. 1984. Copper metallothionein of yeast, structure of the gene, and regulation of expression. Proc Natl Acad Sci U S A. 81(11):3332–3336.

Cataldo VF, López J, Cárcamo M, Agosin E. 2016. Chemical vs. biotechnological synthesis of C13-apocarotenoids: current methods, applications and perspectives. Appl Microbiol Biotechnol. 100(13):5703–5718. doi:10.1007/s00253-016-7583-8.

Chen X, Shukal S, Zhang C. 2019. Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production. J Agric Food Chem. 67(49):13451–13459. doi:10.1021/acs.jafc.9b00860.

Chen X, Zhang C, Lindley ND. 2020. Metabolic Engineering Strategies for Sustainable Terpenoid Flavor and Fragrance Synthesis. J Agric Food Chem. 68(38):10252–10264. doi:10.1021/acs.jafc.9b06203.

Ducat DC, Avelar-Rivas JA, Way JC, Silver PA. 2012. Rerouting carbon flux to enhance photosynthetic productivity. Appl Environ Microbiol. 78(8):2660–2668. doi:10.1128/AEM.07901-11.

Hassan MN ul, Zainal Z, Ismail I. 2015. Green leaf volatiles: biosynthesis, biological functions and their applications in biotechnology. Plant Biotechnology Journal. 13(6):727–739. doi:https://doi.org/10.1111/pbi.12368.

Hays SG, Yan LLW, Silver PA, Ducat DC. 2017. Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction. J Biol Eng. 11(1):4. doi:10.1186/s13036-017-0048-5.

Huang F-C, Schwab W. 2011. Cloning and characterization of a 9-lipoxygenase gene induced by pathogen attack from Nicotiana benthamianafor biotechnological application. BMC Biotechnology. 11(1):30. doi:10.1186/1472-6750-11-30.

Jodlbauer J, Rohr T, Spadiut O, Mihovilovic MD, Rudroff F. 2021. Biocatalysis in Green and Blue: Cyanobacteria. Trends in Biotechnology. 39(9):875–889. doi:10.1016/j.tibtech.2020.12.009.

Knoot CJ, Biswas S, Pakrasi HB. 2020. Tunable Repression of Key Photosynthetic Processes Using Cas12a CRISPR Interference in the Fast-Growing Cyanobacterium Synechococcus sp. UTEX 2973. ACS Synth Biol. 9(1):132–143. doi:10.1021/acssynbio.9b00417.

Li S, Sun T, Xu C, Chen L, Zhang W. 2018. Development and optimization of genetic toolboxes for a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Metabolic Engineering. 48:163–174. doi:10.1016/j.ymben.2018.06.002.

Li T, Li C-T, Butler K, Hays S, Guarnieri M, Oyler G, Betenbaugh M. 2017. Mimicking lichens: Incorporation of yeast strains together with sucrose-secreting cyanobacteria improves survival, growth, ROS removal, and lipid production in a stable mutualistic co-culture production platform. Biotechnology for Biofuels. 10. doi:10.1186/s13068-017-0736-x.

Lin P-C, Zhang F, Pakrasi HB. 2020. Enhanced production of sucrose in the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973. Scientific Reports. 10(1):390. doi:10.1038/s41598-019-57319-5.

Liu J, Wang T, Jiang Y, Liu Z, Tian P, Wang F, Deng L. 2020. Harnessing β-estradiol inducible expression system to overproduce nervonic acid in Saccharomyces cerevisiae. Process Biochemistry. 92. doi:10.1016/j.procbio.2020.02.032.

López J, Bustos D, Camilo C, Arenas N, Saa PA, Agosin E. 2020. Engineering Saccharomyces cerevisiae for the Overproduction of β-Ionone and Its Precursor β-Carotene. Front Bioeng Biotechnol. 8. doi:10.3389/fbioe.2020.578793. [accessed 2021 Jan 30]. https://www.frontiersin.org/articles/10.3389/fbioe.2020.578793/full.

Löwe H, Hobmeier K, Moos M, Kremling A, Pflüger-Grau K. 2017. Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB. Biotechnology for Biofuels. 10(1):190. doi:10.1186/s13068-017-0875-0.

McCarty NS, Ledesma-Amaro R. 2019. Synthetic Biology Tools to Engineer Microbial Communities for Biotechnology. Trends in Biotechnology. 37(2):181–197. doi:10.1016/j.tibtech.2018.11.002.

Moser S, Pichler H. 2019. Identifying and engineering the ideal microbial terpenoid production host. Appl Microbiol Biotechnol. 103(14):5501–5516. doi:10.1007/s00253-019-09892-y.

Nakahira Y, Ogawa A, Asano H, Oyama T, Tozawa Y. 2013. Theophylline-dependent riboswitch as a novel genetic tool for strict regulation of protein expression in Cyanobacterium Synechococcus elongatus PCC 7942. Plant Cell Physiol. 54(10):1724–1735. doi:10.1093/pcp/pct115.

Nozzi NE, Oliver JWK, Atsumi S. 2013. Cyanobacteria as a Platform for Biofuel Production. Front Bioeng Biotechnol. 0. doi:10.3389/fbioe.2013.00007. [accessed 2021 Aug 4]. https://www.frontiersin.org/articles/10.3389/fbioe.2013.00007/full.

Ortiz-Marquez JCF, Do Nascimento M, Zehr JP, Curatti L. 2013. Genetic engineering of multispecies microbial cell factories as an alternative for bioenergy production. Trends in Biotechnology. 31(9):521–529. doi:10.1016/j.tibtech.2013.05.006.

Rabeharindranto H, Castaño-Cerezo S, Lautier T, Garcia-Alles LF, Treitz C, Tholey A, Truan G. 2019. Enzyme-fusion strategies for redirecting and improving carotenoid synthesis in S. cerevisiae. Metabolic Engineering Communications. 8:e00086. doi:10.1016/j.mec.2019.e00086.

Santos-Merino M, Singh AK, Ducat DC. 2019. New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering. Front Bioeng Biotechnol. 7. doi:10.3389/fbioe.2019.00033. [accessed 2021 Apr 8]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400836/.

Silva CSP, Silva-Stenico ME, Fiore MF, de Castro HF, Da Rós PCM. 2014. Optimization of the cultivation conditions for Synechococcus sp. PCC7942 (cyanobacterium) to be used as feedstock for biodiesel production. Algal Research. 3:1–7. doi:10.1016/j.algal.2013.11.012.

Stovicek V, Borja GM, Forster J, Borodina I. 2015. EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains. J Ind Microbiol Biotechnol. 42(11):1519–1531. doi:10.1007/s10295-015-1684-8.

Ungerer J, Wendt KE, Hendry JI, Maranas CD, Pakrasi HB. 2018. Comparative genomics reveals the molecular determinants of rapid growth of the cyanobacterium Synechococcus elongatus UTEX 2973. PNAS. 115(50):E11761–E11770. doi:10.1073/pnas.1814912115.

Vasudevan R, Gale GAR, Schiavon AA, Puzorjov A, Malin J, Gillespie MD, Vavitsas K, Zulkower V, Wang B, Howe CJ, et al. 2019. CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax1[OPEN]. Plant Physiol. 180(1):39–55. doi:10.1104/pp.18.01401.

Vickers CE, Williams TC, Peng B, Cherry J. 2017. Recent advances in synthetic biology for engineering isoprenoid production in yeast. Curr Opin Chem Biol. 40:47–56. doi:10.1016/j.cbpa.2017.05.017.

Vincenti S, Mariani M, Alberti J-C, Jacopini S, Brunini-Bronzini de Caraffa V, Berti L, Maury J. 2019. Biocatalytic Synthesis of Natural Green Leaf Volatiles Using the Lipoxygenase Metabolic Pathway. Catalysts. 9(10):873. doi:10.3390/catal9100873.

Wang F, Gao Y, Yang G. 2020. Recent advances in synthetic biology of cyanobacteria for improved chemicals production. Bioengineered. 11(1):1208–1220. doi:10.1080/21655979.2020.1837458.

Werner N, Ramirez-Sarmiento CA, Agosin E. 2019. Protein engineering of carotenoid cleavage dioxygenases to optimize β-ionone biosynthesis in yeast cell factories. Food Chemistry. 299:125089. doi:10.1016/j.foodchem.2019.125089.

Yazawa H, Iwahashi H, Kamisaka Y, Kimura K, Uemura H. 2009. Production of polyunsaturated fatty acids in yeast Saccharomyces cerevisiae and its relation to alkaline pH tolerance. Yeast. 26(3):167–184. doi:10.1002/yea.1659.

Yu J, Liberton M, Cliften P, Head R, Jacobs J, Smith R, Koppenaal D, Brand J, Pakrasi H. 2015. Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO₂. Scientific reports. 5:8132. doi:10.1038/srep08132.

Zhang C. 2018. Biosynthesis of Carotenoids and Apocarotenoids by Microorganisms and Their Industrial Potential. Zhang C, Chen X, Lindley ND, Too H-P. 2018. A “plug-n-play” modular metabolic system for the production of apocarotenoids. Biotechnology and Bioengineering. 115(1):174–183. doi:10.1002/bit.26462.

Zhang C, Hong K. 2020. Production of Terpenoids by Synthetic Biology Approaches. Front Bioeng Biotechnol. 8:347. doi:10.3389/fbioe.2020.00347.

Zhang L, Chen L, Diao J, Song X, Shi M, Zhang W. 2020. Construction and analysis of an artificial consortium based on the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce the platform chemical 3-hydroxypropionic acid from CO₂. Biotechnology for Biofuels. 13(1):82. doi:10.1186/s13068-020-01720-0.

Zhang X, Liao S, Cao F, Zhao L, Pei J, Tang F. 2018. Cloning and characterization of enoate reductase with high β-ionone to dihydro-β-ionone bioconversion productivity. BMC Biotechnol. 18(1):26. doi:10.1186/s12896-018-0438-x. "

Go to Materials