# Synthetic, marine, light-driven, autotroph-heterotroph co-culture system for sustainable β-caryophyllene production

Wenchao Chen, Young-Kyoung Park, Lucie Studená, David Bell, Piotr Hapeta, Jing Fu, Peter Nixon, Rodrigo Ledesma-Amaro

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## To cite this version:

Wenchao Chen, Young-Kyoung Park, Lucie Studená, David Bell, Piotr Hapeta, et al.. Synthetic, marine, light-driven, autotroph-heterotroph co-culture system for sustainable β-caryophyllene production. Bioresource Technology, 2024, pp.131232. 10.1016/j.biortech.2024.131232. hal-04669846

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https://hal.science/hal-04669846v1

Submitted on 7 Jul 2025

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# Synthetic, marine, light-driven, autotroph-heterotroph co-culture system for sustainable β-caryophyllene production

Wenchao Chen<sup>a, b, 1</sup>, Young-Kyoung Park<sup>b, c, 1</sup>, Lucie Studená<sup>b</sup>, David Bell<sup>d</sup>, Piotr Hapeta<sup>b</sup>, Jing Fu<sup>b</sup>, Peter J. Nixon<sup>e</sup>, Rodrigo Ledesma-Amaro<sup>b, *</sup>

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## HIGHLIGHTS

* *Synechococcus* sp. PCC 7002 was engineered to accumulate and secrete sucrose.

* *Yarrowia lipolytica* was engineered to produce β-caryophyllene from sucrose.

* β-Caryophyllene was produced from the co-culture of *Synechococcus* and *Yarrowia*.

* NaCl, sucrose, and nitrogen affected the β-caryophyllene yield in co-culture.

## GRAPHICAL ABSTRACT

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## ARTICLE INFO

**Keywords:**

Artificial light-driven consortia

Marine cyanobacteria

*Yarrowia lipolytica*

Sucrose

β-Caryophyllene

Metabolic engineering

Synthetic biology

Engineering biology

Bioproduction

Precision fermentation

## ABSTRACT

Applying low-cost substrate is critical for sustainable bioproduction. Co-culture of phototrophic and heterotrophic microorganisms can be a promising solution as they can use CO₂ and light as feedstock. This study aimed to create a light-driven consortium using a marine cyanobacterium *Synechococcus* sp. PCC 7002 and an industrial yeast *Yarrowia lipolytica*. First, the cyanobacterium was engineered to accumulate and secrete sucrose by regulating the expression of genes involved in sucrose biosynthesis and transport, resulting in 4.0 g/L of sucrose secretion. Then, *Yarrowia lipolytica* was engineered to efficiently use sucrose and produce β-caryophyllene that has various industrial applications. Then, co- and sequential-culture were optimized with different induction conditions and media compositions. A maximum β-caryophyllene yield of 14.1 mg/L was obtained from the co-culture. This study successfully established an artificial light-driven consortium based on a marine cyanobacterium and *Y. lipolytica*, and provides a foundation for sustainable bioproduction from CO₂ and light through co-culture systems.

<footnote><sup>a</sup> Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, PR China</footnote>

<footnote><sup>b</sup> Department of Bioengineering, Bezos Centre for Sustainable Protein, Microbial Food Hub and Centre for Synthetic Biology, Imperial College London, London SW72AZ, UK</footnote>

<footnote><sup>c</sup> Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350 Jouy-en-Josas, France</footnote>

<footnote><sup>d</sup> SynbiCITE Innovation and Knowledge Centre, Imperial College London, London SW7 2AZ, UK</footnote>

<footnote><sup>e</sup> Department of Life Sciences, Imperial College London, London SW7 2AZ, UK</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: r.ledesma-amaro@imperial.ac.uk (R. Ledesma-Amaro).</footnote>

<footnote n="1">¹ Authors equally contributed to this study.</footnote>
# 1. Introduction

Bioproduction is a promising alternative to fossil fuels-derived products. However, it is often inefficient due to its high cost of production compared to conventional processes. One way to reduce production costs during bioproduction is the use of low-cost carbon sources. In this regard, phototrophic organisms have advantages over heterotrophs due to their capability to use light and CO₂ as carbon and energy sources (Yamada et al., 2023). However, the production of valuable molecules from phototrophs such as cyanobacteria and microalgae at industrial scale is still very limited, among other factors because of relatively low yields, limited genetic engineering tools and their slow growth (Dias et al., 2019). In the case of cyanobacteria, it also shows limited abilities to direct fluxes towards acetyl-CoA and the TCA cycle, which constrains the range of products that can be made efficiently in this host (Zhao et al., 2022). Microbial co-culture of autotrophic and heterotrophic species has the potential to keep the advantages of both autotrophs (using CO₂ and light) and heterotrophs (more efficient genetic engineering and production yields) (Christie-Oleza et al., 2017). These co-cultures use division of labour, where one fix CO₂ and the other convert a carbon molecule derived from CO₂ into the final product.

Cyanobacteria species used for creating synthetic co-culture are limited to a few strains. The freshwater species *Synechococcus elongatus* PCC 7942 (Syn7942) and *S. elongatus* UTEX 2973 (Syn2973) have been mainly used (Fedeson et al., 2020; Gao et al., 2022; Li et al., 2023; Ma et al., 2022; Wang and Dai, 2023; Weiss et al., 2017; Zhang et al., 2020). Apart from it, marine cyanobacteria can also be considered as a promising phototroph in the co-culture. Because marine cyanobacteria grow well in seawater, which is abundant and affordable, its use can further help to reduce the cost of bioprocesses. In addition, it is known that salt stress induces sucrose production as an osmoprotectant, but it can also be used by the heterotroph as carbon sources (Gao et al., 2022; Lin et al., 2020; Weiss et al., 2017; Zhang et al., 2020). Other molecules rather than sucrose can also be used as bridge intermediates as shown in other studies with appropriate strain engineering (Therien et al., 2014).

As a partner of cyanobacteria, several heterotrophs have been evaluated in co-culture system. *Escherichia coli* was mainly used in a co-culture for producing molecules such as 3-hydroxypropionic acid, ethylene and isoprene, (Zhang et al., 2020, Cui et al., 2022). Polyketide production was shown in the co-culture of *S. elongatus* and *Aspergillus nidulans* (Feng et al., 2023). Yeasts including *Cryptococcus curvatus*, *Rhodotorula glutinis*, and *Saccharomyces cerevisiae* were co-cultured with *S. elongatus* (Li et al., 2017). Studies on bioproduction or establishment of co-culture with cyanobacteria and yeast, especially with the biotechnological workhorse *Y. lipolytica*, are still limited.

This work aimed to create a synthetic, marine, light-driven consortium based on the marine cyanobacterium *Synechococcus* sp. PCC 7002 (Syn7002), which is capable of living photoautotrophically, mixotrophically, and heterotrophically, and can survive under a wide spectrum of salt concentrations and temperatures (Kachel and Mack, 2020). It exhibits an exceptional tolerance to high light irradiation and is fast-growing (Kachel and Mack, 2020; Li et al., 2023). As a heterotrophic

pair, the industrial yeast *Yarrowia lipolytica* was selected due to its efficient genetic engineering tools, ability to produce a wide range of molecules, robust growth in diverse environments, and other advantages (Park and Ledesma-Amaro, 2023; Andreishcheva et al., 1999). In the designed co-culture, the engineered Syn7002 can use CO₂, light, and micronutrients to grow and provide sucrose to *Y. lipolytica*, which can convert it into value-added metabolites (Fig. 1). As a proof of concept, the production of β-caryophyllene, which has applications in pharmaceutical, cosmetics, and fuel industries (Yang and Nie, 2016; Cheng et al., 2022) was evaluated. In order to establish a stable co-culture between two species, media composition including NaCl and nitrogen, induction and inoculating conditions were also optimized.

# 2. Materials and methods

## 2.1. Strains, media, and growth conditions

Standard molecular cloning techniques were used (Sambrook and Green, 2012) and *E. coli* Top10 strain was used to amplify the recombinant plasmid DNA. *E. coli* strains were grown at 37 °C in LB medium (Lysogeny Broth) supplemented with appropriate antibiotics (kanamycin, 50 µg/mL; ampicillin, 100 µg/mL). All plasmids used in this study are listed in Table 1. Heterologous genes were codon-optimized to Syn7002 and *Y. lipolytica*, respectively. Gene sequences and their original sources used in this study are listed in Supplementary Table 1.

*Synechococcus* sp. PCC 7002 (Syn7002) and its engineered strains were routinely grown in either AD7 or MAD medium (Wlodarczyk et al., 2020, Supplementary Table 2). For induction of gene expression, 1 mM of IPTG was added to the media. All cultivations were performed at 30 °C at 150 rpm under continuous white LED illumination.

*Y. lipolytica* strains were grown in YPG (10 g/L of yeast extract, 20 g/L of peptone, and 20 g/L of glucose) or YNBG (1.7 g/L of yeast nitrogen base without amino acids and ammonium sulfate (YNB), 50 mM of phosphate buffer at pH 6.8 (17.1 g/L of KH₂PO₄ and 17.6 g/L of Na₂HPO₄), 5 g/L of NH₄Cl, and 20 g/L of glucose). Monocultures of *Y. lipolytica* were performed at 30 °C at 150 rpm.

## 2.2. Transformation and segregation of cyanobacteria

Cyanobacterial vectors were constructed using In-fusion™ kit (TakaraBio). Knock-out was designed by replacing the original gene sequence by antibiotics resistance while the insertions were joined in another vector to be expressed from a single promoter as an operon.

Cyanobacteria were grown in liquid AD7 medium until 0D<sub>730</sub> = 0.5, centrifuged at 5,000 rpm for 5 min and resuspended in AD7 to reach 0D<sub>730</sub> = 5.1–10 µg of plasmid was mixed with 200 µl of cell suspension and incubated at 29 °C with illumination (5 µmol photons m<sup>-2</sup>s<sup>-1</sup>) for 6 h with agitation at 200 rpm. Then, the transformation mixture was plated onto a 2 µm cellulose nitrate membrane filter (Schleicher & Schuell MicroScience GmbH, Germany) on an AD7 plate and the membrane was transferred after 24–36 h on an antibiotics plate and grown under illumination (20 µmol photons m<sup>-2</sup>s<sup>-1</sup>). For knock-outs, resistant

<figure><img src="image_6.png" /><figcaption>**Fig.1.** An artificial light-driven co-culture system based on marine cyanobacterium Syn7002 and the yeast *Y. lipolytica* for biosynthesis of β-caryophyllene.</figcaption></figure>
<table><caption>**Table 1**
Plasmids and strains used in this study.</caption><thead><tr><th>Plasmid name</th><th>Description</th><th>Reference</th></tr></thead><tbody><tr><td>pLS054</td><td>pJET1.2-PamsA-KanR-TamsA</td><td>This study</td></tr><tr><td>pSZT001</td><td>pSZT001</td><td>Selão et al. (2019)</td></tr><tr><td>pSZT002-plac-gfp</td><td>pSZT002-plac-gfp</td><td>Feng (2020)</td></tr><tr><td>pLS055</td><td>pSZT002-plac-cscB</td><td>This study</td></tr><tr><td>pLS057</td><td>pSZT002-plac-cscB-spsA-sppA-ugp</td><td>This study</td></tr><tr><td>RLA P1182</td><td>JMP62-URA3 ex-pTEF-ScSUC2</td><td>Lazar et al. (2013)</td></tr><tr><td>RLA P2441</td><td>JMP62-LEU2 ex-pTEF-QHS1-pTEF-HMG</td><td>This study</td></tr><tr><td colspan="3"><strong>Cyanobacterium</strong></td></tr><tr><td>RLAS1154</td><td>Synechococcus sp. PCC 7002 WT</td><td>This study</td></tr><tr><td>RLAS1010</td><td>Syn7002 plac-cscB</td><td>This study</td></tr><tr><td>RLAS1014</td><td>Syn7002 ΔamsA plac-cscB-spsA-sppA-ugp</td><td>This study</td></tr><tr><td colspan="3"><strong>Yarrowia lipolytica</strong></td></tr><tr><td>Pold</td><td>Mata, leu2-270, ura3-302, xpr2-322</td><td>Barth and Gaillardin (1996)</td></tr><tr><td>RLAS2873</td><td>Mata, leu2-270, ura3-302, xpr2-322 URA3 ex-pTEF-ScSUC2, LEU2 ex-pTEF-QHS1-pTEF-HMG</td><td>This study</td></tr></tbody></table>

colonies were re-streaked at least 3 times every 7 to 10 days, selecting 3–5 colonies each time. After several passages under selection, segregation of the non-transformed genome copies was confirmed using colony PCR using the Phire Plant Direct PCR Master Mix (Thermo Fisher Scientific, USA) using custom primers.

Syn7002 strains were grown at 30 °C and in constant atmospheric level of CO₂. In the preliminary experiment and when pre-grown for a transformation, the strains were grown at 100 rpm in tissue culture flasks (Corning, Inc., USA) with filter caps (0.2 µm).

## 2.3. Transformation of *Y. lipolytica*

Freshly grown *Y. lipolytica* on YPG plate was used for transformation. A loop of cells was resuspended with the transformation mix (50 % PEG MW4000, 90 µl; 2 M LiAc pH = 6, 5 µl; 2 M DTT, 5 µl). After adding the 1 µg of DNA cassettes and 5 µl of carrier DNA (10 mg/ml), cells were incubated for 30 min at 30 °C, then the heat shock was carried out for 10 min at 42 °C. The cells were spread on the selective media depending on their auxotrophy and grown for 2–3 days. The genes were randomly integrated into the genome, and the integration of heterologous gene was verified by colony PCR with Phire Plant Direct PCR Master Mix.

## 2.4. Co-culture for caryophyllene production

The medium for co-culture was modified from AD7 media with different salt concentration (0, 100, 200 and 308 mM of NaCl) and YN supplements (Both 1.7 g/L of YNB and 5 g/L of NH₄Cl). Co-culture was performed at 30 °C with 150 rpm under LED lights.

β-Caryophyllene is produced intracellularly in *Y. lipolytica*, so its extraction is required to quantify it. To facilitate the extraction of caryophyllene, we used a widely used method based on the *in situ* extraction during the fermentation. This was achieved by adding an organic solvent (dodecane 10 % (v/v)) to the medium.

## 2.5. Analysis of microbial growth, sucrose and β-caryophyllene

Growth of strains was assessed using a spectrophotometer (WPA Biowave II) by measuring the optical density (OD). The OD readings were taken at 600 nm for *Y. lipolytica* and 730 nm for Syn7002.

Sucrose in the culture supernatants was quantified by HPLC (the UltiMate 3000 system, Thermo Fisher Scientific, USA) equipped with an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-RAD, USA), UV detector, and Refractive index detector (Shodex, Japan). The mobile phase used was 0.01 N H₂SO₄ with a flow rate of 0.6 mL/min and the column temperature was 30 °C. Sucrose concentrations in the samples were determined using a calibration curve prepared with pure sucrose

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Construction of a marine cyanobacterium for sucrose accumulation and secretion. (A) The engineering strategy for sucrose biosynthesis and secretion in Syn7002. The blue arrows represent overexpression of genes, while the red circle indicates a gene deletion. (B) Sucrose secretion by engineered strains in AD7 and MAD media. (C-D) Growth of RLAS1014 in AD7 and MAD medium. The initial optical density at 730 nm (OD₇₃₀) was 0.05. All strains were cultivated for 14 days with IPTG induction using AD7 and MAD media. The values presented in the figures represent the averages of three biological replicates.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig.3.** Effect of monoculture condition of the engineered Syn7002 strain (RLAS1014) on sucrose secretion. Effect of different NaCl concentrations (0, 100, 200 and 308 mM) on sucrose production (A) and growth (B). Effect of different initial OD~730~ (2, 4, 6 and 8) on sucrose production (C) and growth (D). All experiments were conducted in AD7 medium at a temperature of 30 °C with agitation at 150 rpm. The values presented in the figures represent the averages of three biological replicates.</figcaption></figure>

(Sigma-Aldrich) at concentrations of 0.1, 0.2, 0.5, 2.5, 5, and 10 g/L.

β-Caryophyllene in the dodecane phase was quantified by GC-MS with a standard curve of (–)-*trans*-caryophyllene (GC grade, Sigma-Aldrich). After centrifuging the culture, the dodecane phase was directly injected into the GC-MS. The following parameters were used for the analysis: The substances were measured using the instrument Agilent 5977A GC-MSD and column Agilent DB5-MS. The injection parameters were: 240 °C, a split ratio of 20:1, and 1 µl of sample solution injected. The following temperature program was used: 100 °C for 2 mins; 165 °C at 10 °C/min; 170 at 1.5 °C/min; 170 °C for 2 mins; 187 °C for 1.5 °C/min; 280 at 30 °C/min; 280 °C for 2 mins; mass spectra: EI, positive, *m/z* 40–600, 1.3 spectra/sec. Three biological replicates were tested and calculated average and standard deviation values.

**3. Results and discussion**

**3.1. Engineering the marine cyanobacteria Syn7002 for sucrose secretion**

The first goal was to engineer the marine cyanobacterium Syn7002 to produce sucrose, which can serve as a carbon source for the partner species in co-culture. The complete engineering strategy is illustrated in Fig. 2A. Initially, candidate genes for overexpression and deletion were selected based on previous research on sucrose-producing freshwater cyanobacteria (Gao et al., 2022; Lin et al., 2020). The *cscB* gene from *E. coli*, known for its role in sucrose secretion (Ducat et al., 2012), was heterologously expressed in Syn7002 after codon optimization. Two native genes, *spsA* (sucrose phosphate synthase) and *sppA* (sucrose phosphate phosphatase), were overexpressed to enhance intracellular sucrose accumulation (Cumino et al., 2010; Ehira et al., 2014). Additionally, the *ugp* (UDP-glucose pyrophosphorylase) gene, essential for sucrose production in *Synechocystis* sp. PCC 6803 (Syn6803) (Kirsch et al., 2018), was heterologously expressed. The expression of genes was controlled by clac143 promoter (Markley et al., 2015) and the

expression cassettes were integrated into a neutral site (NSI) (Davis et al., 2014). Subsequently, *amsA*, a gene encoding amylosucrase, which converts sucrose into glucose and fructose, was deleted to increase sucrose accumulation (Perez-Cenci and Salerno, 2014; Kirsch et al., 2018; Ducat et al., 2012).

The engineered strains were cultivated in two different media, AD7 and modified AD7 (MAD) with higher NaNO₃ content (Włodarczyk et al., 2020), to evaluate the production of sucrose. As shown in Fig. 2B, the strain expressing *cscB* (RLAS1010) secreted approximately 0.6 g/L of sucrose after 14 days of fermentation. The RLAS1014 strain, which additionally carried the cassette for overexpressing *spsA*, *sppA* and *ugp* in the *asmA* knock-out background, exhibited further improvement in sucrose concentration, reaching 2.6 g/L in AD7 medium. This is more than 2 times higher sucrose secretion compared to the previous study that engineered Syn7002 strain (Δ*ggpS*, *sps6803-spp6803*, *cscB*, Δ*amsA*) (Han et al., 2023). Cultivation in MAD medium resulted in lower production of sucrose (1.8 g/L) than that in AD7 medium despite showing a higher biomass (Fig. 2C–D, Fig. S1). The lower nitrogen levels in AD7 compared to MAD medium seem to lead to a higher production of sucrose, in accordance with previous studies studying nitrogen limitation or depletion conditions in cyanobacteria (Arias et al., 2021). Consequently, the AD7 medium was chosen for subsequent experiments.

**3.2. Optimizing the culture condition of the engineered cyanobacteria for sucrose secretion**

It is known that sucrose acts as an osmoprotectant under salt stress in cyanobacteria (Santos-Merino et al., 2023). Therefore, sucrose production and secretion can be modulated by increasing osmotic pressure as shown in previous studies (Gao et al., 2022; Lin et al., 2020; Weiss et al., 2017; Zhang et al., 2020). The impact of different NaCl concentrations on growth and sucrose yield of the engineered strain, RLAS1014, was evaluated (Fig. 3A and B). Interestingly, RLAS1014 was capable of
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Engineering of *Y. lipolytica* for producing β-caryophyllene from sucrose. (A) Biosynthetic pathway of β-caryophyllene from sucrose in *Y. lipolytica*. Overexpression is indicated by blue arrow and gene names in blue, multiple reactions are indicated as dashes arrows. (B) Growth of the control and RLAS2873 in glucose medium (YNBG) and sucrose medium (YNBS). (C) Sucrose consumption of RLAS2873. (D) β-Caryophyllene production of the control and RLAS2873 on sucrose medium (YNBS). (E) Effects of YNB or/and NH₄Cl on the growth of RLAS2873 in AD7-based medium with 20 g/L sucrose. Control represented no supplementation. (F) Effects of different initial sucrose and NaCl concentrations on β-caryophyllene yield in AD7 + YN-based media. The values are averages of three biological replicates.</figcaption></figure>

exporting sucrose even without NaCl supplementation, reaching 0.8 g/L after 24 days of IPTG induction. The optimal NaCl concentration was determined to be 100 mM, which was subsequently utilized in the following experiments. Under this condition, sucrose concentrations in the media reached 0.6, 1.7, and 3.6 g/L at 8, 16, and 24 days after IPTG induction, respectively. It has been previously reported that *S. elongatus* PCC 7942 from freshwater exhibits slower growth as NaCl concentrations increase (Abramson et al., 2016). However, the growth of RLAS1014 did not appear to be influenced by the NaCl concentration (Fig. 3B). This distinction from freshwater cyanobacteria could be considered an advantage in the context of sucrose production.

While previous studies have reported significant sucrose production by freshwater cyanobacteria during the stationary growth phase (Lin et al., 2020), it is revealed that sucrose accumulation initiated during the exponential growth stage, and the rate of accumulation was positively correlated with cell density with marine cyanobacteria in this study. To enhance the efficiency of sucrose accumulation, the effect of initial cell density on sucrose yield was investigated (Fig. 3C). Higher initial cell densities markedly accelerated sucrose accumulation in the media. The average rates of sucrose accumulation during the first eight days with different initial OD<sub>730</sub> values (2, 4, 6 and 8) were 70, 90, 110, and 120 mg/L/d, respectively. The production rates of sucrose for cultures with initial OD<sub>730</sub> values of 6 and 8 remained relatively stable over the subsequent 16 days. On the other hand, cultures with initial OD<sub>730</sub> values of 2 and 4 exhibited an increasing trend, with the rate rising from 170 mg/L/d to 240 mg/L/d, before and after 16 days of induction, respectively. Interestingly, after 24 days of induction, the accumulation of sucrose appeared to be similar across the four different initial cell concentration conditions. These results suggest that the initial cell density primarily influences the initial rate of sucrose accumulation, while it may not significantly affect the final total sucrose production.

Furthermore, the effect of initial OD<sub>730</sub> on the growth of RLAS1014 was also assessed (Fig. 3D). IPTG induction showed a slight inhibitory effect on cyanobacterial growth. Increasing the initial cell density appeared to be a viable strategy for enhancing sucrose secretion rates.

However, it should be noted that increasing the initial cell density also accelerated the entry of cells into the stationary growth phase. This explains why the strain with an initial OD<sub>730</sub> at 8 exhibited a decrease in OD<sub>730</sub> after 16 days, while strains with lower initial OD<sub>730</sub> values were still in the logarithmic growth phase. Upon combined analysis of the growth curve and sucrose production curve, an initial OD<sub>730</sub> value of 6 was selected for subsequent experiments.

### 3.3. Constructing a *Y. lipolytica* strain able to produce β-caryophyllene from sucrose

*Y. lipolytica* is well-known for its ability to accumulate high levels of lipids, which is attributed to its substantial pool of acetyl-CoA (Park and Ledesma-Amaro, 2023). This acetyl-CoA abundance creates a favorable environment for terpene production through mevalonate pathway (Larroude et al., 2018; Zhang et al., 2023). In this study, β-caryophyllene, a sesquiterpene, was selected as a proof-of-concept product. While β-caryophyllene has been successfully produced heterologously in model microorganisms such as *E. coli* and *S. cerevisiae* (Cheng et al., 2022; Yang and Nie, 2016), there is currently no report on its synthesis in *Y. lipolytica*. The pathway leading to β-caryophyllene via the mevalonate pathway in *Y. lipolytica* is depicted in Fig. 4A. In this study, the engineered strain by overexpressing the β-caryophyllene synthase gene (QHS) from *Artemisia annua* and the HMG-CoA reductase (HMG) gene from *S. cerevisiae* catalyzing the rate-limiting step in terpene synthesis (Li et al., 2021) under the strong constitutive pTEF promoter. The parental strain used in all experiments, Po1d, had already been engineered to utilize sucrose (Lazar et al., 2013). However, previous observations by Lazar et al. (2013) indicated limited growth of Po1d on sucrose when compared to a longer version of the *ScSUC2* gene (inversion from *S. cerevisiae*). Therefore, the longer version of *ScSUC2* was additionally overexpressed under the pTEF promoter. By combining these two strategies, an engineered *Y. lipolytica* strain (RLAS2873) capable of producing a similar amount of β-caryophyllene (about 28 mg/L) from 20 g/L of either sucrose or glucose as a carbon source was
<figure><img src="image_10.png" /><figcaption>**Fig.5.** Establishment of a cyanobacteria- *Y. lipolytica* symbiotic system. (A) A brief scheme of sequential and co-culture with different factors for the optimization. (B) Effect of different initial *Y. lipolytica* density in co-culture system on β-caryophyllene yield. Effects of different IPTG induction time (0, 8, 16 and 24 d) on β-caryophyllene yield by sequential culture (C) and co-culture (D). The values are averages of three biological replicates.</figcaption></figure>

successfully constructed (Fig. 4D). Moreover, RLAS2873 strain showed no prolonged lag phase on sucrose that was shown in Po1d, indicating successful engineering of the strain for efficient sucrose consumption (Fig. 4B). β-Caryophyllene production can be further improved by engineering precursor pathway (mevalonate) as well as multi-copy expression, organelle compartmentalization, balancing co-factors, and other metabolic engineering strategies (Yang et al., 2024).

When considering the optimization of culture conditions for the co-culture system, several factors need to be considered. Two important factors are the nitrogen source and the microelements. Therefore, different nutrient supplementation in AD7 media was evaluated to assess their impact on the growth of RLAS2873. The strategies evaluated were as follows: no additional supplementation (as control), supplementation with 1.7 g/L yeast nitrogen base without amino acids and ammonium sulfate (YNB, as microelements), 5 g/L NH₄Cl, or both YNB and NH₄Cl (YN). As shown in Fig. 4E, it was evident that the nitrogen-containing compounds present in AD7 medium, such as NaNO₃, vitamin B₁₂, and Na₂EDTA, were insufficient to support the growth of RLAS2873. It is observed that the addition of both YNB and NH₄Cl (YN) yielded the best growth, and this formulation (AD7 + YN) was chosen for subsequent experiments. Furthermore, the growth of the original strain Po1d and RLAS2873 was compared in two different media: AD7 + YN + G and AD7 + YN + S (Fig. 4B). Similar to the trend in YNBG and YNBS, (i) the growth curves of RLAS2873 in AD7 + YN + G and AD7 + YN + S media were almost identical; (ii) the growth on glucose (YNBG or AD7 + YN + G), RLAS2873 showed slower growth, which could be associated with gene expression burden (Cui et al., 2021). RLAS2873 strain completely degraded 20 g/L of sucrose into glucose and fructose after 24 h of cultivation, and around 2 g/L of fructose was left at the end of cultivation (Fig. 4C).

Then, the relationship between initial sucrose concentration and β-caryophyllene yield was evaluated in selected medium AD7 + YN + S

(Fig. 4F). It is observed that β-caryophyllene production was gradually increased according to the increased sucrose concentration from 2.5 to 10 g/L (Fig. 4F), which indicates that sucrose concentration is an important factor influencing β-caryophyllene yield. Additionally, the effect of NaCl concentration, a key factor for cyanobacteria growth and sucrose production, was also investigated. *Y. lipolytica* exhibited high tolerance to various NaCl concentrations from 100 to 308 mM in AD7 + YN+ media (Fig. 4F), consistent with previous reports (Mamaev and Zbyagilskaya, 2021). Although β-caryophyllene yield showed a slight increase with decreasing NaCl concentration, the effect was not significant. In addition, the AD7 + YN+ medium appeared slightly more favorable than MAD + YN+ for β-caryophyllene production (Fig. 4F). Therefore, AD7-based medium with 100 mM NaCl was selected in the following co-culture experiments.

### 3.4. Establishing a marine cyanobacteria- *Y. lipolytica* symbiotic synthetic community

In synthetic communities for biosynthesis, optimizing inoculation ratios often has shown to be important (Wang et al., 2020; Yuan et al., 2020). Different *Y. lipolytica* inoculation ratios were evaluated for β-caryophyllene production. For this, we tested one of the fermentation broths of cyanobacteria (OD₇₃₀ = 13.8, 0.9 g/L of sucrose at the harvest). Different initial ODs of *Y. lipolytica* were inoculated into the broth from 0.1 to 0.8. β-Caryophyllene production was gradually increased from 0.2 to 2.4 mg/L by increasing initial OD (Fig. 5B). Therefore, an OD₆₀₀ of 0.8 was selected for *Y. lipolytica* inoculation in the co-culture.

Next, optimization of other culture parameters was performed in the co-culture. Different times for IPTG induction (0, 8, 16, 24 days) led to different final OD₇₃₀ values (6, 13.8, 21.0, 23.8) and sucrose concentrations (0, 0.9, 2.6, 4.0 g/L). Starting the co-culture at these different points resulted in different production of β-caryophyllene in two
<figure><img src="image_11.png" /><figcaption>**Fig.6.** Effect of YP and YN supplementation on caryophyllene production. (A) Effect of different nitrogen source on β-caryophyllene production under different cultivation condition. (B) Color changes of fermentation broths with different nitrogen source under different cultivation conditions. The values are averages of three biological replicates.</figcaption></figure>

different experimental setups (Fig. 5A). While using the cyanobacteria-
filtrated culture broth for inoculating *Y. lipolytica* in the sequential
culture, *Y. lipolytica* was directly added into cyanobacteria culture in the
co-culture. The factors including induction time and YN supplementa-
tion were tested in both conditions.

In the sequential culture, 4.3 and 8.6 mg/L of β-caryophyllene were
obtained without and with YN supplementation, respectively (Fig. 5C).
In the co-culture, 1.8-fold higher β-caryophyllene was produced without
YN supplementation (Fig. 5D). Similarly, a bit higher production was
shown in the co-culture with YN supplementation. It is revealed that
additional nitrogen source is crucial for β-caryophyllene from
*Y. lipolytica* in the AD7-based co-culture. In addition, it is observed a
linear relationship between the initial sucrose concentration and β-car-
yophyllene production by either co- or sequential-cultivation (Fig. S2)
indicating the importance of initial sucrose concentration in co-culture
performance.

Having seen the importance of the nutrient supplements in β-car-
yophyllene production, the addition of yeast extract and peptone (YP:
10 g/L yeast extract and 20 g/L peptone) was evaluated in both co-
cultivation and sequential cultivation at the best performing condi-
tion: IPTG induction for 24 days (Fig. 6). Notably, the highest yield of
β-caryophyllene was obtained with YP supplementation, reaching 14.1
mg/L by co-culture which is 31 % higher than the one from sequential
culture (Fig. 6A). After 6 days of co-cultivation, the color of the
fermentation broth changed from green to white, which was distin-
guishable from the other fermentation broths without nitrogen supple-
mentation (Fig. 6B). This change indicated that YP further supported the
growth of *Y. lipolytica*, leading to an increase in yeast biomass and
β-caryophyllene production in both sequential and co-culture (Fig. S3).
However, it is important to note that the YP is a non-defined, complex
media supplement which may also contain sucrose or other carbon
sources, which may affect on *Y. lipolytica* growth and β-caryophyllene
production.

4. Conclusion

Using low-cost substrates is of interest in a bioproduction for
economical feasibility and sustainability. In this study, we established
the co-culture between *Synechococcus* sp. PCC 7002 and *Y. lipolytica* to
convert CO₂ into value-added compound. Both strains were respectively
engineered to secrete high amount of sucrose and to produce β-car-
yophyllene from sucrose. The optimization of the induction, media
composition (salt, nitrogen), and inoculation amount resulted in 14.1
mg/L of β-caryophyllene from the co-culture. Our study builds the
foundations of further improvement in cyanobacterial-yeast co-culture
that can be applied to bioproduction at industrial scale in the future.

CRediT authorship contribution statement

**Wenchao Chen:** Writing – original draft, Methodology, Investiga-
tion, Formal analysis. **Young-Kyoung Park:** Writing – review & editing,
Supervision, Methodology. **Lucie Studená:** Methodology, Investigation,
Formal analysis. **David Bell:** Methodology, Formal analysis. **Piotr
Hapeta:** Writing – review & editing, Methodology, Formal analysis.
**Jing Fu:** Methodology, Formal analysis. **Peter J. Nixon:** Writing – re-
view & editing, Resources, Methodology. **Rodrigo Ledesma-Amaro:**
Writing – review & editing, Supervision, Resources, Project adminis-
tration, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Data availability

Data will be made available on request.

Acknowledgements

We appreciate Professor Don Bryant for kindly providing the *Syn-
echococcus* sp. PCC 7002 WT strain. R.L-A. received funding from BBSRC
(BB/R01602X/1, BB/T013176/1, BB/T011408/1 - 19-ERACoBioTech-
33 SyColim, BB/X01911X/1, BB/Y008510/1 – Engineering Biology
Hub for Microbial Foods), EPSRC (AI-4-EB BB/W013770/1, and EEBio
Programme Grant EP/Y014073/1), Yeast4Bio Cost Action 18229, Eu-
ropean Research Council (ERC) (DEUSBIO - 949080), the Bio-based
Industries Joint (PERFECOAT- 101022370) under the European
Union's Horizon 2020 research and innovation programme and the
European Innovation Council (EIC) under grant agreement No.
101098826 (SKINDEV). Imperial College London UKRI Impact Accel-
eration Account (EPSRC-EP/X52556X/1, BBSRC-BB/X511055/1). Y.-
K.P. received the funding from the Bio-based Industries Joint (PERFE-
COAT- 101022370) under the European Union's Horizon 2020 research
and innovation programme. We acknowledge that the synthetic gene
QHS1 was obtained from the plasmid constructed by Videra Bio.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2024.131232.
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