Goal： The carbon sequestration capacity of kelp is enormous, and it can be produced continuously in a sustainable manner. However, the carbon-sequestered material is not yet fully utilized, and there remains significant potential for its application, which limits the larger-scale development of kelp. Through analysis of kelp composition, we have found that it contains a variety of nutrients, which can make it a food source for microbial strains, unlocking even more value. We plan to engineer a strain of Saccharomyces cerevisiae capable of utilizing kelp as a carbon source and design a process route that converts the kelp-derived carbon into the product, limonene.

Figure 1. Target: Construct a Saccharomyces cerevisiae strain capable of utilizing kelp-derived carbon sources.

Figure 2. Target：Design a process pathway from kelp-derived carbon sources to the product—limonene.

Limonene is a natural monoterpenoid compound, a type of terpenoid, widely found in the peels of citrus fruits such as lemons, oranges, and grapefruits, and is known for its distinctive fresh citrus scent. There are two main isomers of limonene: limonene and L-limonene, with limonene being the most common form and the main component responsible for the citrus aroma. Limonene production in the downstream section of the MVA (Mevalonate Pathway) occurs through two pathways.

The first is the traditional pathway, where IPP and DMAPP are catalyzed by the ERG20 enzyme to synthesize GPP. GPP is then converted into limonene by an exogenous enzyme expressed by ls (limonene synthase).

The second is a more widely used strategy that introduces exogenous ndps1, expressing the NDPS1 enzyme, which catalyzes IPP and DMAPP to synthesize NPP (an isomer of GPP). NPP is then converted to limonene by the enzyme expressed by d-ls (limonene synthase).

Figure 3. Dual pathways for the synthesis of limonene

Since the second pathway is more efficient in producing limonene, we introduce both exogenous ndps1 and ls genes to create a dual-pathway synthesis of limonene in yeast. The ndps1 and ls genes we selected are based on the most optimal combination from the literature^[1], where ndps1 is derived from tomato and d-ls from citrus fruits (hereafter, unless otherwise specified, d-ls and ls refer to limonene synthase).

Since different combinations of gene expression levels significantly affect yield, we need to select multiple promoter and terminator combinations (referred to as Promoter1, Terminator1, Promoter2, and Terminator2, abbreviated as PTPT) for ndps1 and ls, constructing expression vectors to test them. The goal is to find the optimal PTPT for further experiments.

Figure 3.Parameters of the kelp growth function

Vector construction involves three main steps: exogenous gene optimization and synthesis, amplification, and assembly.

First Step:
Since ndps1 and ls are derived from Solanum lycopersicum and citrus respectively, their codon preferences differ from those of Saccharomyces cerevisiae. Therefore, we optimized the codons for yeast and had the genes synthesized by GENEWIZ Biotechnology (Suzhou, China). The synthesized genes were constructed into the pUC series vectors.

Second Step:
We need to obtain the ndps1 and ls target fragments. The two recombinant plasmids (pUC-GW-Kan-ndps1 and pUC-GW-Kan-limonene synthase) were transformed into E. coli. Positive transformants were selected from the Patch (K+) plate and cultured overnight in LB liquid medium (K+), after which the plasmid was extracted. Using pUC-GW-Kan-ndps1 and pUC-GW-Kan-limonene synthase as templates, ndps1-F/R and ls-F/R primers were used with PrimeStar Mix polymerase for PCR amplification to obtain the ndps1 and ls target fragments, followed by PCR product purification.

Next, different promoters and terminators were assembled onto the ends of the ndps1 and ls target fragments. Since there are multiple PTPT combinations, here we take PTPT = (CCW12p, RPL3t, TDH3p, FBA1t) as an example. Primers were designed based on the PTPT combination (with correct homologous arms). Six fragments were amplified using PrimeStar Mix polymerase. CCW12p, ndps1, and RPL3t were assembled by homologous recombination, as were TDH3p, ls, and FBA1t, resulting in two fragments. The two fragments were then recombined to form one.

Next, we need to obtain the backbone of plasmid 352. Using YEp352 as a template, the Backbone-F/R primers and PrimeStar Mix polymerase were used to perform PCR, yielding a linearized YEp352 backbone.

Figure 5. Backbone Plasmid Map

Third Step:
The CCW12p-ndps1-RPL3t-TDH3p-ls-FBA1t fragment was recombined with the linearized YEp352 backbone through homologous recombination, and the resulting product was transformed into E. coli. Positive transformants from the Patch (A+) plate were selected and cultured overnight in LB medium(A+), after which the plasmid was extracted. The plasmid was then transformed into yeast, and successfully transformed yeast was activated in SDΔU. Activated yeast was preserved in 60% glycerol. (Refer to the protocol for transformation, LB (A+) medium preparation, plasmid extraction, PCR amplification, PCR product purification and homologous recombination).

As an example, the constructed plasmid with PTPT = (CCW12p, RPL3t, TDH3p, FBA1t) is shown below:

Figure 6. Constructed Expression Plasmid Map

Following the steps in 1.2, multiple PTPT_ndps1-ls vectors were constructed. After transforming them into yeast, shake flask fermentation tests were conducted. Since limonene is sparingly soluble in water (13.8 mg/L) and highly volatile, we adopted a two-phase fermentation system consisting of 10 mL SDΔU and 2 mL IPM (isopropyl myristate). IPM is a colorless, transparent oily liquid, insoluble in water, and capable of absorbing volatile limonene, which facilitates subsequent gas-phase detection. The culture conditions were 30°C, 220 rpm for 48 hours with an initial OD₆₀₀ of 0.05. (Refer to the protocol for SDΔU medium preparation, gas-phase detection methods, and the creation of a standard limonene content curve).

The results are as follows:

Figure 7. The yields of different PTPT of expression plasmid

The two-phase fermentation results showed that PTPT = (CCW12p, RPL3t, TDH3p, FBA1t) performed best among the six combinations. Unless otherwise specified, p352-IV plasmid with PTPT = (CCW12p, RPL3t, TDH3p, FBA1t) will be used in the subsequent sections 2, 3, and 4.

Enhancing the ability to synthesize more limonene from kelp-derived carbon sources is crucial. This would allow for a higher yield of limonene from the same amount of kelp during fermentation, improving carbon conversion efficiency and fully utilizing kelp. In metabolic pathways, limonene synthesis primarily depends on the MVA pathway. Since the flux of the MVA pathway in wild-type yeast is relatively low, this may result in kelp-derived carbon being consumed by other pathways. Therefore, we use CRISPR/Cas9 technology to insert key enzyme genes from the MVA pathway into the yeast genome, achieving multiple copies of these key enzymes. Since inserting new genes into ORF sites would disrupt the function of the original genes (equivalent to knockout), we also knock out two key global regulatory repressor genes simultaneously.

Figure 8. Key steps of the MVA pathway

ROX1 is a transcriptional repressor mainly responsible for inhibiting the expression of hypoxic response genes under aerobic conditions. It binds to the promoter regions of target genes, preventing their transcription. After ROX1 is knocked out, this repression is lifted, allowing the cell to adapt to low-oxygen environments.

tHMG1 (truncated HMG-CoA reductase 1) is a shortened version of HMG-CoA reductase. HMG-CoA reductase is the key rate-limiting enzyme in the MVA pathway, catalyzing the reduction of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) to mevalonate. tHMG1, as a truncated version of HMG1, has lost part of its regulatory domain and is typically used to enhance metabolic flux when overexpressed, increasing the production of terpenoid compounds.

IDI1 (Isopentenyl-diphosphate Δ-isomerase 1) encodes IPP isomerase, a key enzyme in the mevalonate pathway responsible for converting IPP (isopentenyl diphosphate) to DMAPP (dimethylallyl diphosphate). IPP and DMAPP are the two fundamental building blocks in isoprenoid biosynthesis. IPP isomerase maintains the balance of these molecules, providing essential precursors for the synthesis of terpenoids and isoprenoid compounds. Overexpression of IDI1 can enhance the production of downstream MVA pathway products.

Therefore, we insert tHMG1 and IDI1 at the ROX1 locus. This transformation requires three elements: linear donor DNA (tHMG1, IDI1), a Cas9 protein plasmid, and a ROX1 gRNA plasmid.

Figure 9. Genome Editing Experimental Route

Taking ΔROX: tHMG1, IDI1 as an example. Acquisition of linear donor DNA (tHMG1, IDI1): The sequences of tHMG1 and IDI1 can be obtained from https://yeastgenome.org. The SGD ID for HMG1 is _S000004540, and IDI1, it is _S000006038, respectively. After obtaining the sequences, primers with homologous arms are designed to amplify the target fragments via yeast PCR. Finally, homologous recombination is performed, following the same method for promoter and terminator combinations as in section 1.2, resulting in the donor fragment of tHMG1-IDI1.

Acquisition of Cas9 protein: The Cas9 protein plasmid is first transformed into yeast. Since the Cas9 protein plasmid carries the Trp synthesis gene, the engineered strain (ΔTrp) can be cultured in SDΔT medium to maintain the plasmid.

Acquisition of ROX1 gRNA plasmid: Various PAM sites (NGG) can be found on the yeast genome. After selecting an appropriate PAM (with a high score and proximity to the start codon), the first 20 bp is chosen as the guide RNA sequence. Using an existing gRNA plasmid as a template, a non-guide fragment is selected as the backbone. Primers are then designed, and the target guide RNA sequence is added to one side of the primer, with homologous arms (40 bp upstream and downstream of the guide RNA and PAM sequence in the yeast genome) designed at both ends.

Once the three elements are ready, the donor DNA and gRNA plasmid are co-transformed into yeast (which already contains the Cas9 protein plasmid). Positive transformants are selected and streaked on Patch plates. Positive Patches are subjected to yeast colony PCR, and the desired fragments are picked for transfer to YPD media. After 24 hours, the strain is preserved using 60% glycerol. The PCR product from yeast colonies is purified and sequence verified by Sangon Biotech Co., Ltd (Shanghai).

The yeast colony PCR results are as follows:

Figure 10. Results of the agarose gel electrophoresis

The specific function of YPL062W in yeast has not been fully determined, and it is currently classified as a hypothetical or unknown function protein. However, studies have shown that ΔYPL062W increases the production of all major terpenoids (C₁₀, C₁₅, C₂₀, C₃₀, and C₄₀).

ERG12 (mevalonate kinase) is another key enzyme in the MVA pathway, catalyzing the conversion of mevalonate to mevalonate-5-phosphate, a necessary step toward isoprene biosynthesis. Overexpression of ERG12 can accelerate the MVA pathway flux.

The steps for this genetic modification are similar to those in section 2.1. The SGD ID for ERG12 is _S000004821.

The yeast colony PCR results were as follows:

Figure 11. Results of the agarose gel electrophoresis

From the metabolic pathway, it is clear that the downstream of the MVA pathway not only leads to limonene but also to squalene.

Squalene, as an important lipid precursor in organisms, is a key intermediate in the synthesis of sterols (including cholesterol) and other biologically active molecules. It is further converted into sterols (e.g., ergosterol), which is a crucial component of yeast cell membranes, essential for maintaining membrane fluidity and integrity. Therefore, the synthesis of squalene is tightly coupled with cell growth.

Consequently, prematurely synthesizing limonene during the fermentation process may create excessive competition between limonene synthesis and strain growth, leading to slower growth in the early stages, which in turn reduces production efficiency. To address this, we designed a dynamic regulatory gene circuit to decouple limonene synthesis from strain growth.

Figure 12. Limonene biosynthesis pathway and ERG20

Additionally, through liquid-phase analysis using the sodium tetraborate sulfuric acid-hydroxybiphenyl method to analyze the composition of kelp hydrolysate, we found that kelp (dry weight) contains approximately 0.4 g/g alginate, 0.15 g/g mannitol, and 0.02 g/g glucose. The composition content also serves as an important basis for designing the regulatory circuit.

Figure 13. Ingredients of Kelp

ERG20 is one of the key enzymes in the MVA pathway in yeast and is a bifunctional enzyme. It is responsible for synthesizing GPP from IPP and DMAPP, and further converting GPP into FPP. Typically, the ERG20 promoter (ERG20p) is a constitutive promoter.

The HXT1 promoter (HXT1p) is highly sensitive to external glucose concentrations. It is strongly activated under high glucose conditions, driving high-level expression of downstream genes. When glucose levels are low, the activity of the HXT1p promoter decreases.

Based on the basic principles of these elements, we designed the following strategy: Replace the constitutive ERG20p element with the glucose-inducible HXT1p element. The design rationale is that during the early stages of fermentation, the engineered yeast can rapidly grow using the glucose present in the kelp hydrolysate. However, the glucose in the kelp hydrolysate will be quickly depleted. At this point, the yeast will switch to using the more abundant mannitol in the kelp as a carbon source, with energy and resources increasingly directed toward limonene synthesis.

After knocking in the gene using CRISPR/Cas9 technology, the results of yeast colony PCR are as follows:

Figure 14. Results of the agarose gel electrophoresis

The ERG20F96W mutation significantly alters the substrate preference of farnesyl pyrophosphate synthase, changing its affinity for other substrates during the conversion of IPP and DMAPP into FPP. This mutation favors the production of GPP over FPP. As a result, it reduces the competition between limonene synthesis and cell growth, promoting limonene production.

Figure 16.Limonene biosynthesis pathway and the action point of ERG20p and ERG20^F96W After knocking in the gene using CRISPR/Cas9 technology, the results of yeast colony PCR are as follows:

Figure 15. Results of the agarose gel electrophoresis

The fermentation test results of 1, 2, and 3 are as follows:

Figure 16. Limonene production of different strains

The result shows that the strain's limonene production capacity has steadily improved (in SDΔU medium). Limonene yield increased from undetectable levels in the wild type to 88.2 mg/L in strain AG04.

Based on the PTPT-ndps1-ls plasmid constructed in section 1.3, we integrated PTPT-ndps1-ls fragment from the plasmid level to the genome to prevent plasmid loss when grown in kelp hydrolysate medium without selection markers. This ensures that the strain can stably and permanently synthesize limonene in kelp hydrolysate medium. GuideRNA plasmid design and primer design refer to section 2.1.

Figure 17. Design of the donor DNA

Figure 18. Results of the agarose gel electrophoresis

Adaptive Laboratory Evolution (ALE) is a method of long-term cultivation and evolution in a lab environment to select yeast strains with specific target traits, such as resistance, metabolic capacity, or environmental adaptability. Since mannitol is the most abundant utilizable component in kelp, we chose mannitol as the sole carbon source for ALE. We aim to enhance yeast's ability to utilize kelp hydrolysates through ALE.

Figure 19. Schematic diagram of Mannitol-ALE

We used a two-phase ALE system, consisting of 10 mL SDΔT and 2 mL IPM. The cultivation conditions were 30°C, 220 rpm, and an initial OD of 0.1. Each time the OD reached 3, the culture was transferred to the next generation.

Figure 20. Growth of the yeast after 3 generations of mannitol ALE

Due to the thick cell walls of kelp, the release of its nutrient content is hindered. Additionally, the polysaccharides in kelp are difficult for yeast to utilize directly. Therefore, we implemented the following treatment process to transform kelp into a more nutrient-rich medium.

Figure 21. Kelp decomposition and fermentation process

Our approach starts by drying untreated kelp to reduce its moisture content for easier handling and processing. The dried kelp is then ground into powder to increase its surface area, facilitating subsequent chemical reactions. Next, the powdered kelp undergoes acid hydrolysis, which breaks down the polysaccharides and other complex molecular structures into smaller molecules. After acid hydrolysis, the kelp is neutralized and subjected to enzymatic hydrolysis, further breaking down molecules into simpler sugars and other fermentable substances through enzymatic action. Finally, the hydrolyzed kelp is filtered to create the kelp-based medium.

Since the amount of enzyme used directly affects the breakdown of kelp cell walls and the hydrolysis of polysaccharides into usable sugars, and the concentration of the medium influences the concentration of nutrients, these factors need to be optimized. Additionally, as the engineered strain is deficient in uracil, tryptophan, and histidine, kelp might lack these essential amino acids.

Therefore, we explored the following variables in the process:
1. Amino acid supplementation;
2. Enzyme quantity;
3. Kelp hydrolysate medium concentration.

Amino Acid Supplementation Investigation:
Amino acid supplementation: For every 100 mL of kelp-based medium, 1 mL of amino acid stock solution was added. The stock solution formula is as follows: 10 mL water, 0.62g Drop-out supplement (ΔLeu/ΔTrp/ΔUra), 0.06g Leu, 0.04g Ura, 0.06g Trp.
Based on fermentation tests, we found that the addition of amino acids enhanced the strain's ability to utilize kelp for limonene synthesis. Fermentation conditions: The media were 10 mL SM, kelp hydrolysate (without amino acid supplementation), and kelp hydrolysate (with amino acid supplementation). 2 mL IPM was overlaid. Cultivation conditions were 30°C, 220 rpm, initial OD=0.05. Due to the presence of flocculants in the kelp hydrolysate, OD could not be measured, so no OD data was available.

Figure 22. Effects of amino acid supplementation on limonene production in kelp

The results showed that in the kelp hydrolysate medium without amino acid supplementation, the yeast barely grew, and limonene production was negligible. However, after amino acid supplementation, the limonene yield surpassed that in the SD medium. This suggests that although kelp contains sufficient sugars, it lacks some essential amino acids, which hinder yeast growth. Our earlier experiments overlooked this information, delaying progress.

About the amount of enzymes used:

Figure 23. The composition of kelp hydrolysate corresponding to different enzyme amounts

Investigation of Kelp Hydrolysate Medium Concentration on Fermentation:

Figure 24. 3% enzyme, unconcentrated

Figure 25. 3% enzyme, concentrated 2x

Results of fermentation with kelp hydrolysate medium with different concentrations:

Figure 26. Fermentation results of different culture media

The results indicated that the concentrated (2x) kelp hydrolysate with amino acid supplementation led to higher yields compared to the non-concentrated kelp hydrolysate. Furthermore, it was found that the limonene yield in the concentrated kelp hydrolysate with amino acid supplementation was still in a rapid growth phase at 96 hours.

The fermentation system consists of 200 mL of kelp-based medium (3% enzymatic hydrolysis, unconcentrated, supplemented with amino acids) overlaid with 40 mL IPM. The culture conditions are 30°C, 220 rpm for 144 hours. Every 48 hours, 500 µL of the upper IPM layer is sampled to measure limonene content, and 20 mL of kelp hydrolysate medium (3% enzymatic hydrolysis, 2x concentrated, supplemented with amino acids) is added. After 144 hours, another 500 µL of the upper IPM layer is sampled to measure limonene content.

Figure 27. Fed-batch fermentation

The fermentation results are as follows:

Figure 28. Results of fed-batch fermentation

Upon reviewing the literature, we found no established process for isolating and purifying limonene from IPM. Therefore, we designed the following separation and purification protocol. First, we separated the organic phase from the upper IPM layer obtained in Section 7.2. This was followed by vacuum rotary evaporation.

Figure 29. Schematic diagram of vacuum rotary evaporation device

Table 1. Physicochemical properties of limonene and IPM

Substance	d- limonene	IPM
Molecular formula	C10H16	C17H34O2
Density g/mL	0.844	0.864
M.W g/mol	136.24	270.45
Boiling point ℃（1 bar）	178	319.92

In preliminary experiments, due to insufficient limonene production, we were unable to achieve effective separation and purification. We attempted to separate limonene from the upper IPM layer but were unsuccessful.
To test the separation and purification process under current conditions, we used a simulated solution for separation. The simulated solution consisted of 0.3 mol limonene and 0.7 mol IPM.
First, we used ASPEN V14 software to predict the binary phase diagram of the limonene and IPM mixture at 0.08 bar (the lowest achievable pressure with our lab vacuum pump). However, we found that the azeotrope boiling point at 0.08 bar was 105°C, which exceeds the temperature range of the water bath. To resolve this, we added water as an entrainer to lower the boiling point of the ternary system.

Figure 30. ASPEN software predicts the phase diagram of limonene-IPM two components

Setup: We used a vacuum rotary evaporator and loaded the organic phase sample containing limonene into a 1000 mL distillation flask. The liquid volume did not exceed one-third of the flask capacity. Following the specified setup procedure, we ensured proper connection and stabilization of the condenser, and all glassware used was of thick-wall type to prevent breakage.
Operation: After confirming that the setup was correct, we started the rotary evaporator and gradually increased the rotation rate to form a liquid film. The vacuum pump was activated, and we adjusted the back-pressure valve and heating temperature until the liquid in the distillation flask reached a steady boil, with liquid droplets forming at the condenser outlet. The heating temperature was gradually increased to the maximum (100°C) until the distillation was complete.
Finalization: After distillation was finished, we stopped the vacuum pump, disassembled the apparatus, and collected the product from the round-bottom flask. Typically, this resulted in a two-phase liquid, with water and an oily limonene layer.
Analysis: The residue and product were separated using a glass cylinder for measurement of weight and volume. Samples were diluted and analyzed by gas chromatography to determine the results (as detailed in the protocol).

Figure 31. Rotary vacuum evaporation device

The results of simulated fermentation organic phase after vacuum rotary evaporation are as follows:

Figure 32. Limonene content in each part

Figure 33. Product volume recovery

Material refers to the 0.3:0.7 simulated solution before vacuum rotary evaporation; Product is the upper oily liquid layer recovered from the rotary evaporator; Residue is the remaining IPM in the distillation flask. Standard refers to the commercially purchased limonene standard sample.
The results showed that the purity of the recovered product was nearly four times higher than before, approaching the purity of the standard sample. Only trace amounts of limonene were found in the residual IPM, and the volume recovery rate reached 81%, with mass recovery between 75-82.83%. This is a fairly good outcome. Moreover, since the residual IPM contained very little limonene, it can be reused, thus conserving resources.

The distilled product was packaged in 50 mL centrifuge tube. Limonene is prone to photochemical degradation due to free radical reactions under light exposure, which can altered its molecular structure. Additionally, its double bonds are easily oxidized, altering its chemical properties and affecting its efficacy and fragrance. Therefore, we stored the product under low light and low-temperature conditions (4°C) to better preserve its original aroma and biological activity. (This process complies with iGEM Safety guidelines, the product will not leave the lab, and no human testing has been conducted.)

Figure 34. Our Product

We successfully achieved both of our goals!

Goal 1: Constructed a strain of Saccharomyces cerevisiae capable of utilizing kelp-derived carbon sources!

Goal 2: Successfully designed a process pathway from kelp carbon sources to limonene production!

This project aimed to explore the carbon-sequestration potential of kelp and enhance its utilization value. Kelp, as a carbon sink resource, possesses significant carbon fixation capacity and can be sustainably produced on a large scale. However, current applications of carbon-sequestered materials from kelp remain limited, constraining its further development. By analyzing the components of kelp, we found that it is rich in various nutrients, making it a promising carbon source for microbial use. Our experiments provide a new approach for the deep development and utilization of kelp resources and open up new pathways for the efficient use of marine carbon sink resources.

1. Improving kelp carbon source utilization: We observed that the amount of limonene produced was quite low, suggesting that the yeast’s ability to utilize mannitol or other carbon sources from kelp has not been fully optimized. In the kelp processing procedure, we discarded a significant amount of kelp residue. If we could use the entire kelp biomass (including insoluble solids) as a culture medium and modify the strain to break down polysaccharides and utilize previously inaccessible monosaccharides, we could greatly enhance yeast’s capacity to utilize kelp carbon sources.

2. Expanding product range: Our chassis strain produces limonene, which is a precursor for various terpenoids. By introducing other terpenoid synthases, we could expand the yeast’s ability to produce additional terpenoid products. Additionally, our experiments revealed that kelp contains a high content of alginate, reaching up to 0.4 g/g. Research shows that alginate degradation products, such as alginate oligosaccharides, are highly valuable compounds. If we could degrade alginate during fermentation to produce alginate oligosaccharides, we could generate a broader range of products and improve the carbon conversion rate of kelp carbon sources.

3. Optimizing the process to reduce carbon emissions: During the preparation of the kelp-based medium, we found that the acid hydrolysis, enzymatic hydrolysis, and filtration steps consumed significant time and energy, adding to the cost. This is not conducive to greenhouse gas reduction. Therefore, in future steps, introducing new lytic enzymes (e.g., alginate lyase, laminarinase) that can break down the cell walls under milder conditions and release nutrients would eliminate the need for time- and energy-intensive processes like acid hydrolysis, enzymatic hydrolysis, and filtration. This would help us move closer to our goal of utilizing kelp carbon sources more effectively and mitigating greenhouse gas emissions.