Utilizing kelp as a raw material for limonene fermentation is an effective way to expand the use of blue carbon resources, promote the development of third-generation biomass resources, and provide an environmentally friendly and efficient method for producing high-value terpenoids, such as limonene.

To better achieve this goal, we designed our experiments around four key processes: dual expression cassette plasmid screening, synthetic pathway modification, optimization of kelp component utilization, and product purification. This page outlines the design principles and related achievements of each section.

For detailed steps and methods, please refer to the experiment and protocol page.

(1) Promoter and Terminator Screening

The ndps1 gene and limonene synthase gene are integrated into a plasmid, and various combinations of promoters and terminators are tested to find the optimal dual expression cassette that maximizes the efficiency of limonene synthase production.

(2) YJL064w::ndps1-(R)-ls

The YJL064w gene does not encode a functional protein in yeast, but studies have shown that its knockout can effectively enhance the synthesis of downstream terpenoids. After screening the optimal promoter and terminator combination, we insert the YJL064w locus into this expression cassette to shift the expression of limonene synthase from the plasmid to the genome level, preventing plasmid loss and enabling the strain to permanently synthesize limonene.

(1) The ROX1 gene is a transcriptional repressor in yeast that primarily regulates genes related to anaerobic growth. Knockout of this gene can alter yeast metabolism under anaerobic conditions. Studies have shown that rox1 gene deletion significantly increases the expression of intermediates mevalonic acid in the MVA pathway, and other research has found that rox1 acts as a transcriptional regulator that represses gene expression in the MVA pathway and ergosterol biosynthesis. Its deletion enhances MVA pathway flux.

(2) tHMG1 is a homolog of the human HMG-CoA reductase gene, which catalyzes the reduction of HMG-CoA (3-hydroxy-3-methyl glutaryl coenzyme A) to mevalonate. Overexpression of this gene can increase metabolic flux, enhancing terpene production.

(3) IDI1 (isopentenyl-diphosphate delta-isomerase 1) catalyzes the interconversion of isopentenyl diphosphate (IPP) and its electrophilic isomer dimethylallyl diphosphate (DMAPP) in cells. Overexpression of this gene increases the synthesis of limonene precursors, thus boosting limonene production.

(1) The ERG20 gene encodes farnesyl diphosphate (FPP) synthase, which synthesizes FPP from GPP and IPP. FPP synthesis is essential for yeast growth, but this metabolic pathway competes with limonene synthesis for substrates.

(2) HXT1p encodes a glucose transporter protein responsible for glucose transport in yeast cells. Replacing the native promoter of ERG20 with the HXT1 promoter allows regulation of ERG20 expression, thereby controlling carbon source utilization of yeast—favoring rapid growth in the early stages and directing more carbon to limonene synthesis later.

(3) Substituting phenylalanine (Phe) at position 96 of the ERG20 gene with tryptophan (Trp) affects the activity or properties of FPP synthase. This mutation shifts carbon flux from FPP to GPP, significantly influencing terpenoid production in yeast.

(1) The function of YPL062W in yeast is not well characterized, but studies have shown that its deletion upregulates the pyruvate dehydrogenase bypass and the mevalonate pathway, increasing the production and accumulation of acetyl-CoA, thereby promoting terpenoid production.

(2) ERG12 encodes mevalonate kinase, which is involved in the mevalonate pathway. This enzyme is responsible for the biosynthesis of IPP. Overexpression of this enzyme in yeast promotes IPP synthesis.

(1) Mannitol Adaptive Laboratory Evolution

Analysis shows that mannitol accounts for about 15%-30% of the dry weight of kelp. Mannitol, as a non-fermentable carbon source, has many favorable characteristics. To enhance yeast's ability to utilize mannitol in kelp, we subjected the strain to long-term adaptive laboratory evolution (ALE) using SM△T medium.

(2) Optimization of Kelp Hydrolysate Medium

Analysis shows that mannitol accounts for about 15%-30% of the dry weight of kelp. Mannitol, as a non-fermentable carbon source, has many favorable characteristics. To enhance yeast's ability to utilize mannitol in kelp, we subjected the strain to long-term adaptive laboratory evolution (ALE) using SM△T medium.engi

After performing small-scale (10 ml) biphasic fermentation, we scaled up the fermentation. To obtain high-purity limonene for industrial production, we used vacuum rotary evaporation based on the boiling point characteristics of limonene and the organic phase (IPM) to separate and purify the organic phase obengitained after fermentation.