β-carotene, a crucial precursor of vitamin A, has extensive applications in food, feed, and nutritional supplements [1]. As a Class A nutritional food fortification agent, β-carotene plays a vital role in maintaining normal bodily functions, vision development, and protecting the retina from UV light and oxidative damage [2,3]. Its antioxidant and anti-inflammatory properties contribute to strengthening the immune system and preventing infections [4].

Figure 1 The sources and function of β-carotene [5].

While β-carotene can be obtained through chemical synthesis, physical extraction, and microbial synthesis, each method has its limitations. Physical extraction is constrained by geographical factors and low content in plant materials, while chemical synthesis raises concerns about carcinogenicity and environmental pollution [6]. Microbial synthesis, however, offers higher safety, stability, and quality, making it an increasingly attractive approach. As a terpenoid with promising applications, microbial fermentation production of β-carotene is the most promising production method for application, but there is still much room for improvement in the current yield [7].

Figure 2 Synthesis of β-carotene using microbial cell factories [8].

Saccharomyces cerevisiae (S. cerevisiae), the first eukaryotic organism with a fully sequenced genome, has emerged as a preferred chassis for metabolic engineering due to its clear genetic background, strong genetic operability, and excellent fermentation performance [8]. However, as more and more compounds are synthesized in S. cerevisiae, the process of cell factory construction and pathway optimization has revealed its flaws and limitations. With the rapid development of gene editing technology, early gene editing techniques based on recombinase and homologous recombination are gradually replaced by novel gene editing systems [9].

The CRISPR/Cas9 system is a revolutionary tool for genome editing in S. cerevisiae, enabling gene knockdown, integration, transcriptional interference, and activation. Most metabolic engineering manipulations can now be achieved with CRISPR/Cas9. The system uses a single guide RNA (sgRNA) to direct the Cas9 nuclease to cleave the genome near the protospacer adjacent motif (PAM) sequence, creating a double-strand break (DSB). When donor DNA with homologous sequences is present, precise gene editing occurs in S. cerevisiae [9].

Figure 3 Schematic diagram of the CRISPR/Cas9 mechanism in yeast [9].

Numerous studies have demonstrated the potential of S. cerevisiae in the efficient production of β-carotene, a valuable compound with applications in food, pharmaceuticals, and cosmetics. These findings have inspired our efforts to construct high β-carotene-yielding strains of S. cerevisiae. By optimizing the metabolic pathways and genetic modifications within these yeast strains, we aim to significantly boost β-carotene production. This enhancement not only has the potential to increase yields but also makes the process more viable for large-scale industrial applications, thereby meeting the growing demand for sustainable and cost-effective production methods.

In this project, based on the concept of synthetic biology, we use molecular biology techniques to integrate genes of the β-carotene synthesis pathway from different species into the chassis organism, i.e., S. cerevisiae. Moreover, we will also test the β-carotene production capacity of the engineered strain to verify whether the gene combination we employed improves the ability of S. cerevisiae to synthesize β-carotene. Our design idea is described below (Figure 4).

Figure 4 Schematic design for constructing S. cerevisiae strains with high β-carotene production.

(1) Integration plasmid construction

• Obtain genes for β-carotene biosynthesis from different species sources (Table 1).
• Link the target gene to the integrative plasmid backbone.
• Construct the integration plasmid in E. coli DH5α.

Table 1 Genes for β-carotene biosynthesis from different species sources

(2) Integration of target genes into the yeast genome

• Obtain the integrating fragment by enzymatic cleavage.
• Integrate the target fragment into the yeast genome by CRISPR/Cas9 technology to obtain yeast strains with three gene combinations (Table 1).

(3) Qualitative and quantitative analysis of yeast strains

• Identify the expression of target genes on the yeast genome by qPCR.
• Shake flask culture of three yeast strains, extract β-carotene content determination

We aimed to integrate key genes in the β-carotene synthesis pathway into the genome of S. cerevisiae by gene editing technology, so as to obtain strains with high efficiency and stable production of β-carotene. The development of these S. cerevisiae strains offers great potential for industrial production and can be used in a number of applications in the future. For example, the food and beverage industry could utilize such β-carotene-enriched yeast strains to produce nutrient-rich functional foods and beverages that meet consumer demand for healthy and natural ingredients. The cosmetics industry can also benefit from this technology by producing high-purity, contamination-free β-carotene through fermentation for the development of beauty products with antioxidant and skin-care properties. In addition, these strains can be used in the pharmaceutical industry to provide a stable source of β-carotene for the production of drugs such as vitamin A supplements. With further optimization of the technology, these gene-edited yeast strains can not only reduce production costs, but also enable sustainable industrial production and promote the large-scale application of natural beta-carotene products.

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