As the most commonly used eukaryotic unicellular microorganisms, yeasts exhibit rapid growth and reproduction, and relatively simple genetic manipulation. Compared with prokaryotic expression hosts, Yeasts have greater advantages in heterologous enzyme expression, thus attracting extensive attention from researchers in the application of biosynthesis. In contrast to Saccharomyces cerevisiae, non-conventional yeasts possess unique physiological and metabolic advantages as well as excellent environmental tolerance, such as high temperature resistance and low pH tolerance. Yeasts also have a broad substrate utilization spectrum, a trait that distinguishes them from many other microbial expression systems and highlights the versatility of Yeasts in industrial bioprocesses.
These characteristics endow non-conventional yeasts with distinctive advantages over Saccharomyces cerevisiae in the synthesis of some special natural products. With the rapid development of synthetic biology technology, efficient and convenient genetic manipulation methods have been established in various non-conventional yeasts, providing effective tools for the assembly of synthetic pathways and the optimization of metabolic engineering. The continuous expansion of these tools further enhances the competitiveness of Yeasts as chassis organisms in the field of natural product biosynthesis.
01 Yarrowia lipolytica
Yarrowia lipolytica belongs to the phylum Ascomycota, order Saccharomycetales, and genus Yarrowia. It is recognized as a Generally Recognized as Safe (GRAS) microorganism by the U.S. Food and Drug Administration (FDA). Among industrial Yeasts, it is commonly found in fermented foods such as dairy products and bread, as well as in ecological environments including soil, marine habitats, and oil-contaminated areas. Yarrowia lipolytica is a dimorphic yeast that can grow as round multipolar budding cells, pseudohyphae, or hyphae with septa, and its specific morphology depends on the growth conditions.
It has strong environmental adaptability, capable of growing under high osmotic pressure and relatively extreme pH conditions (2.5–9.5), with a maximum tolerable temperature of 38 °C. Yarrowia lipolytica requires simple culture conditions for growth and has a broad substrate utilization spectrum. Metabolic engineering modification can further expand its carbon source utilization range. Through rational artificial modification, it can utilize xylose, galactose, inulin, lignocellulose, and other substrates for growth and metabolism.
A variety of endogenous promoters of Yarrowia lipolytica have been characterized so far. Constitutive promoters include PTEF1, PTDH1, PFBA1, and PGPM1. Among inducible promoters, many are derived from lipid metabolic pathways, such as PPOX2, PPOT1, and PLIP2 induced by oleic acid. In addition, there are Cu²-inducible promoters PMT1-6 and erythritol-inducible promoter PEYK1. On this basis, artificially designed hybrid promoters have also been developed to meet customized gene expression requirements. The successive development of numerous transcription elements in Yarrowia lipolytica and the application of CRISPR-mediated regulatory systems have also accelerated the development of its genetic manipulation technology system, laying a solid foundation for the synthesis and production of products.
As a natural oleaginous microorganism, the metabolic system of Yarrowia lipolytica is very suitable for the production of oils and fatty acids. The high intracellular lipid content also provides unique conditions for the synthesis and storage of other hydrophobic natural products. Yarrowia lipolytica is a typical Crabtree-negative yeast. Compared with Saccharomyces cerevisiae, it basically does not produce ethanol during cultivation, which can avoid the impact of ethanol accumulation on products during fermentation and has great industrial application potential.
In addition, Yarrowia lipolytica has multiple intracellular acetyl-CoA synthesis pathways, which can provide sufficient precursors for the synthesis of various products. At present, Yarrowia lipolytica has been widely used in the synthesis of various natural products such as lipids, terpenoids, and flavonoids, with considerable yields of some products.
02 Pichia pastoris
Pichia pastoris belongs to the phylum Ascomycota, order Saccharomycetales, and genus *Komagataella*. Among expression Yeasts, it is widely used due to its advantages such as high cell density, moderate post-translational modification of proteins, strong protein secretion capacity, and simple culture process, Pichia pastoris has gradually become the most commonly used eukaryotic expression platform for heterologous proteins among industrial Yeasts.
Pichia pastoris is recognized as a GRAS strain by the FDA and approved for use in the pharmaceutical and food industries. It is reported that more than 5,000 proteins have been expressed using Pichia pastoris, among which more than 70 protein products have entered the market. As a typical methylotrophic yeast, Pichia pastoris can grow using methanol as the sole carbon source, with an optimal growth temperature of 28–30 °C and a tolerable pH range of 3.0–7.0.
Its natural alcohol oxidase promoter PAOX1 has extremely high initiation activity and is strictly induced by methanol. The methanol-induced culture system has also developed into the most commonly used fermentation production process for Pichia pastoris. In addition to methanol, glucose, glycerol, ethanol, sorbitol, and other substances are also available carbon sources for Pichia pastoris. Various related natural promoters have been well applied in Pichia pastoris, including inducible promoters and constitutive promoters with different strengths.
Promoter mutation libraries constructed based on natural promoters and artificially designed synthetic promoters have further enriched the transcriptional regulation tool library of Pichia pastoris. In contrast, the development of terminators is relatively limited, and the alcohol oxidase terminator AOX1tt is still the main one at present.
In recent years, several endogenous terminators have been identified one after another, and some exogenous terminators have also been proven to function in Pichia pastoris. At present, a variety of commercial expression vectors for Pichia pastoris are available for direct use, such as the pPIC3.5K, pPIC9K, and pPICZ series. In recent years, significant progress has also been made in the development of synthetic biology tools in Pichia pastoris, including Golden-Gate assembly, Cre-loxP recombination, and CRISPR/Cas9-based gene editing technology, which have greatly reduced the difficulty of genetic manipulation for pathway assembly and metabolic reconstruction in Pichia pastoris.
The synthesis of natural products by Pichia pastoris is mainly focused on polyketides and terpenoids, in addition to flavonoids, polysaccharides, and fatty acid derivatives. Methanol, glycerol, or glucose are generally used as the main carbon sources.
In recent years, ethanol has also been used as a carbon source and shown obvious advantages in the synthesis of polyketides and flavonoids. In addition, many metabolic engineering regulation strategies such as enhancing precursor supply, pathway compartmentalization, cofactor engineering, and fine pathway regulation have been extensively studied, which can effectively promote product synthesis in Pichia pastoris.
03 Kluyveromyces marxianus
Kluyveromyces marxianus belongs to the phylum Ascomycota, order Saccharomycetales, and genus Kluyveromyces. It was initially isolated from grapes and is widely present in plants and dairy products. The aromatic compounds it produces can add special flavors to dairy products and alcoholic beverages. Kluyveromyces marxianus is not only a GRAS-level microorganism certified by the FDA but also has passed the safety certification of the European Food Safety Authority (EFSA). In 2013, it was approved as a new food raw material by the National Health and Family Planning Commission of China.
The unique physiological characteristics of Kluyveromyces marxianus are mainly reflected in high temperature resistance, high growth rate, and the ability to utilize a variety of carbon sources. Strains can generally grow at 40 °C, with a growth rate of 0.86–0.99/h, which is much higher than that of other yeasts. Some strains can even tolerate temperatures above 50 °C.
High-temperature fermentation can significantly reduce cooling costs and the risk of contamination, and is more conducive to the catalytic reaction of some enzymes with better activity at high temperatures. In addition to glucose, Kluyveromyces marxianus can also use a variety of other sugars as the sole carbon source for growth, including fructose, xylose, arabinose, galactose, lactose, and inulin. Therefore, many cheap agricultural and food industry by-products derived from these sugars can be used as its fermentation carbon sources.
Many promoters derived from Saccharomyces cerevisiae can function in Kluyveromyces marxianus, mainly constitutive promoters. In view of the thermotolerance of Kluyveromyces marxianus, the strength of some endogenous promoters is also affected and regulated by temperature. The natural homologous recombination efficiency of Kluyveromyces marxianus is very low, and homologous arms of more than 500 bp are usually required to achieve gene replacement or deletion.
At present, both Cre-loxP and CRISPR systems have been successfully applied for gene knockout in Kluyveromyces marxianus. Natural Kluyveromyces marxianus can produce compounds such as phenylethanol and ethyl acetate. Through genetic engineering modification, Kluyveromyces marxianus can also produce fructose syrup, astaxanthin, and triacetic acid lactone. Due to the limitations of molecular manipulation technology and related metabolic background, the commonly used strategies for improving product yields are still overexpression of key genes and knockout of bypass genes.
04 Rhodosporidium toruloides
Rhodosporidium toruloides belongs to the phylum Basidiomycota, order Sporidiobolales, and genus Rhodosporidium. It is widely distributed in nature and has strong stress resistance. It can utilize a variety of cheap substrates, making it a promising candidate among industrial Yeasts for sustainable bioproduction. It can utilize the waste from the production of starch, sugarcane molasses, and rice husk waste.
As a microorganism with strong lipid accumulation capacity, its intracellular lipid content under different culture conditions can account for 20%–79% of the dry weight, making it a potential microorganism for the production of edible oils and biodiesel raw materials. In addition, Rhodosporidium is also used for the synthesis of various carotenoids, fatty acid derivatives, and terpenoids.
Since 2011, the whole-genome sequencing of several strains of Rhodosporidium toruloides has been completed. Subsequently, multi-omics analysis has been widely used to explore its carbon source utilization, stress response, and lipid-related metabolism. On this basis, several research teams have constructed and improved the detailed lipid metabolic network of Rhodosporidium toruloides, providing a good metabolic background for further optimizing lipid production of the strain. Traditional chemical and physical mutagenesis methods have always been commonly used to obtain high-yield Rhodosporidium toruloides strains. Classic metabolic engineering strategies are also often combined with Agrobacterium tumefaciens-mediated transformation (ATMT) to improve the yields of lipids and carotenoids.
However, the extremely low homologous recombination efficiency of Rhodosporidium toruloides itself has greatly limited its development in strain modification and heterologous synthesis. With the development of gene manipulation technology, several research groups have successfully established the CRISPR/Cas9 system in Rhodosporidium in recent years. At present, technologies such as ATMT and CRISPR can all realize gene deletion in Rhodosporidium toruloides, and RNAi can also achieve gene transcription inhibition. The establishment of these tools has further promoted the development and application of Rhodosporidium toruloides in compound production.
Outlook
Based on the current situation, to give full play to the potential of non-conventional yeasts as chassis for natural product synthesis, it is necessary to conduct more research and exploration on the background of cellular metabolic regulation and carefully explore the special advantages that hosts can bring in the synthesis of specific products.
On the other hand, it is still necessary to further develop synthetic biology tools for non-conventional yeasts, including efficient genome manipulation systems and flexible expression regulation tools. On this basis, starting from the unique physiological and metabolic mechanisms of non-conventional yeasts themselves, rationally design synthetic pathways, and combine advanced biological technologies such as multi-omics analysis, high-throughput screening, and machine learning to optimize product synthesis, so as to realize the efficient, economical, and industrial application of non-conventional yeasts in natural product synthesis.
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