As the most commonly used eukaryotic single-cell microorganism, yeast grows and reproduces rapidly and has relatively simple genetic manipulation. It has more advantages in expressing heterologous enzymes than prokaryotic expression hosts, and has attracted much attention from researchers in biosynthetic applications. Compared with Saccharomyces cerevisiae, unconventional yeasts have unique physiological metabolic advantages and good environmental tolerance, such as high temperature resistance and low pH tolerance, and most of them also have a wide substrate utilization spectrum. These characteristics give unconventional yeasts unique advantages over Saccharomyces cerevisiae in the synthesis of some special natural products. With the rapid development of synthetic biotechnology, efficient and convenient gene manipulation methods have been established in a variety of unconventional yeasts, providing effective tools for the assembly of synthetic pathways and the optimization of metabolic engineering.

◆Yarrowia lipolytica

Yarrowia lipolytica belongs to the phylum Ascomycota, the order Saccharomyces, and the genus Yarrowia. It is recognized as a generally recognized safe microorganism by the U.S. Food and Drug Administration. It is usually found in fermented foods such as dairy products and bread, as well as in soil, oceans and oils. in ecological environments such as quality pollution. Yarrowia lipolytica is a dimorphic yeast that can grow as round multipolar budding cells, pseudohyphae, or mycelium with septate hyphae, depending on growth conditions. It has strong environmental adaptability and can grow under high osmotic pressure and extreme pH conditions (2.5–9.5). The maximum temperature it can tolerate is 38°C. The culture conditions required for the growth of Yarrowia lipolytica are simple and have a wide substrate utilization spectrum. Metabolic engineering modification can further expand the carbon source utilization spectrum of Yarrowia lipolytica. Through rational artificial modification, it can use xylose, galactose, inulin, lignocellulose, etc. for growth and metabolism.

Yarrowia lipolytica has currently characterized a variety of endogenous promoters. The constitutive promoters include PTEF1, PTDH1, PFBA1 and PGPM1. Many of the inducible promoters are derived from lipid metabolism pathways, such as those induced by oleic acid. PPOX2, PPOT1 and PLIP2, in addition to Cu2+ inducible promoter PMT1-6, erythritol inducible promoter PEYK1, etc. On this basis, there are also artificially designed hybrid promoters to achieve customized gene expression. need. The successive development of many transcription elements and the application of CRISPR-mediated regulatory systems in Yarrowia lipolytica have also accelerated the development of its genetic manipulation technology system, laying a solid foundation for product synthesis and production.

As a natural oil-producing microorganism, the metabolic system of Yarrowia lipolytica is very suitable for the production of oils and fatty acids. The high concentration of 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 the culture process. It can avoid the impact of ethanol accumulation on the product during the fermentation process and has good industrial application potential. In addition, there are multiple acetyl-CoA synthesis pathways in Yarrowia lipolytica cells, which can provide sufficient precursors for the synthesis of various products. At present, Yarrowia lipolytica has been widely used in the synthesis of many types of natural products such as lipids, terpenoids, and flavonoids, and the yields of some products are considerable.

◆Pichia pastoris

Pichia pastoris belongs to the phylum Ascomycota, the order Saccharomycota, and the genus Komagataella. Pichia pastoris has gradually become the most commonly used eukaryotic expression platform for heterologous proteins due to its advantages such as high growth density, moderate protein post-translational modification, strong protein secretion ability, and simple culture process. Pichia pastoris is recognized by the FDA as a GRAS strain and is approved for use in the pharmaceutical and food industries. According to reports, more than 5,000 proteins have been expressed using Pichia pastoris, of which more than 70 protein products have entered the market. As a typical methylotrophic yeast, Pichia pastoris can grow on methanol as the sole carbon source, with an optimal growth temperature of 28–30°C and a tolerated pH range of 3.0–7.0.

Its natural alcohol oxidase promoter PAOX1 has very efficient startup ability and is strictly induced by methanol. The methanol-induced culture system has also developed into the most commonly used fermentation production process of Pichia pastoris. In addition to methanol, glucose, glycerol, ethanol, sorbitol, etc. are also available carbon source substrates for Pichia pastoris. Various related natural promoters are also well used in Pichia pastoris, including those with different strengths. Inducible promoters and constitutive promoters, etc. 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 currently the alcohol oxidase terminator AOX1tt is still the main one. In recent years, some endogenous terminators have been identified, and some exogenous terminators have also been confirmed to function in Pichia pastoris. Currently, Pichia pastoris has a variety of commercial expression vectors available for direct use, such as pPIC3.5K, pPIC9K, pPICZ series, etc. In recent years, important progress has 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 has greatly reduced the complexity of pathway assembly in Pichia pastoris. Difficulty of genetic manipulation of metabolic remodeling.

In terms of natural product synthesis, Pichia pastoris mainly produces polyketides and terpenes, and others also include flavonoids, polysaccharides and fatty acid derivatives. Methanol, glycerol or glucose are generally used as the main carbon source substrates. In recent years, ethanol has also been used as a carbon source and has shown obvious advantages in the synthesis of polyketides and flavonoids. In addition, there have been many studies on metabolic engineering regulatory strategies such as enhanced precursor supply, pathway compartmentalization, cofactor engineering, and fine regulation of pathways, which can effectively promote product synthesis in Pichia pastoris.

◆Kluyveromyces marxianus

Kluyveromyces marxianus belongs to the phylum Ascomycota, the order Saccharomyces, and the genus Kluyveromyces. It was originally isolated from grapes. It is widely found in plants and dairy products. The aromatic compounds it produces can be added to dairy products and wine. Special flavor. Kluyveromyces marxianus is not only a GRAS-level microorganism certified by the FDA, but also passed the safety certification of the European Food Safety Authority (EFSA), and was approved as a new food raw material by the

National Health and Family Planning Commission of China in 2013.

The unique physiological characteristics of Kluyveromyces marxianus are mainly reflected in high temperature resistance, high growth rate and the ability to utilize multiple carbon sources. Strains can generally grow at 40°C, with a growth rate of 0.86–0.99/h, which is much higher than other yeasts. Some strains can tolerate temperatures above 50°C. High-temperature fermentation can significantly reduce cooling costs and the risk of bacterial contamination, and is more conducive to catalytic reactions by enzymes with better activity at high temperatures. In addition to glucose, Kluyveromyces marxianus can also use some other sugars as a single carbon source for growth, including fructose, xylose, arabinose, galactose, lactose and inulin, etc., so many cheap agricultural sources of these sugars and food industry by-products can be used as carbon sources for fermentation.

Many promoters derived from Saccharomyces cerevisiae can function in Kluyveromyces marxianus, mainly constitutively. Given the thermotolerant nature of Kluyveromyces marxianus, the strength of some endogenous promoters will also be affected and regulated by temperature. The natural homologous recombination efficiency in Kluyveromyces marxianus is very low, and homology arms of more than 500 bp are usually required to achieve gene replacement or deletion. Currently, Cre-loxP and CRISPR systems have been successfully used to achieve gene knockout in Kluyveromyces marxianus. Natural Kluyveromyces marxianus can produce compounds such as phenylethyl alcohol and ethyl acetate. Through genetic engineering, Kluyveromyces marxianus can also produce fructose syrup, astaxanthin, triacetolactone, etc. Due to limitations of molecular manipulation technology and related metabolic background, currently commonly used product improvement strategies still rely on overexpression of key genes and Mainly knockout of bypass genes.

◆Rhodosporium toruloides

Rhodosporidium toruloides, belonging to the phylum Basidiomycota, the order Sclerochydomonas, and the genus Rhodosporium toruloides, is widely distributed in nature and has strong stress resistance. It can use a variety of industrial and agricultural wastes as carbon dioxide. Sources, such as crude glycerol, lignocellulose hydrolysates, volatile fatty acids, sugarcane molasses and rice husk waste, etc. As a microorganism with strong oil accumulation ability, its cell oil content can account for 20%-79% of dry weight under different culture conditions. It is a potential microorganism for the production of edible oils and biodiesel raw materials. In addition, Rhodosporium spores is also used to synthesize a variety of carotenoids, fatty acid derivatives, and terpenoids. Since 2011, many strains of Rhodosporidium toruloides have completed whole-genome sequencing. Subsequently, multi-omics analysis has been widely used to explore its carbon source utilization, stress response, and related metabolism of lipids. On this basis, multiple research teams have constructed and improved the detailed lipid metabolism network of Rhodosporium toruloides, providing a good metabolic background foundation for further optimizing the strain’s lipid production. Traditional chemical and physical mutagenesis methods have always been common methods to obtain high-yield Rhodosporidium toruloides strains, and classic metabolic engineering strategies are often combined with ATMT to improve oil and carotenoid production. However, the homologous recombination efficiency of Rhodosporium toruloides itself is too low, which greatly limits its development in strain modification and heterologous synthesis. With the development of gene manipulation technology, multiple research groups have successfully established the CRISPR/Cas9 system in red yeast in recent years. At present, technologies such as ATMT and CRISPR can achieve gene deletion operations in Rhodosporium toruloides, and RNAi can also inhibit gene transcription. The establishment of these tools further promotes the development and application of Rhodosporium toruloides in compound production.