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Development and Utilization of Yeast Resources

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A special issue of Journal of Fungi (ISSN 2309-608X).

Deadline for manuscript submissions: closed (31 October 2023) | Viewed by 37027

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Prof. Dr. Xin-Qing Zhao

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Guest Editor

School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
Interests: yeast; synthetic biology; metabolic engineering; biofuels; organic acids; genome mining
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Qi-Ming Wang

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Guest Editor

School of Life Sciences, Hebei University, Baoding 071002, China
Interests: yeast systematics & phylogenomic; evolutionary & population genetics; systems & synthetic biology; fungal evolution

Special Issue Information

Dear Colleagues,

Yeasts are simple and single cellular fungi that are widely distributed and have been widely used in various traditional industries. Yeasts are also being investigated as microbial cell factories in novel applications in medicine, agriculture, and biorefinery of lignocellulosic biomass. Among various yeasts, budding yeast is efficient to ferment sugars into alcohol, and has been used in the production of wine, beer, beverage and biofuels. In recent years, non-conventional yeasts, such as Yarrowia lipolytica, Kluyveromyces marxianus, Komagataella phaffii (also known as Pichia pastoris) have emerged as attracting cell factories to produce organic acids, natural products from plants, vaccines and antibodies. The development of yeast strains benefits not only their applications but also the discovery of novel mechanisms that provide a basis for studying other more complicated eukaryotic systems, including human being. In the past few years, great progress has been made in the characterization of novel yeast species or strains, metabolic engineering and genome editing of yeasts, as well as exploration of both wild yeasts and engineered yeast strains in various applications. In this special issue, we would like to present valuable latest findings in the development and utilization of yeast resources. Both dedicated review and research articles are welcome for the special issue. We welcome articles related but not limited to the following contents:

Yeast diversity and its potential in industrial applications;
Advanced technologies for the development of yeast strains;
Metabolic engineering of yeast strains for bioproduction;
Synthetic biology and artificial intelligence of yeast host.

We wish that this special issue contributes to summarizing the latest progress in the related fields, which would promote the utilization of yeast resources for efficient biological manufacturing.

Prof. Dr. Xin-Qing Zhao
Prof. Dr. Qi-Ming Wang
Guest Editors

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Journal of Fungi is an international peer-reviewed open access monthly journal published by MDPI.

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Keywords

Yeast
diversity
metabolic engineering
gene editing
synthetic biology
cell factory
artificial intelligence
lignocellulosic biomass
biofuels
biorefinery

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Published Papers (11 papers)

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Research

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14 pages, 4208 KiB

Open AccessArticle

Targeting Mosquitoes through Generation of an Insecticidal RNAi Yeast Strain Using Cas-CLOVER and Super PiggyBac Engineering in Saccharomyces cerevisiae

by Corey Brizzee, Keshava Mysore, Teresia M. Njoroge, Seth McConnell, Majidah Hamid-Adiamoh, Akilah T. M. Stewart, J. Tyler Kinder, Jack Crawford and Molly Duman-Scheel

J. Fungi 2023, 9(11), 1056; https://doi.org/10.3390/jof9111056 - 27 Oct 2023

Cited by 3 | Viewed by 2567

Abstract

The global deployment of RNAi yeast insecticides involves transitioning from the use of laboratory yeast strains to more robust strains that are suitable for scaled fermentation. In this investigation, the RNA-guided Cas-CLOVER system was used in combination with Piggybac transposase to produce robust Saccharomyces cerevisiae strains with multiple integrated copies of the Sh.463 short hairpin RNA (shRNA) insecticide expression cassette. This enabled the constitutive high-level expression of an insecticidal shRNA corresponding to a target sequence that is conserved in mosquito Shaker genes, but which is not found in non-target organisms. Top-expressing Cas-CLOVER strains performed well in insecticide trials conducted on Aedes, Culex, and Anopheles larvae and adult mosquitoes, which died following consumption of the yeast. Scaled fermentation facilitated the kilogram-scale production of the yeast, which was subsequently heat-killed and dried. These studies indicate that RNAi yeast insecticide production can be scaled, an advancement that may one day facilitate the global distribution of this new mosquito control intervention. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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26 pages, 4610 KiB

Open AccessArticle

Production of Phenylacetylcarbinol via Biotransformation Using the Co-Culture of Candida tropicalis TISTR 5306 and Saccharomyces cerevisiae TISTR 5606 as the Biocatalyst

by Anbarasu Kumar, Charin Techapun, Sumeth Sommanee, Chatchadaporn Mahakuntha, Juan Feng, Su Lwin Htike, Julaluk Khemacheewakul, Kritsadaporn Porninta, Yuthana Phimolsiripol, Wen Wang, Xinshu Zhuang, Wei Qi, Kittisak Jantanasakulwong, Rojarej Nunta and Noppol Leksawasdi

J. Fungi 2023, 9(9), 928; https://doi.org/10.3390/jof9090928 - 14 Sep 2023

Cited by 3 | Viewed by 1699

Abstract

Phenylacetylcarbinol (PAC) is a precursor for the synthesis of several pharmaceuticals, including ephedrine, pseudoephedrine, and norephedrine. PAC is commonly produced through biotransformation using microbial pyruvate decarboxylase (PDC) in the form of frozen–thawed whole cells. However, the lack of microorganisms capable of high PDC activity is the main factor in the production of PAC. In addition, researchers are also looking for ways to utilize agro-industrial residues as an inexpensive carbon source through an integrated biorefinery approach in which sugars can be utilized for bioethanol production and frozen–thawed whole cells for PAC synthesis. In the present study, Candida tropicalis, Saccharomyces cerevisiae, and the co-culture of both strains were compared for their biomass and ethanol concentrations, as well as for their volumetric and specific PDC activities when cultivated in a sugarcane bagasse (SCB) hydrolysate medium (SCBHM). The co-culture that resulted in a higher level of PAC (8.65 ± 0.08 mM) with 26.4 ± 0.9 g L⁻¹ ethanol production was chosen for further experiments. Biomass production was scaled up to 100 L and the kinetic parameters were studied. The biomass harvested from the bioreactor was utilized as frozen–thawed whole cells for the selection of an initial pyruvate (Pyr)-to-benzaldehyde (Bz) concentration ([Pyr]/[Bz]) ratio suitable for the PAC biotransformation in a single-phase emulsion system. The initial [Pyr]/[Bz] at 100/120 mM resulted in higher PAC levels with 10.5 ± 0.2 mM when compared to 200/240 mM (8.60 ± 0.01 mM). A subsequent two-phase emulsion system with Pyr in the aqueous phase, Bz in the organic phase, and frozen–thawed whole cells of the co-culture as the biocatalyst produced a 1.46-fold higher PAC level when compared to a single-phase emulsion system. In addition, the cost analysis strategy indicated preliminary costs of USD 0.82 and 1.01/kg PAC for the single-phase and two-phase emulsion systems, respectively. The results of the present study suggested that the co-culture of C. tropicalis and S. cerevisiae can effectively produce bioethanol and PAC from SCB and would decrease the overall production cost on an industrial scale utilizing the two-phase emulsion system with the proposed multiple-pass strategy. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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20 pages, 3711 KiB

Open AccessArticle

A Potentially Practicable Halotolerant Yeast Meyerozyma guilliermondii A4 for Decolorizing and Detoxifying Azo Dyes and Its Possible Halotolerance Mechanisms

by Yue Feng, Jingru Cui, Bingwen Xu, Yifan Jiang, Chunqing Fu and Liang Tan

J. Fungi 2023, 9(8), 851; https://doi.org/10.3390/jof9080851 - 15 Aug 2023

Cited by 2 | Viewed by 1497

Abstract

In this study, a halotolerant yeast that is capable of efficiently decolorizing and detoxifying azo dyes was isolated, identified and characterized for coping with the treatment of azo-dye-containing wastewaters. A characterization of the yeast, including the optimization of its metabolism and growth conditions, its detoxification effectiveness and the degradation pathway of the target azo dye, as well as a determination of the key activities of the enzyme, was performed. Finally, the possible halotolerance mechanisms of the yeast were proposed through a comparative transcriptome analysis. The results show that a halotolerant yeast, A4, which could decolorize various azo dyes, was isolated from a marine environment and was identified as Meyerozyma guilliermondii. Its optimal conditions for dye decolorization were ≥1.0 g/L of sucrose, ≥0.2 g/L of (NH₄)₂SO₄, 0.06 g/L of yeast extract, pH 6.0, a temperature of 35 °C and a rotation speed of ≥160 rpm. The yeast, A4, degraded and detoxified ARB through a series of steps, relying on the key enzymes that might be involved in the degradation of azo dye and aromatic compounds. The halotolerance of the yeast, A4, was mainly related to the regulation of the cell wall components and the excessive uptake of Na⁺/K⁺ and/or compatible organic solutes into the cells under different salinity conditions. The up-regulation of genes encoding Ca²⁺-ATPase and casein kinase II as well as the enrichment of KEGG pathways associated with proteasome and ribosome might also be responsible for its halotolerance. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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16 pages, 6058 KiB

Open AccessArticle

Metabolic Engineering of Saccharomyces cerevisiae for Efficient Retinol Synthesis

by Xuan Wang, Xianhao Xu, Jiaheng Liu, Yanfeng Liu, Jianghua Li, Guocheng Du, Xueqin Lv and Long Liu

J. Fungi 2023, 9(5), 512; https://doi.org/10.3390/jof9050512 - 26 Apr 2023

Cited by 7 | Viewed by 3824

Abstract

Retinol, the main active form of vitamin A, plays a role in maintaining vision, immune function, growth, and development. It also inhibits tumor growth and alleviates anemia. Here, we developed a Saccharomyces cerevisiae strain capable of high retinol production. Firstly, the de novo synthesis pathway of retinol was constructed in S. cerevisiae to realize the production of retinol. Second, through modular optimization of the metabolic network of retinol, the retinol titer was increased from 3.6 to 153.6 mg/L. Then, we used transporter engineering to regulate and promote the accumulation of the intracellular precursor retinal to improve retinol production. Subsequently, we screened and semi-rationally designed the key enzyme retinol dehydrogenase to further increase the retinol titer to 387.4 mg/L. Lastly, we performed two-phase extraction fermentation using olive oil to obtain a final shaking flask retinol titer of 1.2 g/L, the highest titer reported at the shake flask level. This study laid the foundation for the industrial production of retinol. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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13 pages, 1855 KiB

Open AccessArticle

Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone

by Linjuan Feng, Junhao Xu, Cuifang Ye, Jucan Gao, Lei Huang, Zhinan Xu and Jiazhang Lian

J. Fungi 2023, 9(4), 494; https://doi.org/10.3390/jof9040494 - 20 Apr 2023

Cited by 1 | Viewed by 2736

Abstract

Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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17 pages, 7248 KiB

Open AccessArticle

Proposal of Four New Aureobasidium Species for Exopolysaccharide Production

by Feng Wu, Zixuan Feng, Manman Wang and Qiming Wang

J. Fungi 2023, 9(4), 447; https://doi.org/10.3390/jof9040447 - 6 Apr 2023

Cited by 2 | Viewed by 2946

Abstract

In this study, 99 strains of Aureobasidium species were isolated from various samples collected from different locations in China, among which 14 isolates showed different morphological characteristics to other strains identified as known Aureobasidium species. Based on morphological characteristics, those 14 strains were classified into four groups, represented by stains of KCL139, MDSC−10, XZY411−4, and MQL9−100, respectively. Molecular analysis of the internal transcriptional spacer (ITS) and part of the large ribosome subunit (D1/D2 domains) indicated that those four groups represent four new species in the Aureobasidium. Therefore, the names Aureobasidium insectorum sp. nov., A. planticola sp. nov., A. motuoense sp. nov., and A. intercalariosporum sp. nov. are proposed for KCL139, MDSC−10, XZY411−4, and MQL9−100, respectively. We also found that there were differences in the yield of exopolysaccharides (EPS) among and within species, indicating strain-related exopolysaccharide-producing diversity. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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16 pages, 1756 KiB

Open AccessArticle

Improving Methanol Utilization by Reducing Alcohol Oxidase Activity and Adding Co-Substrate of Sodium Citrate in Pichia pastoris

by Shufan Liu, Haofan Dong, Kai Hong, Jiao Meng, Liangcai Lin and Xin Wu

J. Fungi 2023, 9(4), 422; https://doi.org/10.3390/jof9040422 - 29 Mar 2023

Cited by 5 | Viewed by 3352

Abstract

Methanol, which produced in large quantities from low-quality coal and the hydrogenation of CO₂, is a potentially renewable one-carbon (C1) feedstock for biomanufacturing. The methylotrophic yeast Pichia pastoris is an ideal host for methanol biotransformation given its natural capacity as a methanol assimilation system. However, the utilization efficiency of methanol for biochemical production is limited by the toxicity of formaldehyde. Therefore, reducing the toxicity of formaldehyde to cells remains a challenge to the engineering design of a methanol metabolism. Based on genome-scale metabolic models (GSMM) calculations, we speculated that reducing alcohol oxidase (AOX) activity would re-construct the carbon metabolic flow and promote balance between the assimilation and dissimilation of formaldehyde metabolism processes, thereby increasing the biomass formation of P. pastoris. According to experimental verification, we proved that the accumulation of intracellular formaldehyde can be decreased by reducing AOX activity. The reduced formaldehyde formation upregulated methanol dissimilation and assimilation and the central carbon metabolism, which provided more energy for the cells to grow, ultimately leading to an increased conversion of methanol to biomass, as evidenced by phenotypic and transcriptome analysis. Significantly, the methanol conversion rate of AOX-attenuated strain PC110-AOX1-464 reached 0.364 g DCW/g, representing a 14% increase compared to the control strain PC110. In addition, we also proved that adding a co-substrate of sodium citrate could further improve the conversion of methanol to biomass in the AOX-attenuated strain. It was found that the methanol conversion rate of the PC110-AOX1-464 strain with the addition of 6 g/L sodium citrate reached 0.442 g DCW/g, representing 20% and 39% increases compared to AOX-attenuated strain PC110-AOX1-464 and control strain PC110 without sodium citrate addition, respectively. The study described here provides insight into the molecular mechanism of efficient methanol utilization by regulating AOX. Reducing AOX activity and adding sodium citrate as a co-substrate are potential engineering strategies to regulate the production of chemicals from methanol in P. pastoris. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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18 pages, 3232 KiB

Open AccessArticle

Engineering Flocculation for Improved Tolerance and Production of d-Lactic Acid in Pichia pastoris

by Kittapong Sae-Tang, Pornsiri Bumrungtham, Wuttichai Mhuantong, Verawat Champreda, Sutipa Tanapongpipat, Xin-Qing Zhao, Chen-Guang Liu and Weerawat Runguphan

J. Fungi 2023, 9(4), 409; https://doi.org/10.3390/jof9040409 - 27 Mar 2023

Cited by 5 | Viewed by 2703

Abstract

d-lactic acid, a chiral organic acid, can enhance the thermal stability of polylactic acid plastics. Microorganisms such as the yeast Pichia pastoris, which lack the natural ability to produce or accumulate high amounts of d-lactic acid, have been metabolically engineered to produce it in high titers. However, tolerance to d-lactic acid remains a challenge. In this study, we demonstrate that cell flocculation improves tolerance to d-lactic acid and increases d-lactic acid production in Pichia pastoris. By incorporating a flocculation gene from Saccharomyces cerevisiae (ScFLO1) into P. pastoris KM71, we created a strain (KM71-ScFlo1) that demonstrated up to a 1.6-fold improvement in specific growth rate at high d-lactic acid concentrations. Furthermore, integrating a d-lactate dehydrogenase gene from Leuconostoc pseudomesenteroides (LpDLDH) into KM71-ScFlo1 resulted in an engineered strain (KM71-ScFlo1-LpDLDH) that could produce d-lactic acid at a titer of 5.12 ± 0.35 g/L in 48 h, a 2.6-fold improvement over the control strain lacking ScFLO1 expression. Transcriptomics analysis of this strain provided insights into the mechanism of increased tolerance to d-lactic acid, including the upregulations of genes involved in lactate transport and iron metabolism. Overall, our work represents an advancement in the efficient microbial production of d-lactic acid by manipulating yeast flocculation. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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Review

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19 pages, 2246 KiB

Open AccessReview

Advances in Metabolic Engineering of Pichia pastoris Strains as Powerful Cell Factories

by Jian Zha, Dan Liu, Juan Ren, Zhijun Liu and Xia Wu

J. Fungi 2023, 9(10), 1027; https://doi.org/10.3390/jof9101027 - 19 Oct 2023

Cited by 8 | Viewed by 5908

Abstract

Pichia pastoris is the most widely used microorganism for the production of secreted industrial proteins and therapeutic proteins. Recently, this yeast has been repurposed as a cell factory for the production of chemicals and natural products. In this review, the general physiological properties of P. pastoris are summarized and the readily available genetic tools and elements are described, including strains, expression vectors, promoters, gene editing technology mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, and adaptive laboratory evolution. Moreover, the recent achievements in P. pastoris-based biosynthesis of proteins, natural products, and other compounds are highlighted. The existing issues and possible solutions are also discussed for the construction of efficient P. pastoris cell factories. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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18 pages, 2118 KiB

Open AccessReview

Recent Advances in the Biosynthesis of Natural Sugar Substitutes in Yeast

by Jian Li, Honghao Li, Huayi Liu and Yunzi Luo

J. Fungi 2023, 9(9), 907; https://doi.org/10.3390/jof9090907 - 7 Sep 2023

Viewed by 2878

Abstract

Natural sugar substitutes are safe, stable, and nearly calorie-free. Thus, they are gradually replacing the traditional high-calorie and artificial sweeteners in the food industry. Currently, the majority of natural sugar substitutes are extracted from plants, which often requires high levels of energy and causes environmental pollution. Recently, biosynthesis via engineered microbial cell factories has emerged as a green alternative for producing natural sugar substitutes. In this review, recent advances in the biosynthesis of natural sugar substitutes in yeasts are summarized. The metabolic engineering approaches reported for the biosynthesis of oligosaccharides, sugar alcohols, glycosides, and rare monosaccharides in various yeast strains are described. Meanwhile, some unresolved challenges in the bioproduction of natural sugar substitutes in yeast are discussed to offer guidance for future engineering. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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21 pages, 688 KiB

Open AccessReview

Advances in the Application of the Non-Conventional Yeast Pichia kudriavzevii in Food and Biotechnology Industries

by Yunfei Chu, Mengmeng Li, Jiahui Jin, Xiameng Dong, Ke Xu, Libo Jin, Yanming Qiao and Hao Ji

J. Fungi 2023, 9(2), 170; https://doi.org/10.3390/jof9020170 - 27 Jan 2023

Cited by 27 | Viewed by 5149

Abstract

Pichia kudriavzevii is an emerging non-conventional yeast which has attracted increased attention for its application in food and biotechnology areas. It is widespread in various habitats and often occurs in the spontaneous fermentation process of traditional fermented foods and beverages. The contributions of P. kudriavzevii in degrading organic acid, releasing various hydrolase and flavor compounds, and displaying probiotic properties make it a promising starter culture in the food and feed industry. Moreover, its inherent characteristics, including high tolerance to extreme pH, high temperature, hyperosmotic stress and fermentation inhibitors, allow it the potential to address technical challenges in industrial applications. With the development of advanced genetic engineering tools and system biology techniques, P. kudriavzevii is becoming one of the most promising non-conventional yeasts. This paper systematically reviews the recent progress in the application of P. kudriavzevii to food fermentation, the feed industry, chemical biosynthesis, biocontrol and environmental engineering. In addition, safety issues and current challenges to its use are discussed. Full article

(This article belongs to the Special Issue Development and Utilization of Yeast Resources)

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