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Bioengineering
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From Yeast to Biotechnology
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From Yeast to Biotechnology

A special issue of Bioengineering (ISSN 2306-5354).

Deadline for manuscript submissions: closed (30 September 2020) | Viewed by 63962

Special Issue Editor

Dr. Eva Garcia Ruiz

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Guest Editor
Department of Biocatalysis, Institute of Catalysis and Petrochemistry, Spanish National Research Council (ICP-CSIC), 28049 Madrid, Spain
Interests: synthetic biology; directed evolution; metabolic engineering; sustainable green processes; yeast
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Yeast is regarded as a versatile tool for biotechnological purposes. For thousands of years, yeasts have been exploited by mankind for the production of food and beverages. Their metabolic versatility, robustness under fermentation conditions, generally recognized as safe (GRAS) status, ease of culture, and the availability of biomolecular tools have turned yeasts into one of the main workhorses in biotechnology. Recent advances in genomics, metabolic engineering, and synthetic biology have enabled the engineering of yeasts to function as microfactories for the production of relevant compounds such as pharmaceuticals, biofuels, fine chemicals, or proteins. Yeasts also have a great potential in molecular biology as a biomolecular toolbox. Among the various yeast species, Saccharomyces cerevisiae stands out for its high fidelity and high frequency of homologous DNA recombination, which has been successfully exploited in the synthesis of large DNA fragments (as whole genomes) and engineering of enzymes by directed evolution through so-called in vivo DNA shuffling.

Although S. cerevisiae is the best genetically and biologically characterized yeast and that most widely used in industrial applications, new players are rapidly emerging, such as Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Kluyveromyces marxianus, to name but a few.

For this Special Issue “From Yeast to Biotechnology” we invite authors to submit articles covering all aspects of this theme, including, but not limited to, yeast engineering for high-value compound production, yeast genome engineering, computational design/modeling/analysis applied to yeast biotechnology, methods to characterize yeast, yeast as biomolecular toolbox, yeast biosensors, and/or yeast industrial fermentation processes.

Dr. Eva Garcia Ruiz
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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. Bioengineering is an international peer-reviewed open access monthly journal published by MDPI.

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Published Papers (12 papers)

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Editorial

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4 pages, 198 KiB  
Editorial
Engineering Biology of Yeast for Advanced Biomanufacturing
by Wei Jiang, Yanjun Li and Huadong Peng
Bioengineering 2023, 10(1), 10; https://doi.org/10.3390/bioengineering10010010 - 21 Dec 2022
Cited by 1 | Viewed by 2240
Abstract
Advanced biomanufacturing has been widely involved in people’s daily life, such as the production of molecules used as pharmaceuticals, in foods and beverages, and in bio-fuels [...] Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)

Research

Jump to: Editorial, Review

15 pages, 4397 KiB  
Article
SCRaMbLE: A Study of Its Robustness and Challenges through Enhancement of Hygromycin B Resistance in a Semi-Synthetic Yeast
by Jun Yang Ong, Reem Swidah, Marco Monti, Daniel Schindler, Junbiao Dai and Yizhi Cai
Bioengineering 2021, 8(3), 42; https://doi.org/10.3390/bioengineering8030042 - 23 Mar 2021
Cited by 9 | Viewed by 6486
Abstract
Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration [...] Read more.
Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed “essential raft”, where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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17 pages, 3538 KiB  
Article
Saccharomyces cerevisiae Concentrates Subtoxic Copper onto Cell Wall from Solid Media Containing Reducing Sugars as Carbon Source
by Lavinia L. Ruta and Ileana C. Farcasanu
Bioengineering 2021, 8(3), 36; https://doi.org/10.3390/bioengineering8030036 - 6 Mar 2021
Cited by 3 | Viewed by 3573
Abstract
Copper is essential for life, but it can be deleterious in concentrations that surpass the physiological limits. Copper pollution is related to widespread human activities, such as viticulture and wine production. To unravel aspects of how organisms cope with copper insults, we used [...] Read more.
Copper is essential for life, but it can be deleterious in concentrations that surpass the physiological limits. Copper pollution is related to widespread human activities, such as viticulture and wine production. To unravel aspects of how organisms cope with copper insults, we used Saccharomyces cerevisiae as a model for adaptation to high but subtoxic concentrations of copper. We found that S. cerevisiae cells could tolerate high copper concentration by forming deposits on the cell wall and that the copper-containing deposits accumulated predominantly when cells were grown statically on media prepared with reducing sugars (glucose, galactose) as sole carbon source, but not on media containing nonreducing carbon sources, such as glycerol or lactate. Exposing cells to copper in liquid media under strong agitation prevented the formation of copper-containing deposits at the cell wall. Disruption of low-affinity copper intake through the plasma membrane increased the potential of the cell to form copper deposits on the cell surface. These results imply that biotechnology problems caused by high copper concentration can be tackled by selecting yeast strains and conditions to allow the removal of excess copper from various contaminated sites in the forms of solid deposits which do not penetrate the cell. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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13 pages, 2345 KiB  
Article
Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene
by Yu Zhang, Tsan-Yu Chiu, Jin-Tao Zhang, Shu-Jie Wang, Shu-Wen Wang, Long-Ying Liu, Zhi Ping, Yong Wang, Ao Chen, Wen-Wei Zhang, Tai Chen, Yun Wang and Yue Shen
Bioengineering 2021, 8(1), 14; https://doi.org/10.3390/bioengineering8010014 - 19 Jan 2021
Cited by 11 | Viewed by 4472
Abstract
Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved [...] Read more.
Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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24 pages, 2434 KiB  
Article
Depletion of Boric Acid and Cobalt from Cultivation Media: Impact on Recombinant Protein Production with Komagataella phaffii
by Alexander Pekarsky, Sophia Mihalyi, Maximilian Weiss, Andreas Limbeck and Oliver Spadiut
Bioengineering 2020, 7(4), 161; https://doi.org/10.3390/bioengineering7040161 - 13 Dec 2020
Cited by 5 | Viewed by 4025
Abstract
The REACH regulation stands for “Registration, Evaluation, Authorization and Restriction of Chemicals” and defines certain substances as harmful to human health and the environment. This urges manufacturers to adapt production processes. Boric acid and cobalt dichloride represent such harmful ingredients, but are commonly [...] Read more.
The REACH regulation stands for “Registration, Evaluation, Authorization and Restriction of Chemicals” and defines certain substances as harmful to human health and the environment. This urges manufacturers to adapt production processes. Boric acid and cobalt dichloride represent such harmful ingredients, but are commonly used in yeast cultivation media. The yeast Komagataella phaffii (Pichia pastoris) is an important host for heterologous protein production and compliance with the REACH regulation is desirable. Boric acid and cobalt dichloride are used as boron and cobalt sources, respectively. Boron and cobalt support growth and productivity and a number of cobalt-containing enzymes exist. Therefore, depletion of boric acid and cobalt dichloride could have various negative effects, but knowledge is currently scarce. Herein, we provide an insight into the impact of boric acid and cobalt depletion on recombinant protein production with K. phaffii and additionally show how different vessel materials affect cultivation media compositions through leaking elements. We found that boric acid could be substituted through boron leakiness from borosilicate glassware. Furthermore, depletion of boric acid and cobalt dichloride neither affected high cell density cultivation nor cell morphology and viability on methanol. However, final protein quality of three different industrially relevant enzymes was affected in various ways. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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15 pages, 586 KiB  
Article
Inference of Regulatory System for TAG Biosynthesis in Lipomyces starkeyi
by Sachiyo Aburatani, Koji Ishiya, Toshikazu Itoh, Toshihiro Hayashi, Takeaki Taniguchi and Hiroaki Takaku
Bioengineering 2020, 7(4), 148; https://doi.org/10.3390/bioengineering7040148 - 19 Nov 2020
Cited by 5 | Viewed by 3100
Abstract
Improving the bioproduction ability of efficient host microorganisms is a central aim in bioengineering. To control biosynthesis in living cells, the regulatory system of the whole biosynthetic pathway should be clearly understood. In this study, we applied our network modeling method to infer [...] Read more.
Improving the bioproduction ability of efficient host microorganisms is a central aim in bioengineering. To control biosynthesis in living cells, the regulatory system of the whole biosynthetic pathway should be clearly understood. In this study, we applied our network modeling method to infer the regulatory system for triacylglyceride (TAG) biosynthesis in Lipomyces starkeyi, using factor analyses and structural equation modeling to construct a regulatory network model. By factor analysis, we classified 89 TAG biosynthesis-related genes into nine groups, which were considered different regulatory sub-systems. We constructed two different types of regulatory models. One is the regulatory model for oil productivity, and the other is the whole regulatory model for TAG biosynthesis. From the inferred oil productivity regulatory model, the well characterized genes DGA1 and ACL1 were detected as regulatory factors. Furthermore, we also found unknown feedback controls in oil productivity regulation. These regulation models suggest that the regulatory factor induction targets should be selected carefully. Within the whole regulatory model of TAG biosynthesis, some genes were detected as not related to TAG biosynthesis regulation. Using network modeling, we reveal that the regulatory system is helpful for the new era of bioengineering. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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15 pages, 5796 KiB  
Article
Proteomics Answers Which Yeast Genes Are Specific for Baking, Brewing, and Ethanol Production
by Svetlana Davydenko, Tatiana Meledina, Alexey Mittenberg, Sergey Shabelnikov, Maksim Vonsky and Artyom Morozov
Bioengineering 2020, 7(4), 147; https://doi.org/10.3390/bioengineering7040147 - 18 Nov 2020
Cited by 8 | Viewed by 4431
Abstract
Yeast strains are convenient models for studying domestication processes. The ability of yeast to ferment carbon sources from various substrates and to produce ethanol and carbon dioxide is the core of brewing, winemaking, and ethanol production technologies. The present study reveals the differences [...] Read more.
Yeast strains are convenient models for studying domestication processes. The ability of yeast to ferment carbon sources from various substrates and to produce ethanol and carbon dioxide is the core of brewing, winemaking, and ethanol production technologies. The present study reveals the differences among yeast strains used in various industries. To understand this, we performed a proteomic study of industrial Saccharomyces cerevisiae strains followed by a comparative analysis of available yeast genetic data. Individual protein expression levels in domesticated strains from different industries indicated modulation resulting from response to technological environments. The innovative nature of this research was the discovery of genes overexpressed in yeast strains adapted to brewing, baking, and ethanol production, typical genes for specific domestication were found. We discovered a gene set typical for brewer’s yeast strains. Baker’s yeast had a specific gene adapted to osmotic stress. Toxic stress was typical for yeast used for ethanol production. The data obtained can be applied for targeted improvement of industrial strains. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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13 pages, 989 KiB  
Article
Combinatorial Metabolic Engineering in Saccharomyces cerevisiae for the Enhanced Production of the FPP-Derived Sesquiterpene Germacrene
by Jan Niklas Bröker, Boje Müller, Dirk Prüfer and Christian Schulze Gronover
Bioengineering 2020, 7(4), 135; https://doi.org/10.3390/bioengineering7040135 - 24 Oct 2020
Cited by 11 | Viewed by 4871
Abstract
Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing [...] Read more.
Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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Review

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21 pages, 2821 KiB  
Review
Pichia pastoris (Komagataella phaffii) as a Cost-Effective Tool for Vaccine Production for Low- and Middle-Income Countries (LMICs)
by Salomé de Sá Magalhães and Eli Keshavarz-Moore
Bioengineering 2021, 8(9), 119; https://doi.org/10.3390/bioengineering8090119 - 31 Aug 2021
Cited by 27 | Viewed by 7913
Abstract
Vaccination is of paramount importance to global health. With the advent of the more recent pandemics, the urgency to expand the range has become even more evident. However, the potential limited availability and affordability of vaccines to resource low- and middle-income countries has [...] Read more.
Vaccination is of paramount importance to global health. With the advent of the more recent pandemics, the urgency to expand the range has become even more evident. However, the potential limited availability and affordability of vaccines to resource low- and middle-income countries has created a need for solutions that will ensure cost-effective vaccine production methods for these countries. Pichia pastoris (P. pastoris) (also known as Komagataella phaffii) is one of the most promising candidates for expression of heterologous proteins in vaccines development. It combines the speed and ease of highly efficient prokaryotic platforms with some key capabilities of mammalian systems, potentially reducing manufacturing costs. This review will examine the latest developments in P. pastoris from cell engineering and design to industrial production systems with focus on vaccine development and with reference to specific key case studies. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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17 pages, 2248 KiB  
Review
The Promise of Optogenetics for Bioproduction: Dynamic Control Strategies and Scale-Up Instruments
by Sylvain Pouzet, Alvaro Banderas, Matthias Le Bec, Thomas Lautier, Gilles Truan and Pascal Hersen
Bioengineering 2020, 7(4), 151; https://doi.org/10.3390/bioengineering7040151 - 24 Nov 2020
Cited by 38 | Viewed by 6849
Abstract
Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable [...] Read more.
Progress in metabolic engineering and synthetic and systems biology has made bioproduction an increasingly attractive and competitive strategy for synthesizing biomolecules, recombinant proteins and biofuels from renewable feedstocks. Yet, due to poor productivity, it remains difficult to make a bioproduction process economically viable at large scale. Achieving dynamic control of cellular processes could lead to even better yields by balancing the two characteristic phases of bioproduction, namely, growth versus production, which lie at the heart of a trade-off that substantially impacts productivity. The versatility and controllability offered by light will be a key element in attaining the level of control desired. The popularity of light-mediated control is increasing, with an expanding repertoire of optogenetic systems for novel applications, and many optogenetic devices have been designed to test optogenetic strains at various culture scales for bioproduction objectives. In this review, we aim to highlight the most important advances in this direction. We discuss how optogenetics is currently applied to control metabolism in the context of bioproduction, describe the optogenetic instruments and devices used at the laboratory scale for strain development, and explore how current industrial-scale bioproduction processes could be adapted for optogenetics or could benefit from existing photobioreactor designs. We then draw attention to the steps that must be undertaken to further optimize the control of biological systems in order to take full advantage of the potential offered by microbial factories. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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21 pages, 3943 KiB  
Review
Genetic Engineering and Synthetic Genomics in Yeast to Understand Life and Boost Biotechnology
by Daniel Schindler
Bioengineering 2020, 7(4), 137; https://doi.org/10.3390/bioengineering7040137 - 29 Oct 2020
Cited by 23 | Viewed by 9284
Abstract
The field of genetic engineering was born in 1973 with the “construction of biologically functional bacterial plasmids in vitro”. Since then, a vast number of technologies have been developed allowing large-scale reading and writing of DNA, as well as tools for [...] Read more.
The field of genetic engineering was born in 1973 with the “construction of biologically functional bacterial plasmids in vitro”. Since then, a vast number of technologies have been developed allowing large-scale reading and writing of DNA, as well as tools for complex modifications and alterations of the genetic code. Natural genomes can be seen as software version 1.0; synthetic genomics aims to rewrite this software with “build to understand” and “build to apply” philosophies. One of the predominant model organisms is the baker’s yeast Saccharomyces cerevisiae. Its importance ranges from ancient biotechnologies such as baking and brewing, to high-end valuable compound synthesis on industrial scales. This tiny sugar fungus contributed greatly to enabling humankind to reach its current development status. This review discusses recent developments in the field of genetic engineering for budding yeast S. cerevisiae, and its application in biotechnology. The article highlights advances from Sc1.0 to the developments in synthetic genomics paving the way towards Sc2.0. With the synthetic genome of Sc2.0 nearing completion, the article also aims to propose perspectives for potential Sc3.0 and subsequent versions as well as its implications for basic and applied research. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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18 pages, 1521 KiB  
Review
Next Generation Winemakers: Genetic Engineering in Saccharomyces cerevisiae for Trendy Challenges
by Patricia Molina-Espeja
Bioengineering 2020, 7(4), 128; https://doi.org/10.3390/bioengineering7040128 - 14 Oct 2020
Cited by 10 | Viewed by 4394
Abstract
The most famous yeast of all, Saccharomyces cerevisiae, has been used by humankind for at least 8000 years, to produce bread, beer and wine, even without knowing about its existence. Only in the last century we have been fully aware of the [...] Read more.
The most famous yeast of all, Saccharomyces cerevisiae, has been used by humankind for at least 8000 years, to produce bread, beer and wine, even without knowing about its existence. Only in the last century we have been fully aware of the amazing power of this yeast not only for ancient uses but also for biotechnology purposes. In the last decades, wine culture has become and more demanding all over the world. By applying as powerful a biotechnological tool as genetic engineering in S. cerevisiae, new horizons appear to develop fresh, improved, or modified wine characteristics, properties, flavors, fragrances or production processes, to fulfill an increasingly sophisticated market that moves around 31.4 billion € per year. Full article
(This article belongs to the Special Issue From Yeast to Biotechnology)
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