Yeast with an over half synthetic genome is created in the lab

Researchers have combined over seven synthetic chromosomes that were made in the lab into a single yeast cell, resulting in a strain with more than 50% synthetic DNA that survives and replicates similarly to wild yeast strains. The team present the half-synthetic yeast November 8 in the journal Cell as part of a collection of papers across Cell, Molecular Cell,and Cell Genomics that showcase the Synthetic Yeast Genome Project (Sc2.0), a global consortium working to develop the first synthetic eukaryote genome from scratch. The team has now synthesized and debugged all sixteen yeast chromosomes.

"Our motivation is to understand the first principles of genome fundamentals by building synthetic genomes," says co-author and synthetic biologist Patrick Yizhi Cai of the University of Manchester, who is also senior author of two other papers in the collection. "The team has now re-written the operating system of the budding yeast, which opens up a new era of engineering biology -- moving from tinkering a handful of genes to de novo design and construction of entire genomes."

Though bacterial and viral genomes have been synthesized previously, this would be the first synthetic eukaryote genome, which introduces the complication of multiple chromosomes. The synthetic yeast is also a "designer" genome that differs substantially from the natural Saccharomyces cerevisiae (brewer's or baker's yeast) genome on which it is based.

"We decided that it was important to produce something that was very heavily modified from nature's design," says senior author and Sc2.0 leader Jef Boeke, a synthetic biologist at NYU Langone Health. "Our overarching aim was to build a yeast that can teach us new biology."

To this end, the researchers removed chunks of non-coding DNA and repetitive elements that could be considered "junk," added new snippets of DNA to help them more easily distinguish between synthesized and native genes, and introduced a built-in diversity generator called "SCRaMbLE" that shuffles the order of genes within and between chromosomes.

To increase genome stability, the team also removed many of the genes that encode transfer RNA (tRNA) and relocated them to an entirely new "neochromosome" consisting only of tRNA genes. "The tRNA neochromosome is the world's first completely de novo synthetic chromosome," says Cai. "Nothing like this exists in nature."

Since the yeast genome is organized into sixteen chromosomes, the researchers began by assembling each chromosome independently to create sixteen partially synthetic yeast strains that each contained 15 natural chromosomes and one synthetic chromosome. The next challenge was to begin combining these synthetic chromosomes into a single yeast cell.

To do this, Boeke's team started by using a method reminiscent of Mendel's peas: essentially, the researchers interbred different partially synthetic yeast strains and then searched amongst their progeny for individuals carrying both synthetic chromosomes. Though effective, this method is very slow, but the team gradually consolidated all previously synthesized chromosomes -- six full chromosomes and one chromosome arm -- into a single cell. The resulting yeast strain was more than 31% synthetic, had normal morphology, and showed only slight growth defects compared to wild-type yeast.

To more efficiently transfer specific chromosomes between yeast strains, the researchers developed a new method called chromosome substitution that is discussed in another paper in the new collection. As a proof of concept, they used chromosome substitution to transfer a newly synthesized chromosome (chromosome IV, the largest of all the synthetic chromosomes), resulting in a yeast cell with 7.5 synthetic chromosomes that is more than 50% synthetic.

When the synthetic chromosomes were consolidated into a single yeast strain, the team detected several genetic defects or "bugs" that were invisible in yeast strains that only carried one synthetic chromosome. "We knew in principle that this might happen -- that we might have a huge number of things that had tiny little effects and that, when you put them all together, it might result in death by a thousand cuts," says Boeke.

Some of these bugs were simply due to the additive impact of having many tiny defects within the genome, while others involved genetic interactions between genes on the different synthetic chromosomes. The researchers were able to map and fix several of these bugs and increase the synthetic yeast's fitness by using a method based on CRISPR/Cas9.

"We've now shown that we can consolidate essentially half of the genome with good fitness, which suggests that this is not going to be a big problem," says Boeke. "And from debugging, we learn new twists on the rules of life."

The next step will be to integrate the remaining synthetic chromosomes. "Now we're just this far from the finish line of having all 16 chromosomes in a single cell," says Boeke. "I like to call this the end of the beginning, not the beginning of the end, because that's when we're really going to be able to start shuffling that deck and producing yeast that can do things that we've never seen before."

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Cans or bottles: What's better for a fresh, stable beer?

The flavor of beer begins to change as soon as it's packaged, prompting a debate among afficionados: Does the beverage stay fresher in a bottle or a can? Now, researchers report in ACS Food Science & Technology that the answer is, well, complicated, and depends on the type of beer. An amber ale stayed fresher in bottles, whereas container choice made much less difference to the stability of an India Pale Ale (IPA).

In addition to water and ethanol, beer contains thousands of flavor compounds, which are metabolites produced by yeast, hops and other ingredients. During storage, chemical reactions break down some of those components while forming others. This reduces the content of some tasty flavors while generating unappetizing ones, contributing to the aging, or staling, of beer. To help brewers prolong shelf life, researchers have studied beer aging, but they've concentrated on light lagers and a limited group of chemicals. Jessica Prenni and colleagues wanted to extend that work to amber ale and IPA, as well as additional compounds. The team also wanted to conduct the first stability comparison of beer packaged in glass bottles versus aluminum cans.

Cans and brown bottles of amber ale and IPA were chilled for a month and then kept at room temperature for five months to mimic typical storage conditions. Every two weeks, the researchers analyzed the metabolites in newly opened containers. Throughout this time, the concentration of certain metabolites in amber ale -- including some amino acids and esters -- differed significantly depending on whether it was packaged in a bottle or can. IPA, however, was much less sensitive to packaging type, possibly because of its higher concentration of polyphenols from hops. These compounds not only prevent oxidation but also bind to amino acids, thus retaining them in the beer rather than allowing them to get stuck to the inside of a container.

The researchers also found that the metabolic profile of both amber ale and IPA changed over time, whether packaged in a can or bottle. However, amber ale in cans showed the greatest variation during aging. Once scientists find out how all of these changes affect flavor, brewers will be able to make more-informed decisions about the best type of packaging for their particular type of beer.

From Science Daily

Blowing bubbles in dough to bake perfect yeast-free pizza

In typical breads, yeast produces bubbles via a biochemical process, causing dough to rise and develop into light, airy, and tasty treats. Without that yeast, it is difficult to make morsels with the same characteristic taste and texture. The perfect, yeast-free pizza, as such a food, presents an important challenge for bakers and yeast-intolerant crust enthusiasts across the globe.

In Physics of Fluids, by AIP Publishing, researchers from the University of Naples Federico II developed a method to leaven pizza dough without yeast.

The team, which included its very own professional pizza-maker/graduate student, prepared the dough by mixing water, flour, and salt and placing it in a hot autoclave, an industrial device designed to raise temperature and pressure.

From there, the process is like the one used to produce carbonation in soda. Gas is dissolved into the dough at high pressure, and bubbles form in the dough as pressure is released during baking. In comparison to other scientific experiments, the pressures involved were mild. They can be obtained by a typical at-home coffee maker.

However, the scientists-turned-bakers had to be cautious with the pressure release. Compared to soda, pizza dough does not respond as nicely to an abrupt change in pressure.

"The key to the process is to design the pressure release rate not to stress the dough, which likes to expand gently," said author Ernesto Di Maio.

The authors evaluated their dough with rheology, which measures the flow and deformation of a material. Fine-tuning the pressure release through rheological analysis made it possible to gently inflate bubbles to the desired extent.

"We mainly studied how dough behaves with and without yeast. How the softness changes with leavening, and how the dough responds to a temperature program during baking," said author Rossana Pasquino. "This was fundamental to designing the pressure protocol for the dough without yeast."

After many unofficial taste tests, the researchers are purchasing a larger, food-grade autoclave that will make full-sized pizzas in future experiments. They hope to see their idea used in pizza shops.

"We had a lot of fun applying things we know well to delicious polymers, instead of our typical and sometimes boring smelly plastics," said Pasquino. "The idea of approaching food samples with the same technologies and knowledge used for thermoplastic polymers was surprisingly successful!"

As a person with a yeast allergy, Di Maio is also excited about applications for other leavened products like bread, cakes, and snacks.

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Harvesting baker's yeast for aging-related therapeutics

Around the world, more people are growing older. According to the World Health Organisation, 1 in 6 people in the world will be aged 60 years or over by 2030. By 2050, the world's population of people aged 60 years and older will double to 2.1 billion. The number of persons aged 80 years or older is expected to triple between 2020 and 2050 to reach 426 million.

In line with the growing number of seniors, the number of people living with age-related diseases such as dementia, including Alzheimer's Disease and Parkinson's Disease is also expected to increase exponentially. These age-related diseases are an emerging impediment to healthy and functional aging.

A class of medicine used in the treatment of neuro-cognitive diseases and other neurological ailments (migraines, headaches, etc) are currently obtained from extracts of the ergot fungus. However, continued cultivation of the ergot fungus for medicine is not sustainable as industrial agriculture is one of the largest contributors to carbon emissions worldwide.

To meet the global demand for such medication, between 10-15 tons of D-lysergic acid (DLA), an ingredient used in producing the medicine, are produced each year. The ergot fungi are parasites to cereal crops such as rye, and their cultivation entails growing them on top of fields of such crops that could otherwise be used for food production. In order to reduce the use of arable land to produce such medicine, a group of researchers from the Yong Loo Lin School of Medicine at the National University of Singapore (NUS Medicine) and Imperial College London have trialled an alternative way of producing DLA.

Using yeast commonly known to make bread, and synthetic biology techniques, the team introduced the enzymes from the ergot fungus into baker's yeast, which also happens to be another fungus. Through a process known as fermentation, the modified yeast was then grown using sugar to produce DLA. Natural fermentation has been used throughout human history for food production, most notably in the production of bread and beer. Just like how baker's yeast has been used to produce the alcohol and flavours in beer, fermentation using the modified yeast can now produce DLA.

The study was published in Nature Communications on 7 Feb 2022.

"It is possible to produce up to five tons of DLA annually using the current yeast strain; and with further optimisation, commercial production levels could be attainable," explained Associate Professor Yew Wen Shan from the Department of Biochemistry at NUS Medicine and the co-lead Principal Investigator of the study. "This research builds upon the growing body of work that use microbes such as yeast for the sustainable production of medicine and functional food ingredients."

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An easy relationship between a beetle and its yeast symbiont

Japanese lizard beetle larvae feed on yeast injected from their mothers' abdomens into the bamboo stems they are growing in. Now, scientists at Nagoya University have made a surprising discovery: the yeast can digest some complex sugars in the bamboo woody tissue, but it doesn't. Instead, it consumes much simpler and more available sugar sources.

"This was a real surprise," says Nagoya University bioagricultural researcher Wataru Toki. "While yeast can indeed decompose those indigestible components, our analysis shows the yeast actually grows on small molecule monosaccharides." The results are published in the journal Scientific Reports.

Female Japanese lizard beetles carry the yeast Wickerhamomyces anomalus in a specialised pocket-like organ. In spring, they dig holes in bamboo and insert their eggs and the yeast. W. anomalus grows into a sort of fungal garden that the very hungry beetle larvae munch on as soon as they hatch.

In other symbiotic relationships, fungi typically break down complex sugars into more digestible chunks that their host insects can feed on. Toki and his colleague, Dan Aoki, wanted to know whether this was also the case in the relationship between the Japanese lizard beetle and W. anomalus.

Their research suggests not. The scientists used a technique called ion exchange chromatography to analyse and compare the sugar content of fresh bamboo pith, and pith colonized by yeast alone or by yeast and beetle larvae. The comparison revealed that the yeast mostly ate the simple free sugars glucose and fructose.

This surprised the scientists because further tests showed that the yeast can actually digest some complex, indigestible sugars if necessary.

"Bamboo is not only a farm for the yeast but also a house for the larvae. So the larvae can live in a strong house safely because the house is not decomposed by the food," explains Toki.

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Japanese sake: the new pick-me-up? Yeast strain makes fatigue-fighting ornithine

 Fans of sake, the traditional Japanese alcoholic beverage, may have even more reason to enjoy it now: Japanese scientists have discovered that a mutant strain of sake yeast produces high levels of the amino acid ornithine.

In a study published this month in Metabolic Engineering, researchers from the Nara Institute of Science and Technology and the Nara Prefecture Institute of Industrial Development have revealed that a mutant strain of sake yeast produces 10 times the amount of the amino acid ornithine compared with the parent yeast strain.

Ornithine is a non-protein-making amino acid and a precursor to two amino acids -- arginine and proline. It has been found to perform several physiological functions, such as reducing fatigue and improving sleep quality.

"We wanted to obtain sake yeast strains with improved ethanol tolerance," says a first author of this article, Masataka Ohashi. "During sake fermentation, the yeast is exposed to high concentrations of ethanol, which impedes yeast cell growth, viability and fermentation. Increased ethanol tolerance in sake yeast strains could improve ethanol production and reduce fermentation time."

To find ethanol-tolerant yeast strains, the researchers isolated mutants that accumulated proline, which can alleviate ethanol toxicity, using a conventional mutagenesis (i.e., one that doesn't involve genetic modification). They also conducted whole genome sequencing analysis, and performed brewing tests with sake yeast strains. Then they identified and analyzed a new mutation in a gene that encodes a variant of N-acetyl glutamate kinase that increases intracellular ornithine level.

"We previously constructed self-cloning industrial yeast strains that accumulate proline to increase ethanol tolerance and productivity of yeast," explains Prof. Hiroshi Takagi, a corresponding author. "But those yeasts have not been yet acceptable to consumers because they're considered to be genetically modified, even though a self-cloning yeast has no foreign genes or DNA sequences -- they only have yeast DNA."

The researchers successfully isolated non-genetically modified yeasts that produced 10 times the amount of ornithine compared with the parent strain, which is widely used in Japanese sake breweries, and the sake brewed with them contained 4-5 times more ornithine.

The results of this study will contribute to the development of improved yeast strains for production of high levels of ornithine, and the strain obtained in this study could be readily applied to sake, wine, and beer brewing. Ornithine-accumulating yeast strains could also be used in the production of ornithine-rich dietary supplements made from these yeasts and their products.

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The secret of classic Belgian beers? Medieval super yeasts!

An international team of scientists, led by Prof. Kevin Verstrepen (VIB-KU-Leuven) and Prof. Steven Maere (VIB-UGent), has discovered that some of the most renowned classic Belgian beers, including Gueuze and Trappist ales, are fermented with a rare and unusual form of hybrid yeasts. These yeasts combine DNA of the traditional ale yeast, Saccharomyces cerevisiae, with that of more stress-resistant feral yeasts such as Saccharomyces kudriavzevii.

Mixed origins

"These yeasts are hybrids between two completely different species" says Dr. Jan Steensels (VIB -- KU Leuven Center for Microbiology), who coordinated the lab work of this study. "Think of lions and tigers making a super-baby."

Such interspecific hybridizations are rare and seem to be favored by the domestication process. In this case, the new hybrid yeasts combined important characteristics of both parental species, with the fermentation capacity of normal beer yeasts and the stress tolerance and capacity to form special aromas of more feral ancient yeasts like S. kudriavzevii that haphazardly made their way into the brewery.

The team, from the VIB-KU Leuven Center for Microbiology and the University of Munich, supported by industrial partners, has spent five years characterizing the different yeasts used in today's production of beer, wine, bread and biofuels. The genetic analysis of these yeasts was quite a piece of work, because none of the existing pipelines for DNA sequencing can deal with such mixed origins.

For this the team could, surprisingly, count on the plant expertise of professor Steven Maere, a bioinformatics expert from the VIB-UGent Center for Plant Systems Biology. Maere explains: "Plants have some of the most complex genomes of all living organisms. It is fascinating that complex interspecific hybrids with doubled genomes feature prominently both among domesticated yeasts and domesticated plants."

A surprise in DNA

"It was a bit of a surprise for us" says Dr. Brigida Gallone (VIB-KU Leuven Center for Microbiology), the lead author on the paper that appeared today in Nature Ecology and Evolution. "In 2016, we reported that most industrial yeasts belong to, or arose from the species Saccharomyces cerevisiae, the traditional baker's and brewer's yeast. We found that these industrial yeasts are quite different from their wild progenitors, with different subfamilies having adapted to beer, wine and bakery environments. We also noticed that some of the yeasts that were isolated from ancient Belgian beer styles, like Gueuze and Trappist beers, are even more unusual and contained DNA of two different yeast species."

"It really seems that these unique natural yeasts allowed the development of some of the most renowned beers that Belgium is so famous for," says Dr. Philippe Malcorps, Senior Scientist at the Global Innovation and Technology Center of AB InBev, the world's largest brewer. The team of Malcorps helped with the isolation of yeasts from some of their spontaneous fermentation beer cellars. Those natural super-yeasts are living witnesses of brewing from pre-industrial ages, adapted to harsh conditions of fermentation of the strong Trappist beers, or survival in the long lagering typical for Gueuze beers.

"One could say that the unique habitat in wooden fermentation barrels created by adventurous Medieval Belgian brewers allowed these new species to thrive until today," says Prof. Kevin Verstrepen (VIB-KU Leuven Center for Microbiology).

A history of yeasts

Apart from the special Belgian yeasts, the team also collected a large number of hybrids from S. eubayanus and S. cerevisiae, or from S. uvarum strongly adapted to cold fermentation. While it was already known that lager yeasts were hybrids, the complete DNA analysis of a large number of these yeasts showed how these specific hybrids originated in medieval Germany and later spread across different European breweries as the pilsner beers grew more popular.

"It is no coincidence that the origin of today's beer yeasts lies in Belgium and Germany, arguably the two countries that are most associated with the art of brewing," says Prof. Mathias Hutzler (TU Munich).

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