BER Research Highlights

Search Date: March 22, 2019

3 Records match the search term(s):


January 11, 2019

New Method Knocks Out Yeast Genes with Single-Point Precision

Researchers can precisely study how different genes affect key properties in a yeast used industrially to produce fuel and chemicals.

The Science
How do you make yeast work harder? Not to make bread, but in processes that yield chemicals and pharmaceuticals. Industries currently use a yeast called Saccharomyces cerevisiae. They’d like it to work better. The answer is in manipulating the yeast’s genetic code. To get at that code, researchers developed a method that turns off targeted genes in the yeast, introducing mutations. The team’s approach deletes specific points in the DNA sequence. They study how each deletion affects the yeast. Does a deletion cause the yeast to stop working in certain chemicals? Does a deletion make the yeast grow more slowly? The team’s approach lets them study each gene, as well as in combination with other genes. With this approach, scientists can construct libraries of mutants for use in discovering how each gene works.

The Impact
Libraries of genetic mutations have so far only been achieved in simpler organisms, specifically prokaryotes. Now, scientists can build such libraries for more complex organisms. The new technique lets scientists rapidly engineer tens of thousands of genes. They can target the genes with 98 percent efficiency. The results ease identifying and isolating mutant strains that show desired traits, such as tolerance to toxic compounds necessary to produce industrial products.

Summary
Researchers developed a method called CRISPRCas9- and homology-directed-repair-assisted genome-scale engineering (CHAnGE) using libraries of synthetic oligonucleotides (cassettes) containing a CRISPR guide sequence, gene-specific sequences to target homologous recombination to those selected genes, and unique barcodes to track each mutant strain. The oligonucleotide library was cloned into a plasmid and introduced into Saccharomyces cerevisiae. Nearly 25,000 sequences representing almost every one of the 6,500 yeast open reading frames were synthesized. More than 98 percent of the CHAnGE cassettes resulted in mutations in the target genes at least 82 percent of the time, demonstrating a high editing efficiency. The technology proved to be effective for the introduction of both small deletions and single-base mutations, as well as for saturation mutagenesis of a single gene or domain. CHAnGE was successfully applied to engineer yeast strains that are tolerant to furfural, indicating that it could be used to engineer industrially relevant eukaryotes to advance toward renewable production of biofuels and valuable chemicals.

Contact
Program Manager
Pablo Rabinowicz
Department of Energy, Office of Science, Biological and Environmental Research
pablo.rabinowicz@science.doe.gov

Principal Investigator
Huimin Zhao
University of Illinois at Urbana-Champaign 
zhao5@illinois.edu  

Funding
This work was supported by the Office of Biological and Environmental Research within the Department of Energy’s Office of Science and the Carl R. Woese Institute for Genomic Biology at the University of Illinois at Urbana-Champaign.

Publications
Z. Bao, M. HamediRad, P. Xue, H. Xiao, I. Tasan, R. Chao, J. Liang, and H. Zhao, “Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision.” Nature Biotechnology 36, 505 (2018). [DOI: 10.1038/nbt.4132]

Related Links
University of Illinois press release: New CRISPR technology ‘knocks out’ yeast genes with single-point precision

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 09, 2019

How Plants Regulate Sugar Deposition in Cell Walls

Identified genes involved in plant cell wall polysaccharide production and restructuring could aid in engineering bioenergy crops.

The Science
Ultimately, researchers want to engineer bioenergy crops to accumulate large amounts of easy-to-use sugars. Researchers from the Great Lakes Bioenergy Research Center identified a major part of the sugar production process in a model leafy grass. They discovered a transcription factor, which turns a gene on and off. The gene triggers the synthesis of a sugar, called mixed-linkage glucan (MLG). Characterizing downstream genes regulated by this transcription factor provides insight into how plants make MLG. This information is vital to overcoming growth defects associated with engineering plants to produce large quantities of MLG.

The Impact
To make fuels from grasses or other plants, scientists often focus on certain sugars, such as mixed-linkage glucan. Understanding the genes that produce and restructure such sugars should lead to a better understanding of how the bioenergy grass sorghum stores sugar in cell walls. With such information, Great Lakes Bioenergy Research Center researchers aim to engineer bioenergy crops like sorghum to accumulate large amounts of the sugar in the stem. They aim to do it without disrupting plant growth.

Summary
Mixed-linkage glucan (MLG) is an energy-rich polysaccharide found at high levels in some grass endosperm cell walls and at lower amounts in other tissues. Cellulose synthase-like F and cellulose synthase-like H genes synthesize MLG, but it is unknown if other genes participate in the production and restructuring of MLG. Working with the model grass Brachypodium distachyon, GLBRC researchers identified a trihelix family transcription factor (BdTHX1) that is highly co-expressed with the BdCSLF6 gene and which appears to help regulate MLG biosynthesis. They showed that BdTHX1 protein can bind with high affinity to BdCSLF6 as well as BdXTH8, which encodes a grass-specific endotransglucosylase, an enzyme involved in cell wall structuring. The team found that BdXTH8 preferentially interacts with MLG and xyloglucans, suggesting it may mediate their binding in plant tissues. In addition, B. distachyon shoots grown from cells overexpressing BdTHX1 showed abnormal growth and early death. These results indicate that the transcription factor BdTHX1 likely plays an important role in MLG biosynthesis and restructuring by regulating the expression of BdCSLF6 and BdXTH8. This knowledge will be instrumental for engineering the bioenergy grass sorghum to accumulate large amounts of MLG in its stem tissue.

Contact
Program Manager
N. Kent Peters
Department of Energy, Office of Science, Office of Biological and Environmental Research
kent.peters@science.doe.gov; (301) 903-5549

Principal Investigator
Curtis Gene Wilkerson
Michigan State University 
wilker13@msu.edu

Funding
This work was supported by the Department of Energy Great Lakes Bioenergy Research Center and the United Kingdom Biotechnology and Biological Sciences Research Council.

Publications
M. Fan, K. Herburger, J.K. Jensen, S. Zemelis-Durfee, F. Brandizzi, S.C. Fry, and C.G. Wilkerson, “A trihelix family transcription factor is associated with key genes in mixed-linkage glucan accumulation.” Plant Physiology 178, 1207 (2018). [DOI: 10.1104/pp.18.00978]

Related Links
Plant Physiology article: A Trihelix Family Transcription Factor Is Associated with Key Genes in Mixed-Linkage Glucan Accumulation

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER


January 09, 2019

Scientists Identify Gene Cluster in Budding Yeasts with Major Implications for Renewable Energy

How yeast partition carbon into a metabolite may offer insights into boosting production for biofuels.

The Science
Yeasts are complex organisms that may become the workhorses of biofuel production. To move yeasts into this larger role, scientists need to understand the genetic machinery that leads to the production of complex molecules like the iron-binding molecule pulcherrimin in budding yeasts. Scientists revealed a four-gene cluster associated with pulcherrimin production. Further tests revealed likely functions for each of the genes: two biosynthetic enzymes, a transporter, and a transcription factor involved in both biosynthesis and transport.

The Impact
Yeasts use the same pathway to make pulcherrimin and an alcohol, isobutanol, of interest for biofuels. Some yeast strains direct a significant amount of carbon into pulcherrimin. Since both pulcherrimin and isobutanol are made from a common pathway, this suggests that the metabolic control of high pulcherrimin producers may be harnessed for increased isobutanol production in yeast. This could help engineer yeast to make larger quantities of isobutanol.

Summary
Despite the discovery of an iron-binding pigment known as pulcherrimin 65 years ago, the genes responsible for its biosynthesis remained uncharacterized. Using a comparative genomics approach among 90 genomes from the budding yeast subphylum Saccharomycotina, researchers from the Great Lakes Bioenergy Research Center identified the first yeast secondary metabolite gene cluster and showed that it’s responsible for pulcherrimin biosynthesis. Targeted gene disruptions in Kluyveromyces lactis identified putative functions for each of the four genes: two pulcherriminic acid biosynthesis enzymes, a pulcherrimin transporter, and a transcription factor involved in both biosynthesis and transport. The requirement of a functional putative transporter to utilize extracellular pulcherrimin-complexed iron demonstrates that pulcherriminic acid is a siderophore, an iron-chelating compound secreted by microorganisms. This research also characterized and named two genes that previously lacked assigned functions in the fuel-producing model yeast Saccharomyces cerevisiae. The evolution of this gene cluster in budding yeast suggests an ecological role for pulcherrimin akin to other microbial public goods systems. Because some yeasts species are particularly adept at funneling carbon into pulcherrimin, studying how high-level pulcherrimin producing strains are altered in their metabolic control may inform strategies for increased biofuel production in model organisms.

Contact
Program Manager
N. Kent Peters
Department of Energy, Office of Science, Office of Biological and Environmental Research
kent.peters@science.doe.gov; (301) 903-5549

Principal Investigator
Chris Todd Hittinger
University of Wisconsin-Madison
cthittinger@wisc.edu

Funding
This material is based upon work supported by the Department of Energy, Office of Science, Office of Biological and Environmental Research, as well as the National Science Foundation. Additional funding was provided by the Pew Charitable Trusts and the Vilas Trust Estate.

Publications
D.J. Krause, J Kominek, D.A. Opulente, X. Shen, X. Zhou, Q.K. Langdon, J. DeVirgilio, A.B. Hulfachor, C.P. Kurtzman, A. Rokas and C.T. Hittinger, “Functional and evolutionary characterization of a secondary metabolite gene cluster in budding yeasts.” Proceedings of the National Academy of Sciences USA 115(43), 11030 (2018). [DOI: 10.1073/pnas.1806268115]

Related Links
Great Lakes Bioenergy Research Center news release: Red-hued yeasts hold clues to producing better biofuels

Topic Areas:

Division: SC-23.2 Biological Systems Science Division, BER