Study investigates the role of essential genes in acetic acid tolerance

Saccharomyces cerevisiae, also known as Baker’s yeast, is industrially utilized for the production of different biochemicals. These biochemicals can be produced from waste material from the forest or agricultural industry (second-generation biomass).

Vaskar Mukherjee, researcher, and Yvonne Nygård, Associate Professor, both at the Division of Industrial Biotechnology, are two of the researchers behind the study where a CRISPRi library of 9,000 yeast strains was used to investigate the role of essential genes in acetic acid tolerance. Image Credit: Chalmers University of Technology.

Acetic acid is released at the time of mechanical and enzymatic degradation of biomass. It hinders the growth and the biochemical production rate of yeast. Recently, scientists from Chalmers employed high-resolution CRISPRi library screening to offer a novel understanding of the stress response of yeast. They also identified novel target genes for the bioengineering of efficient industrial yeast.

We are presenting a massive dataset that offers an extraordinary resolution of the functional contribution of essential genes in baker’s yeast under acetic acid stress. This was never attempted before.”

Vaskar Mukherjee, Study First Author and Researcher, Division of Industrial Biotechnology, Chalmers University of Technology

According to Yvonne Nygård, study last author and an Associate Professor at Chalmers, “In the strain library we screened, the expression of all essential genes was altered, something which was very difficult to do before the discovery of the CRISPR-Cas9-technology.”

Reduced expression of essential genes using CRISPRi

CRISPR interference (CRISPRi) is a robust tool to analyze cellular physiology under various growth conditions. Using this derivative of the Nobel prize-winning CRISPR-Cas9 technology, genes are neither inserted nor deleted, but the regulation of the target gene is altered.

The scientists employed CRISPRi technology to decrease the expression of the essential genes (genes when deleted result in the death of the organism) and thereby reduce the level of the protein coded by the target gene.

For most of the essential genes, this keeps the organism viable, and we also get to see the functional contribution of that gene at different expression levels under different nutrient or environmental conditions, in this case under acetic acid stress.”

Vaskar Mukherjee, Study First Author and Researcher, Division of Industrial Biotechnology, Chalmers University of Technology

Proteosomal genes involved in acetic acid tolerance

The researchers utilized a CRISPRi library containing over 9,000 yeast strains and targeted more than 98% of all vital and respiratory growth-essential genes. The findings indicate that refining the expression of proteasomal genes leads to higher tolerance to acetic acid.

The proteosome is a protein complex that degrades damaged or excess proteins by spending ATP—an organic compound that supplies energy to induce numerous mechanisms in living cells and is specifically necessary for huge amounts in yeast cells to deal with acetic acid stress.

The researchers put forth that adaptation of proteasomal degradation of oxidized proteins safeguards ATP and thus increases tolerance to acetic acid. The findings are of great interest, indicating that these genes can be targeted for bioengineering of enhanced industrial cells.

Our results allowed us to build rational mechanistic models that expand our current understanding of molecular biology of yeast under acetic acid stress. I am sure our footsteps will be followed by many researchers to screen essential genes under many other different conditions. I believe our dataset will be used by academia or industries to identify novel genetic candidates to bioengineer robust acetic acid-tolerant yeast strains.”

Vaskar Mukherjee, Study First Author and Researcher, Division of Industrial Biotechnology, Chalmers University of Technology

More research on yeast and second-generation biomass

Scientists from Chalmers are currently experimenting on three various projects employing similar technologies. One of the projects deals with the use of CRISPRi technology to pinpoint new bioengineering genetic candidates to enhance co-utilization of glucose and xylose at the time of biochemical fermentation employing second-generation biomass.

Wild S. cerevisiae is not capable of metabolizing xylose, and a xylose-utilizing engineered strain of S. cerevisiae favors glucose over xylose as the primary carbon source. Consequently, xylose consumption is mostly incomplete in industrial second-generation biochemical fermentation and persists as a major bottleneck for the commercial production of second-generation biochemicals.