Development of an Haa1-based biosensor for acetic acid sensing in Saccharomyces cerevisiae

Acetic acid is one of the main inhibitors of lignocellulosic hydrolysates and acetic acid tolerance is crucial for the development of robust cell factories for conversion of biomass. As a precursor of acetyl-coenzyme A, it also plays an important role in central carbon metabolism. Thus, monitoring acetic acid levels is a crucial aspect when cultivating yeast. Transcription factor-based biosensors represent useful tools to follow metabolite concentrations. Here, we present the development of an acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1 that upon binding to acetic acid relocates to the nucleus. In the biosensor, a synthetic transcription factor consisting of Haa1 and BM3R1 from Bacillus megaterium was used to control expression of a reporter gene under a promoter containing BM3R1 binding sites. The biosensor did not drive expression under a promoter containing Haa1 binding sites and responded to acetic acid over a linear range spanning from 10 to 60 mM. To validate its applicability, the biosensor was integrated into acetic acid-producing strains. A direct correlation between biosensor output and acetic acid production was detected. The developed biosensor enables high-throughput screening of strains producing acetic acid and could also be used to investigate acetic acid-tolerant strain libraries

Adaptation during propagation improves Clostridium autoethanogenum tolerance towards benzene, toluene and xylenes during gas fermentation

Benzene, toluene and xylenes (BTX) are a group of compounds detected in many crude syngas mixtures. However, BTX have been identified to negatively affect microorganisms, including acetogenic species that are capable of fermenting syngas into valuable biocommodities. In order to overcome BTX inhibitory effects, we describe stepwise adaptation in Clostridium autoethanogenum that leads to tolerance to up to 0.5 mM benzene, 0.21 mM toluene and 0.07 mM xylenes. This is equivalent to eightfold of that which is found in a wood gasification plant syngas stream. Fully adapted cultures matched growth, acetate and ethanol product concentrations, and CO consumption compared to the control. The results demonstrate an efficient route towards producing a highly tolerant, industrially relevant acetogenic strain

A CRISPR activation and interference toolkit for industrial Saccharomyces cerevisiae strain KE6-12

Recent advances in CRISPR/Cas9 based genome editing have considerably advanced genetic engineering of industrial yeast strains. In this study, we report the construction and characterization of a toolkit for CRISPR activation and interference (CRISPRa/i) for a polyploid industrial yeast strain. In the CRISPRa/i plasmids that are available in high and low copy variants, dCas9 is expressed alone, or as a fusion with an activation or repression domain; VP64, VPR or Mxi1. The sgRNA is introduced to the CRISPRa/i plasmids from a double stranded oligonucleotide by in vivo homology-directed repair, allowing rapid transcriptional modulation of new target genes without cloning. The CRISPRa/i toolkit was characterized by alteration of expression of fluorescent protein-encoding genes under two different promoters allowing expression alterations up to similar to 2.5-fold. Furthermore, we demonstrated the usability of the CRISPRa/i toolkit by improving the tolerance towards wheat straw hydrolysate of our industrial production strain. We anticipate that our CRISPRa/i toolkit can be widely used to assess novel targets for strain improvement and thus accelerate the design-build-test cycle for developing various industrial production strains

Clostridium ljungdahlii as a biocatalyst in microbial electrosynthesis – Effect of culture conditions on product formation

Microbial electrosynthesis enables the production of value-added chemicals from CO2 and electrons provided by an electrode. Clostridium ljungdahlii is an electroactive acetogen that potentially could be used in microbial electrosynthesis systems. However, the optimal operational parameters for microbial electrosynthesis using C. ljungdahlii are not known. Here, we explored the effects of yeast extract, pH, and cathode potential. A low initial pH increased the rate of acetate production from CO2 and H2 in serum bottle cultures. When cultivated in bioelectrochemical systems, the optimal coulombic efficiency (i.e. close to 100 %) was observed at a cathode potential between −0.8 V and −1.0 V, while the highest productivity was reached at −1.0 V. Addition of yeast extract to the medium was needed to ensure reproducible results. Using cyclic voltammetry, we detected hydrogen-mediated extracellular electron transfer of C. ljungdahlii during growth on CO2 in a bioelectrochemical system. These results show that operational parameters should be chosen carefully to maximise the efficiency of microbial electrosynthesis

RNA sequencing reveals metabolic and regulatory changes leading to more robust fermentation performance during short-term adaptation of Saccharomyces cerevisiae to lignocellulosic inhibitors

Background: The limited tolerance of Saccharomyces cerevisiae to inhibitors is a major challenge in second-generation bioethanol production, and our understanding of the molecular mechanisms providing tolerance to inhibitor-rich lignocellulosic hydrolysates is incomplete. Short-term adaptation of the yeast in the presence of dilute hydrolysate can improve its robustness and productivity during subsequent fermentation. Results: We utilized RNA sequencing to investigate differential gene expression in the industrial yeast strain CR01 during short-term adaptation, mimicking industrial conditions for cell propagation. In this first transcriptomic study of short-term adaption of S. cerevisiae to lignocellulosic hydrolysate, we found that cultures respond by fine-tuned up- and down-regulation of a subset of general stress response genes. Furthermore, time-resolved RNA sequencing allowed for identification of genes that were differentially expressed at 2 or more sampling points, revealing the importance of oxidative stress response, thiamin and biotin biosynthesis. furan-aldehyde reductases and specific drug:H+ antiporters, as well as the down-regulation of certain transporter genes. Conclusions: These findings provide a better understanding of the molecular mechanisms governing short-term adaptation of S. cerevisiae to lignocellulosic hydrolysate, and suggest new genetic targets for improving fermentation robustness

Nutrient-supplemented propagation of Saccharomyces cerevisiae improves its lignocellulose fermentation ability

Propagation conditions have been shown to be of considerable importance for the fermentation ability of Saccharomyces cerevisiae. The limited tolerance of yeast to inhibitors present in lignocellulosic hydrolysates is a major challenge in second-generation bioethanol production. We have investigated the hypothesis that the addition of nutrients during propagation leads to yeast cultures with improved ability to subsequently ferment lignocellulosic materials. This hypothesis was tested with and without short-term adaptation to wheat straw or corn stover hydrolysates during propagation of the yeast. The study was performed using the industrial xylose-fermenting S. cerevisiae strain CR01. Adding a mixture of pyridoxine, thiamine, and biotin to unadapted propagation cultures improved cell growth and ethanol yields during fermentation in wheat straw hydrolysate from 0.04\ua0g\ua0g−1 to 0.19\ua0g\ua0g−1 and in corn stover hydrolysate from 0.02\ua0g\ua0g−1 to 0.08\ua0g\ua0g−1. The combination of short–term adaptation and supplementation with the vitamin mixture during propagation led to ethanol yields of 0.43\ua0g\ua0g−1 in wheat straw hydrolysate fermentation and 0.41\ua0g\ua0g−1 in corn stover hydrolysate fermentation. These ethanol yields were improved compared to ethanol yields from cultures that were solely short-term adapted (0.37 and 0.33\ua0g g−1). Supplementing the propagation medium with nutrients in combination with short-term adaptation was thus demonstrated to be a promising strategy to improve the efficiency of industrial lignocellulosic fermentation

Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library

BACKGROUND: Acetic acid tolerance is crucial for the development of robust cell factories for conversion of lignocellulosic hydrolysates that typically contain high levels of acetic acid. Screening mutants for growth in medium with acetic acid is an attractive way to identify sensitive variants and can provide novel insights into the complex mechanisms regulating the acetic acid stress response. RESULTS: An acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1, was used to screen a CRISPRi yeast strain library where dCas9-Mxi was set to individually repress each essential or respiratory growth essential gene. Fluorescence-activated cell sorting led to the enrichment of a population of cells with higher acetic acid retention. These cells with higher biosensor signal were demonstrated to be more sensitive to acetic acid. Biosensor-based screening of the CRISPRi library strains enabled identification of strains with increased acetic acid sensitivity: strains with gRNAs targeting TIF34, MSN5, PAP1, COX10 or TRA1. CONCLUSIONS: This study demonstrated that biosensors are valuable tools for screening and monitoring acetic acid tolerance in yeast. Fine-tuning the expression of essential genes can lead to altered acetic acid tolerance

A CRISPR Interference Screen of Essential Genes Reveals that Proteasome Regulation Dictates Acetic Acid Tolerance in Saccharomyces cerevisiae

CRISPR interference (CRISPRi) is a powerful tool to study cellular physiology under different growth conditions, and this technology provides a means for screening changed expression of essential genes. In this study, a Saccharomyces cerevisiae CRISPRi library was screened for growth in medium supplemented with acetic acid. Acetic acid is a growth inhibitor challenging the use of yeast for the industrial conversion of lignocellulosic biomasses. Tolerance to acetic acid that is released during biomass hydrolysis is crucial for cell factories to be used in biorefineries. The CRISPRi library screened consists of .9,000 strains, where .98% of all essential and respiratory growth-essential genes were targeted with multiple guide RNAs (gRNAs). The screen was performed using the high-throughput, high-resolution Scan-o-matic platform, where each strain is analyzed separately. Our study identified that CRISPRi targeting of genes involved in vesicle formation or organelle transport processes led to severe growth inhibition during acetic acid stress, emphasizing the importance of these intracellular membrane structures in maintaining cell vitality. In contrast, strains in which genes encoding subunits of the 19S regulatory particle of the 26S proteasome were downregulated had increased tolerance to acetic acid, which we hypothesize is due to ATP salvage through an increased abundance of the 20S core particle that performs ATP-independent protein degradation. This is the first study where high-resolution CRISPRi library screening paves the way to understanding and bioengineering the robustness of yeast against acetic acid stress

CRISPRi screen highlights chromatin regulation to be involved in formic acid tolerance in Saccharomyces cerevisiae

Formic acid is one of the main weak acids in lignocellulosic hydrolysates that is known to be inhibitory to yeast growth even at low concentrations. In this study, we employed a CRISPR interference (CRISPRi) strain library comprising >9000 strains encompassing >98% of all essential and respiratory growth-essential genes, to study formic acid tolerance in Saccharomyces cerevisiae. To provide quantitative growth estimates on formic acid tolerance, the strains were screened individually on solid medium supplemented with 140 mM formic acid using the Scan-o-Matic platform. Selected resistant and sensitive strains were characterized in liquid medium supplemented with formic acid and in synthetic hydrolysate medium containing a combination of inhibitors. Strains with gRNAs targeting genes associated with chromatin remodeling were significantly enriched for strains showing formic acid tolerance. In line with earlier findings on acetic acid tolerance, we found genes encoding proteins involved in intracellular vesicle transport enriched among formic acid sensitive strains. The growth of the strains in synthetic hydrolysate medium followed the same trend as when screened in medium supplemented with formic acid. Strains sensitive to formic acid had decreased growth in the synthetic hydrolysate and all strains that had improved growth in the presence of formic acid also grew better in the hydrolysate medium. Systematic analysis of CRISPRi strains allowed identification of genes involved in tolerance mechanisms and provided novel engineering targets for bioengineering strains with increased resistance to inhibitors in lignocellulosic hydrolysates

CRISPR interference technology for development of more tolerant industrial yeast strains (Gothenburg)

Second generation bioethanol using lignocellulosic biomass as raw material is a promising alternative to bioethanol produced from sugar-based feedstocks. In addition to sugars, lignocellulosic hydrolysates also contain inhibitors that impair microbial growth. One way to tackle the low productivities is to develop new strains with increased tolerance towards inhibitors.Over the past few years, different CRISPR technologies have been developed to accelerate the construction of new strains. The CRISPR interference (CRISPRi) technology utilizes a catalytically inactive Cas9 (dCas9) to modulate the expression of genes targeted by a sgRNA, allowing the alteration of essential genes and the manipulation of multiple traits without altering the target sequence.In the present work, our goal was to use CRISPRi to improve the inhibitor tolerance of a polyploid industrial yeast strain. We explored different strategies to overcome the challenges of implementing CRISPRi in an industrial strain. As a proof of concept, the expression of a gene encoding a fluorescent protein was modulated using dCas9 with different activation or repression domains. Changes in fluorescence were measured by flow cytometry and changes in expression were verified by qPCR, validating the use of CRISPRi for alteration of gene expression in an industrial yeast strain.Subsequently, a number of genes previously identified to be involved in inhibitor tolerance were selected as targets for CRISPRi. The performance of the novel strains during growth in the presence of different inhibitors was analysed in a high-throughput platform, leading to identification of strains where the altered gene expression led to improved tolerance.This work shows that the CRISPRi technology can be used to accelerate the development of more robust, industrial production hosts