Elucidation of weak organic acid resistance mechanisms in non-Saccharomyces yeast: a case study of Zygosaccharomyces parabailii and Kluyveromyces marxianus

The efficient implementation of biorefineries to produce bio-based chemicals and fuels requires sustainable source of feedstock and robust microbial factories. Among others, lignocellulose and whey, which are residual wastes deriving from wood/agriculture and dairy industries, represent cheap, sugar-enriched feedstocks. The conversion of lignocellulose and whey into the desired products using microbial cell factories is a promising option to replace the fossil based petrochemical refinery. Different bacteria, algae and yeasts are currently used as microbial hosts, and their number is predicted to increase over next years. Minimum nutritional requirements and robustness have made yeasts a class of microbial hosts widely employed in industrial biotechnology, exploiting their natural abilities as well as genetically acquired pathways for production of natural and recombinant products, including bulk chemicals such as organic acids. However, efficient and economically viable production of organic acids has to face problems related to low productivity/titer and toxicity of the final product. Therefore, the exploration of yeast biodiversity to exploit unique native features and the understanding of mechanisms to endure harsh conditions are essential to develop ultraefficient and robust industrial yeast with novel properties. The aim of the research thesis is to evaluate the mechanism of weak acid stress response in the non-Saccharomyces yeasts Zygosaccharomyces parabailii and Kluyveromyces marxianus. To better understand the weak acid stress response of Z. parabailii, we summarized recent finding on the species. Knowing the relevant scientific reports, the next study was focused on the effect of lactic acid stress on Z. parabailii. This organic acid can be used as monomer for the production of biodegradable bioplastic polymers, such as poly lactic acid (PLA). The study revealed that cells are able to tolerate 40g/l of lactic acid without inducing a lag phase of growth and exhibit a negligible percentage of dead cells. More importantly, during lactic acid exposure, we observed structural modifications at the level of cell wall and membrane. These findings confirmed the peculiar ability of Z. parabailii to adapt to weak organic acids via remodeling of cellular components. The lack of a complete genome assembly and annotation encouraged us to perform a genome sequencing and genome study of our Z. parabailii strain. The results revealed that Z. parabailii is undergoing fertility restoration after interspecies hybridization event, which may shed a light to the process of whole genome duplication. The availability of Z. parabailii complete genome information allowed us to perform the first RNA-sequencing analysis on the species exposed to lactic acid stress. The results showed upregulation of mitochondrial and oxidative stress genes, and downregulation of a subset of cell wall genes, in addition to other specific regulation related to redox balance and ion homeostasis. Remarkably, several differentially regulated genes differ significantly from the S. cerevisiae counterpart or, in some cases, even seem not to have a homologue. Increased interest of K. marxianus application in industrial biotechnology led us to study its multidrug resistance transporters during acetic and lactic acid stress, the first being a contaminant related to the use of lignocellulose as feedstocks, while the second as final product of interest, as mentioned above. The results showed a strain-specific response to weak organic acid stress, and a possible involvement of KmPDR12 in acetic and lactic acid stress resistance, opening potential for future discoveries and novel studies. Overall, this work contributes to the vast array of studies that are shedding light on yeasts biodiversity, both as a way for understanding their natural potential and as an instrument for tailoring novel cell factories

Sugar transporter engineering in yeast to enable simultaneous co-utilization of sugars prevalent in cellulosic hydrolysates

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Transcriptional response to lactic acid stress in the hybrid yeast Zygosaccharomyces parabailii

Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH, and a fermentative metabolism with a fast growth rate. Here we used RNA-seq to analyze Z. parabailii's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than in control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/2, previously described as lactic acid-responsive in Saccharomyces cerevisiae. Furthermore, formate dehydrogenase (FDH) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii as compared to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production.Importance Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression in conditions of interest, and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains

Improved xylose fermentation by expression of a putative xylose transporter encoding gene HXT2.4 in Saccharomyces cerevisiae

Saccharomyces cerevisiae is considered one of the promising microorganisms in lignocellulosic bioethanol production. Unfortunately S. cerevisiae cannot consume xylose, a pentose sugar which comprises almost 30% of lignocellulosic biomass. Metabolic and genetic engineering methods were used to develop S. cerevisiae that could consume xylose. However in S. cerevisiae, pentose sugars can only enter the cell through native hexose transporters which have two orders of magnitude lower affinities toward pentose sugar than hexose sugar. Thus pentose uptake is a limiting step in xylose fermentation using S. cerevisiae. In order to solve this problem, we introduced putative xylose transporter gene HXT2.4 from natural xylose consuming Scheffersomyces stipitis into engineered xylose consuming S. cerevisiae. Xylose consumption by the HXT2.4 expressing S. cerevisiae was tested through fermentation. To prove that HXT2.4 indeed enhanced the flux of xylose into the yeast cell, intracellular xylose and xylitol concentrations were measured using 100% methanol quenching and extraction. The results showed that the HXT2.4 expressing S. cerevisiae could accumulate 10% more xylose and 40% more xylitol than the control strain. Sugar uptake kinetic parameters were determined using 14C-labeled xylose. The results showed higher Vmax of HXT2.4 expressing S. cerevisiae than control strain. Introduction of HXT2.4 may improve xylose fermentation by engineered S. cerevisiae depending on strain background. More efficient transport of pentose sugar can improve the utilization of xylose, which will allow the development of an efficient xylose fermenting S. cerevisiae for the production of bioethanol from lignocellulose

Compositional and temporal division of labor modulates mixed sugar fermentation by an engineered yeast consortium

Abstract Synthetic microbial communities have emerged as an attractive route for chemical bioprocessing. They are argued to be superior to single strains through microbial division of labor (DOL), but the exact mechanism by which DOL confers advantages remains unclear. Here, we utilize a synthetic Saccharomyces cerevisiae consortium along with mathematical modeling to achieve tunable mixed sugar fermentation to overcome the limitations of single-strain fermentation. The consortium involves two strains with each specializing in glucose or xylose utilization for ethanol production. By controlling initial community composition, DOL allows fine tuning of fermentation dynamics and product generation. By altering inoculation delay, DOL provides additional programmability to parallelly regulate fermentation characteristics and product yield. Mathematical models capture observed experimental findings and further offer guidance for subsequent fermentation optimization. This study demonstrates the functional potential of DOL in bioprocessing and provides insight into the rational design of engineered ecosystems for various applications

Evolutionary restoration of fertility in an interspecies hybrid yeast, by whole-genome duplication after a failed mating-type switch - Fig 5

<p>(A) Organization of <i>MAT</i>, <i>HML</i>, and <i>HMR</i> loci in <i>Z</i>. <i>parabailii</i> ATCC60483. The genome contains 6 <i>MAT</i>-related regions, with 1 <i>MAT</i>, 1 <i>HML</i>, and 1 <i>HMR</i> locus derived from each of the A and B parents. Pink and green backgrounds indicate sequences from the A- and B-subgenomes, respectively. The <i>MAT</i> locus in the A-subgenome (position 294 kb on chromosome 7) is intact and expressed. The <i>MAT</i> locus of the B-subgenome has been broken into 2 parts by cleavage by HO endonuclease. All 6 copies of the X repeat region (654 bp) are identical in sequence, as are all 6 copies of the Z repeat region (266 bp). Gray triangles indicate the disruption of the splicing of intron 2 in <i>MAT</i>α2 and <i>HML</i>α2 of the B-subgenome. The binding sites for primers A–F used for PCR amplification are indicated by gray arrows. (B) Sequences at the <i>MAT</i> locus breakpoint. Red, <i>MAT</i>α1-derived sequences. The HO cleavage site (CGCAGCA, giving a 4-nucleotide 3′ overhang) is highlighted in gray. Blue, the <i>GDA1-YEF1</i> intergenic region from the equivalent region of <i>Z</i>. <i>bailii</i> CLIB213^T and homologous sequences from the A-subgenome on <i>Z</i>. <i>parabailii</i> chromosomes (chrs.) 2 and 16. A 5-bp sequence (ACAAC) that became duplicated during the rearrangement is underlined. (C) Sequences of <i>MAT</i>α2 intron 2 (lowercase) from the A- and B-subgenomes. An AG-to-AC mutation (red) at the 3′ end of the intron moved the splice site by 2 bp in the B-subgenome, causing a frameshift and premature translation termination. The splice sites in both genes were identified from RNA sequencing (RNA-Seq) data.</p

Cartoon of key steps in the origin of the <i>Z</i>. <i>parabailii</i> genome.

<p>Chromosome regions (thick bars) are colored according to their location in <i>Z</i>. <i>bailii</i> (magenta outlines). The corresponding homeologous regions are scrambled in Parent B (green outlines). Circles represent centromeres. (i) Interspecies mating occurred between Parent A (<i>Z</i>. <i>bailii</i>) and Parent B. The genomes differed by about 34 rearrangement breakpoints and 7% nucleotide sequence divergence. The resulting zygote was unable to form viable spores because of the noncollinearity of its chromosomes. (ii) Expression of HO endonuclease in the zygote, due to the absence of <b>a</b>1-α2, resulted in cleavage of the B-copy of the <i>MAT</i> locus and ectopic recombination with the <i>GDA1-YEF1</i> region of the A-subgenome, causing a reciprocal translocation. (iii) The resulting genome has only 1 functional <i>MAT</i> locus and behaves as a haploid. Recombinations and other exchanges between homeologous regions of the 2 subgenomes, such as those that exchanged the <i>HML</i>/<i>HMR</i> regions, occurred but are not shown here for simplicity. (iv) The current life cycle of <i>Z</i>. <i>parabailii</i> involves mating between 16-chromosome haploids to form 32-chromosome diploids, which immediately sporulate to regenerate 16-chromosome haploids. <i>Z</i>. <i>parabailii</i> is homothallic because it contains an intact <i>HO</i> gene, which allows interconversion between <i>MAT</i><b>a</b> and <i>MAT</i>α haploids and hence autodiploidization. chrs., chromosomes.</p

Circos plot of relationships among the <i>Z</i>. <i>parabailii</i> ATCC60483 chromosomes.

<p>In the outer arcs, purple and green coloring indicates A- and B-genes on the Watson and Crick strands of each chromosome. Arcs in the center of the diagram link homeologous (A:B) gene pairs.</p

Subgenome and duplication status of each <i>Z</i>. <i>parabailii</i> gene.

<p>Each gene was classified into 1 of 7 categories and color-coded as shown in the legend. For each chromosome, 7 rows were then drawn, showing the locations of genes in each category (the 7 rows appear in the same order from top to bottom as in the legend). “R” shows the locations of ribosomal DNA (rDNA clusters). “M” and “H” indicate the locations of <i>MAT</i> and <i>HML</i>/<i>HMR</i> loci. Circles with arrows mark the 3 chromosome ends where our sequence is incomplete due to break-induced replication (BIR); in each case, the missing sequence is apparently identical to the end of another chromosome, as shown. For example, we infer that at the right end of chromosome 14, our assembly artefactually lacks a second copy of the genes that are labeled as “A unique” on the right end of chromosome 9. The high sequence identity of the chromosome 9 and 14 copies of this region caused them to coassemble, and the coassembled contig was arbitrarily assigned to chromosome 9.</p

Dot-matrix plot between <i>Z</i>. <i>bailii</i> CLIB213^T scaffolds [38] and <i>Z</i>. <i>parabailii</i> ATCC60483 chromosomes.

<p>Each dot is a protein-coding gene (purple: A-genes; green, B-genes). Red triangles indicate chromosome ends that appear unpaired due to break-induced replication (BIR). “M” and “m” indicate the active and broken <i>MAT</i> loci of <i>Z</i>. <i>parabailii</i>, respectively.</p