Kluyveromyces marxianus is a promising host for producing bioethanol and heterologous proteins. It displays many superior traits to a conventional industrial yeast species, Saccharomyces cerevisiae, including fast growth, thermotolerance and the capacity to assimilate a wider variety of sugars. However, little is known about the mechanisms underlying the fast-growing feature of K. marxianus. In this study, we performed a comparative genomic analysis between K. marxianus and other Saccharomycetaceae species. Genes involved in flocculation, iron transport, and biotin biosynthesis have particularly high copies in K. marxianus. In addition, 60 K. marxianus specific genes were identified, 45% of which were upregulated during cultivation in rich medium and these genes may participate in glucose transport and mitochondrion related functions. Furthermore, the transcriptomic analysis revealed that under aerobic condition, normalized levels of genes participating in TCA cycles, respiration chain and ATP biosynthesis in the lag phase were higher in K. marxianus than those in S. cerevisiae. Levels of highly copied genes, genes involved in the respiratory chain and mitochondrion assembly, were upregulated in K. marxianus, but not in S. cerevisiae, in later time points during cultivation compared with those in the lag phase. Notably, during the fast-growing phase, genes involved in the respiratory chain, ATP synthesis and glucose transport were co-upregulated in K. marxianus. A few shared motifs in upstream sequences of relevant genes might result in the co-upregulation. Specific features in the co-regulations of gene expressions might contribute to the fast-growing phenotype of K. marxianus. Our study underscores the importance of genome-wide rewiring of the transcriptional network during evolution.
The growth and tolerance of Kluyveromyces marxianus at high temperatures decreased significantly in the synthetic medium (SM), which is commonly used in industrial fermentations. After 100 days of adaptive laboratory evolution, a strain named KM234 exhibited excellent tolerance at a high temperature, without loss of its growth ability at a moderate temperature. Transcriptomic analysis revealed that the KM234 strain decreased the expression of the ammonium (NH 4 + ) transporter gene MEP3 and increased the synthesis of the amino acid carbon backbone, which may contribute greatly to the high‐temperature growth phenotype. High NH 4 + content in SM significantly increased the reactive oxygen species (ROS) production at high temperatures and thus caused toxicity to yeast cells. Replacing NH 4 + with organic nitrogen sources or increasing the concentration of potassium ions (K + ) in the medium restored the growth of the wild‐type K. marxianus at a high temperature in SM. We also showed that the NH 4 + toxicity mitigated by K + might closely depend on the KIN1 gene. Our results provide a practical solution to industrial fermentation under high‐temperature conditions.
Background Using yeast fermentation to produce bioethanol, is an economic and renewable way to tackle the rapid increase in fuel consumption. Faster cell growth rate guarantees the superior result of fermentation course. The “non-conventional” yeast Kluyveromyces marxianus is the known fastest-growing eukaryote on the earth. Although its wide application in industry, the molecular mechanisms for its fast growth have seldom been discovered.Results We first carried out a comparative genome content analysis for K. marxianus evolution in Saccharomycetaceae and identified the gain/lost genes as well as highly copied genes during K. marxianus speciation. Then RNA-seq analyses for K. marxianus and S. cerevisiae at different time points along cultivation were performed, to infer the function of K. marxianus-specific genes and to find out the difference in homologous gene expression patterns between the two species. RNA-seq results were further validated with RT-qPCR analysis. Genome content analysis shows the highly intense events of genes’ gain/loss happened at the occurrence of Saccharomycetaceae and Kluyveromyces, and K. marxianus has particularly high copy numbers of genes participating in iron transport and biotin biosynthesis. The RNA-seq analysis reveals 40% of K. marxianus-specific genes were up-regulated and may participate in glucose transport and mitochondrial function. Furthermore, compared to S. cerevisiae, K. marxianus has developed two features in homologous gene expressions to ensure its fast growth: (1) enhanced fundamental expressions of TCA cycle and respiratory chain genes at the beginning of cultivation; (2) tightly co-up-regulated expressions of respiratory chain, F0F1 ATPase, and glucose transporter genes at its fastest growth phase. Those co-expressions are mainly ascribed to the higher number of significant motifs in the upstream sequences of involved genes in K. marxianus than in S. cerevisiae, indicating the importance of transcriptional network remodelling during evolution.Conclusions This study gives insights into the possible mechanisms of K. marxianus’ fast growth trait, via efficiently supplying sufficient energy for cell growth, meanwhile reinforcing glucose transport to guarantee the competition for environmental resources. Our findings present a theoretical support for K. marxianus’ prospective application in industry, and give clues for further rational construction of fast-growing yeast strains.
Kluyveromyces marxianus is the fastest-growing eukaryote and a promising host for producing bioethanol and heterologous proteins. To perform a laboratory evolution of thermal tolerance in K. marxianus, diploid, triploid and tetraploid strains were constructed, respectively. Considering the genetic diversity caused by genetic recombination in meiosis, we established an iterative cycle of “diploid/polyploid - meiosis - selection of spores at high temperature” to screen thermotolerant strains. Results showed that the evolution of thermal tolerance in diploid strain was more efficient than that in triploid and tetraploid strains. The thermal tolerance of the progenies of diploid and triploid strains after a two-round screen was significantly improved than that after a one-round screen, while the thermal tolerance of the progenies after the one-round screen was better than that of the initial strain. After a two-round screen, the maximum tolerable temperature of Dip2-8, a progeny of diploid strain, was 3°C higher than that of the original strain. Whole-genome sequencing revealed nonsense mutations of PSR1 and PDE2 in the thermotolerant progenies. Deletion of either PSR1 or PDE2 in the original strain improved thermotolerance and two deletions displayed additive effects, suggesting PSR1 and PDE2 negatively regulated the thermotolerance of K. marxianus in parallel pathways. Therefore, the iterative cycle of “meiosis - spore screening” developed in this study provides an efficient way to perform the laboratory evolution of heat resistance in yeast.
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