The flocculant Saccharomyces cerevisiae strain gains robustness via alteration of the cell wall hydrophobicity.

When lignocellulosic biomass is utilized as a fermentative substrate to produce biochemicals, the existence of a yeast strain resistant to inhibitory chemical compounds (ICCs) released from the biomass becomes critical. To achieve the purpose, in this study, Saccharomyces yeast strains from a NBRC yeast culture collection were used for exploration and evaluated in two different media containing ICCs that mimic one another but resemble the hydrolysate of real biomass. Among them, S. cerevisiae F118 strain shows robustness upon the fermentation with unique flocculation trait that was strongly responsive to ICC stress. When this strain was cultured in the presence of ICCs, its cell wall hydrophobicity increased dramatically, and reduced significantly when the ICCs were depleted, demonstrating that cell-surface hydrophobicity can also act as an adaptive response to the ICCs. Cells from the strain with the highest cell-wall hydrophobicity displayed progressively stronger flocculation, indicating that the F118 strain is having unique robustness under ICC stress. Gene expression perturbation analysis revealed that mot3 gene encoding regulatory Mot3p from the F118 strain was expressed in response to the concentration of ICCs. This gene was found to control expression of ygp1 gene that encoding Ygp1p, one of cell wall proteins. Deep sequencing analysis revealed that the Mot3p of the F118 strain features a unique insertion and deletion of nucleotides that encode glutamine or asparagine residues, particularly in N-terminal domain, as determined by comparison to the Mot3p sequence from the S288c strain, which was employed as a control strain. Furthermore, the cell wall hydrophobicity of the S288c strain was greatly enhanced and became ICC-responsive after gene swapping with the mot3 gene from the F118 strain. The gene-swapped S288c strain fermented 6-fold faster than the wild-type strain, producing 14.5 g/L of ethanol from 30 g/L of glucose consumed within 24 h in a medium containing the ICCs. These such modifications to Mot3p in unique locations in its sequence have a potential to change the expression of a gene involved in cell wall hydrophobicity and boosted the flocculation response to ICC stress, allowing for the acquisition of extraordinary robustness.

Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi.

Oleaginous microbes can convert substrates such as carbon dioxide, sugars, and organic acids to single-cell oils (SCOs). Among the oleaginous microorganisms, Lipomyces starkeyi is a particularly well-suited host given its impressive native abilities, including the capability to utilize a wide variety of carbon sources. In this work, the potential of L. starkeyi NBRC10381 to produce SCOs in a synthetically nitrogen-limited mineral medium (-NMM) was investigated by differing the inoculum size using glucose and/or xylose as a carbon source. Fermentation using glucose and xylose as mixed carbon sources generated the highest production of biomass at 40.8 g/L, and achieved a lipid content of 84.9% (w/w). When either glucose or xylose was used separately, the totals for achieved lipid content were 79.6% (w/w) and 85.1% (w/w), respectively. However, biomass production was higher for glucose than for xylose (30.3 vs. 28.7 g/L, respectively). This study describes the first simultaneous achievement of higher levels of cell mass and lipid production using glucose and/or xylose as the carbon sources in different inoculum sizes.

Challenges of non-flocculating Saccharomyces cerevisiae haploid strain against inhibitory chemical complex for ethanol production.

This study provides insight observation based on the gene expression and the metabolomic analysis of the natural robust yeast Saccharomyces cerevisiae NBRC849 during the fermentation in the medium containing inhibitory chemical complexes (ICC) at different concentrations. The tolerance mechanisms involved in the strain might have existed through the upregulation of genes involved in NAD(H)/NADP(H) cofactors generations (ALD6, ZWF1, GND1), membrane robustness for efflux pump (YOR1, PDR5, TPO3) and cation/polyamine transport (TPO3). The alteration of metabolic flux to the shikimic pathway was also found in this strain, resulted in the enhanced formation of aromatic amino acid required for cell survival. Enhanced expression of these genes as well as the increase of metabolic flux to shikimic pathway were suggested to result in the robustness of non-flocculating S. cerevisiae haploid strain.

Pyruvate dehydrogenase complex regulator (PdhR) gene deletion boosts glucose metabolism in Escherichia coli under oxygen-limited culture conditions.

Pyruvate dehydrogenase complex regulator (PdhR) is a transcriptional regulator that negatively regulates formation of pyruvate dehydrogenase complex (PDHc), NADH dehydrogenase (NDH)-2, and cytochrome bo3 oxidase in Escherichia coli. To investigate the effects of a PdhR defect on glucose metabolism, a pdhR deletion mutant was derived from the wild-type E. coli W1485 strain by λ Red-mediated recombination. While no difference in the fermentation profiles was observed between the two strains under oxygen-sufficient conditions, under oxygen-limited conditions, the growth level of the wild-type strain was significantly decreased with retarded glucose consumption accompanied by by-production of substantial amounts of pyruvic acid and acetic acid. In contrast, the mutant grew and consumed glucose more efficiently than did the wild-type strain with enhanced respiration, little by-production of pyruvic acid, less production yield and rates of acetic acid, thus displaying robust metabolic activity. As expected, increased activities of PDHc and NDH-2 were observed in the mutant. The increased activity of PDHc may explain the loss of pyruvic acid by-production, probably leading to decreased acetic acid formation, and the increased activity of NDH-2 may explain the enhanced respiration. Measurement of the intracellular NAD+/NADH ratio in the mutant revealed more oxidative or more reductive intracellular environments than those in the wild-type strain under oxygen-sufficient and -limited conditions, respectively, suggesting another role of PdhR: maintaining redox balance in E. coli. The overall results demonstrate the biotechnological advantages of pdhR deletion in boosting glucose metabolism and also improve our understanding of the role of PdhR in bacterial physiology.

Alterations of glucose metabolism in Escherichia coli mutants defective in respiratory-chain enzymes.

The effects of reduced efficiency of proton-motive force (pmf) generation on glucose metabolism were investigated in Escherichia coli respiratory-chain mutants. The respiratory chain of E. coli consists of two NADH dehydrogenases and three terminal oxidases, all with different abilities to generate a pmf. The genes for isozymes with the highest pmf-generating capacity (NADH dehydrogenase-1 and cytochrome bo3 oxidase) were knocked out singly or in combination, using a wild-type strain as the parent. Analyses of glucose metabolism by jar-fermentation revealed that the glucose consumption rate per cell increased with decreasing efficiency of pmf generation, as determined from the growth parameters of the mutants. The highest rate of glucose metabolism was observed in the double mutant, and the lowest was observed in the wild-type strain. The respiration rates of the single-knockout mutants were comparable to that of the wild-type strain, and that of the double mutant was higher, apparently as a result of the upregulation of the remaining respiratory chain enzymes. All of the strains excreted 2-oxoglutaric acid as a product of glucose metabolism. Additionally, all of the mutants excreted pyruvic acid and/or acetic acid. Interestingly, the double mutant excreted L-glutamic acid. Alterations of the fermentation profiles provide clues regarding the metabolic regulation in each mutant.