Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export

BACKGROUND: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is becoming a well established concept. For the production of important metabolites, but also for a better understanding of the fundamentals of cell biology, detailed studies are required. In this work we analysed the lactate production from metabolic engineered Saccharomyces cerevisiae cells expressing a heterologous lactate dehydrogenase (LDH) gene. The LDH gene expression in a budding yeast cell introduces a novel and alternative pathway for the NAD(+ )regeneration, allowing a direct reduction of the intracellular pyruvate to lactate, leading to a simultaneous accumulation of lactate and ethanol. RESULTS: Four different S. cerevisiae strains were transformed with six different wild type and one mutagenised LDH genes, in combination or not with the over-expression of a lactate transporter. The resulting yield values (grams of lactate produced per grams of glucose consumed) varied from as low as 0,0008 to as high as 0.52 g g(-1). In this respect, and to the best of our knowledge, higher redirections of the glycolysis flux have never been obtained before without any disruption and/or limitation of the competing biochemical pathways. CONCLUSION: In the present work it is shown that the redirection of the pathway towards the lactate production can be strongly modulated by the genetic background of the host cell, by the source of the heterologous Ldh enzyme, by improving its biochemical properties as well as by modulating the export of lactate in the culture media

Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation

BACKGROUND: Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. RESULTS: We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified. CONCLUSIONS: We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase

Systems-level organization of yeast methylotrophic lifestyle

BACKGROUND: Some yeasts have evolved a methylotrophic lifestyle enabling them to utilize the single carbon compound methanol as a carbon and energy source. Among them, Pichia pastoris (syn. Komagataella sp.) is frequently used for the production of heterologous proteins and also serves as a model organism for organelle research. Our current knowledge of methylotrophic lifestyle mainly derives from sophisticated biochemical studies which identified many key methanol utilization enzymes such as alcohol oxidase and dihydroxyacetone synthase and their localization to the peroxisomes. C1 assimilation is supposed to involve the pentose phosphate pathway, but details of these reactions are not known to date. RESULTS: In this work we analyzed the regulation patterns of 5,354 genes, 575 proteins, 141 metabolites, and fluxes through 39 reactions of P. pastoris comparing growth on glucose and on a methanol/glycerol mixed medium, respectively. Contrary to previous assumptions, we found that the entire methanol assimilation pathway is localized to peroxisomes rather than employing part of the cytosolic pentose phosphate pathway for xylulose-5-phosphate regeneration. For this purpose, P. pastoris (and presumably also other methylotrophic yeasts) have evolved a duplicated methanol inducible enzyme set targeted to peroxisomes. This compartmentalized cyclic C1 assimilation process termed xylose-monophosphate cycle resembles the principle of the Calvin cycle and uses sedoheptulose-1,7-bisphosphate as intermediate. The strong induction of alcohol oxidase, dihydroxyacetone synthase, formaldehyde and formate dehydrogenase, and catalase leads to high demand of their cofactors riboflavin, thiamine, nicotinamide, and heme, respectively, which is reflected in strong up-regulation of the respective synthesis pathways on methanol. Methanol-grown cells have a higher protein but lower free amino acid content, which can be attributed to the high drain towards methanol metabolic enzymes and their cofactors. In context with up-regulation of many amino acid biosynthesis genes or proteins, this visualizes an increased flux towards amino acid and protein synthesis which is reflected also in increased levels of transcripts and/or proteins related to ribosome biogenesis and translation. CONCLUSIONS: Taken together, our work illustrates how concerted interpretation of multiple levels of systems biology data can contribute to elucidation of yet unknown cellular pathways and revolutionize our understanding of cellular biology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12915-015-0186-5) contains supplementary material, which is available to authorized users

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength

Production of l-Ascorbic Acid by Metabolically Engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii

Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, l-galactono-1,4-lactone, or l-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and d-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme l-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous d-arabinono-1,4-lactone oxidase as well as l-galactose dehydrogenase are capable of producing about 100 mg of l-ascorbic acid liter(−1), converting 40% (wt/vol) of the starting compound l-galactose

Improvement of Lactic Acid Production in Saccharomyces cerevisiae by Cell Sorting for High Intracellular pH

Yeast strains expressing heterologous l-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pH(i)) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pH(i). During batch culture, we observed in both strains a reduction of the mean pH(i) and the appearance of a subpopulation of cells with low pH(i). Simultaneous analysis of pH(i) and viability proved that the cells with low pH(i) were dead. Based on the observation that the better lactic-acid-producing strain had a higher pH(i) and that the cells with low pH(i) were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pH(i) range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pH(i) range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants

Mattanovich D Improvement of lactic acid production in Saccharomyces cerevisiae by cell sorting for high intracellular pH. Appl Environ Microbiol 2006;72:5492–9

Yeast strains expressing heterologous L-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pH i ) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pH i . During batch culture, we observed in both strains a reduction of the mean pH i and the appearance of a subpopulation of cells with low pH i . Simultaneous analysis of pH i and viability proved that the cells with low pH i were dead. Based on the observation that the better lactic-acid-producing strain had a higher pH i and that the cells with low pH i were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pH i range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pH i range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acidproducing strain was identified within the screening of CEN.PK m850 mutants