Enhancing metabolic efficiency via novel constitutive promoters to produce protocatechuic acid in Escherichia coli.

The antioxidant molecule protocatechuic acid (PCA) can also serve as a precursor for polymer building blocks. PCA can be produced in Escherichia coli overexpressing 3-dehydroshikimate dehydratase (DSD), an enzyme that catalyses the transformation of 3-dehydroshikimate to PCA. Nevertheless, optimizing the expression rate of recombinant enzymes is a key factor in metabolic engineering when producing biobased chemicals. In this study, a degenerate synthetic promoter approach was investigated to improve further the production of PCA. By limited screening of a randomized promoter library made using pSEVA221 plasmid in E. coli, three novel synthetic constitutive promoters were selected that increased the PCA yield from glucose by 10-21% compared to the inducible T7-promoter. RT-qPCR analysis showed that the DSD gene, regulated by the synthetic promoters, had high expression during the exponential phase, albeit the gene expression level dropped 250-fold during stationary phase. Besides the increased product yield, the synthetic promoters avoided the need for a costly inducer for gene expression. Screening of the entire promoter library is likely to provide more positive hits. The study also shows that E. coli transformed with the DSD gene on either pSEVA221 or pCDFDuet plasmids exhibit background PCA levels (~ 0.04 g/L) in the absence of a transcriptional regulatory element. KEY POINTS: • Degenerate synthetic promoters are remarkable tools to produce protocatechuic acid. • The constitutive synthetic promoters did not affect the growth rate of the bacterial host. • The use of constitutive synthetic promoters avoids the need for the costly inducer.

Polygenic Analysis of Tolerance to Carbon Dioxide Inhibition of Isoamyl Acetate "Banana" Flavor Production in Yeast Reveals MDS3 as Major Causative Gene.

The introduction in modern breweries of tall cylindroconical fermentors, replacing the traditional open fermentation vats, unexpectedly revealed strong inhibition of flavor production by the high CO2 pressure in the fermentors. We have screened our collection of Saccharomyces cerevisiae strains for strains displaying elevated tolerance to inhibition of flavor production by +0.65 bar CO2, using a laboratory scale CO2 pressurized fermentation system. We focused on the production of isoamyl acetate, a highly desirable flavor compound conferring fruity banana flavor in beer and other alcoholic beverages, from its precursor isoamyl alcohol (IAAc/Alc ratio). We selected the most tolerant Saccharomyces cerevisiae strain, saké yeast Kyokai no. 1, isolated a stable haploid segregant seg63 with the same high IAAc/Alc ratio under CO2 pressure, crossed seg63 with the unrelated inferior strain ER7A and phenotyped 185 haploid segregants, of which 28 displaying a high IAAc/Alc ratio were pooled. Mapping of Quantitative Trait Loci (QTLs) by whole-genome sequence analysis based on SNP variant frequency revealed two QTLs. In the major QTL, reciprocal hemizygosity analysis identified MDS3 as the causative mutant gene, a putative member of the TOR signaling pathway. The MDS3Seg.63 allele was dominant and contained a single causative point mutation, T2171C, resulting in the F274S substitution. Introduction of MDS3Seg.63 in an industrial tetraploid lager yeast with CRISPR/Cas9 enhanced isoamyl acetate production by 145% under CO2 pressure. This work shows the strong potential of polygenic analysis and targeted genetic modification for creation of cisgenic industrial brewer's yeast strains with specifically improved traits. IMPORTANCE The upscaling of fermentation to very tall cylindroconical tanks is known to negatively impact beer flavor. Most notably, the increased CO2 pressure in such tanks compromises production by the yeast of the desirable fruity "banana" flavor (isoamyl acetate). The cause of the CO2 inhibition of yeast flavor production has always remained enigmatic. Our work has brought the first insight into its molecular-genetic basis and provides a specific gene tool for yeast strain improvement. We first identified a yeast strain with superior tolerance to CO2 inhibition of flavor production, and applied polygenic analysis to identify the responsible gene. We narrowed down the causative element to a single nucleotide difference, MDS3T2171C, and showed that it can be engineered into brewing yeast to obtain strains with superior flavor production in high CO2 pressure conditions, apparently without affecting other traits relevant for beer brewing. Alternatively, such a strain could be obtained through marker-assisted breeding.

"This is not an apple"-yeast mutualism in codling moth.

The larva of codling moth Cydia pomonella (Tortricidae, Lepidoptera) is known as the worm in the apple, mining the fruit for food. We here show that codling moth larvae are closely associated with yeasts of the genus Metschnikowia. Yeast is an essential part of the larval diet and further promotes larval survival by reducing the incidence of fungal infestations in the apple. Larval feeding, on the other hand, enables yeast proliferation on unripe fruit. Chemical, physiological and behavioral analyses demonstrate that codling moth senses and responds to yeast aroma. Female moths are attracted to fermenting yeast and lay more eggs on yeast-inoculated than on yeast-free apples. An olfactory response to yeast volatiles strongly suggests a contributing role of yeast in host finding, in addition to plant volatiles. Codling moth is a widely studied insect of worldwide economic importance, and it is noteworthy that its association with yeasts has gone unnoticed. Tripartite relationships between moths, plants, and microorganisms may, accordingly, be more widespread than previously thought. It, therefore, is important to study the impact of microorganisms on host plant ecology and their contribution to the signals that mediate host plant finding and recognition. A better comprehension of host volatile signatures also will facilitate further development of semiochemicals for sustainable insect control.

Candida albicans--a pre-whole genome duplication yeast--is predominantly aerobic and a poor ethanol producer.

Yeast species belonging to the lineage that underwent the whole genome duplication (WGD), and including Saccharomyces cerevisiae, can grow under anaerobiosis and accumulate ethanol in the presence of glucose and oxygen. The pre-WGD yeasts, which branched from the S. cerevisiae lineage just before the WGD event, including Kluyveromyces lactis, are more dependent on oxygen and do not accumulate large amounts of ethanol in the presence of excess oxygen. Yeasts that belong to the so-called 'lower branches' of the yeast phylogenetic tree and diverged from S. cerevisiae more than 200 million years ago have so far not been thoroughly investigated for their physiology and carbon metabolism. Here, we have studied several isolates of Candida albicans and Debaryomyces hansenii for their dependence on oxygen. Candida albicans grew very poorly at an oxygen concentration <1 p.p.m. and D. hansenii could not grow at all. In aerobic batch cultivations, C. albicans exhibited a predominantly aerobic metabolism, accumulating only small amounts of ethanol (0.01-0.09 g g(-1) glucose). Apparently, C. albicans and several other pre-WGD yeasts still exhibit the original traits of the yeast progenitor: poor accumulation of ethanol under aerobic conditions and strong dependence on the presence of oxygen.

Chemical signaling and insect attraction is a conserved trait in yeasts.

Yeast volatiles attract insects, which apparently is of mutual benefit, for both yeasts and insects. However, it is unknown whether biosynthesis of metabolites that attract insects is a basic and general trait, or if it is specific for yeasts that live in close association with insects. Our goal was to study chemical insect attractants produced by yeasts that span more than 250 million years of evolutionary history and vastly differ in their metabolism and lifestyle. We bioassayed attraction of the vinegar fly Drosophila melanogaster to odors of phylogenetically and ecologically distinct yeasts grown under controlled conditions. Baker's yeast Saccharomyces cerevisiae, the insect-associated species Candida californica, Pichia kluyveri and Metschnikowia andauensis, wine yeast Dekkera bruxellensis, milk yeast Kluyveromyces lactis, the vertebrate pathogens Candida albicans and Candida glabrata, and oleophilic Yarrowia lipolytica were screened for fly attraction in a wind tunnel. Yeast headspace was chemically analyzed, and co-occurrence of insect attractants in yeasts and flowering plants was investigated through a database search. In yeasts with known genomes, we investigated the occurrence of genes involved in the synthesis of key aroma compounds. Flies were attracted to all nine yeasts studied. The behavioral response to baker's yeast was independent of its growth stage. In addition to Drosophila, we tested the basal hexapod Folsomia candida (Collembola) in a Y-tube assay to the most ancient yeast, Y. lipolytica, which proved that early yeast signals also function on clades older than neopteran insects. Behavioral and chemical data and a search for selected genes of volatile metabolites underline that biosynthesis of chemical signals is found throughout the yeast clade and has been conserved during the evolution of yeast lifestyles. Literature and database reviews corroborate that yeast signals mediate mutualistic interactions between insects and yeasts. Moreover, volatiles emitted by yeasts are commonly found also in flowers and attract many insect species. The collective evidence suggests that the release of volatile signals by yeasts is a widespread and phylogenetically ancient trait, and that insect-yeast communication evolved prior to the emergence of flowering plants. Co-occurrence of the same attractant signals in yeast and flowers suggests that yeast-insect communication may have contributed to the evolution of insect-mediated pollination in flowers.

A study on the fundamental mechanism and the evolutionary driving forces behind aerobic fermentation in yeast.

Baker's yeast Saccharomyces cerevisiae rapidly converts sugars to ethanol and carbon dioxide at both anaerobic and aerobic conditions. The later phenomenon is called Crabtree effect and has been described in two forms, long-term and short-term effect. We have previously studied under fully controlled aerobic conditions forty yeast species for their central carbon metabolism and the presence of long-term Crabtree effect. We have also studied ten steady-state yeast cultures, pulsed them with glucose, and followed the central carbon metabolism and the appearance of ethanol at dynamic conditions. In this paper we analyzed those wet laboratory data to elucidate possible mechanisms that determine the fate of glucose in different yeast species that cover approximately 250 million years of evolutionary history. We determine overflow metabolism to be the fundamental mechanism behind both long- and short-term Crabtree effect, which originated approximately 125-150 million years ago in the Saccharomyces lineage. The "invention" of overflow metabolism was the first step in the evolution of aerobic fermentation in yeast. It provides a general strategy to increase energy production rates, which we show is positively correlated to growth. The "invention" of overflow has also simultaneously enabled rapid glucose consumption in yeast, which is a trait that could have been selected for, to "starve" competitors in nature. We also show that glucose repression of respiration is confined mainly among S. cerevisiae and closely related species that diverged after the whole genome duplication event, less than 100 million years ago. Thus, glucose repression of respiration was apparently "invented" as a second step to further increase overflow and ethanol production, to inhibit growth of other microbes. The driving force behind the initial evolutionary steps was most likely competition with other microbes to faster consume and convert sugar into biomass, in niches that were semi-anaerobic.

Analysis of the yeast short-term Crabtree effect and its origin.

The short-term Crabtree effect is defined as the immediate occurrence of aerobic alcoholic fermentation in response to provision of a pulse of excess sugar to sugar-limited yeast cultures. Here we have characterized ten yeast species with a clearly defined phylogenetic relationship. Yeast species were cultivated under glucose-limited conditions, and we studied their general carbon metabolism in response to a glucose pulse. We generated an extensive collection of data on glucose and oxygen consumption, and ethanol and carbon dioxide generation. We conclude that the Pichia, Debaryomyces, Eremothecium and Kluyveromyces marxianus yeasts do not exhibit any significant ethanol formation, while Kluyveromyces lactis behaves as an intermediate yeast, and Lachancea, Torulaspora, Vanderwaltozyma and Saccharomyces yeasts exhibit rapid ethanol accumulation. Based on the present data and our previous data relating to the presence of the long-term Crabtree effect in over 40 yeast species, we speculate that the origin of the short-term effect may coincide with the origin of the long-term Crabtree effect in the Saccharomycetales lineage, occurring ~ 150 million years ago.

Yeast "make-accumulate-consume" life strategy evolved as a multi-step process that predates the whole genome duplication.

When fruits ripen, microbial communities start a fierce competition for the freely available fruit sugars. Three yeast lineages, including baker's yeast Saccharomyces cerevisiae, have independently developed the metabolic activity to convert simple sugars into ethanol even under fully aerobic conditions. This fermentation capacity, named Crabtree effect, reduces the cell-biomass production but provides in nature a tool to out-compete other microorganisms. Here, we analyzed over forty Saccharomycetaceae yeasts, covering over 200 million years of the evolutionary history, for their carbon metabolism. The experiments were done under strictly controlled and uniform conditions, which has not been done before. We show that the origin of Crabtree effect in Saccharomycetaceae predates the whole genome duplication and became a settled metabolic trait after the split of the S. cerevisiae and Kluyveromyces lineages, and coincided with the origin of modern fruit bearing plants. Our results suggest that ethanol fermentation evolved progressively, involving several successive molecular events that have gradually remodeled the yeast carbon metabolism. While some of the final evolutionary events, like gene duplications of glucose transporters and glycolytic enzymes, have been deduced, the earliest molecular events initiating Crabtree effect are still to be determined.

Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila.

Adaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii. The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner-Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro, may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA (sigma28) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival.