ME2 Deficiency Is Associated With Recessive Neurodevelopmental Disorder.

Malate is an important dicarboxylic acid produced from fumarate in the tricarboxylic acid cycle. Deficiencies of fumarate hydrolase (FH) and malate dehydrogenase (MDH), responsible for malate formation and metabolism, respectively, are known to cause recessive forms of neurodevelopmental disorders (NDDs). The malic enzyme isoforms, malic enzyme 1 (ME1) and 2 (ME2), are required for the conversion of malate to pyruvate. To date, there have been no reports linking deficiency of either malic enzyme isoforms to any Mendelian disease in humans. We report a patient presenting with NDD, subtle dysmorphic features, resolved dilated cardiomyopathy, and mild blood lactate elevation. Whole exome sequencing (WES) revealed a homozygous frameshift variant (c.1379_1380delTT, p.Phe460fs*22) in the malic enzyme 2 (ME2) gene resulting in truncated and unstable ME2 protein in vitro. Subsequent deletion of the yeast ortholog of human ME2 (hME2) resulted in growth arrest, which was rescued by overexpression of hME2, strongly supporting an important role of ME2 in mitochondrial function. Our results also support the pathogenicity and candidacy of the ME2 gene and variant in association with NDD. To our knowledge, this is the first report of a Mendelian human disease resulting from a biallelic variant in the ME encoding gene. Future studies are warranted to confirm ME2-associated recessive NDD.

Development of vitamin B12 dependency in Saccharomyces cerevisiae.

For decades, the industrial vitamin B12 (cobalamin) production has been based on bacterial producer strains. Due to limited methods for strain optimization and difficult strain handling, the desire for new vitamin B12-producing hosts has risen. As a vitamin B12-independent organism with a big toolbox for genomic engineering and easy-to-handle cultivation conditions, Saccharomyces cerevisiae has high potential for heterologous vitamin B12 production. However, the B12 synthesis pathway is long and complex. To be able to easily engineer and evolve B12-producing recombinant yeast cells, we have developed an S. cerevisiae strain whose growth is dependent on vitamin B12. For this, the B12-independent methionine synthase Met6 of yeast was replaced by a B12-dependent methionine synthase MetH from Escherichia coli. Adaptive laboratory evolution, RT-qPCR, and overexpression experiments show that additional high-level expression of a bacterial flavodoxin/ferredoxin-NADP+ reductase (Fpr-FldA) system is essential for in vivo reactivation of MetH activity and growth. Growth of MetH-containing yeast cells on methionine-free media is only possible with the addition of adenosylcobalamin or methylcobalamin. A heterologous vitamin B12 transport system turned out to be not necessary for the uptake of cobalamins. This strain should be a powerful chassis to engineer B12-producing yeast cells.

A yeast-based in vivo assay system for analyzing efflux of sugars mediated by glucose and xylose transporters.

Sugar transporter research focuses on the sugar uptake into cells. Under certain physiological conditions, however, the intracellular accumulation and secretion of carbohydrates (efflux) are relevant processes in many cell types. Currently, no cell-based system is available for specifically investigating glucose efflux. Therefore, we designed a system based on a hexose transporter-deficient Saccharomyces cerevisiae strain, in which the disaccharide maltose is provided as a donor of intracellular glucose. By deleting the hexokinase genes, we prevented the metabolization of glucose, and thereby achieved the accumulation of growth-inhibitory glucose levels inside the cells. When a permease mediating glucose efflux is expressed in this system, the inhibitory effect is relieved proportionally to the capacity of the introduced transporter. The assay is thereby suitable for screening of transporters and quantitative analyses of their glucose efflux capacities. Moreover, by simultaneous provision of intracellular glucose and extracellular xylose, we investigated how each sugar influences the transport of the other one from the opposite side of the membrane. Thereby, we could show that the xylose transporter variant Gal2N376F is insensitive not only to extracellular but also to intracellular glucose. Considering the importance of sugar transporters in biotechnology, the assay could facilitate new developments in a variety of applications.

Glucose-induced internalization of the S. cerevisiae galactose permease Gal2 is dependent on phosphorylation and ubiquitination of its aminoterminal cytoplasmic tail.

The hexose permease Gal2 of Saccharomyces cerevisiae is expressed only in the presence of its physiological substrate galactose. Glucose tightly represses the GAL2 gene and also induces the clearance of the transporter from the plasma membrane by ubiquitination and subsequent degradation in the vacuole. Although many factors involved in this process, especially those responsible for the upstream signaling, have been elucidated, the mechanisms by which Gal2 is specifically targeted by the ubiquitination machinery have remained elusive. Here, we show that ubiquitination occurs within the N-terminal cytoplasmic tail and that the arrestin-like proteins Bul1 and Rod1 are likely acting as adaptors for docking of the ubiquitin E3-ligase Rsp5. We further demonstrate that phosphorylation on multiple residues within the tail is indispensable for the internalization and possibly represents a primary signal that might trigger the recruitment of arrestins to the transporter. In addition to these new fundamental insights, we describe Gal2 mutants with improved stability in the presence of glucose, which should prove valuable for engineering yeast strains utilizing complex carbohydrate mixtures present in hydrolysates of lignocellulosic or pectin-rich biomass.

Transcriptomic response of Saccharomyces cerevisiae to octanoic acid production.

The medium-chain fatty acid octanoic acid is an important platform compound widely used in industry. The microbial production from sugars in Saccharomyces cerevisiae is a promising alternative to current non-sustainable production methods, however, titers need to be further increased. To achieve this, it is essential to have in-depth knowledge about the cell physiology during octanoic acid production. To this end, we collected the first RNA-Seq data of an octanoic acid producer strain at three time points during fermentation. The strain produced higher levels of octanoic acid and increased levels of fatty acids of other chain lengths (C6-C18) but showed decreased growth compared to the reference. Furthermore, we show that the here analyzed transcriptomic response to internally produced octanoic acid is notably distinct from a wild type's response to externally supplied octanoic acid as reported in previous publications. By comparing the transcriptomic response of different sampling times, we identified several genes that we subsequently overexpressed and knocked out, respectively. Hereby we identified RPL40B, to date unknown to play a role in fatty acid biosynthesis or medium-chain fatty acid tolerance. Overexpression of RPL40B led to an increase in octanoic acid titers by 40%.

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations.

The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields.

Improving 3-methylphenol (m-cresol) production in yeast via in vivo glycosylation or methylation.

Heterologous expression of 6-methylsalicylic acid synthase (MSAS) together with 6-MSA decarboxylase enables de novo production of the platform chemical and antiseptic additive 3-methylphenol (3-MP) in the yeast Saccharomyces cerevisiae. However, toxicity of 3-MP prevents higher production levels. In this study, we evaluated in vivo detoxification strategies to overcome limitations of 3-MP production. An orcinol-O-methyltransferase from Chinese rose hybrids (OOMT2) was expressed in the 3-MP producing yeast strain to convert 3-MP to 3-methylanisole (3-MA). Together with in situ extraction by dodecane of the highly volatile 3-MA this resulted in up to 211 mg/L 3-MA (1.7 mM) accumulation. Expression of a UDP-glycosyltransferase (UGT72B27) from Vitis vinifera led to the synthesis of up to 533 mg/L 3-MP as glucoside (4.9 mM). Conversion of 3-MP to 3-MA and 3-MP glucoside was not complete. Finally, deletion of phosphoglucose isomerase PGI1 together with methylation or glycosylation and feeding a fructose/glucose mixture to redirect carbon fluxes resulted in strongly increased product titers, with up to 897 mg/L 3-MA/3-MP (9 mM) and 873 mg/L 3-MP/3-MP as glucoside (8.1 mM) compared to less than 313 mg/L (2.9 mM) product titers in the wild type controls. The results show that methylation or glycosylation are promising tools to overcome limitations in further enhancing the biotechnological production of 3-MP.

An artificial transport metabolon facilitates improved substrate utilization in yeast.

Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.

Engineering of hydroxymandelate synthases and the aromatic amino acid pathway enables de novo biosynthesis of mandelic and 4-hydroxymandelic acid with Saccharomyces cerevisiae.

Mandelic acid (MA) and 4-hydroxymandelic acid (HMA) are valuable specialty chemicals used as precursors for flavors as well as for cosmetic and pharmaceutical purposes. Today they are mainly synthesized chemically. Their synthesis through microbial fermentation would allow for environmentally sustainable production. In this work, we engineered the yeast Saccharomyces cerevisiae for high-level production of MA and HMA. Expressing the hydroxymandelate synthase from Amycolatopsis orientalis in a yeast wild type strain resulted in the production of 119mg/L HMA from glucose. As the enzyme also accepts phenylpyruvate as a substrate aside from its native substrate 4-hydroxyphenylpyruvate, 0.7mg/L MA was also produced. Preventing binding of 4-hydroxyphenylpyruvate to the hydroxymandelate synthase by introducing a S201V replacement in its substrate binding site nearly completely prevented HMA production but increased MA production only 3.5-fold. To further increase HMA and MA production, the aromatic amino acid pathway was engineered. We increased the precursor supply by introducing modifications in the shikimic acid pathway (ARO1↑, ARO3K222L↑, ARO4K220L↑) and reducing flux into the Ehrlich pathway (aro10Δ), and thereby enhanced the HMA titer to 465mg/L and the MA titer to 2.9mg/L. A further increase in HMA and MA titers was achieved by replacing the hydroxymandelate synthase from A. orientalis with the corresponding enzyme from Nocardia uniformis. Subsequently, we introduced additional deletions to block the competing tryptophan branch (trp2Δ), to further decrease flux into the Ehrlich pathway (pdc5Δ) and to avoid transamination of phenylpyruvate and 4-hydroxyphenylpyruvate (aro8Δ, aro9Δ). We achieved more than 1g/L 4-hydroxymandelate when additionally preventing formation of phenylpyruvate by deleting PHA2. When deleting TYR1 to prevent formation of 4-hydroxyphenylpyruvate instead, an MA titer of 236mg/L was achieved. This is a more than 200-fold increase in MA production compared to the wild type strain expressing the hydroxymandelate synthase from A. orientalis. Finally, we showed that S. cerevisiae tolerates HMA and MA to concentrations as high as 3g/L and 7.5g/L, respectively. Our results demonstrate that S. cerevisiae is a promising host for sustainable MA and HMA production.

An expanded enzyme toolbox for production of cis, cis-muconic acid and other shikimate pathway derivatives in Saccharomyces cerevisiae.

A wide range of commercially relevant aromatic chemicals can be synthesized via the shikimic acid pathway. Thus, this pathway has been the target of diverse metabolic engineering strategies. In the present work, an optimized yeast strain for production of the shikimic acid pathway intermediate 3-dehydroshikimate (3-DHS) was generated, which is a precursor for the production of the valuable compounds cis, cis-muconic acid (CCM) and gallic acid (GA). Production of CCM requires the overexpression of the heterologous enzymes 3-DHS dehydratase AroZ, protocatechuic acid (PCA) decarboxylase AroY and catechol dioxygenase CatA. The activity of AroY limits the yield of the pathway. This repertoire of enzymes was expanded by a novel fungal decarboxylase. Introducing this enzyme into the pathway in the optimized strain, a titer of 1244 mg L-1 CCM could be achieved, yielding 31 mg g-1 glucose. This represents the highest yield of this compound reported in Saccharomyces cerevisiae to date. To demonstrate the applicability of the optimized strain for production of other compounds from 3-DHS, we overexpressed AroZ together with a mutant of a para-hydroxybenzoic acid hydroxylase with improved substrate specificity for PCA, PobAY385F. Thereby, we could demonstrate the production of GA for the first time in S. cerevisiae.

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.

Biotechnological production of cis,cis-muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis,cis-muconic acid has already been established in the host organism Saccharomyces cerevisiae, the generation of industrially relevant amounts of cis,cis-muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit Ciso (AroY-Ciso), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-Ciso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1, which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-Ciso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-Ciso, although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-Ciso, suggesting that mitochondrial localization has no major impact on cofactor biosynthesis.IMPORTANCE In Saccharomyces cerevisiae, the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis,cis-muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain.

Secretion of 2,3-dihydroxyisovalerate as a limiting factor for isobutanol production in Saccharomyces cerevisiae.

Isobutanol is a superior biofuel compared to ethanol, and it is naturally produced by yeasts. Previously, Saccharomyces cerevisiae has been genetically engineered to improve isobutanol production. We found that yeast cells engineered for a cytosolic isobutanol biosynthesis secrete large amounts of the intermediate 2,3-dihydroxyisovalerate (DIV). This indicates that the enzyme dihydroxyacid dehydratase (Ilv3) is limiting the isobutanol pathway and/or yeast exhibit effective transport systems for the secretion of the intermediate, competing with isobutanol synthesis. Moreover, we found that DIV cannot be taken up by the cells again. To identify the responsible transporters, microarray analysis was performed with a DIV producing strain compared to a wild type. Altogether, 19 genes encoding putative transporters were upregulated under DIV-producing conditions. Thirteen of these were deleted together with five homologous genes. A yro2 mrh1 deletion strain showed reduced DIV secretion, while a hxt5 deletion mutant showed increased isobutanol production. However, a strain deleted for all the 18 genes secreted even slightly increased amounts of the intermediates and less isobutanol. The lactate transporter Jen1 turned out to transport the intermediate 2-ketoisovalerate, but not DIV. The results suggest that the transport of DIV is a rather complex process and several unspecific transporters seem to be involved.

Pathway engineering for the production of heterologous aromatic chemicals and their derivatives in Saccharomyces cerevisiae: bioconversion from glucose.

Saccharomyces cerevisiae has been extensively engineered for optimising its performance as a microbial cell factory to produce valuable aromatic compounds and their derivatives as bulk and fine chemicals. The production of heterologous aromatic molecules in yeast is achieved via engineering of the aromatic amino acid biosynthetic pathway. This pathway is connected to two pathways of the central carbon metabolism, and is highly regulated at the gene and protein level. These characteristics impose several challenges for tailoring it, and various modifications need to be applied in order to redirect the carbon flux towards the production of the desired compounds. This minireview addresses the metabolic engineering approaches targeting the central carbon metabolism, the shikimate pathway and the tyrosine and phenylalanine biosynthetic pathway of S. cerevisiae for biosynthesis of aromatic chemicals and their derivatives from glucose.

Optimisation of trans-cinnamic acid and hydrocinnamyl alcohol production with recombinant Saccharomyces cerevisiae and identification of cinnamyl methyl ketone as a by-product.

Trans-cinnamic acid (tCA) and hydrocinnamyl alcohol (HcinOH) are valuable aromatic compounds with applications in the flavour, fragrance and cosmetic industry. They can be produced with recombinant yeasts from sugars via phenylalanine after expression of a phenylalanine ammonia lyase (PAL) and an aryl carboxylic acid reductase. Here, we show that in Saccharomyces cerevisiae a PAL enzyme from the bacterium Photorhabdus luminescens was superior to a previously used plant PAL enzyme for the production of tCA. Moreover, after expression of a UDP-glucose:cinnamate glucosyltransferase (FaGT2) from Fragaria x ananassa, tCA could be converted to cinnamoyl-D-glucose which is expected to be less toxic to the yeast cells. Production of tCA and HcinOH from glucose could be increased by eliminating feedback-regulated steps of aromatic amino acid biosynthesis and diminishing the decarboxylation step of the competing Ehrlich pathway. Finally, an unknown by-product resulting from further metabolisation of a carboligation product of cinnamaldehyde (cinALD) with activated acetaldehyde, mediated by pyruvate decarboxylases, could be identified as cinnamyl methyl ketone providing a new route for the biosynthesis of precursors, such as (2S,3R) 5-phenylpent-4-ene-2,3-diol, necessary for the chemical synthesis of specific biologically active drugs such as daunomycin.

Polymorphisms in the LAC12 gene explain lactose utilisation variability in Kluyveromyces marxianus strains.

Kluyveromyces marxianus is a safe yeast used in the food and biotechnology sectors. One of the important traits that sets it apart from the familiar yeasts, Saccharomyces cerevisiae, is its capacity to grow using lactose as a carbon source. Like in its close relative, Kluyveromyces lactis, this requires lactose transport via a permease and intracellular hydrolysis of the disaccharide. Given the importance of the trait, it was intriguing that most, but not all, strains of K. marxianus are reported to consume lactose efficiently. In this study, primarily through heterologous expression in S. cerevisiae and K. marxianus, it was established that a single gene, LAC12, is responsible for lactose uptake in K. marxianus. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, rendering the protein non-functional for lactose transport. Genome analysis showed that the LAC12 gene is present in four copies in the subtelomeric regions of three different chromosomes but only the ancestral LAC12 gene encodes a functional lactose transporter. Other copies of LAC12 may be non-functional or have alternative substrates. The analysis raises some interesting questions regarding the evolution of sugar transporters in K. marxianus.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose.

All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growth-based screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.

Parallelised online biomass monitoring in shake flasks enables efficient strain and carbon source dependent growth characterisation of Saccharomyces cerevisiae.

BACKGROUND: Baker's yeast, Saccharomyces cerevisiae, as one of the most often used workhorses in biotechnology has been developed into a huge family of application optimised strains in the last decades. Increasing numbers of strains render their characterisation highly challenging, even with the simple methods of growth-based analytics. Here we present a new sensor system for the automated, non-invasive and parallelisable monitoring of biomass in continuously shaken shake flask cultures, called CGQ ("cell growth quantifier"). The CGQ implements a dynamic approach of backscattered light measurement, allowing for efficient and accurate growth-based strain characterisation, as exemplarily demonstrated for the four most commonly used laboratory and industrial yeast strains, BY4741, W303-1A, CEN.PK2-1C and Ethanol Red. RESULTS: Growth experiments revealed distinct carbon source utilisation differences between the investigated S. cerevisiae strains. Phenomena such as diauxic shifts, morphological changes and oxygen limitations were clearly observable in the growth curves. A strictly monotonic non-linear correlation of OD600 and the CGQ's backscattered light intensities was found, with strain-to-strain as well as growth-phase related differences. The CGQ measurements showed high resolution, sensitivity and smoothness even below an OD600 of 0.2 and were furthermore characterised by low background noise and signal drift in combination with high reproducibility. CONCLUSIONS: With the CGQ, shake flask fermentations can be automatically monitored regarding biomass and growth rates with high resolution and parallelisation. This makes the CGQ a valuable tool for growth-based strain characterisation and development. The exceptionally high resolution allows for the identification of distinct metabolic differences and shifts as well as for morphologic changes. Applications that will benefit from that kind of automatized biomass monitoring include, amongst many others, the characterization of deregulated native or integrated heterologous pathways, the fast detection of co-fermentation as well as the realisation of rational and growth-data driven evolutionary engineering approaches.

The genome sequence of the popular hexose-transport-deficient Saccharomyces cerevisiae strain EBY.VW4000 reveals LoxP/Cre-induced translocations and gene loss.

Saccharomyces cerevisiae harbours a large group of tightly controlled hexose transporters with different characteristics. Construction and characterization of S. cerevisiae EBY.VW4000, a strain devoid of glucose import, was a milestone in hexose-transporter research. This strain has become a widely used platform for discovery and characterization of transporters from a wide range of organisms. To abolish glucose uptake, 21 genes were knocked out, involving 16 successive deletion rounds with the LoxP/Cre system. Although such intensive modifications are known to increase the risk of genome alterations, the genome of EBY.VW4000 has hitherto not been characterized. Based on a combination of whole genome sequencing, karyotyping and molecular confirmation, the present study reveals that construction of EBY.VW4000 resulted in gene losses and chromosomal rearrangements. Recombinations between the LoxP scars have led to the assembly of four neo-chromosomes, truncation of two chromosomes and loss of two subtelomeric regions. Furthermore, sporulation and spore germination are severely impaired in EBY.VW4000. Karyotyping of the EBY.VW4000 lineage retraced its current chromosomal architecture to four translocations events occurred between the 6th and the 12th rounds of deletion. The presented data facilitate further studies on EBY.VW4000 and highlight the risks of genome alterations associated with repeated use of the LoxP/Cre system.

On the role of GAPDH isoenzymes during pentose fermentation in engineered Saccharomyces cerevisiae.

In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway.