Production of D-glucaric acid with phosphoglucose isomerase-deficient Saccharomyces cerevisiae.

D-Glucaric acid is a potential biobased platform chemical. Previously mainly Escherichia coli, but also the yeast Saccharomyces cerevisiae, and Pichia pastoris, have been engineered for conversion of D-glucose to D-glucaric acid via myo-inositol. One reason for low yields from the yeast strains is the strong flux towards glycolysis. Thus, to decrease the flux of D-glucose to biomass, and to increase D-glucaric acid yield, the four step D-glucaric acid pathway was introduced into a phosphoglucose isomerase deficient (Pgi1p-deficient) Saccharomyces cerevisiae strain. High D-glucose concentrations are toxic to the Pgi1p-deficient strains, so various feeding strategies and use of polymeric substrates were studied. Uniformly labelled 13C-glucose confirmed conversion of D-glucose to D-glucaric acid. In batch bioreactor cultures with pulsed D-fructose and ethanol provision 1.3 g D-glucaric acid L-1 was produced. The D-glucaric acid titer (0.71 g D-glucaric acid L-1) was lower in nitrogen limited conditions, but the yield, 0.23 g D-glucaric acid [g D-glucose consumed]-1, was among the highest that has so far been reported from yeast. Accumulation of myo-inositol indicated that myo-inositol oxygenase activity was limiting, and that there would be potential to even higher yield. The Pgi1p-deficiency in S. cerevisiae provides an approach that in combination with other reported modifications and bioprocess strategies would promote the development of high yield D-glucaric acid yeast strains.

Enhanced Triacylglycerol Production With Genetically Modified Trichosporon oleaginosus.

Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21-0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.

Production of ethylene glycol or glycolic acid from D-xylose in Saccharomyces cerevisiae.

The important platform chemicals ethylene glycol and glycolic acid were produced via the oxidative D-xylose pathway in the yeast Saccharomyces cerevisiae. The expression of genes encoding D-xylose dehydrogenase (XylB) and D-xylonate dehydratase (XylD) from Caulobacter crescentus and YagE or YjhH aldolase and aldehyde dehydrogenase AldA from Escherichia coli enabled glycolic acid production from D-xylose up to 150 mg/L. In strains expressing only xylB and xylD, 29 mg/L 2-keto-3-deoxyxylonic acid [(S)-4,5-dihydroxy-2-oxopentanoic acid] (2K3DXA) was produced and D-xylonic acid accumulated to ca. 9 g/L. A significant amount of D-xylonic acid (ca. 14%) was converted to 3-deoxypentonic acid (3DPA), and also, 3,4-dihydroxybutyric acid was formed. 2K3DXA was further converted to glycolaldehyde when genes encoding by either YagE or YjhH aldolase from E. coli were expressed. Reduction of glycolaldehyde to ethylene glycol by an endogenous aldo-keto reductase activity resulted further in accumulation of ethylene glycol of 14 mg/L. The possibility of simultaneous production of lactic and glycolic acids was evaluated by expression of gene encoding lactate dehydrogenase ldhL from Lactobacillus helveticus together with aldA. Interestingly, this increased the accumulation of glycolic acid to 1 g/L. The D-xylonate dehydratase activity in yeast was notably low, possibly due to inefficient Fe-S cluster synthesis in the yeast cytosol, and leading to D-xylonic acid accumulation. The dehydratase activity was significantly improved by targeting its expression to mitochondria or by altering the Fe-S cluster metabolism of the cells with FRA2 deletion.

A design-build-test cycle using modeling and experiments reveals interdependencies between upper glycolysis and xylose uptake in recombinant S. cerevisiae and improves predictive capabilities of large-scale kinetic models.

BACKGROUND: Recent advancements in omics measurement technologies have led to an ever-increasing amount of available experimental data that necessitate systems-oriented methodologies for efficient and systematic integration of data into consistent large-scale kinetic models. These models can help us to uncover new insights into cellular physiology and also to assist in the rational design of bioreactor or fermentation processes. Optimization and Risk Analysis of Complex Living Entities (ORACLE) framework for the construction of large-scale kinetic models can be used as guidance for formulating alternative metabolic engineering strategies. RESULTS: We used ORACLE in a metabolic engineering problem: improvement of the xylose uptake rate during mixed glucose-xylose consumption in a recombinant Saccharomyces cerevisiae strain. Using the data from bioreactor fermentations, we characterized network flux and concentration profiles representing possible physiological states of the analyzed strain. We then identified enzymes that could lead to improved flux through xylose transporters (XTR). For some of the identified enzymes, including hexokinase (HXK), we could not deduce if their control over XTR was positive or negative. We thus performed a follow-up experiment, and we found out that HXK2 deletion improves xylose uptake rate. The data from the performed experiments were then used to prune the kinetic models, and the predictions of the pruned population of kinetic models were in agreement with the experimental data collected on the HXK2-deficient S. cerevisiae strain. CONCLUSIONS: We present a design-build-test cycle composed of modeling efforts and experiments with a glucose-xylose co-utilizing recombinant S. cerevisiae and its HXK2-deficient mutant that allowed us to uncover interdependencies between upper glycolysis and xylose uptake pathway. Through this cycle, we also obtained kinetic models with improved prediction capabilities. The present study demonstrates the potential of integrated "modeling and experiments" systems biology approaches that can be applied for diverse applications ranging from biotechnology to drug discovery.

Yeast as a tool to express sugar acid transporters with biotechnological interest.

Sugar acids can be used as platform chemicals to generate primary building blocks of industrially relevant products. Microbial production of these organic compounds at high yields requires the engineering of the enzymatic machinery and the presence of plasma membrane transporters able to export them outside the cells. In this study, several yeast carboxylic acid transporters belonging to the Jen family were screened for the transport of biotechnologically relevant sugar acids, namely gluconic, saccharic, mucic, xylaric and xylonic acid, and functionally characterised in Saccharomyces cerevisiae. We show that Jen permeases are capable of transporting most of these sugar acids, although with different specificities. Saccharate is a substrate of the transporters ScJen1-S271Q and KlJen2, gluconate of CaJen2 and KlJen2, and xylarate and mucate of CaJen2. A molecular docking approach of these transporters identified the residues that play a major role in the substrate binding of these sugar acids, namely R188 (ScJen1), R122 (CaJen2) and R127 (KlJen2), all equivalent residues (TMS II). The identification of Jen members as sugar acid transporters can contribute to engineering efficient microbial cell factories with increased sugar acid production, as the ScJen1 is able to promote substrate efflux.

Maltose and maltotriose utilisation by group I strains of the hybrid lager yeast Saccharomyces pastorianus.

Brewer's wort is a challenging environment for yeast as it contains predominantly α-glucoside sugars. There exist two subgroups of the lager yeast Saccharomyces pastorianus which differ in sugar utilisation. We performed wort fermentations and compared representative strains from both groups with respect to their ability to transport and ferment maltose and maltotriose. Additionally, we mapped the transporters MALx1, AGT1, MPHx and MTT1 by Southern blotting. Contrary to previous observations, group I comprises a diverse set of strains, with varying ability to transport and ferment maltotriose. Of the eight group I strains, three efficiently utilised maltotriose, a property enabled by the presence of transmembrane transporters SeAGT1 and MTT1 A58, a variant of the group I type strain (CBS1513) performed particularly well, taking up maltotriose at a higher rate than maltose and retaining significant transport activity at temperatures as low as 0°C. Analysis of transporter distribution in this strain revealed an increased copy number of the MTT1 gene, which encodes the only permease known with higher affinity for maltotriose than maltose and low temperature dependence for transport. We propose that much of the variation in lager yeast fermentation behaviour is determined by the presence or absence of specific transmembrane transporters.

Engineering Aspergillus nidulans for heterologous ent-kaurene and gamma-terpinene production.

Terpenes are a large and varied group of natural products with a wide array of bioactivities and applications. The chemical production of industrially relevant terpenes can be expensive and time-consuming due to the structural complexity of these compounds. Here, we studied Aspergillus nidulans as a heterologous host for monoterpene and diterpene production. Previously, we identified a novel diterpene gene cluster in A. nidulans and showed that overexpression of the cluster-specific transcription factor (pbcR) led to ent-pimara-8(14),15-diene (PD) production. We report further characterization of the A. nidulans PD synthase gene (pbcA). In A. nidulans, overexpression of pbcA resulted in PD production, while deletion of pbcA abolished PD production. Overexpression of Fusarium fujikuroi ent-kaurene synthase (cps/ks) and Citrus unshiu gamma-terpinene synthase resulted in ent-kaurene and gamma-terpinene production, respectively. A. nidulans is a fungal model organism and a close relative to other industrially relevant Aspergillus species. A. nidulans is a known producer of many secondary metabolites, but its ability to produce heterologous monoterpene and diterpene compounds has not been characterized. Here, we show that A. nidulans is capable of heterologous terpene production and thus has potential as a production host for industrially relevant compounds. The genetic engineering principles reported here could also be applied to other Aspergilli.

Xylose-induced dynamic effects on metabolism and gene expression in engineered Saccharomyces cerevisiae in anaerobic glucose-xylose cultures.

Xylose is present with glucose in lignocellulosic streams available for valorisation to biochemicals. Saccharomyces cerevisiae has excellent characteristics as a host for the bioconversion, except that it strongly prefers glucose to xylose, and the co-consumption remains a challenge. Further, since xylose is not a natural substrate of S. cerevisiae, the regulatory response it induces in an engineered strain cannot be expected to have evolved for its utilisation. Xylose-induced effects on metabolism and gene expression during anaerobic growth of an engineered strain of S. cerevisiae on medium containing both glucose and xylose medium were quantified. The gene expression of S. cerevisiae with an XR-XDH pathway for xylose utilisation was analysed throughout the cultivation: at early cultivation times when mainly glucose was metabolised, at times when xylose was co-consumed in the presence of low glucose concentrations, and when glucose had been depleted and only xylose was being consumed. Cultivations on glucose as a sole carbon source were used as a control. Genome-scale dynamic flux balance analysis models were simulated to analyse the metabolic dynamics of S. cerevisiae. The simulations quantitatively estimated xylose-dependent flux dynamics and challenged the utilisation of the metabolic network. A relative increase in xylose utilisation was predicted to induce the bi-directionality of glycolytic flux and a redox challenge even at low glucose concentrations. Remarkably, xylose was observed to specifically delay the glucose-dependent repression of particular genes in mixed glucose-xylose cultures compared to glucose cultures. The delay occurred at a cultivation time when the metabolic flux activities were similar in the both cultures.

A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.

Identification of novel isoprene synthases through genome mining and expression in Escherichia coli.

Isoprene is a naturally produced hydrocarbon emitted into the atmosphere by green plants. It is also a constituent of synthetic rubber and a potential biofuel. Microbial production of isoprene can become a sustainable alternative to the prevailing chemical production of isoprene from petroleum. In this work, sequence homology searches were conducted to find novel isoprene synthases. Candidate sequences were functionally expressed in Escherichia coli and the desired enzymes were identified based on an isoprene production assay. The activity of three enzymes was shown for the first time: expression of the candidate genes from Ipomoea batatas, Mangifera indica, and Elaeocarpus photiniifolius resulted in isoprene formation. The Ipomoea batatas isoprene synthase produced the highest amounts of isoprene in all experiments, exceeding the isoprene levels obtained by the previously known Populus alba and Pueraria montana isoprene synthases that were studied in parallel as controls.

L-arabinose/D-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of L-arabinose to L-arabonate with Saccharomyces cerevisiae.

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for L-arabonate production from L-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a D-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a L-arabinose/D-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP(+) but uses also NAD(+) as a cofactor, and showed highest catalytic efficiency (k cat/K m) towards L-arabinose, D-galactose and D-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of L-arabinose, and the stable oxidation product detected is L-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear L-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for L-arabinose uptake, resulted in production of 18 g of L-arabonate per litre, at a rate of 248 mg of L-arabonate per litre per hour, with 86 % of the provided L-arabinose converted to L-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for L-arabonate production in yeast.

Transcriptome of Saccharomyces cerevisiae during production of D-xylonate.

BACKGROUND: Production of D-xylonate by the yeast S. cerevisiae provides an example of bioprocess development for sustainable production of value-added chemicals from cheap raw materials or side streams. Production of D-xylonate may lead to considerable intracellular accumulation of D-xylonate and to loss of viability during the production process. In order to understand the physiological responses associated with D-xylonate production, we performed transcriptome analyses during D-xylonate production by a robust recombinant strain of S. cerevisiae which produces up to 50 g/L D-xylonate. RESULTS: Comparison of the transcriptomes of the D-xylonate producing and the control strain showed considerably higher expression of the genes controlled by the cell wall integrity (CWI) pathway and of some genes previously identified as up-regulated in response to other organic acids in the D-xylonate producing strain. Increased phosphorylation of Slt2 kinase in the D-xylonate producing strain also indicated that D-xylonate production caused stress to the cell wall. Surprisingly, genes encoding proteins involved in translation, ribosome structure and RNA metabolism, processes which are commonly down-regulated under conditions causing cellular stress, were up-regulated during D-xylonate production, compared to the control. The overall transcriptional responses were, therefore, very dissimilar to those previously reported as being associated with stress, including stress induced by organic acid treatment or production. Quantitative PCR analyses of selected genes supported the observations made in the transcriptomic analysis. In addition, consumption of ethanol was slower and the level of trehalose was lower in the D-xylonate producing strain, compared to the control. CONCLUSIONS: The production of organic acids has a major impact on the physiology of yeast cells, but the transcriptional responses to presence or production of different acids differs considerably, being much more diverse than responses to other stresses. D-Xylonate production apparently imposed considerable stress on the cell wall. Transcriptional data also indicated that activation of the PKA pathway occurred during D-xylonate production, leaving cells unable to adapt normally to stationary phase. This, together with intracellular acidification, probably contributes to cell death.

L-lactic acid production from D-xylose with Candida sonorensis expressing a heterologous lactate dehydrogenase encoding gene.

BACKGROUND: Bioplastics, like polylactic acid (PLA), are renewable alternatives for petroleum-based plastics. Lactic acid, the monomer of PLA, has traditionally been produced biotechnologically with bacteria. With genetic engineering, yeast have the potential to replace bacteria in biotechnological lactic acid production, with the benefits of being acid tolerant and having simple nutritional requirements. Lactate dehydrogenase genes have been introduced to various yeast to demonstrate this potential. Importantly, an industrial lactic acid producing process utilising yeast has already been implemented. Utilisation of D-xylose in addition to D-glucose in production of biochemicals such as lactic acid by microbial fermentation would be beneficial, as it would allow lignocellulosic raw materials to be utilised in the production processes. RESULTS: The yeast Candida sonorensis, which naturally metabolises D-xylose, was genetically modified to produce L-lactic acid from D-xylose by integrating the gene encoding L-lactic acid dehydrogenase (ldhL) from Lactobacillus helveticus into its genome. In microaerobic, CaCO3-buffered conditions a C. sonorensis ldhL transformant having two copies of the ldhL gene produced 31 g l-1 lactic acid from 50 g l-1 D-xylose free of ethanol.Anaerobic production of lactic acid from D-xylose was assessed after introducing an alternative pathway of D-xylose metabolism, i.e. by adding a xylose isomerase encoded by XYLA from Piromyces sp. alone or together with the xylulokinase encoding gene XKS1 from Saccharomyces cerevisiae. Strains were further modified by deletion of the endogenous xylose reductase encoding gene, alone or together with the xylitol dehydrogenase encoding gene. Strains of C. sonorensis expressing xylose isomerase produced L-lactic acid from D-xylose in anaerobic conditions. The highest anaerobic L-lactic acid production (8.5 g l-1) was observed in strains in which both the xylose reductase and xylitol dehydrogenase encoding genes had been deleted and the xylulokinase encoding gene from S. cerevisiae was overexpressed. CONCLUSIONS: Integration of two copies of the ldhL gene in C. sonorensis was sufficient to obtain good L-lactic acid production from D-xylose. Under anaerobic conditions, the ldhL strain with exogenous xylose isomerase and xylulokinase genes expressed and the endogenous xylose reductase and xylitol dehydrogenase genes deleted had the highest L- lactic acid production.

Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae.

D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by (1)H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo(1)H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15-60min of D-xylose provision. During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.

Effect of oxygenation and temperature on glucose-xylose fermentation in Kluyveromyces marxianus CBS712 strain.

BACKGROUND: The yeast Kluyveromyces marxianus features specific traits that render it attractive for industrial applications. These include production of ethanol which, together with thermotolerance and the ability to grow with a high specific growth rate on a wide range of substrates, could make it an alternative to Saccharomyces cerevisiae as an ethanol producer. However, its ability to co-ferment C5 and C6 sugars under oxygen-limited conditions is far from being fully characterized. RESULTS: In the present study, K. marxianus CBS712 strain was cultivated in defined medium with glucose and xylose as carbon source. Ethanol fermentation and sugar consumption of CBS712 were investigated under different oxygen supplies (1.75%, 11.00% and 20.95% of O2) and different temperatures (30°C and 41°C). By decreasing oxygen supply, independently from the temperature, both biomass production as well as sugar utilization rate were progressively reduced. In all the tested conditions xylose consumption followed glucose exhaustion. Therefore, xylose metabolism was mainly affected by oxygen depletion. Loss in cell viability cannot explain the decrease in sugar consumption rates, as demonstrated by single cell analyses, while cofactor imbalance is commonly considered as the main cause of impairment of the xylose reductase (KmXR) - xylitol dehydrogenase (KmXDH) pathway. Remarkably, when these enzyme activities were assayed in vitro, a significant decrease was observed together with oxygen depletion, not ascribed to reduced transcription of the corresponding genes. CONCLUSIONS: In the present study both oxygen supply and temperature were shown to be key parameters affecting the fermentation capability of sugars in the K. marxianus CBS712 strain. In particular, a direct correlation was observed between the decreased efficiency to consume xylose with the reduced specific activity of the two main enzymes (KmXR and KmXDH) involved in its catabolism. These data suggest that, in addition to the impairment of the oxidoreductive pathway being determined by the cofactor imbalance, post-transcriptional and/or post-translational regulation of the pathway enzymes contributes to the efficiency of xylose catabolism in micro-aerobic conditions. Overall, the presented work provides novel information on the fermentation capability of the CBS712 strain that is currently considered as the reference strain of the genus K. marxianus.

The diverse role of Pdr12 in resistance to weak organic acids.

Resistance to weak organic acids is important relative to both weak organic acid preservatives and the development of inhibitor tolerant yeast as industrial production organisms. The ABC transporter Pdr12 is important for resistance to sorbic and propionic acid, but its role in tolerance to other weak organic acids with industrial relevance is not well established. In this study, yeast strains with altered expression of PDR12 and/or CMK1, a protein kinase associated with post-transcriptional negative regulation of Pdr12, were exposed to seven weak organic acids: acetic, formic, glycolic, lactic, propionic, sorbic and levulinic acid. These are widely used as preservatives, present in lignocellulosic hydrolysates or attractive as chemical precursors. Overexpression of PDR12 increased tolerance to acids with longer chain length, such as sorbic, propionic and levulinic acid, whereas deletion of PDR12 increased tolerance to the shorter acetic and formic acid. The viability of all strains decreased dramatically in acetic or propionic acid, but the Δpdr12 strains recovered more rapidly than other strains in acetic acid. Furthermore, our results indicated that Cmk1 plays a role in weak organic acid tolerance, beyond its role in regulation of Pdr12, since deletion of both Cmk1 and Pdr12 resulted in different responses to exposure to acids than were explained by deletion of Pdr12 alone.

Overexpression of NADH-dependent fumarate reductase improves D-xylose fermentation in recombinant Saccharomyces cerevisiae.

Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.

Single-cell measurements of enzyme levels as a predictive tool for cellular fates during organic acid production.

Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as "acidified"). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This "switch-like" relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways.

Production of L-lactic acid by the yeast Candida sonorensis expressing heterologous bacterial and fungal lactate dehydrogenases.

BACKGROUND: Polylactic acid is a renewable raw material that is increasingly used in the manufacture of bioplastics, which offers a more sustainable alternative to materials derived from fossil resources. Both lactic acid bacteria and genetically engineered yeast have been implemented in commercial scale in biotechnological production of lactic acid. In the present work, genes encoding L-lactate dehydrogenase (LDH) of Lactobacillus helveticus, Bacillus megaterium and Rhizopus oryzae were expressed in a new host organism, the non-conventional yeast Candida sonorensis, with or without the competing ethanol fermentation pathway. RESULTS: Each LDH strain produced substantial amounts of lactate, but the properties of the heterologous LDH affected the distribution of carbon between lactate and by-products significantly, which was reflected in extra-and intracellular metabolite concentrations. Under neutralizing conditions C. sonorensis expressing L. helveticus LDH accumulated lactate up to 92 g/l at a yield of 0.94 g/g glucose, free of ethanol, in minimal medium containing 5 g/l dry cell weight. In rich medium with a final pH of 3.8, 49 g/l lactate was produced. The fermentation pathway was modified in some of the strains studied by deleting either one or both of the pyruvate decarboxylase encoding genes, PDC1 and PDC2. The deletion of both PDC genes together abolished ethanol production and did not result in significantly reduced growth characteristic to Saccharomyces cerevisiae deleted of PDC1 and PDC5. CONCLUSIONS: We developed an organism without previous record of genetic engineering to produce L-lactic acid to a high concentration, introducing a novel host for the production of an industrially important metabolite, and opening the way for exploiting C. sonorensis in additional biotechnological applications. Comparison of metabolite production, growth, and enzyme activities in a representative set of transformed strains expressing different LDH genes in the presence and absence of a functional ethanol pathway, at neutral and low pH, generated a comprehensive picture of lactic acid production in this yeast. The findings are applicable in generation other lactic acid producing yeast, thus providing a significant contribution to the field of biotechnical production of lactic acid.

Low pH D-xylonate production with Pichia kudriavzevii.

D-xylonic acid is one of the top 30 most desirable chemicals to be derived from biomass sugars identified by the US Department of Energy, being applicable as a non-food substitute for D-gluconic acid and as a platform chemical. We engineered the non-conventional yeast Pichia kudriavzevii VTT C-79090T to express a D-xylose dehydrogenase coding gene from Caulobacter crescentus. With this single modification the recombinant P. kudriavzevii strain produced up to 171 g L(-1) of D-xylonate from 171 g L(-1) D-xylose at a rate of 1.4 g L(-1) h(-1) and yield of 1.0 g [g substrate consumed](-1), which was comparable with D-xylonate production by Gluconobacter oxydans or Pseudomonas sp. The productivity of the strain was also remarkable at low pH, producing 146 g L(-1) D-xylonate at 1.2 g L(-1) h(-1) at pH 3.0. This is the best low pH production reported for D-xylonate. These results encourage further development towards industrial scale production.