Enhancing a Sphaerobacter thermophilus ω-transaminase for kinetic resolution of ß- and γ-amino acids.

Sphaerobacter thermophilus synthesizes an ω-transaminase (ω-TA) that allows the production of enantiomerically pure ß-amino acids. To obtain ω-TA variants with a higher activity and more favorable properties for industrial use, we modified critical amino acid residues either in the catalytic center or in a previously proposed signature motif critical for aromatic ß-amino acid ω-TAs. Seventeen different variants of this enzyme were generated and their activity was examined with four ß-amino acids and one γ-amino acid, and compared with the wildtype's activity. Among all variants, seven showed up to ninefold higher activity with at least one of the tested substrates. For most of these seven variants, the temperature optimum was even lower as in the wild type enzyme, with keeping a high temperature stability, making them more valuable for industrial purposes. Our results indicate that for the production of enantiomerically pure ß-amino acids replacement of critical amino acid residues in the proposed signature motif of ω-TAs is a more effective strategy than modifying their catalytic center. Another finding was, that the proposed motif is not only suitable for aromatic amino acid ω-TAs, because some of the variants have a higher activity with ß-alanine or ß-leucine than with aromatic ß-amino acids.

A transaminase with ß-activity from Variovorax boronicumulans for the production of enantiopure ß-amino acids.

Enantioselective transamination of amino acids is a great challenge in biotechnology as suitable enzymes with wide substrate spectrum are rare. Here, we present a new transaminase from Variovorax boronicumulans (VboTA, Variovorax boronicumulansω-transaminase) which is specific for ß-amino acids. The amino acid sequence of VboTA is similar to an ω-transaminase from Variovorax paradoxus, for which a crystal-structure is available. This similarity is allowing us to classify VboTA as a fold type 1 ω-transaminase (ω-TA). Although both enzymes have a high sequence similarity (86% identities, 92% positives), there are differences in the active center, which allow VboTA to accept a broader substrate spectrum. Both enzymes have also a different temperature stability and temperature optimum. VboTA deaminates the D-form of aromatic ß-amino acids, such as ß-homophenylalanine and ß-phenylalanine as well as aliphatic ß-amino acids, such as ß-homoalanine and ß-leucine. The optimal reaction conditions turned out to be 32 °C and pH 9. Kinetic resolution lead to high enantiomeric excess of 86.6% to >99.9%, depending on the amino donor/acceptor pair. In contrast to many other ω-TAs, VboTA has a broad substrate spectrum and uses both aromatic or aliphatic amino acids. With γ-amino acids as substrates, VboTA showed no activity at all.

Cutinase ACut2 from Blastobotrysraffinosifermentans for the Selective Desymmetrization of the Symmetric Diester Diethyl Adipate to the Monoester Monoethyl Adipate.

Monoethyl adipate (MEA) is a highly valuable monoester for activating resistance mechanisms and improving protective effects in pathogen-attacked plants. The cutinase ACut2 from the non-conventional yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) was used for its synthesis by the desymmetrization of dicarboxylic acid diester diethyl adipate (DEA). Up to 78% MEA with 19% diacid adipic acid (AA) as by-product could be synthesized by the unpurified ACut2 culture supernatant from the B. raffinosifermentans overexpression strain. By adjusting pH and enzyme concentration, the selectivity of the free ACut2 culture supernatant was increased, yielding 95% MEA with 5% AA. Selectivity of the carrier immobilized ACut2 culture supernatant was also improved by pH adjustment during immobilization, as well as carrier enzyme loading, ultimately yielding 93% MEA with an even lower AA concentration of 3-4%. Thus, optimizations enabled the selective hydrolysis of DEA into MEA with only a minor AA impurity. In the up-scaling, a maximum of 98% chemical and 87.8% isolated MEA yield were obtained by the adsorbed enzyme preparation with a space time yield of 2.6 g L-1 h-1. The high monoester yields establish the ACut2-catalyzed biosynthesis as an alternative to existing methods.

Characterization of Catechol-1,2-Dioxygenase (Acdo1p) From Blastobotrys raffinosifermentans and Investigation of Its Role in the Catabolism of Aromatic Compounds.

Gallic acid, protocatechuic acid, catechol, and pyrogallol are only a few examples of industrially relevant aromatics. Today much attention is paid to the development of new microbial factories for the environmentally friendly biosynthesis of industrially relevant chemicals with renewable resources or organic pollutants as the starting material. The non-conventional yeast, Blastobotrys raffinosifermentans, possesses attractive properties for industrial bio-production processes such as thermo- and osmotolerance. An additional advantage is its broad substrate spectrum, with tannins at the forefront. The present study is dedicated to the characterization of catechol-1,2-dioxygenase (Acdo1p) and the analysis of its function in B. raffinosifermentans tannic acid catabolism. Acdo1p is a dimeric protein with higher affinity for catechol (K M = 0.004 ± 0.001 mM, k cat = 15.6 ± 0.4 s-1) than to pyrogallol (K M = 0.1 ± 0.02 mM, k cat = 10.6 ± 0.4 s-1). It is an intradiol dioxygenase and its reaction product with catechol as the substrate is cis,cis-muconic acid. B. raffinosifermentans G1212/YIC102-AYNI1-ACDO1-6H, which expresses the ACDO1 gene under the control of the strong nitrate-inducible AYNI1 promoter, achieved a maximum catechol-1,2-dioxygenase activity of 280.6 U/L and 26.9 U/g of dry cell weight in yeast grown in minimal medium with nitrate as the nitrogen source and 1.5% glucose as the carbon source. In the same medium with glucose as the carbon source, catechol-1,2-dioxygenase activity was not detected for the control strain G1212/YIC102 with ACDO1 expression under the regulation of its respective endogenous promoter. Gene expression analysis showed that ACDO1 is induced by gallic acid and protocatechuic acid. In contrast to the wild-type strain, the B. raffinosifermentans strain with a deletion of the ACDO1 gene was unable to grow on medium supplemented with gallic acid or protocatechuic acid as the sole carbon source. In summary, we propose that due to its substrate specificity, its thermal stability, and its ability to undergo long-term storage without significant loss of activity, B. raffinosifermentans catechol-1,2-dioxygenase (Acdo1p) is a promising enzyme candidate for industrial applications.

A new lipase (Alip2) with high potential for enzymatic hydrolysis of the diester diethyladipate to the monoester monoethyladipate.

Several putative lipase genes from the genome of the yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) LS3 were overexpressed in the yeast itself and screened for the desymmetrization of the dicarboxylic acid diester diethyl adipate (DEA) into the monoester monoethyl adipate (MEA). MEA can serve as a monomeric spacer group for functional polymers used in medical chemistry and dental applications. The selected lipase Alip2-c6hp was intracellularly located. After overexpression of the corresponding gene, it was purified and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. In fed-batch cultivation with constructed yeast strain B. raffinosifermentans G1212/YRC102-Alip2-c6h for large scale production of the Alip2-c6hp biocatalyst enzyme activities up to 674 U L-1 were reached. Several tested diesters were hydrolyzed selectively to monoesters. Under optimized conditions, the purified enzyme Alip2p-c6h converted 96 % of the substrate DEA to MEA within 30 min incubation, whereby only 1.6 % of the unwanted side-product adipic acid (AA) was formed. At room temperature the dicarboxylic acid esters diethyl malonate (DEM), diethyl succinate (DES), dimethyl adipate (DMA) and dimethyl suberate (DMSub) were completely hydrolyzed to their corresponding monoesters. A high yield of 87 % and 25 % could also be achieved with the dioldiesters 1,4-diacetoxybutane (DAB) and diacetoxyhexane (DAH). In conclusion the potential of the lipase Alip2-c6hp expressed in B. raffinosifermentans is very promising for selective hydrolysis of DEA to MEA as well as for the production of other monoesters.

The Jacalin-Related Lectin HvHorcH Is Involved in the Physiological Response of Barley Roots to Salt Stress.

Salt stress tolerance of crop plants is a trait with increasing value for future food production. In an attempt to identify proteins that participate in the salt stress response of barley, we have used a cDNA library from salt-stressed seedling roots of the relatively salt-stress-tolerant cv. Morex for the transfection of a salt-stress-sensitive yeast strain (Saccharomyces cerevisiae YSH818 Δhog1 mutant). From the retrieved cDNA sequences conferring salt tolerance to the yeast mutant, eleven contained the coding sequence of a jacalin-related lectin (JRL) that shows homology to the previously identified JRL horcolin from barley coleoptiles that we therefore named the gene HvHorcH. The detection of HvHorcH protein in root extracellular fluid suggests a secretion under stress conditions. Furthermore, HvHorcH exhibited specificity towards mannose. Protein abundance of HvHorcH in roots of salt-sensitive or salt-tolerant barley cultivars were not trait-specific to salinity treatment, but protein levels increased in response to the treatment, particularly in the root tip. Expression of HvHorcH in Arabidopsis thaliana root tips increased salt tolerance. Hence, we conclude that this protein is involved in the adaptation of plants to salinity.

The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed.

Blastobotrys raffinosifermentans is an ascomycetous yeast with biotechnological applications, recently shown to be an oleaginous yeast accumulating lipids under nitrogen limitation. Diacylglycerol acyltransferases (DGATs) act in the lipid storage pathway, in the last step of triacylglycerol biosynthesis. Two DGAT families are widespread in eukaryotes. We first checked that B. raffinosifermentans strain LS3 possessed both types of DGAT, and we then overexpressed the native DGAT-encoding genes, DGA1 and DGA2, separately or together. DGA2 (from the DGAT1 family) overexpression was sufficient to increase lipid content significantly in LS3, to up to 26.5% of dry cell weight (DCW), 1.6 times the lipid content of the parental strain (16.90% of DCW) in glucose medium under nitrogen limitation. By contrast, DGA1 (of the DGAT2 type) overexpression led to a large increase (up to 140-fold) in the amount of the corresponding transcript, but had no effect on overall lipid content relative to the parental strain. Analysis of the expression of the native genes over time in the parental strain revealed that DGA2 transcript levels quadrupled between 8 and 24 h in the N-limited lipogenic medium, whereas DGA1 transcript levels remained stable. This survey highlights the predominant role of the DGAT1 family in lipid accumulation and demonstrates the suitability of B. raffinosifermentans for engineering for lipid production.

Effects of Arbuscular Mycorrhization on Fruit Quality in Industrialized Tomato Production.

Industrialized tomato production faces a decrease in flavors and nutritional value due to conventional breeding. Moreover, tomato production heavily relies on nitrogen and phosphate fertilization. Phosphate uptake and improvement of fruit quality by arbuscular mycorrhizal (AM) fungi are well-studied. We addressed the question of whether commercially used tomato cultivars grown in a hydroponic system can be mycorrhizal, leading to improved fruit quality. Tomato plants inoculated with Rhizophagus irregularis were grown under different phosphate concentrations and in substrates used in industrial tomato production. Changes in fruit gene expression and metabolite levels were checked by RNAseq and metabolite determination, respectively. The tests revealed that reduction of phosphate to 80% and use of mixed substrate allow AM establishment without affecting yield. By comparing green fruits from non-mycorrhizal and mycorrhizal plants, differentially expressed genes (DEGs) were found to possibly be involved in processes regulating fruit maturation and nutrition. Red fruits from mycorrhizal plants showed a trend of higher BRIX values and increased levels of carotenoids in comparison to those from non-mycorrhizal plants. Free amino acids exhibited up to four times higher levels in red fruits due to AM, showing the potential of mycorrhization to increase the nutritional value of tomatoes in industrialized production.

Biotransformation of bisphenol A analogues by the biphenyl-degrading bacterium Cupriavidusbasilensis - a structure-biotransformation relationship.

Comparative analyses determined the relationship between the structure of bisphenol A (BPA) as well as of seven bisphenol analogues (bisphenol B (BPB), bisphenol C (BPC), bisphenol E (BPE), bisphenol F (BPF), bisphenol Z (BPZ), bisphenol AP (BPAP), bisphenol PH (BPPH)) and their biotransformability by the biphenyl-degrading bacterium Cupriavidus basilensis SBUG 290. All bisphenols were substrates for bacterial transformation with conversion rates ranging from 6 to 98% within 216 h and 36 different metabolites were characterized. Transformation by biphenyl-grown cells comprised four different pathways: (a) formation of ortho-hydroxylated bisphenols, hydroxylating either one or both phenols of the compounds; (b) ring fission; (c) transamination followed by acetylation or dimerization; and (d) oxidation of ring substituents, such as methyl groups and aromatic ring systems, present on the 3-position. However, the microbial attack of bisphenols by C. basilensis was limited to the phenol rings and its substituents, while substituents on the carbon bridge connecting the rings were not oxidized. All bisphenol analogues with modifications at the carbon bridge could be oxidized up to ring cleavage, while substituents at the 3-position of the phenol ring other than hydroxyl groups did not allow this reaction. Replacing one methyl group at the carbon bridge of BPA by a hydrophobic aromatic or alicyclic ring system inhibited both dimerization and transamination followed by acetylation. While most of the bisphenol analogues exhibited estrogenic activity, four biotransformation products tested were not estrogenically active.

Characterization of recombinant laccase from Trametes versicolor synthesized by Arxula adeninivorans and its application in the degradation of pharmaceuticals.

Recent years have seen an increasing interest in laccase enzymes. Due to their ability of oxidizing various substrates, they are nowadays applied in multiple industrial fields including pulp delignification, textile dye bleaching, and bioremediation. In contrast to laccase production from native sources, with its generally low yield and high cost, heterologous laccase expression is far better suited to meet the growing industrial demands. TVLCC5 gene encoding Trametes versicolor laccase 5 was overexpressed in Arxula adeninivorans using the strong constitutive TEF1 promoter. Recombinant Tvlcc5 protein was purified by immobilized-metal ion affinity chromatography and biochemically characterized using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as substrate for standard activity assays. The enzyme showed the highest activity at 50 °C between pH 4.5-5.5. The half-life of Tvlcc5 at 60 °C was around 20 min. The negative effect of chloride anions on enzyme activity was demonstrated. A fed-batch cultivation of Tvlcc5 producing strain A. adeninivorans G1212/YRC102-TEF1-TVLCC5-6H was performed and resulted in a laccase activity of 4986.3 U L-1. To improve the expression level of recombinant laccase in A. adeninivorans, cultivation conditions were optimized by single factor experiments. Recombinant Tvlcc5 proved to be a promising agent for degradation of pharmaceuticals that are an important source of environmental pollution. Concentration of diclofenac and sulfamethoxazole decreased to 46.8% and 51.1% respectively after 24 h incubation with Tvlcc5. When 1 mM redox mediator ABTS was added complete degradation was obtained within 1 h.

Blastobotrys adeninivorans and B. raffinosifermentans, two sibling yeast species which accumulate lipids at elevated temperatures and from diverse sugars.

BACKGROUND: In the context of sustainable development, yeast are one class of microorganisms foreseen for the production of oil from diverse renewable feedstocks, in particular those that do not compete with the food supply. However, their use in bulk production, such as for the production of biodiesel, is still not cost effective, partly due to the possible poor use of desired substrates or poor robustness in the practical bioconversion process. We investigated the natural capacity of Blastobotrys adeninivorans, a yeast already used in biotechnology, to store lipids under different conditions. RESULTS: The genotyping of seven strains showed the species to actually be composed of two different groups, one that (including the well-known strain LS3) could be reassigned to Blastobotrys raffinosifermentans. We showed that, under nitrogen limitation, strains of both species can synthesize lipids to over 20% of their dry-cell weight during shake-flask cultivation in glucose or xylose medium for 96 h. In addition, organic acids were excreted into the medium. LS3, our best lipid-producing strain, could also accumulate lipids from exogenous oleic acid, up to 38.1 ± 1.6% of its dry-cell weight, and synthesize lipids from various sugar substrates, up to 36.6 ± 0.5% when growing in cellobiose. Both species, represented by LS3 and CBS 8244T, could grow with little filamentation in the lipogenic medium from 28 to 45 °C and reached lipid titers ranging from 1.76 ± 0.28 to 3.08 ± 0.49 g/L in flasks. Under these conditions, the maximum bioconversion yield (Y FA/S = 0.093 ± 0.017) was obtained with LS3 at 37 °C. The presence of genes for predicted subunits of an ATP citrate lyase in the genome of LS3 reinforces its oleaginous character. CONCLUSIONS: Blastobotrys adeninivorans and B. raffinosifermentans, which are known to be xerotolerant and genetically-tractable, are promising biotechnological yeasts of the Saccharomycotina that could be further developed through genetic engineering for the production of microbial oil. To our knowledge, this is the first report of efficient lipid storage in yeast when cultivated at a temperature above 40 °C. This paves the way to help reducing costs through consolidated bioprocessing.

Correction to: (Anti-)estrogenic and (anti-)androgenic effects in wastewater during advanced treatment: comparison of three in vitro bioassays.

Linda Gehrmann and Helena Bielak contributed equally to this work and are regarded as joint first authors.

Selection of the Optimal Yeast Host for the Synthesis of Recombinant Enzymes.

Yeasts, like Arxula adeninivorans, Hansenula polymorpha, Pichia pastoris, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Yarrowia lipolytica, and Saccharomyces cerevisiae are frequently used producers of recombinant enzymes, particularly when posttranslational modifications are mandatory to obtain full functionality. The wide-range transformation/expression platform presented in this chapter can be used to select the optimal yeast host for high-level synthesis of the desired enzyme with favorable biochemical properties. This platform is composed of a selection marker and up to four expression modules in a linearized cassette. Here we describe the protocols for the assembly as well as the transformation of yeast strains with the respective cassettes, screening of transformants, the isolation and biochemical characterization of the enzymes, and finally a simple fermentation strategy to achieve maximal yields of the chosen recombinant enzyme.

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.

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity.

(Anti-)estrogenic and (anti-)androgenic effects in wastewater during advanced treatment: comparison of three in vitro bioassays.

Endocrine-disrupting chemicals are mainly discharged into the environment by wastewater treatment plants (WWTPs) and are known to induce adverse effects in aquatic life. Advanced treatment with ozone successfully removes such organic micropollutants, but an increase of estrogenic effects after the ozonation of hospital wastewater was observed in previous studies. In order to investigate this effect, estrogenic and androgenic as well as anti-estrogenic and anti-androgenic activities were observed during treatment of hospital wastewater using three different effect-based reporter gene bioassays. Despite different matrix influences, sensitivities, and test-specific properties, all assays used obtained comparable results. Estrogenic and androgenic activities were mainly reduced during the biological treatment and further removed during ozonation and sand filtration, resulting in non-detectable agonistic activities in the final effluent. An increased estrogenic activity after ozonation could not be observed in this study. Antagonistic effects were removed in the biological treatment by up to 50 % without further reduction in the advanced treatment. Due to the presence of antagonistic substances within the wastewater, masking effects were probable. Therefore, this study showed the relevance of antagonistic activities at hospital WWTPs and illustrates the need for a better understanding about antagonistic effects.

Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17ß-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17ß-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.

Agdc1p - a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans.

Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid), are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p) which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (Km -0.7 ± 0.2 mM, kcat -42.0 ± 8.2 s-1) than to protocatechuic acid (3,4-dihydroxybenzoic acid) (Km -3.2 ± 0.2 mM, kcat -44.0 ± 3.2 s-1). Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1-AGDC1 and G1212/YRC102, A. adeninivorans G1234 [Δagdc1] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis,cis-muconic acid. However, the protocatechuic acid catabolism via Agdc1p to catechol seems to be not the only degradation pathway.

Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulation in Arxula adeninivorans by stabilization of production.

BACKGROUND: In recent years the production of biobased biodegradable plastics has been of interest of researchers partly due to the accumulation of non-biodegradable plastics in the environment and to the opportunity for new applications. Commonly investigated are the polyhydroxyalkanoates (PHAs) poly(hydroxybutyrate) and poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-V). The latter has the advantage of being tougher and less brittle. The production of these polymers in bacteria is well established but production in yeast may have some advantages, e.g. the ability to use a broad spectrum of industrial by-products as a carbon sources. RESULTS: In this study we increased the synthesis of PHB-V in the non-conventional yeast Arxula adeninivorans by stabilization of polymer accumulation via genetic modification and optimization of culture conditions. An A. adeninivorans strain with overexpressed PHA pathway genes for ß-ketothiolase, acetoacetyl-CoA reductase, PHAs synthase and the phasin gene was able to accumulate an unexpectedly high level of polymer. It was found that an optimized strain cultivated in a shaking incubator is able to produce up to 52.1% of the DCW of PHB-V (10.8 g L-1) with 12.3%mol of PHV fraction. Although further optimization of cultivation conditions in a fed-batch bioreactor led to lower polymer content (15.3% of the DCW of PHB-V), the PHV fraction and total polymer level increased to 23.1%mol and 11.6 g L-1 respectively. Additionally, analysis of the product revealed that the polymer has a very low average molecular mass and unexpected melting and glass transition temperatures. CONCLUSIONS: This study indicates a potential of use for the non-conventional yeast, A. adeninivorans, as an efficient producer of polyhydroxyalkanoates.