Characterization of a thiamin diphosphate‐dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86885/1/j.1742-4658.2011.08103.x.pd

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans . Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 μM at pH 5), high catalytic efficiency (4.75×105 M−1 s−1 at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45–55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host’s transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35±0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.Web of Scienc

Yeast : the soul of beer’s aroma—a review of flavour-active esters and higher alcohols produced by the brewing yeast

Among the most important factors influencing beer quality is the presence of well-adjusted amounts of higher alcohols and esters. Thus, a heavy body of literature focuses on these substances and on the parameters influencing their production by the brewing yeast. Additionally, the complex metabolic pathways involved in their synthesis require special attention. More than a century of data, mainly in genetic and proteomic fields, has built up enough information to describe in detail each step in the pathway for the synthesis of higher alcohols and their esters, but there is still place for more. Higher alcohols are formed either by anabolism or catabolism (Ehrlich pathway) of amino acids. Esters are formed by enzymatic condensation of organic acids and alcohols. The current paper reviews the up-to-date knowledge in the pathways involving the synthesis of higher alcohols and esters by brewing yeasts. Fermentation parameters affecting yeast response during biosynthesis of these aromatic substances are also fully reviewed.Eduardo Pires gratefully acknowledges the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal) for the PhD fellowship support (SFRH/BD/61777/2009). The financial contributions of the EU FP7 project Ecoefficient Biodegradable Composite Advanced Packaging (EcoBioCAP, grant agreement no. 265669) as well as of the Grant Agency of the Czech Republic (project GACR P503/12/1424) are also gratefully acknowledged. The authors thank the Ministry of Education, Youth and Sports of the Czech Republic (MSM 6046137305) for their financial support

Branched-chain and aromatic amino acid catabolism into aroma volatiles in Cucumis melo L. fruit

The unique aroma of melons (Cucumis melo L., Cucurbitaceae) is composed of many volatile compounds biosynthetically derived from fatty acids, carotenoids, amino acids, and terpenes. Although amino acids are known precursors of aroma compounds in the plant kingdom, the initial steps in the catabolism of amino acids into aroma volatiles have received little attention. Incubation of melon fruit cubes with amino acids and α-keto acids led to the enhanced formation of aroma compounds bearing the side chain of the exogenous amino or keto acid supplied. Moreover, L-[13C6]phenylalanine was also incorporated into aromatic volatile compounds. Amino acid transaminase activities extracted from the flesh of mature melon fruits converted L-isoleucine, L-leucine, L-valine, L-methionine, or L-phenylalanine into their respective α-keto acids, utilizing α-ketoglutarate as the amine acceptor. Two novel genes were isolated and characterized (CmArAT1 and CmBCAT1) encoding 45.6 kDa and 42.7 kDa proteins, respectively, that displayed aromatic and branched-chain amino acid transaminase activities, respectively, when expressed in Escherichia coli. The expression of CmBCAT1 and CmArAT1 was low in vegetative tissues, but increased in flesh and rind tissues during fruit ripening. In addition, ripe fruits of climacteric aromatic cultivars generally showed high expression of CmBCAT1 and CmArAT1 in contrast to non-climacteric non-aromatic fruits. The results presented here indicate that in melon fruit tissues, the catabolism of amino acids into aroma volatiles can initiate through a transamination mechanism, rather than decarboxylation or direct aldehyde synthesis, as has been demonstrated in other plants

Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus

BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance. RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (KM 0.06 mM at pH 5), high catalytic efficiencies, pHopt of 5.5 and Topt at 45 degrees C. The enzyme is not thermostable (T of 18 minutes at 60 degrees C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 A for C? when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503. CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.IS

Ca isotope fingerprints of early crust-mantle evolution

Among the most important factors influencing beer quality is the presence of well-adjusted amounts of higher alcohols and esters; as well as the successful reduction of undesirable by-products such as diacetyl. While higher alcohols and esters contribute rather positively to the beer aroma, diacetyl is mostly unwelcome for beer types with lighter taste. Thus, the complex metabolic pathways in yeast responsible for the synthesis of both pleasant and unpleasant by-products of fermentation were given special attention in this last chapter

Determination of soil losses in forest road slopes in semi-arid mountainous areas by UAV and terrestrial photogrammetric methods

Genellikle dağlık alanlarda bulunan ormanların işletmeye açılmasında orman yolları yeterli mühendislik standartlarında ve gerekli koruyucu önlemler alınmadan inşa edildiklerinde bazı olumsuz sonuçlar ortaya çıkarabilmektedirler. Bu olumsuz sonuçların sebep olduğu sorunların başında toprak erozyonu (kayıpları) gelmektedir. Özellikle yarı kurak alanlarda bu konu daha fazla önem arz etmektedir. Bu nedenle orman yolları inşa edildikten sonraki dönemlerde izlenerek sebep oldukları toprak kayıpları ortaya konulmalıdır. Yoğun emek ve iş gücü gerektiren geleneksel toprak kaybı belirleme çalışmalarında; parsel kurulumundaki zorluklar, sisteminin bağlantı noktalarından oluşan kaçaklar, depolama birimlerindeki malzemenin taşması ve meydana gelen yağışların düzenli takip edilmesi vb. gibi sistemin dezavantajları bulunmaktadır. Ancak son yıllarda uzaktan algılama teknolojisinin gelişmesiyle beraber geleneksel ölçümlerin dezavantajlarını ortadan kaldırmaya ve en aza indirmeye imkân veren uzaktan algılama gibi alternatif yöntemler de ortaya çıkmaktadır. Bu tez çalışmasında uzaktan algılama tekniklerinden olan, İHA ve yersel fotogrametrik yöntemlerin kullanım imkânlarını değerlendirmeye yönelik, yarı kurak dağlık alanlarda orman yolu kazı şevlerinde meydana gelen toprak kayıplarının belirlenmesi amacıyla uygulamalar yapılmıştır. Ayrıca yöntemlerin avantaj ve dezavantajları irdelenmiştir. Çalışma alanı olarak Taşlıyayla Orman İşletme Şefliği (Seben/Bolu)sınırları içerisinde yer alan ve 2019 Ağustos ayında yapımı tamamlanan 3,7 km uzunluğundaki 001 kodlu B Tipi orman yolunun 100 metrelik kısmı seçilmiştir. Veri alımları İHA ve yersel fotogrametrik yöntemlerle Mayıs 2020-Kasım 2020 döneminde gerçekleştirilmiştir. Çalışmada; seçilen yolun kazı şevinde birim alandaki alansal erozyon ve birikme büyüklükleri İHA ile 0,080 m2m-2 erozyon, 0,067m2m-2 birikme, yersel fotogrametrik yöntemle 0,127 m2m-2 erozyon, 0,088 m2m-2 birikme tespit edilmiştir. Ayrıca kazı şevinde birim alanda hacimsel erozyon ve birikme miktarları ise İHA ile -0,0060 m3m-2 aşınma miktarı, 0,0046 m3m-2 birikme miktarı, yersel fotogrametrik yöntemle -0,0050 m3m-2 aşınma miktarı, 0,0031 m3m-2 birikme miktarı bulunmuştur. Her iki yöntem zamansal olarak karşılaştırıldığında yersel fotogrametri yönteminde işlemler yaklaşık 2 kat daha uzun sürmüştür. Ayrıca İHA ile üretilen SYM ve ortofotoların yer örneklem mesafesi 2 cm iken, yersel fotogrametri yönteminde 1 cm olarak elde edilmiş, yersel alımlar 2 kat daha yüksek çözünürlüklü bulunmuştur. Elde edilen sonuçlara göre bu gibi çalışmalarda yersel fotogremetrik yöntem daha doğru sonuçlar vermektedir.The construction of forest roads in order to put the mountainous forests into operation brings with it some problems if not careful enough and the necessary protective measures are not taken. Soil erosion comes first among these problems. This issue is especially important in semi-arid ecosystems. The traditional soil loss determination method; difficulty of parcel establishment, leaks from connection points in the system, overflow of collection tank and monitoring of precipitation time etc. has such disadvantages. With the development of remote sensing technology in recent years, access to soil loss data and obtaining results can be economical, easy and fast. In this thesis, an alternative to the traditional soil loss determination method was investigated by UAV and terrestrial photogrammetric methods. The aim of this study is to determine the soil losses in forest road cut slopes in semi-arid mountainous areas by UAV and terrestrial photogrammetric methods. In addition, the advantages and disadvantages of both methods used in the study were examined. In this context, the 100-meter part of the 3,7 km-long Type B forest road with the code 001, whose construction was completed in August 2019, within the boundaries of the Taşlıyayla Forest Management Chief (Seben/Bolu), was chosen as the study area. Data acquisition was carried out by UAV and terrestrial photogrammetric methods in the period of May 2020 - November 2020.In the results of study; degradation per unit was determined by UAVerosion in 0.080 m2m-2 area and accumulation in 0.067 m2m-2 area and by terrestrial photogrammetry erosion in 0,127 m2m-2 area and accumulation in 0,088 m2m-2 area. In addition, according to the volumetric degradation per unit area results, -0.0060 m3 m-2 erosion amount and 0.0046 m3 m-2 accumulation amount were determined in the UAV. In the photogrammetric method, -0.0050 m3 m-2 erosion amount and 0.0031 m3 m-2 accumulation amount were found. When both methods were compared temporally, the processes took approximately 2 times longer in the terrestrial photogrammetry method.In addition, while the ground sampling distance of DEM and orthophoto produced with the UAV was 2 cm, it was obtained as 1 cm in the terrestrial photogrammetry method, terrestrial receptions were found to be 2 times higher resolution. According to the results obtained, the terrestrial photogremetric method gives results that are more accurate in such studies

Engineering of aromatic amino acid metabolism in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a popular industrial microorganism. It has since long been used in bread, beer and wine making. More recently it is also being applied for heterologous protein production and as a target organism for metabolic engineering. The work presented in this thesis describes how S. cerevisiae may be used as a metabolic-engineering platform to produce aromatic compounds such as phenylalanine or its catabolites, phenylethanol and phenylacetate. In this thesis two research lines were followed: The first research line focused on the molecular identity and substrate specificity of 2-oxo-acid decarboxylase in S. cerevisiae whereas the second line of research investigated the elimination of feedback inhibition steps in the phenylalanine biosynthetic pathway in S. cerevisiae. The impact of feedback inhibition on the aromatic biosynthesis pathway was quantified by analyzing intra- and extracellular concentrations of relevant aromatic compounds in glucose-limited chemostat cultures of wild-type and engineered strains.Applied Science

Effect of Elevated CO₂ and Drought on Biomass, Gas Exchange and Wood Structure of <i>Eucalyptus grandis</i>

Juvenile Eucalyptus grandis were exposed to drought and elevated CO2 to evaluate the independent and interactive effects on growth, gas exchange and wood structure. Trees were grown in a greenhouse at ambient and elevated CO2 (aCO2, 410 ppm; eCO2, 950 ppm), in combination with daily irrigation and cyclic drought during one growing season. The results demonstrated that drought stress limited intercellular CO2 concentration, photosynthesis, stomatal conductance, and transpiration, which correlated with a lower increment in height, stem diameter and biomass. Drought also induced formation of frequent and narrow vessels accompanied by a reduction in vessel lumen area. Conversely, elevated CO2 increased intercellular CO2 concentration as well as photosynthesis, and partially closed stomata, leading to a more efficient water use, especially under drought. There was a clear trend towards greater biomass accumulation at eCO2, although the results did not show statistical significance for this parameter. We observed an increase in vessel diameter and vessel lumen area at eCO2, and, contrarily, the vessel frequency decreased. Thus, we conclude that eCO2 delayed the effects of drought and potentialized growth. However, results on vessel anatomy suggest that increasing vulnerability to cavitation due to formation of larger vessels may counteract the beneficial effects of eCO2 under severe drought