Tolerant Yeast Research Articles

Characterization of tolerance and multi-enzyme activities in non-Saccharomyces yeasts isolated from Vidal blanc icewine fermentation.

This work aim to study the tolerance and aroma-related enzymes activities of the non-Saccharomyces yeasts in Vidal blanc icewine from the Huanren region of China. The strains were identified by sequencing internal transcribed spacer (ITS) region and the 26S rDNA D1/D2 domain genes and all representative non-Saccharomyces yeasts were belonged to genera Metschnikowia, Hanseniaspora, Torulaspora, Candida, and Debaryomyces. A total of 28 strains were carried out for tolerance experiments and results suggested that most of them could tolerate 500g/L glucose, 4% ethanol, 20g/L tartaric acid, and 350mg/L SO2 . Finally, a total of 17 strains with better tolerance were carried out for the β-glucosidase, β-xylosidase, and pectinase activities experiments. The results showed that Candida railenensis HC08 and the strains of Hanseniaspora genus have satisfactory multi-enzyme activities, which can be used to design mixed fermentation to produce characteristic icewine. PRACTICAL APPLICATIONS: Non-Saccharomyces yeasts produces a series of hydrolytic enzymes that are thought to have a significant contribution to the aroma complexity of wines, however, are poorly explored in icewine. In this work, most of the non-Saccharomyces yeasts screened from Chinese icewine can adapt well to the high-sugar and high-acid environment of icewine, and can secrete hydrolase. The application of these strains in mixed fermentation could provide a prospect for the production of characteristic icewine.

Proteasome subunit \u03b11 overexpression preferentially drives canonical proteasome biogenesis and enhances stress tolerance in yeast

The 26S proteasome conducts the majority of regulated protein catabolism in eukaryotes. At the heart of the proteasome is the barrel-shaped 20S core particle (CP), which contains two β-rings sandwiched between two α-rings. Whereas canonical CPs contain α-rings with seven subunits arranged α1-α7, a non-canonical CP in which a second copy of the α4 subunit replaces the α3 subunit occurs in both yeast and humans. The mechanisms that control canonical versus non-canonical CP biogenesis remain poorly understood. Here, we have repurposed a split-protein reporter to identify genes that can enhance canonical proteasome assembly in mutant yeast producing non-canonical α4-α4 CPs. We identified the proteasome subunit α1 as an enhancer of α3 incorporation, and find that elevating α1 protein levels preferentially drives canonical CP assembly under conditions that normally favor α4-α4 CP formation. Further, we demonstrate that α1 is stoichiometrically limiting for α-ring assembly, and that enhancing α1 levels is sufficient to increase proteasome abundance and enhance stress tolerance in yeast. Together, our data indicate that the abundance of α1 exerts multiple impacts on proteasome assembly and composition, and we propose that the limited α1 levels observed in yeast may prime cells for alternative proteasome assembly following environmental stimuli.

The zinc-regulated protein (ZIP) family genes and glutathione s-transferase (GST) family genes play roles in Cd resistance and accumulation of pak choi (Brassica campestris ssp. chinensis)

The molecular mechanisms of the differences among the Cd tolerance and accumulation of different pak choi cultivars are essential to further breed Cd-safe genotypes pak choi. In our research, via morphological comparison, qRT-PCR and yeast function complementary approaches, we explored the differences of Cd tolerance and capacity for Cd uptake in nine various pak choi varieties. Results showed that higher expressions of BcZIPs involved in Cd uptake in ‘Kang Re605’ may lead to its higher capacity for Cd accumulation. The lowest expressions of transporter gene in ‘Wu Yueman’ were consistent with its fewest ability to uptake Cd. Beyond that, the difference of resistance was very great among varieties. Meanwhile, the expressions of the BcGSTUs were differentially induced by Cd exposure in different pak choi varieties, and ‘Kang Re605’ performed the highest BcGSTUs expression overall. To verify the role of GSTUs played in Cd resistance of pak choi, four BcGSTUs, BcGSTU4, BcGSTU11, BcGSTU12 and BcGSTU22 in a high-Cd accumulation and tolerance variety ‘Kang Re605’ were cloned, quantitated and transferred to Cd-sensitive yeast mutant strain. And finally found that BcGSTU11 increased the Cd tolerance of yeast, which may associate with a high Cd resistance of ‘Kang Re605’. Simultaneously, less BcGSTUs abundance in ‘Shang Haiqing’ may result in its weak tolerance to Cd. These findings will help us to comprehend the roles of BcZIPs and BcGSTUs in Cd absorption and detoxification as well as promote our understanding of the Cd-resistant and Cd-accumulated mechanisms in pak choi.

Highlight on the expression and the function of a novel MnSOD from diploid wheat (T. monococcum) in response to abiotic stress and heavy metal toxicity

Superoxide dismutases (SODs) play a pivotal role in improving abiotic stress tolerance in plant cells. A novel manganese superoxide dismutase gene, denoted as TmMnSOD, was identified from Triticum monococcum. The encoded protein displayed high sequence identity with MnSOD family members and was highly homologous to TdMnSOD from durum wheat. Furthermore, the 3D structure analysis revealed that TmMnSOD displayed homotetramer subunit organization, incorporating four Mn2+ ions. Notably, TmMnSOD structure contains predominantly alpha helices with three beta sheets. On the other hand, under stress conditions, TmMnSOD transcript level was significantly up-regulated by salt, oxidative and heavy metal stresses. At the functional level, TmMnSOD imparts tolerance of yeast and E. coli cells under diverse stresses. Promoter analysis of TmMnSOD gene showed the presence of a great number of salt and pathogen-responsive cis-regulatory elements, highlighting the interest of this gene in breeding programs towards improved tolerance to salt stress in wheat.

Coordination of the Cell Wall Integrity and High-Osmolarity Glycerol Pathways in Response to Ethanol Stress in Saccharomyces cerevisiae.

During fermentation, a high ethanol concentration is a major stress that influences the vitality and viability of yeast cells, which in turn leads to a termination of the fermentation process. In this study, we show that the BCK1 and SLT2 genes encoding mitogen-activated protein kinase kinase kinase (MAPKKK) and mitogen-activated protein kinase (MAPK) of the cell wall integrity (CWI) pathway, respectively, are essential for ethanol tolerance, suggesting that the CWI pathway is involved in the response to ethanol-induced cell wall stress. Upon ethanol exposure, the CWI pathway induces the expression of specific cell wall-remodeling genes, including FKS2, CRH1, and PIR3 (encoding β-1,3-glucan synthase, chitin transglycosylase, and O-glycosylated cell wall protein, respectively), which eventually leads to the remodeling of the cell wall structure. Our results revealed that in response to ethanol stress, the high-osmolarity glycerol (HOG) pathway plays a collaborative role with the CWI pathway in inducing cell wall remodeling via the upregulation of specific cell wall biosynthesis genes such as the CRH1 gene. Furthermore, the substantial expression of CWI-responsive genes is also triggered by external hyperosmolarity, suggesting that the adaptive changes in the cell wall are crucial for protecting yeast cells against not only cell wall stress but also osmotic stress. On the other hand, the cell wall stress-inducing agent calcofluor white has no effect on promoting the expression of GPD1, a major target gene of the HOG pathway. Collectively, these findings suggest that during ethanol stress, the CWI and HOG pathways collaboratively regulate the transcription of specific cell wall biosynthesis genes, thereby leading to adaptive changes in the cell wall.IMPORTANCE The budding yeast Saccharomyces cerevisiae has been widely used in industrial fermentations, including the production of alcoholic beverages and bioethanol. During fermentation, an increased ethanol concentration is the main stress that affects yeast metabolism and inhibits ethanol production. This work presents evidence that in response to ethanol stress, both CWI and HOG pathways cooperate to control the expression of cell wall-remodeling genes in order to build the adaptive strength of the cell wall. These findings will contribute to a better understanding of the molecular mechanisms underlying adaptive responses and tolerance of yeast to ethanol stress, which is essential for successful engineering of yeast strains for improved ethanol tolerance.

Protein and transcript profiling analysis of the response of Yarrowia lipolytica Y‐2 in the degradation of ochratoxin A

AbstractThe occurrence of ochratoxin A (OTA) has been reported in cereals, feeds and food. Biological control of OTA has been considered as a promising strategy. Our previous study has shown that Yarrowia lipolytica Y‐2 could degrade OTA. In this study, proteome and transcriptome analysis were performed to investigate the possible mechanisms involved in OTA degradation by Y. lipolytica Y‐2 and the responses of this antagonistic yeast to OTA. The results showed that many proteins and genes involved in removal of reactive O2 species were up‐regulated in Y. lipolytica Y‐2 to respond to the oxidative stress caused by OTA. Also, some proteins and genes involved in the removal of generated toxicants under stress condition were up‐regulated to improve tolerance of this antagonistic yeast to OTA stress. Furthermore, some genes encoding transport proteins and multidrug resistance proteins were up‐regulated significantly to transport and expel OTA from cells and alleviate the toxicity to Y. lipolytica Y‐2. Notably, the gene ECM14 (encoding putative metallocarboxypeptidase ECM14) directly involved in OTA degradation was up‐regulated in Y. lipolytica Y‐2 treated with this mycotoxin. To defend against OTA stress, some genes related to tricarboxylic acid cycle were induced by OTA to generate abundant energy for Y. lipolytica Y‐2. Besides, the down‐regulation of some genes involved in DNA replication and repair decreased the ability of Y. lipolytica Y‐2 to repair DNA damage caused by OTA. These results may imply that OTA degradation by this antagonistic yeast was the synergetic effect of multifarious defence responses to OTA stress.

Enhanced acetic acid stress tolerance and ethanol production in Saccharomyces cerevisiae by modulating expression of the de novo purine biosynthesis genes

BackgroundYeast strains that are tolerant to multiple environmental stresses are highly desired for various industrial applications. Despite great efforts in identifying key genes involved in stress tolerance of budding yeast Saccharomyces cerevisiae, the effects of de novo purine biosynthesis genes on yeast stress tolerance are still not well explored. Our previous studies showed that zinc sulfate addition improved yeast acetic acid tolerance, and key genes involved in yeast stress tolerance were further investigated in this study.ResultsThree genes involved in de novo purine biosynthesis, namely, ADE1, ADE13, and ADE17, showed significantly increased transcription levels by zinc sulfate supplementation under acetic acid stress, and overexpression of these genes in S. cerevisiae BY4741 enhanced cell growth under various stress conditions. Meanwhile, ethanol productivity was also improved by overexpression of the three ADE genes under stress conditions, among which the highest improvement attained 158.39% by ADE17 overexpression in the presence of inhibitor mixtures derived from lignocellulosic biomass. Elevated levels of adenine-nucleotide pool “AXP” ([ATP] + [ADP] + [AMP]) and ATP content were observed by overexpression of ADE17, both under control condition and under acetic acid stress, and is consistent with the better growth of the recombinant yeast strain. The global intracellular amino acid profiles were also changed by overexpression of the ADE genes. Among the changed amino acids, significant increase of the stress protectant γ-aminobutyric acid (GABA) was revealed by overexpression of the ADE genes under acetic acid stress, suggesting that overexpression of the ADE genes exerts control on both purine biosynthesis and amino acid biosynthesis to protect yeast cells against the stress.ConclusionWe proved that the de novo purine biosynthesis genes are useful targets for metabolic engineering of yeast stress tolerance. The engineered strains developed in this study with improved tolerance against multiple inhibitors can be employed for efficient lignocellulosic biorefinery to produce biofuels and biochemicals.

Functional characterization of metallothionein-like genes from Physcomitrella patens: expression profiling, yeast heterologous expression, and disruption of PpMT1.2a gene.

Physcomitrella patens contains four metallothionein-like genes. Three were shown to confer metal tolerance in yeast. Transcript profiling suggests their roles in senescence and reproductive development or cadmium and oxidative stress. Metallothioneins (MTs) have been suggested to play various roles including metal detoxification, nutrient remobilization, ROS scavenging, stress tolerance, and plant development. However, little is known about the forms and functions of MTs in bryophytes. The moss Physcomitrella patens genome was found to contain four MT-like genes. Amino acid sequence composition showed that the P. patens MTs (PpMTs) were clustered with Type 1 plant MTs, and could be further classified into two sub-types, herein referred to as sub-type 1: PpMT1.1a and PpMT1.1b and sub-type 2: PpMT1.2a and PpMT1.2b. Transcript abundance of PpMT1.1b and PpMT1.2b was upregulated in the gametophore compared to protonema, and all, except PpMT1.2a, were highly induced in senescing gametophytes. PpMT1.1a and PpMT1.1b transcripts were upregulated in protonema treated with cadmium and hydrogen peroxide. Unlike many higher plant MTs, the PpMT transcript abundance was not strongly induced in response to copper and zinc. These results suggest that PpMTs may play a role in protecting P. patens from cadmium and oxidative stress and may be involved in tissues senescence and reproductive development. The PpMTs, except PpMT1.2b, were also able to confer metal tolerance and accumulation when heterologously expressed in the ∆cup1 yeast. A P. patens mutant lacking PpMT1.2a through targeted gene disruption was generated. However, it did not show any alteration in growth phenotypes under senescence-induced conditions or hypersensitivity to cadmium, copper, zinc, H2O2, and NaCl stresses. Further characterization of additional P. patens mutants lacking single or multiple PpMTs may provide insight into the physiological roles of bryophytic MTs.

Evolutionary engineering in Saccharomyces cerevisiae reveals a TRK1-dependent potassium influx mechanism for propionic acid tolerance

BackgroundPropionic acid (PA), a key platform chemical produced as a by-product during petroleum refining, has been widely used as a food preservative and an important chemical intermediate in many industries. Microbial PA production through engineering yeast as a cell factory is a potentially sustainable alternative to replace petroleum refining. However, PA inhibits yeast growth at concentrations well below the titers typically required for a commercial bioprocess.ResultsAdaptive laboratory evolution (ALE) with PA concentrations ranging from 15 to 45 mM enabled the isolation of yeast strains with more than threefold improved tolerance to PA. Through whole genome sequencing and CRISPR–Cas9-mediated reverse engineering, unique mutations in TRK1, which encodes a high-affinity potassium transporter, were revealed as the cause of increased propionic acid tolerance. Potassium supplementation growth assays showed that mutated TRK1 alleles and extracellular potassium supplementation not only conferred tolerance to PA stress but also to multiple organic acids.ConclusionOur study has demonstrated the use of ALE as a powerful tool to improve yeast tolerance to PA. Potassium transport and maintenance is not only critical in yeast tolerance to PA but also boosts tolerance to multiple organic acids. These results demonstrate high-affinity potassium transport as a new principle for improving organic acid tolerance in strain engineering.

The Goldilocks effect of respiration on canavanine tolerance in Saccharomyces cerevisiae.

When glucose is available, Saccharomyces cerevisiae prefers fermentation to respiration. In fact, it can live without respiration at all. Here, we study the role of respiration in stress tolerance in yeast. We found that colony growth of respiratory-deficient yeast (petite) is greatly inhibited by canavanine, the toxic analog of arginine that causes proteotoxic stress. We found lower amounts of the amino acids involved in arginine biosynthesis in petites compared with WT. This finding may be explained by the fact that petite cells exposed to canavanine show reduction in the efficiency of targeting of proteins required for arginine biosynthesis. The retrograde (RTG) pathway signals mitochondrial stress. It positively controls production of arginine precursors. We show that canavanine abrogates RTG signaling especially in petite cells, and mutants in the RTG pathway are extremely sensitive to canavanine. We suggest that petite cells are naturally ineffective in production of some amino acids; combination of this fact with the effect of canavanine on the RTG pathway is the simplest explanation why petite cells are inhibited by canavanine. Surprisingly, we found that canavanine greatly inhibits colony formation when WT cells are forced to respire. Our research proposes a novel connection between respiration and proteotoxic stress.

Identification and expression analysis of the GDSL esterase/lipase family genes, and the characterization of SaGLIP8 in Sedum alfredii Hance under cadmium stress.

BackgroundThe herb Sedum alfredii (S. alfredii) Hance is a hyperaccumulator of heavy metals (cadmium (Cd), zinc (Zn) and lead (Pb)); therefore, it could be a candidate plant for efficient phytoremediation. The GDSL esterase/lipase protein (GELP) family plays important roles in plant defense and growth. Although the GELP family members in a variety of plants have been cloned and analyzed, there are limited studies on the family’s responses to heavy metal-stress conditions.MethodsMultiple sequence alignments and phylogenetic analyses were performed according to the criteria described. A WGCNA was used to construct co-expression regulatory networks. The roots of S. alfredii seedlings were treated with 100 µM CdCl2 for qRT-PCR to analyze expression levels in different tissues. SaGLIP8 was transformed into the Cd sensitive mutant strain yeast Δycf1 to investigate its role in resistance and accumulation to Cd.ResultsWe analyzed GELP family members from genomic data of S. alfredii. A phylogenetic tree divided the 80 identified family members into three clades. The promoters of the 80 genes contained certain elements related to abiotic stress, such as TC-rich repeats (defense and stress responsiveness), heat shock elements (heat stress) and MYB-binding sites (drought-inducibility). In addition, 66 members had tissue-specific expression patterns and significant responses to Cd stress. In total, 13 hub genes were obtained, based on an existing S. alfredii transcriptome database, that control 459 edge genes, which were classified into five classes of functions in a co-expression subnetwork: cell wall and defense function, lipid and esterase, stress and tolerance, transport and transcription factor activity. Among the hub genes, Sa13F.102 (SaGLIP8), with a high expression level in all tissues, could increase Cd tolerance and accumulation in yeast when overexpressed.ConclusionBased on genomic data of S. alfredii, we conducted phylogenetic analyses, as well as conserved domain, motif and expression profiling of the GELP family under Cd-stress conditions. SaGLIP8 could increase Cd tolerance and accumulation in yeast. These results indicated the roles of GELPs in plant responses to heavy metal exposure and provides a theoretical basis for further studies of the SaGELP family’s functions.

Study on the Soil Fertilizing Activities of Salt Tolerant Yeast Isolates

Study on the Soil Fertilizing Activities of Salt Tolerant Yeast Isolates

SCRaMbLE generates evolved yeasts with increased alkali tolerance

BackgroundStrains with increased alkali tolerance have a broad application in industrial, especially for bioremediation, biodegradation, biocontrol and production of bio-based chemicals. A novel synthetic chromosome recombination and modification by LoxP-mediated evolution (SCRaMbLE) system has been introduced in the synthetic yeast genome (Sc 2.0), which enables generation of a yeast library with massive structural variations and potentially drives phenotypic evolution. The structural variations including deletion, inversion and duplication have been detected within synthetic yeast chromosomes.ResultsHaploid yeast strains harboring either one (synV) or two (synV and synX) synthetic chromosomes were subjected to SCRaMbLE. Seven of evolved strains with increased alkali tolerance at pH 8.0 were generated through multiple independent SCRaMbLE experiments. Various of structural variations were detected in evolved yeast strains by PCRTag analysis and whole genome sequencing including two complex structural variations. One possessed an inversion of 20,743 base pairs within which YEL060C (PRB1) was deleted simultaneously, while another contained a duplication region of 9091 base pairs in length with a deletion aside. Moreover, a common deletion region with length of 11,448 base pairs was mapped in four of the alkali-tolerant strains. We further validated that the deletion of YER161C (SPT2) within the deleted region could increase alkali tolerance in Saccharomyces cerevisiae.ConclusionsSCRaMbLE system provides a simple and efficient way to generate evolved yeast strains with enhanced alkali tolerance. Deletion of YER161C (SPT2) mapped by SCRaMbLE can improve alkali tolerance in S. cerevisiae. This study enriches our understanding of alkali tolerance in yeast and provides a standard workflow for the application of SCRaMbLE system to generate various phenotypes that may be interesting for industry and extend understanding of phenotype-genotype relationship.

DEVELOPMENT OF SOME TOLERANT YEAST (SACCHAROMYCES CEREVISIAE) STRAINS TO HEAT AND SALT STRESSES

All living organisms are subjected to changing in conservational conditions, to which they must adapt to. Stress is defined as a threat refers to the physiological balance of systems critical to survival organisms. Five yeast strains (Saccharomyces cerevisiae) were subjected to different adverse environmental situations, such as thermal, osmotic and oxidative (salt) stresses. The objective of this work was to detect the most tolerant yeast strains under salt or heat stresses. Five yeast strains were exposed in a first experiment to heat stress at 20°C or 40°C beside to the control at 30°C to detect the more tolerant strain. The same yeast strains were subjected in a second experiment to two different concentrations of salt stress (NaCl); 0.5 or 1.0 M, separately, for two days (at 30°C as normal temperature for growth), other strains were exposed to 0.5 M concentration of NaCl for 24 hours, then 1 M for another 24 hours. For the heat stress results, strain S4 was more tolerant at 40oCwith insignificant difference compared to the control (30oC), while it showed significant difference at 20°C. Strain S5 also was more tolerant at 20oC with insignificant difference compared to the control. For salt treatment, the only insignificant value was for strain S3at 0.5M NaCl compared with the control.

A repeat region from the Brassica juncea HMA4 gene BjHMA4R is specifically involved in Cd2+ binding in the cytosol under low heavy metal concentrations

BackgroundHMA4 transporters are involved in the transport and binding of divalent heavy metals (Cd, Zn, Pb [lead] and Co [cobalt]). In general, as efflux pumps, HMA4 transporters can increase the heavy metal tolerance of yeast and Escherichia coli. Additional research has shown that the C-terminus of HMA4 contains a heavy metal-binding domain and that heterologous expression of a portion of peptides from this C-terminal domain in yeast provides a high level of Cd tolerance and Cd hyperaccumulation.ResultsWe cloned BjHMA4 from Brassica juncea, and quantitative real-time PCR analysis revealed that BjHMA4 was upregulated by Zn and Cd in the roots, stems and leaves. Overexpression of BjHMA4 dramatically affects Zn/Cd distribution in rice and wheat seedlings. Interestingly, BjHMA4 contains a repeat region named BjHMA4R within the C-terminal region; this repeat region is not far from the last transmembrane domain. We further characterized the detailed function of BjHMA4R via yeast and E. coli experiments. Notably, BjHMA4R greatly and specifically improved Cd tolerance, and BjHMA4R transformants both grew on solid media that contained 500 μM CdCl2 and presented improved Cd accumulation (approximately twice that of wild-type [WT] strains). Additionally, visualization via fluorescence microscopy indicated that BjHMA4R clearly localizes in the cytosol of yeast. Overall, these findings suggest that BjHMA4R specifically improves Cd tolerance and Cd accumulation in yeast by specifically binding Cd2+ in the cytosol under low heavy metal concentrations. Moreover, similar results in E. coli experiments corroborate this postulation.ConclusionBjHMA4R can specifically bind Cd2+ in the cytosol, thereby substantially and specifically improving Cd tolerance and accumulation under low heavy metal concentrations.

Mitochondrial Pyruvate Carriers Prevent Cadmium Toxicity by Sustaining the TCA Cycle and Glutathione Synthesis.

Cadmium (Cd) is a major heavy metal pollutant, and Cd toxicity is a serious cause of abiotic stress in the environment. Plants protect themselves against Cd stress through a variety of pathways. In a recent study, we found that mitochondrial pyruvate carriers (MPCs) are involved in Cd tolerance in Arabidopsis (Arabidopsis thaliana). Following the identification of MPCs in yeast (Saccharomyces cerevisiae) in 2012, most studies have focused on the function of MPCs in animals, as a possible approach to reduce the risk of cancer developing. The results of this study show that AtMPC protein complexes are required for Cd tolerance and prevention of Cd accumulation in Arabidopsis. AtMPC complexes are composed of two elements, AtMPC1 and AtMPC2 (AtNRGA1 or AtMPC3). When the formation of AtMPCs was interrupted by the loss of AtMPC1, glutamate could supplement the synthesis of acetyl-coenzyme A and sustain the TCA cycle. With the up-regulation of glutathione synthesis following exposure to Cd stress, the supplementary pathway could not efficiently drive the tricarboxylic acid cycle without AtMPC. The ATP content decreased concomitantly with the deletion of tricarboxylic acid activity, which led to Cd accumulation in Arabidopsis. More importantly, ScMPCs were also required for Cd tolerance in yeast. Our results suggest that the mechanism of Cd tolerance may be similar in other species.

Microbiological Quality Assessment of Commercially and Laboratory Prepared Orange Juice

Orange juice has become one of the most widely accepted natural beverages due to its invigorating flavours, source of quick energy in the form of natural sugars and its nutrient which support the growth of acid tolerant bacteria, yeasts and moulds. Present investigation was conducted to study the microbiological examination of commercially and freshly prepared orange juices. Freshly prepared orange juices were pasteurised, carbonated, concentrated and chemically treated with a preservative (sodium metabisulphite) and stored under, freezer, refrigeration and ambient temperature for 90 days. The pH ranged from 3.0 to 4.5 for the commercially, fresh and stored samples respectively. The total bacterial load ranged from 1.6x105 to 3.6x106 and the total yeast cell counts ranged from 1.7x104 to 4.8x106 cfu/ml for the commercially, freshly and stored samples. Twenty-two microbial species including 5 bacterial isolates, 6 yeast isolates and 11 isolates of mould were isolated from the orange juices. The bacterial isolates identified from the samples were Bacillus megaterium, Bacillus cereus, Bacillus pantothenticus, Bacillus aeruginosa and Escherichia coli. The fungal isolates were Saccharomyces cerevisiae, Saccharomyces rouxii, Saccharomyces telluris, Blastomyces sp, Aspergillus sp, Mycelia sp, Chrysosporium sp, and Trichoderma sp. Among the bacterial isolates, Bacillus sp was the predominant, while the fungi Saccharomyces sp and Mycelia sp were the predominant fungi and accounted for five out of 11 isolates. The isolates of bacteria, yeasts and moulds appear to be persistent throughout the period of this study and could be used as indicators of microbial quality. A safe microbial load and reduction in contamination of orange juice /fruit juices can be achieved by combination of processing methods such as chemical preservatives with pasteurisation,concentration with carbonation under controlled microbiological environments such as freezer and refrigeration temperatures.

Molecular Identification and Potential Ethanol Production of Long-term Thermo-tolerant Yeast Candida Tropicalis

Candida tropicalis is an organism capable of promoting the fermentation process of starch media supplementation with α-amylase sufficient to produce ethanol. This study aims to identify Candida tropicalis yeast and the potential for ethanol production which is tolerant at high temperatures. Molecular identification methods using Molecular rDNA sequencing and measurement of ethanol production at the incubation rate of yeast growth at 38°C, 39°C and 40°C. The best isolates used were JCBI-23 and JCBI-24 from Indonesia and DMKU-3 thermo tolerant yeast control isolates Kluyveromyces marxianus. The results showed that molecular identification of JCBI-23 isolate was Candida tropicalis culture CBS: 2424 large subunit of ribosome RNA gene with an identification value of 99% and JCBI-24 isolate was Candida tropicalis isolate 4kr27 26S ribosome RNA gene with an identification value of 96%. Based on the growth resistance test, isolates showed that optimum growth occurred during seven-day incubation period and tend to decrease its growth on day 14, both at 37°C, 38°C and 39°C. The best ethanol production at 37°C and not significantly different from the controlled isolates, and showed ethanol production tend to be constant on day 48, both at 37 and 40°C.

Identification and application of novel low pH-inducible promoters for lactic acid production in the tolerant yeast Candida glycerinogenes

Bioproduction of organic acids under low pH condition without adding inducer and neutralizer is an economical craft to decrease downstream cost. Candida glycerinogenes had higher tolerances to low pH and lactic acid than Saccharomyces cerevisiae. QRT-PCR analysis showed that four of fifteen candidates functioned as potentially acid-inducible promoters in C. glycerinogenes. In particular, PCggmt1 showed the strongest induction ability at pH 2.5. Fluorescence analysis indicated that the induction ability of PCggmt1 gradually increased as the pH decreased. In addition, PCggmt1 had the binding sites of Msn2p/4p, Azf1p, and Nrg1p. PCggmt1 was further used to control the expression of lactate dehydrogenase gene from Rhizopus oryzae in C.glycerinogenes. Compared with pH 5.5, the specific activity of lactate dehydrogenase and lactic acid titer at pH 2.5 were increased by 229% and 218%, reaching 13.8mU/mg and 12.3g/L, respectively. These results presented here showed a potential to produce organic acid economically at low pH by the stress tolerant C.glycerinogenes and the novel low-pH inducible promoter PCggmt1.

The maize late embryogenesis abundant protein ZmDHN13 positively regulates copper tolerance in transgenic yeast and tobacco

Late embryogenesis abundant (LEA) proteins accumulate in the late stage of plant seed development, and are upregulated in most plants during drought, cold, heat, or salinity stress. LEA proteins can be classified by amino-acid sequence into seven groups. Dehydrins belong to LEA protein group II. In previous studies, the maize KS type dehydrin ZmDHN13 increased the tolerance of transgenic tobacco to oxidative stress. In the present study, ZmDHN13 was identified under copper stress conditions, and the protein was then characterized using transgenic yeast and tobacco plants to investigate its functions. ZmDHN13 bound Cu2+. Its overexpression in transgenic tobacco conferred tolerance to copper stress by binding metals and reducing the accumulation of reactive oxygen species (ROS). Three conserved domains displayed a cooperative effect under copper stress conditions.