Construction and application of multi-host integrative vector system for xylose-fermenting yeast.

The xylose-fermenting yeasts (CTG clade yeasts, e.g. Scheffersomyces stipitis, Spathaspora passalidarum, Candida amazonensis and Candida jeffriesii) have the potential to be superior platforms for the conversion of lignocellulosic hydrolysate into fuel-grade ethanol and other chemical products. Here, a genetic expression system compatible with the genetic coding characteristics of CTG clade yeasts was constructed for use in xylose-fermenting yeasts. The pRACTH-gfpm plasmid based on an 18S rDNA shuttle vector was capable of stable integration into the genomes of a wide range of heterologous hosts. Green fluorescent protein was transformed and functionally expressed in S. stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and Saccharomyces cerevisiae under control of the SpADH1 promoter and SpCYC1 terminator. Finally, the expression system was useful in multiple yeast hosts for construction of the plasmid pRACTH-ldh. Scheffersomyces stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and S. cerevisiae were enabled to produce lactate from glucose or xylose by pRACTH-based expression of a heterologous lactate dehydrogenase. Among them, C. amazonensis (pRACTH-ldh) exhibited the highest lactate fermentation capacity, which reached a maximum of 44 g L-1 of lactate with a yield of 0.85 g lactate/g xylose.

Metabolic engineering of Escherichia coli W3110 for the production of L-methionine.

In this study, we constructed an L-methionine-producing recombinant strain from wild-type Escherichia coli W3110 by metabolic engineering. To enhance the carbon flux to methionine and derepression met regulon, thrBC, lysA, and metJ were deleted in turn. Methionine biosynthesis obstacles were overcome by overexpression of metA Fbr (Fbr, Feedback resistance), metB, and malY under control of promoter pN25. Recombinant strain growth and methionine production were further improved by attenuation of metK gene expression through replacing native promoter by metK84p. Blocking the threonine pathway by deletion of thrBC or thrC was compared. Deletion of thrC showed faster growth rate and higher methionine production. Finally, metE, metF, and metH were overexpressed to enhance methylation efficiency. Compared with the original strain E. coli W3110, the finally obtained Me05 (pETMAFbr-B-Y/pKKmetH) improved methionine production from 0 to 0.65 and 5.62 g/L in a flask and a 15-L fermenter, respectively.

Omics analysis of acetic acid tolerance in Saccharomyces cerevisiae.

Acetic acid is an inhibitor in industrial processes such as wine making and bioethanol production from cellulosic hydrolysate. It causes energy depletion, inhibition of metabolic enzyme activity, growth arrest and ethanol productivity losses in Saccharomyces cerevisiae. Therefore, understanding the mechanisms of the yeast responses to acetic acid stress is essential for improving acetic acid tolerance and ethanol production. Although 329 genes associated with acetic acid tolerance have been identified in the Saccharomyces genome and included in the database ( http://www.yeastgenome.org/observable/resistance_to_acetic_acid/overview ), the cellular mechanistic responses to acetic acid remain unclear in this organism. Post-genomic approaches such as transcriptomics, proteomics, metabolomics and chemogenomics are being applied to yeast and are providing insight into the mechanisms and interactions of genes, proteins and other components that together determine complex quantitative phenotypic traits such as acetic acid tolerance. This review focuses on these omics approaches in the response to acetic acid in S. cerevisiae. Additionally, several novel strains with improved acetic acid tolerance have been engineered by modifying key genes, and the application of these strains and recently acquired knowledge to industrial processes is also discussed.

Genetic dissection of acetic acid tolerance in Saccharomyces cerevisiae.

Dissection of the hereditary architecture underlying Saccharomyces cerevisiae tolerance to acetic acid is essential for ethanol fermentation. In this work, a genomics approach was used to dissect hereditary variations in acetic acid tolerance between two phenotypically different strains. A total of 160 segregants derived from these two strains were obtained. Phenotypic analysis indicated that the acetic acid tolerance displayed a normal distribution in these segregants, and suggested that the acetic acid tolerant traits were controlled by multiple quantitative trait loci (QTLs). Thus, 220 SSR markers covering the whole genome were used to detect QTLs of acetic acid tolerant traits. As a result, three QTLs were located on chromosomes 9, 12, and 16, respectively, which explained 38.8-65.9 % of the range of phenotypic variation. Furthermore, twelve genes of the candidates fell into the three QTL regions by integrating the QTL analysis with candidates of acetic acid tolerant genes. These results provided a novel avenue to obtain more robust strains.

Metabolic engineering of Escherichia coli for the production of phenylpyruvate derivatives.

Phenylpyruvate derivatives (PPD), such as phenylpropanoids, DL-phenylglycine, dl-phenylalanine, and styrene, are biosynthesized using phenylpyruvate as the precursor. They are widely used in human health and nutrition products. Recently, metabolic engineering provides effective strategies to develop PPD producers. Based on phenylpyruvate-producing chassis, genetically defined PPD producers have been successfully constructed. In this work, the most recent information on genetics and on the molecular mechanisms regulating phenylpyruvate synthesis pathways in Escherichia coli are summarized, and the engineering strategies to construct the PPD producers are also discussed. The enzymes and pathways are proposed for PPD-producer constructions, and potential difficulties in strain construction are also identified and discussed. With respect to recent advances in synthetic biology, future strategies to construct efficiently producers are discussed.

Genetically switched D-lactate production in Escherichia coli.

During a fermentation process, the formation of the desired product during the cell growth phase competes with the biomass for substrates or inhibits cell growth directly, which results in a decrease in production efficiency. A genetic switch is required to precisely separate growth from production and to simplify the fermentation process. The ldhA promoter, which encodes the fermentative D-lactate dehydrogenase (LDH) in the lactate producer Escherichia coli CICIM B0013-070 (ack-pta pps pflB dld poxB adhE frdA), was replaced with the λ p(R) and p(L) promoters (as a genetic switch) using genomic recombination and the thermo-controllable strain B0013-070B (B0013-070, ldhAp::kan-cI(ts)857-p(R)-p(L)), which could produce two-fold higher LDH activity at 42°C than the B0013-070 strain, was created. When the genetic switch was turned off at 33°C, strain B0013-070B produced 10% more biomass aerobically than strain B0013-070 and produced only trace levels of lactate which could reduce the growth inhibition caused by oxygen insufficiency in large scale fermentation. However, 42°C is the most efficient temperature for switching on lactate production. The volumetric productivity of B0013-070B improved by 9% compared to that of strain B0013-070 when it was grown aerobically at 33°C with a short thermo-induction at 42°C and then switched to the production phase at 42°C. In a bioreactor experiment using scaled-up conditions that were optimized in a shake flask experiment, strain B0013-070B produced 122.8 g/l D-lactate with an increased oxygen-limited productivity of 0.89 g/g·h. The results revealed the effectiveness of using a genetic switch to regulate cell growth and the production of a metabolic compound.

Improvement of D-lactate productivity in recombinant Escherichia coli by coupling production with growth.

Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA pta pps pflB dld poxB adhE frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity of lactic acid compared to aerobic and O(2)-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O(2)-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results have revealed an approach for improving production of fermentative products in E. coli.

Fine tuning the transcription of ldhA for D-lactate production.

Fine tuning of the key enzymes to moderate rather than high expression levels could overproduce the desired metabolic products without inhibiting cell growth. The aims of this investigation were to regulate rates of lactate production and cell growth in recombinant Escherichia coli through promoter engineering and to evaluate the transcriptional function of the upstream region of ldhA (encoding fermentative lactate dehydrogenase in E. coli). Twelve ldhA genes with sequentially shortened chromosomal upstream regions were cloned in an ldhA deletion, E. coli CICIM B0013-080C (ack-pta pps pflB dld poxB adhE frdA ldhA). The varied ldhA upstream regions were further analyzed using program NNPP2.2 (Neural Network Promoter Prediction 2.2) to predict the possible promoter regions. Two-phase fermentations (aerobic growth and oxygen-limited production) of these strains showed that shortening the ldhA upstream sequence from 291 to 106 bp successively reduced aerobic lactate synthesis and the inhibition effect on cell growth during the first phase. Simultaneously, oxygen-limited lactate productivity was increased during the second phase. The putative promoter downstream of the -96 site of ldhA could function as a transcriptional promoter or regulator. B0013-080C/pTH-rrnB-ldhA8, with the 72-bp upstream segment of ldhA, could be grown at a high rate and achieve a high oxygen-limited lactate productivity of 1.09 g g(-1) h(-1). No transcriptional promoting region was apparent downstream of the -61 site of ldhA. We identified the latent transcription regions in the ldhA upstream sequence, which will help to understand regulation of ldhA expression.

Minimization of glycerol synthesis in industrial ethanol yeast without influencing its fermentation performance.

To synthesize glycerol, a major by-product during anaerobic production of ethanol, the yeast Saccharomyces cerevisiae would consume up to 4% of the sugar feedstock in typical industrial ethanol processes. The present study was dedicated to decreasing the glycerol production mostly in industrial ethanol producing yeast without affecting its desirable fermentation properties including high osmotic and ethanol tolerance, natural robustness in industrial processes. In the present study, the GPD1 gene, encoding NAD+-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol producing strain of S. cerevisiae, was deleted. Simultaneously, a non-phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus was expressed in the mutant deletion of GPD1. Although the resultant strain AG1A (gpd1△ P(PGK)-gapN) exhibited a 48.7±0.3% (relative to the amount of substrate consumed) lower glycerol yield and a 7.6±0.1% (relative to the amount of substrate consumed) higher ethanol yield compared to the wild-type strain, it was sensitive to osmotic stress and failed to ferment on 25% glucose. However, when trehalose synthesis genes TPS1 and TPS2 were over-expressed in the above recombinant strain AG1A, its high osmotic stress tolerance was not only restored but also improved. In addition, this new recombinant yeast strain displayed further reduced glycerol yield, indistinguishable maximum specific growth rate (µ(max)) and fermentation ability compared to the wild type in anaerobic batch fermentations. This study provides a promising strategy to improve ethanol yields by minimization of glycerol production.

Evaluation of genetic manipulation strategies on D-lactate production by Escherichia coli.

In order to rationally manipulate the cellular metabolism of Escherichia coli for D: -lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding D-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA-pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 (ackA-pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 (ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l D-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of D-lactate production are among the highest reported and the results have revealed which genetic manipulations improved D-lactate production by E. coli.

Improving the ethanol yield by reducing glycerol formation using cofactor regulation in Saccharomyces cerevisiae.

To increase ethanol yield and decrease glycerol production in Saccharomyces cerevisiae, the strategies of direct cofactor-regulation were explored. During anaerobic batch fermentations, the yeast expressing Bacillus cereus gapN gene, encoding non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrognease, produced 73.8 g ethanol l(-1), corresponding to 96% of theoretical maximum yield compared to 92% for the wild type. The yeast expressing Escherichia coli frdA gene encoding the NAD(+)-dependent fumarate reductase, exhibited a 22% (relative to the amount of substrate consumed) increase in glycerol yield in medium containing 2 g fumarate l(-1). The yeast expressing mhpF gene, encoding acetylating NAD(+)-dependent acetaldehyde dehydrogenase, produced 74.5 g ethanol l(-1), corresponding to 97.4% of theoretical maximum yield while glycerol decreased by 40% when acetic acid was added before inoculation. This strain represents a promising alternative for ethanol production with lignocellulosic hydrolysates where acetate is available at significant amounts.

Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast.

The GPD2 gene, encoding NAD(+)-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P (PGK)-gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12% (relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B (gpd2Δ P (PGK)-GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15% (relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly, the maximum specific growth rates (µ (max)) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that the redox imbalance of the mutant could be partially solved by expressing the heterologous genes.

Improving the performance of industrial ethanol-producing yeast by expressing the aspartyl protease on the cell surface.

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell-surface anchored expressed in industrial ethanol-producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild-type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98-99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild-type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity.

Role of the calcium-binding residues Asp231, Asp233, and Asp438 in alpha-amylase of Bacillus amyloliquefaciens as revealed by mutational analysis.

Role of the calcium-binding residues Asp231, Asp233, and Asp438 of Bacillus amyloliquefaciens alpha-amylase (BAA) on the enzyme properties was investigated by site-directed mutagenesis. The calcium-binding residues Asp231, Asp233, and Asp438 were replaced with Asn, Asn, and Gly to produce the mutants D231N, D233N, and D438G, respectively. The mutant amylases were purified to homogeneity and the purified enzymes was estimated to be approximately 58 kDa. The specific activity for the mutant enzyme D233N was decreased by 84.8%, while D231N and D438G showed a decrease of 6.3% and 3.5% to that of the wild-type enzyme, respectively. No significant changes in the K (m) value, thermo-stability, optimum temperature, and optimum pH were observed in the mutations of D231N and D438G, while substitution of Asp233 with Asn resulted in a dramatic reduction in the value of catalytic efficiency (K (cat)/K (m)) and thermo-stability at 60 degrees C. The ranges of optimum temperature and optimum pH for D233N were also reduced to about 10 degrees C and 3-4 units, respectively.

A newly isolated Rhizopus microsporus var. chinensis capable to secrete amyloltic enzymes with raw-starch-digesting activity.

A newly isolated active producer of raw starch digesting amyloltic enzymes, Rhizopus microsporus var. chinensis CICIM-CU F0088 was screened and identified by morphological characteristics and molecular phylogenetic analysis. This fungus was isolated from the soil of Chinese glue pudding mill, and produced high levels of amylolytic activity under solid state fermentation with supplementation of starch and wheat bran. Results of thin layer chromatography showed there are two kinds of amyloltic enzymes formed by this strain, including one alpha-amylase and two glucoamylases. It was found that the two glucoamylases can digest raw corn starch in the electron microscope experiments, and has an optimal temperature of 70 degrees Celsius. These results signified that amyloltic enzymes secreted by strain of Rhizopus microsporus var. chinensis CICIM-CU F0088 were kinds of thermostable amyloltic enzymes and able to digest raw corn starch.

High yield recombinant thermostable alpha-amylase production using an improved Bacillus licheniformis system.

BACKGROUND: Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable alpha-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable. RESULTS: A new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for Bacillus licheniformis alpha-amylase (BLA) production was constructed. The combination of target-directed screening and genetic recombination led to an approximately 26-fold improvement of BLA production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained. CONCLUSION: This production level of BLA by B. licheniformis CBBD302(pHY-amyL) is amongst the highest levels in Gram-positive bacteria reported so far.

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production.

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD(+)-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Delta and gpd2Delta were studied under alcoholic fermentation conditions. gpd1Delta mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2Delta mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1Delta) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1Delta) but a slight decrease in the strain ANG2 (gpd2Delta). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1Delta) and ANG2 (gpd2Delta) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.

A novel constructed SPT15 mutagenesis library of Saccharomyces cerevisiae by using gTME technique for enhanced ethanol production.

During the last few years, the global transcription machinery engineering (gTME) technique has gained more attention as an effective approach for the construction of novel mutants. Genetic strategies (molecular biology methods) were utilized to get mutational for both genes (SPT15 and TAF23) basically existed in the Saccharomyces cerevisiae genome via screening the gTME approach in order to obtain a new mutant S. cerevisiae diploid strain. The vector pYX212 was utilized to transform these genes into the control diploid strain S. cerevisiae through the process of mating between haploids control strains S. cerevisiae (MAT-a [CICC 1374]) and (MAT-α [CICC 31144]), by using the oligonucleotide primers SPT15-EcoRI-FW/SPT15-SalI-RV and TAF23-SalI-FW/TAF23-NheI-RV, respectively. The resultant mutants were examined for a series of stability tests. This study showed how strong the effect of using strategic gTME with the importance of the modification we conducted in Error Prone PCR protocol by increasing MnCl2 concentration instead of MgCl2. More than ninety mutants we obtained in this study were distinguished by a high level production of bio-ethanol as compared to the diploid control strain.

A new agent developed by biotransformation of polyphyllin VII inhibits chemoresistance in breast cancer.

Biotransformation by the endophytes of certain plants changes various compounds, and this 'green' chemistry becomes increasingly important for finding new products with pharmacological activity. In this study, polyphyllin VII (PPL7) was biotransformed by endophytes from the medicinal plant Paris polyphylla Smith, var. yunnanensis. This produced a new compound, ZH-2, with pharmacological activity in vitro and in vivo. ZH-2 was more potent than PPL7 in selectively killing more chemoresistant than chemosensitive breast cancer cells. ZH-2 also re-sensitized chemoresistant breast cancer cells, as evidenced by the improved anti-cancer activity of commonly-used chemotherapeutic agent in vitro, in vivo, and in clinical samples. This anti-chemoresistance effect of ZH-2 was associated with inhibiting the epithelial-mesenchymal transition (EMT) pathway. Taken together, our findings are the first one to link biotransformation with a biomedicine. The results provide insights into developing new pharmacologically-active agents via biotransformation by endophytes.

Sequence-based screening and characterization of cytosolic mandelate oxidase using oxygen as electron acceptor.

Sequence-based screening was carried out to find a type of cytosolic mandelate oxidase that converted l-mandelate to phenylglyoxylate using oxygen as the final electron acceptor. The sequence features of the cytosolic mandelate oxidase were summarized, and were used in the screening process. Mandelate oxidases from Streptomyces coelicolor (HmoSC) and Amycolatopsis orientalis (HmoAO) were screened and then they were heterologously expressed and characterized. At pH 7.3 40 °C, the HmoAO showed kcat and Km values of 140 min(-1) and 10.2 mM, the HmoSC showed kcat and Km values of 105.1 min(-1) and 2.06 mM. The HmoSC was thermal stable and retained its 90% activity at 60 °C for up to 5 h, while HmoAO lost most of its activity at this temperature. The HmoSC could effectively catalyze the conversion of l-mandelate to phenylglyoxylate at higher temperature using oxygen as the final electron acceptor.