Comparative analysis of the transcriptional responses of Acetilactobacillus jinshanensis BJ01 to organic acids.

As the important functional microorganism in the brewing process of Chinese Baijiu, lactic acid bacteria influences the microbial community and production of flavor substances in the Baijiu brewing process. In this study, we first isolated an Acetilactobacillus jinshanensis strain from baijiu fermented grains and named it A. jinshanensis BJ01. Its optimal growth conditions are 30 °C and pH 3.5. In particular, A. jinshanensis BJ01 cannot utilize inorganic acids and most organic acids, except for lactic acid (HL) and acetic acid (HAc). The observed phenotypes showed good growth with HL. When the mixed acid of HL-HAc (V:V = 1:1) was used, the growth rate of A. jinshanensis BJ01 greatly accelerated. Transcriptomic sequencing revealed the specific responses of the strain to the acidulants used. The number of upregulated genes in HL-HAc medium was more than that in single acid medium (HL or HAc). KEGG enrichment analyses indicated that the glycometabolism level of HAc regulation was relatively downregulated. The gene expression of quorum sensing and ABC transporter pathways were remarkably upregulated under HL-HAc regulation. Pyruvate metabolic pathway may be an important reason for the difference in A. jinshanensis BJ01 response to different organic acids. Our study reported a new organic acid-inducible growth type of bacteria mainly depending on the presence of HL and HAc, and was beneficial to the improvement of fermentation technology of Baijiu.

Enhancing the secretion of a feruloyl esterase in Bacillus subtilis by signal peptide screening and rational design.

Feruloyl esterase is a subclass of α/ß hydrolase, which could release ferulic acid from biomass residues for use as an efficient additive in food or pharmaceutical industries. In the present study, a feruloyl esterase with broad substrate specificity was characterised and secreted by Bacillus subtilis WB600. After codon usage optimisation and signal peptide library screening, the secretion amount of feruloyl esterase was enhanced by up to 10.2-fold in comparison with the base strain. The site-specific amino acid substitutions that facilitate protein folding further improved the secretion by about 1.5-fold. The purified rationally designed enzyme exhibited maximal activity against methyl ferulate at pH 6.5 and 65 °C. In the solid-state fermentation, the genetically engineered B. subtilis released about 37% of the total alkali-extractable ferulic acid in maize bran. This study provides a promising candidate for ferulic acid production and demonstrates that the secretion of a heterologous enzyme from B. subtilis can be cumulatively improved by changes in protein sequence features.

Comparative transcriptome analysis of root, stem, and leaf tissues of Entada phaseoloides reveals potential genes involved in triterpenoid saponin biosynthesis.

BACKGROUND: Entada phaseoloides (L.) Merr. is an important traditional medicinal plant. The stem of Entada phaseoloides is popularly used as traditional medicine because of its significance in dispelling wind and dampness and remarkable anti-inflammatory activities. Triterpenoid saponins are the major bioactive compounds of Entada phaseoloides. However, genomic or transcriptomic technologies have not been used to study the triterpenoid saponin biosynthetic pathway in this plant. RESULTS: We performed comparative transcriptome analysis of the root, stem, and leaf tissues of Entada phaseoloides with three independent biological replicates and obtained a total of 53.26 Gb clean data and 116,910 unigenes, with an average N50 length of 1218 bp. Putative functions could be annotated to 42,191 unigenes (36.1%) based on BLASTx searches against the Non-redundant, Uniprot, KEGG, Pfam, GO, KEGG and COG databases. Most of the unigenes related to triterpenoid saponin backbone biosynthesis were specifically upregulated in the stem. A total of 26 cytochrome P450 and 17 uridine diphosphate glycosyltransferase candidate genes related to triterpenoid saponin biosynthesis were identified. The differential expressions of selected genes were further verified by qPT-PCR. CONCLUSIONS: The dataset reported here will facilitate the research about the functional genomics of triterpenoid saponin biosynthesis and genetic engineering of Entada phaseoloides.

Isolation of axenic cyanobacterium and the promoting effect of associated bacterium on axenic cyanobacterium.

BACKGROUND: Harmful cyanobacterial blooms have attracted wide attention all over the world as they cause water quality deterioration and ecosystem health issues. Microcystis aeruginosa associated with a large number of bacteria is one of the most common and widespread bloom-forming cyanobacteria that secret toxins. These associated bacteria are considered to benefit from organic substrates released by the cyanobacterium. In order to avoid the influence of associated heterotrophic bacteria on the target cyanobacteria for physiological and molecular studies, it is urgent to obtain an axenic M. aeruginosa culture and further investigate the specific interaction between the heterotroph and the cyanobacterium. RESULTS: A traditional and reliable method based on solid-liquid alternate cultivation was carried out to purify the xenic cyanobacterium M. aeruginosa FACHB-905. On the basis of 16S rDNA gene sequences, two associated bacteria named strain B905-1 and strain B905-2, were identified as Pannonibacter sp. and Chryseobacterium sp. with a 99 and 97% similarity value, respectively. The axenic M. aeruginosa FACHB-905A (Microcystis 905A) was not able to form colonies on BG11 agar medium without the addition of strain B905-1, while it grew well in BG11 liquid medium. Although the presence of B905-1 was not indispensable for the growth of Microcystis 905A, B905-1 had a positive effect on promoting the growth of Microcystis 905A. CONCLUSIONS: The associated bacteria were eliminated by solid-liquid alternate cultivation method and the axenic Microcystis 905A was successfully purified. The associated bacterium B905-1 has the potentiality to promote the growth of Microcystis 905A. Moreover, the purification technique for cyanobacteria described in this study is potentially applicable to a wider range of unicellular cyanobacteria.

Community composition and cellulase activity of cellulolytic bacteria from forest soils planted with broad-leaved deciduous and evergreen trees.

Cellulolytic bacteria in forest soil provide carbon sources to improve the soil fertility and sustain the nutrient balance of the forest ecological system through the decomposition of cellulosic remains. These bacteria can also be utilized for the biological conversion of biomass into renewable biofuels. In this study, the community compositions and activities of cellulolytic bacteria in the soils of forests planted with broad-leaved deciduous (Chang Qing Garden, CQG) and broad-leaved evergreen (Forest Park, FP) trees in Wuhan, China were resolved through restriction fragment length polymorphism (RFLP) and sequencing analysis of the 16S rRNA gene. All of the isolates exhibited 35 RFLP fingerprint patterns and were clustered into six groups at a similarity level of 50 %. The phylogeny analysis based on the 16S rRNA gene sequence revealed that these RFLP groups could be clustered into three phylogenetic groups and further divided into six subgroups at a higher resolution. Group I consists of isolates from Bacillus cereus, Bacillus subtilis complex (I-A) and from Paenibacillus amylolyticus-related complex (I-B) and exhibited the highest cellulase activity among all of the cellulolytic bacteria isolates. Cluster II consists of isolates belonging to Microbacterium testaceum (II-A), Chryseobacterium indoltheticum (II-B), and Flavobacterium pectinovorum and the related complex (II-C). Cluster III consists of isolates belonging to Pseudomonas putida-related species. The community shift with respect to the plant species and the soil properties was evidenced by the phylogenetic composition of the communities. Groups I-A and I-B, which account for 36.0 % of the cellulolytic communities in the CQG site, are the dominant groups (88.4 %) in the FP site. Alternatively, the ratio of the bacteria belonging to group III (P. putida-related isolates) shifted from 28.0 % in CQG to 4.0 % in FP. The soil nutrient analysis revealed that the CQG site planted with deciduous broad-leaved trees has a richer organic nutrient (total organic carbon and total nitrogen) than the FP site planted with evergreen broad-leaved trees. Against this background, the population density and the diversity of cellulolytic bacteria in the CQG site are clearly higher than those in the FP site, and the latter was dominated with high-cellulase-activity Bacillus- and Paenibacillus-related bacteria. The canonical correspondence analysis further indicated that the distribution of these groups is correlated with the FP site, whereas groups II and III are correlated with the organic nutrient-rich CQG site.

Community composition of nirS-type denitrifier in a shallow eutrophic lake.

Denitrification is a major biological process to reduce nitrate to molecular nitrogen (N2). In shallow eutrophic lakes, this process can remove the largest portion of fixed nitrogen and plays an important role in self-purification of this ecosystem. To understand the structure of denitrifying communities in a shallow eutrophic lake, denitrifier communities in four sub-lakes of East Lake in Wuhan, China, were explored by restriction fragment length polymorphisms (RFLP) analysis and sequencing of nirS gene clone libraries. nirS is a functional marker gene for denitrification encoding cytochrome cd 1-containing nitrite reductase, which catalyzes the reduction of nitrite to nitric oxide. Both RFLP fingerprints clustering analysis and phylogeny analysis based on the amino acid sequences of NirS revealed that NirS-type communities in East Lake sediment could be roughly divided into three clusters. Cluster I accounted for 74-82 % of clones from the moderately eutrophic sub-lakes Tuan, Tang Ling, and Guo Zheng. Cluster II accounted for 76 % of the communities in hypertrophic sub-lake Miao Lake and cluster III as a minor group (7 % of the total), mainly presented in Miao Lake. Phylogenetic analysis revealed that cluster I was related to the reference clones from a broad range of ecological environments, and clusters II and III were more phylogenetically related to the reference clones from entrophic environments. Canonical correspondence analysis indicated that total nitrogen, total phosphate, total organic carbon, and NH4-N and NO2-N were important environmental factors affecting the dispersion of NirS-type denitrifier in the sediments. Cluster I showed a weak relationship with the nutrient content, while cluster II and III were positively related with the nutrient content. Principal coordinates analysis indicated that NirS-type communities from Tuan Lake, Tang Ling Lake, and Guo Zheng Lake sediments were divergent from those found in river, estuary sediment, and forest soil but similar to communities in constructed wetland sediment despite large geographic distances. The communities from the hypertrophic sub-lake Miao Lake deviated from other sub-lakes and the reference communities and clustered independently. Our results support the argument that environmental factors regulate the composition and distribution of the functional bacterial groups.

Recent Advances in the Research on the Anticyanobacterial Effects and Biodegradation Mechanisms of Microcystis aeruginosa with Microorganisms.

Harmful algal blooms (HABs) have attracted great attention around the world due to the numerous negative effects such as algal organic matters and cyanobacterial toxins in drinking water treatments. As an economic and environmentally friendly technology, microorganisms have been widely used for pollution control and remediation, especially in the inhibition/biodegradation of the toxic cyanobacterium Microcystis aeruginosa in eutrophic water; moreover, some certain anticyanobacterial microorganisms can degrade microcystins at the same time. Therefore, this review aims to provide information regarding the current status of M. aeruginosa inhibition/biodegradation microorganisms and the acute toxicities of anticyanobacterial substances secreted by microorganisms. Based on the available literature, the anticyanobacterial modes and mechanisms, as well as the in situ application of anticyanobacterial microorganisms are elucidated in this review. This review aims to enhance understanding the anticyanobacterial microorganisms and provides a rational approach towards the future applications.

An acid and highly thermostable xylanase from Phialophora sp. G5.

An endo-ß-1,4-xylanase gene, designated xyn10G5, was cloned from Phialophora sp. G5 and expressed in Pichia pastoris. The 1,197-bp full-length gene encodes a polypeptide of 399 amino acids consisting of a putative signal peptide at residues 1-20, a family 10 glycoside hydrolase domain, a short Gly/Thr-rich linker and a family 1 carbohydrate-binding module (CBM). The deduced amino acid sequence of XYN10G5 shares the highest identity (53.4%) with a putative xylanase precursor from Aspergillus terreus NIH2624. The purified recombinant XYN10G5 exhibited the optimal activity at pH 4.0 and 70 °C, remained stable at pH 3.0-9.0 (>70% of the maximal activity), and was highly thermostable at 70 °C (retaining ~90% of the initial activity for 1 h). Substrate specificity studies have shown that XYN10G5 had the highest activity on soluble wheat arabinoxylan (350.6 U mg(-1)), and moderate activity to various heteroxylans, and low activity on different types of cellulosic substrates. Under simulated gastric conditions, XYN10G5 was stable and released more reducing sugars from soluble wheat arabinoxylan; when combined with a glucanase (CelA4), the viscosity of barley-soybean feed was significantly reduced. These favorable enzymatic properties make XYN10G5 a good candidate for application in the animal feed industry.

Multiple Batches of Fermentation Promote the Formation of Functional Microbiota in Chinese Miscellaneous-Flavor Baijiu Fermentation.

Baiyunbian baijiu, the most popular miscellaneous-flavor baijiu in China, has been widely consumed for decades. Similar to many Chinese baijiu, Baiyunbian baijiu is fermented in five successive batches every year. Sensory analysis demonstrated that the raw baijiu obtained from the last two fermentation batches always has better quality than that produced from the former three batches. In this study, the microbial compositions of fungi and bacteria in each fermentation batch were investigated via high-throughput sequencing. The results showed that Bacillus, Virgibacillus, and Lactobacillus dominated the bacterial community in the last two batches, and the most prevalent fungi were Paecilomyces, Saccharomyces, and Zygosaccharomyces. In contrast, large percentages of fungi belonging to Thermomyces, Thermoascus, Monascus, and Issatchenkia and prokaryotes belonging to Acetobacter, Lactobacillus, and Thermoactinomyces were observed in the former three fermentation batches. GC-MS analysis revealed that the fermented grains sampled from the latter two batches contained high concentrations of ethyl lactate, 2,3-butanediol and ethyl caproate, which were mainly generated by co-fermentation of Lactobacillus and yeast. The high acidity of the fermented grains in the fourth and fifth fermentation batches as well as the large contents of ethanol and moisture promoted the formation of the functional microbial community. This study provides insight into factors that influenced the baijiu fermentation and is helpful for developing new fermentation techniques with higher baijiu quality.

LncRNA PPP1R14B-AS1 Promotes Tumor Cell Proliferation and Migration via the Enhancement of Mitochondrial Respiration.

PPP1R14B-AS1 is an antisense long non-coding RNA with unknown functions. Herein, gene differential analyses were performed using the data of patients with liver cancer and lung adenocarcinoma (LUAD) from The Cancer Genome Atlas database. PPP1R14B-AS1 was found to be upregulated and also overexpressed in 10 other types of cancers. In addition, PPP1R14B-AS1 overexpression was associated with poor overall prognosis in eight cancers. Furthermore, PPPAR14B-AS1 upregulation was positively associated with worsening development of liver and LUAD cancers and related to poor disease-free survival. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses suggested that PPP1R14B-AS1 strongly participated in regulating cell aerobic respiration processes, such as mitochondrial electron respiration chain and NADH dehydrogenation processes. Cell cytoplasmic and nuclear RNA purification assessment results revealed that PPP1R14B-AS existed in the cell nucleus and cytoplasm. The knockdown of PPP1R14B-AS1 in HepG2 and A549 cells using PPP1R14B-AS1-specific siRNAs decreased mitochondrial respiration as demonstrated by the reduction in basal respiration and ATP production. Moreover, PPP1R14B-AS1 downregulation did not obviously affect cell glycolysis ability. Finally, PPP1R14B-AS1 inhibition inhibited HepG2 and A549 cell migration and proliferation. In summary, our study found for the first time that PPP1R14B-AS1 could be a potential biomarker for cancer diagnosis and that PPP1R14B-AS1 inhibition could be a potentially effective therapy.

[Sub-cellular localization and overexpressing analysis of hydroxylase gene TcCYP725A22 of Taxus chinensis].

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.

Complete Genome of the Chitin-Degrading Bacterium, Paenibacillus xylanilyticus W4.

Chitinases possess an extraordinary ability to directly hydrolyze highly insoluble chitin polymers to low-molecular-weight chito-oligomers, which possess particular biological functions, such as elicitor action and antitumor activity. A novel strain, Paenibacillus xylanilyticus W4, which was isolated from soil, showed strong chitin degradation activity. Here, we first reported the complete genome information of P. xylanilyticus. Paenibacillus xylanilyticus W4 contains a 5,532,141 bp single circular chromosome with 47.33% GC content. The genome contains 5,996 genes, including 39 rRNA- and 109 tRNA-coding genes. Phylogenetic analysis and Genome-to-Genome Distance revealed its taxonomic characterization into a separate family. Six glycoside hydrolase 18 (GH18) and 2 GH23 enzymes involved in chitin degradation. Although many of the chitinases were conserved in Paenibacillus, several GH18 chitinases share high similarity with Bacillus circulans. The genome information provided here could benefit for understanding the chitin-degrading properties of P. xylanilyticus as well as its potential application in biotechnological and pharmaceutical fields.

Comparative Transcriptome Analysis of Pseudomonas putida KT2440 Revealed Its Response Mechanisms to Elevated Levels of Zinc Stress.

The whole-genome transcriptional response of Pseudomonas putida KT2440 to stress-inducing concentrations of zinc was analyzed in this study by RNA sequencing to thoroughly investigate the bacterial cell response to zinc toxicity. The data revealed that different levels of zinc stress strongly affected the transcription of genes from the following categories: metal transport genes, genes involved in membrane homeostasis, oxidative-stress-responding genes, and genes associated with basic cellular metabolism. At the lowest zinc dose, only several genes associated with metal transport and membrane homeostasis were strongly influenced. At the intermediate zinc dose, transcriptional changes of genes belonging to these two categories were highly pronounced. In addition, the intermediate zinc stress produced high levels of oxidative stress, and influenced amino acid metabolism and respiratory chains of P. putida. At the highest zinc dose, the induction of genes responsible for Fe-S cluster biogenesis was the most remarkable feature. Moreover, upregulation of glyoxylate cycle was observed. In summary, the adaptation of the cell envelope, the maintenance of metal homeostasis and intracellular redox status, and the transcriptional control of metabolism are the main elements of stress response, which facilitates the survival of P. putida KT2440 in zinc-polluted environments.

Characterization of Thermotolerant Chitinases Encoded by a Brevibacillus laterosporus Strain Isolated from a Suburban Wetland.

To isolate and characterize chitinases that can be applied with practical advantages, 57 isolates of chitin-degrading bacteria were isolated from the soil of a suburban wetland. 16S rRNA gene analysis revealed that the majority of these strains belonged to two genera, Paenibacillus and Brevibacillus. Taking thermostability into account, the chitinases (ChiA and ChiC) of a B. laterosporus strain were studied further. Ni-NTA affinity-purified ChiA and ChiC were optimally active at pH 7.0 and 6.0, respectively, and showed high temperature stability up to 55 °C. Kinetic analysis revealed that ChiC has a lower affinity and stronger catalytic activity toward colloidal chitin than ChiA. With their stability in a broad temperature range, ChiA and ChiC can be utilized for the industrial bioconversion of chitin wastes into biologically active products.

Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris.

Lipase (EC 3.1.1.3) stands amongst the most important and promising biocatalysts for industrial applications. In this study, in order to realize a high-level expression of the Yarrowia lipolytica lipase gene in Pichia pastoris, we optimized the codon of LIP2 by de novo gene design and synthesis, which significantly improved the lipase expression when compared to the native lip2 gene. We also comparatively analyzed the effects of the promoter types (PAOX1 and PFLD1) and the Pichia expression systems, including the newly developed PichiaPink system, on lipase production and obtained the optimal recombinants. Bench-top scale fermentation studies indicated that the recombinant carrying the codon-optimized lipase gene syn-lip under the control of promoter PAOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262g/L, 38,500U/mL and 2.82g/L, respectively. This study has not only greatly facilitated the bioapplication of lipase in industrial fields but the strategies utilized, such as de novo gene design and synthesis, the comparative analysis among promoters and different generations of Pichia expression systems will also be useful as references for future work in this field.

Medium optimization for the production of anti-cyanobacterial substances by Streptomyces sp. HJC-D1 using response surface methodology.

Bioremediation using isolated anti-cyanobacterial microorganism has been widely applied in harmful algal blooms (HABs) control. In order to improve the secretion of activated anti-cyanobacterial substances, and lower the cost, a sequential optimization of the culture medium based on statistical design was employed for enhancing the anti-cyanobacterial substances production and chlorophyll a (Chl a) removal by Streptomyces sp. HJC-D1 in the paper. Sucrose and KNO3 were selected as the most suitable carbon and nitrogen sources based on the one-at-a-time strategy method, and sucrose, KNO3 and initial pH were found as major factors that affected the anti-cyanobacterial ability of the isolated stain via the Plackett-Burman design. Based on the response surface and canonical analysis, the optimum condition of culture medium was obtained at 22.73 g l(-1) of sucrose, 0.96 g l(-1) of KNO3, and initial pH 8.82, and the Chl a removal efficiency by strain HJC-D1 increased from 63 ± 2 % to 78 ± 2 % on the optimum conditions.

Control of the harmful alga Microcystis aeruginosa and absorption of nitrogen and phosphorus by Candida utilis.

This study is aimed at controlling eutrophication through converting the nutrients such as nitrogen and phosphorus into microbial protein and simultaneously inhibiting the growth of Microcystis aeruginosa by Candida utilis. C. utilis and M. aeruginosa (initial cell density was 2.25 × 10(7) and 4.15 × 10(7) cells·mL(-1)) were cultured together in the absence or presence of a carbon source (glucose) during a 10-day experiment. In the absence of carbon source, the measured removal efficiencies of NH(4) (+)-N and PO(4) (3-)-P were 41.39 ± 2.19 % and 82.93 ± 3.95 %, respectively, at the second day, with the removal efficiency of 67.82 ± 2.29 % for M. aeruginosa at the fourth day. In contrast, the removal efficiencies of NH(4) (+)-N and PO(4) (3-)-P were increased to 87.45 ± 4.25 % and 83.73 ± 3.55 %, respectively, while the removal efficiency of M. aeruginosa decreased to 37.89 ± 8.41 % in the presence of the carbon source (C/N = 2:1). These results showed that the growth of M. aeruginosa was inhibited by C. utilis. Our finding sheds light on a novel potential approach for yeast to consume nutrients and control harmful algal during bloom events.

Physiological responses of Microcystis aeruginosa under the stress of antialgal actinomycetes.

Eutrophication has occurred frequently in various lakes and reservoirs, and the metabolic excretion produced during the algae growth causes serious water pollution and threatens ecological security. Biological control approaches such as screening bacteria with the capability to degrade cyanobacteria are an environment-friendly way. An isolated antialgal strain Streptomyces sp. KY-34, was applied to degrade the cyanobacterium Microcystis aeruginosa, and the possible biodegradation mechanism was investigated. The results showed that the fermentation liquor of Streptomyces sp. KY-34 could inhibit the growth of M. aeruginosa by restrained the synthesis of chlorophyll and photosynthetic pigments, and decreasing the contents of cellular protein and non-protein, accordingly led to the increase of malondialdehyde content, and the activities of superoxide dismutase, catalase and peroxidase in algae cells. In addition, the variation of the cellular ultrastructure indicated a serious change in algal physiology. It's revealed that the biodegradation mechanism of M. aeruginosa should primarily be that Streptomyces sp. KY-34 caused the damage of algae cell membrane and led to the increases of antioxidant enzymes, and then the growth of M. aeruginosawas inhibited.

A simple and accurate two-step long DNA sequences synthesis strategy to improve heterologous gene expression in pichia.

In vitro gene chemical synthesis is a powerful tool to improve the expression of gene in heterologous system. In this study, a two-step gene synthesis strategy that combines an assembly PCR and an overlap extension PCR (AOE) was developed. In this strategy, the chemically synthesized oligonucleotides were assembled into several 200-500 bp fragments with 20-25 bp overlap at each end by assembly PCR, and then an overlap extension PCR was conducted to assemble all these fragments into a full length DNA sequence. Using this method, we de novo designed and optimized the codon of Rhizopus oryzae lipase gene ROL (810 bp) and Aspergillus niger phytase gene phyA (1404 bp). Compared with the original ROL gene and phyA gene, the codon-optimized genes expressed at a significantly higher level in yeasts after methanol induction. We believe this AOE method to be of special interest as it is simple, accurate and has no limitation with respect to the size of the gene to be synthesized. Combined with de novo design, this method allows the rapid synthesis of a gene optimized for expression in the system of choice and production of sufficient biological material for molecular characterization and biotechnological application.

Nesterenkonia alba sp. nov., an alkaliphilic actinobacterium isolated from the black liquor treatment system of a cotton pulp mill.

An alkaliphilic actinobacterium, designated strain CAAS 252(T), was isolated from the black liquor treatment system of a cotton pulp mill in Wuhan, China. Cells of strain CAAS 252(T) were Gram-positive, non-motile, non-endospore-forming, short rod-shaped, and grew optimally at 42 degrees C and pH 9-10 in the presence of 3 % (w/v) NaCl. Strain CAAS 252(T) contained MK-7, MK-8 and MK-9 as the major menaquinones and anteiso-C(17 : 0), anteiso-C(15 : 0) and C(16 : 0) as the predominant cellular fatty acids and had a peptidoglycan type of A4alpha, Lys-Gly-d-Asp. The DNA G+C content was 60.2 mol%. Based on analysis of 16S rRNA gene sequences (94.7-96.8 % similarity), DNA-DNA hybridization (<70 % relatedness) and chemotaxonomic characteristics, strain CAAS 252(T) belonged to the genus Nesterenkonia, but differed from all recognized species. Therefore, it is proposed that strain CAAS 252(T) represents a novel species of the genus Nesterenkonia, for which the name Nesterenkonia alba sp. nov. is proposed. The type strain is CAAS 252(T) (=CCTCC AB 207011(T)=DSM 19423(T)).