Development of a Lipase Fermentation Process That Uses a Recombinant Pseudomonas alcaligenes Strain

Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions. A fed-batch fermentation process based on the secretion of the alkaline lipase from P. alcaligenes was developed. Due to the inability of P. alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from P. alcaligenes was isolated and characterized. Amplification of lipase gene copies in P. alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number. It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (>20) of the lipase structural gene, lipA. In strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only. During scaling up of the fermentation process to 100 m³, severe losses in lipase productivity were observed. Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses. A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.     

Gene cloning and molecular characterization of the Talaromyces thermophilus lipase catalyzed efficient hydrolysis and synthesis of esters

A genomic bank from Talaromyces thermophilus fungus was constructed and screened using a previously isolated fragment lipase gene as probe. From several clones isolated, the nucleotide sequence of the lipase gene (TTL gene) was completed and sequenced. The TTL coding gene consists of an open reading frame (ORF) of 1083 bp encoding a protein of 269 Aa with an estimated molecular mass of 30 kDa. The TTL belongs to the same gene family as Thermomyces lanuginosus lipase (TLL, Lipolase®), a well known lipase with multiple applications. The promoter sequence of the TTL gene showed the conservation of known consensus sequences PacC, CreA, Hap2-3-4 and the existence of a particular sequence like the binding sites of Oleate Response Element (ORE) and Fatty acids Responsis Element (FARE) which are similar to that already found to be specific of lipolytic genes in Candida and Fusarium, respectively. Northern blot analysis showed that the TTL expression was much higher on wheat bran than on olive oil as sole carbon source. Compared to the Lipolase®, this enzyme was found to be more efficient for the hydrolysis and the synthesis of esters; and its synthetic efficiency even reached 91.6% from Waste Cooking Oil triglycerides.     

Use of the glyceraldehyde-3-phosphate dehydrogenase promoter from a thermotolerant yeast,Pichia thermomethanolica,for heterologous gene expression,especially at elevated temperature

The glyceraldehyde-3-phosphate dehydrogenase (GAP) gene from the thermotolerant yeast strain Pichia thermomethanolica BCC16875 was characterized. To investigate the efficiency of the GAP promoter for heterologous expression, especially at high temperature in various carbon sources, the promoter was employed for constitutive expression of a phytase reporter gene. The results showed that this promoter was able to drive efficient expression of phytase at 30 °C; the native promoter was highly robust compared with the heterologous GAP promoter from Pichia pastoris. More importantly, the GAP promoter was shown to be able to function at higher temperatures up to 42 °C, which could be useful for large-scale protein production to help reduce cooling costs in the fermenter. Expression in different carbon sources revealed that the GAP promoter was functional in glucose-, glycerol-, and methanol-containing media, with the highest level of expression in YPD medium. This strong promoter will help promote high expression of heterologous protein expression in P. thermomethanolica, especially in large-scale fermentation. In addition, a new tool for heterologous expression in yeast has been gained.     

Conditional UAS-targeted repression in Drosophila

The Gal4–UAS enhancer trap system is useful for driving gene expression in various tissues. A new tool that extends Gal4 technology is described here. A fusion protein containing the Gal4 binding domain and the repression domain of the isolator suppressor of hairy wing was placed under the control of a heat shock-inducible promoter. The construct mediates the conditional repression of genes located downstream of a UAS sequence. The repressive effects of the chimeric protein on fasII gene expression were tested by western-blot analysis and in brain sections of adult Drosophila. Owing to the increasing number of Gal4 and UAS transgenic lines, this versatile system will facilitate the study of gene function.     

The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

Regulated expression systems for the development of whole-cell biocatalysts expressing oxidative enzymes in a sequential manner

This work reports the preparation of two recombinant strains each containing two enzymatic activities mutually expressed through regulated systems for production of functionalized epoxides in one-pot reactions. One strain was Pseudomonas putida PaW340, containing the gene coding for styrene monooxygenase (SMO) from Pseudomonas fluorescens ST under the auto-inducing Ptou promoter and the TouR regulator of Pseudomonas sp. OX1 and the gene coding for naphthalene dihydrodiol dehydrogenase (NDDH) from P. fluorescens N3 under the Ptac promoter inducible by IPTG. The second strain was Escherichia coli JM109, in which the expression of SMO was under the control of the Pnah promoter and the NahR regulator of P. fluorescens N3 inducible by salicylate, while the gene expressing NDDH was under the control of the Plac promoter inducible by IPTG. SMO and NDDH activities were tested in bioconversion experiments using cinnamyl alcohol as reference substrate. The application that we selected is one example of the sequential use of the two enzymatic activities which require a temporal control of the expression of both genes.     

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 (P_AOX1 and P_FLD1) 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 P_AOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96 h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262 g/L, 38,500 U/mL and 2.82 g/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.     

Xanthomonas campestris as a host for the production of recombinantPseudomonas aeruginosa lipase

Recombinant plasmid pBP13, which expresses the alkaline lipase fromPseudomonas aeruginosa IGB83 under thetac promoter was transferred toXanthomonas campestris pvcampestris IBT148. Different fermentation conditions were tested for lipase productivity by strain IBT148 carrying plasmid pBP13, and a fermentation process was established in an instrumented bioreactor, where lipase production was increased more than 12-fold with respect to the initial culture conditions in shake flasks. Xanthan gum stabilized the activity of the alkaline lipase.     

Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was Isolated as an Intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd30₁. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPH1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex plants in two such constructs BSL 12–1 and BSL 12–3.     

Arsenic Methylation and Volatilization by Arsenite S-Adenosylmethionine Methyltransferase in Pseudomonas alcaligenes NBRC14159

Inorganic arsenic (As) is highly toxic and ubiquitous in the environment. Inorganic As can be transformed by microbial methylation, which constitutes an important part of the As biogeochemical cycle. In this study, we investigated As biotransformation by Pseudomonas alcaligenes NBRC14159. P. alcaligenes was able to methylate arsenite [As(III)] rapidly to dimethylarsenate and small amounts of trimethylarsenic oxide. An arsenite S-adenosylmethionine methyltransferase, PaArsM, was identified and functionally characterized. PaArsM shares low similarities with other reported ArsM enzymes (<55%). When P. alcaligenes arsM gene (PaarsM) was disrupted, the mutant lost As methylation ability and became more sensitive to As(III). PaarsM was expressed in the absence of As(III) and the expression was further enhanced by As(III) exposure. Heterologous expression of PaarsM in an As-hypersensitive strain of Escherichia coli conferred As(III) resistance. Purified PaArsM protein methylated As(III) to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases. We propose that PaArsM plays a role in As methylation and detoxification of As(III) and could be exploited in bioremediation of As-contaminated environments.     

Repeated Family of Genes Controlling Maltose Fermentation in Saccharomyces carlsbergensis

Maltose fermentation in Saccharomyces spp. requires the presence of any one of five unlinked genes: MAL1, MAL2, MAL3, MAL4, or MAL6. Although the genes are functionally equivalent, their natures and relationships to each other are not known. At least three proteins are necessary for maltose fermentation: maltase, maltose permease, and a regulatory protein. The MAL genes may code for one or more of these proteins. Recently a DNA fragment containing a maltase structural gene has been cloned from a MAL6 strain, CB11, to produce plasmid pMAL9-26. We have conducted genetic and physical analyses of strain CB11. The genetic analysis has demonstrated the presence of two cryptic MAL genes in CB11, MAL1g and MAL3g (linked to MAL1 and to MAL3, respectively), in addition to the MAL6 locus. The physical analysis, which used a subclone of plasmid pMAL9-26 as a probe, detected three HindIII genomic fragments with homology to the probe. Each fragment was shown to be linked to one of the MAL loci genetically demonstrated to be present in CB11. Our results indicate that the cloned maltase structural gene in plasmid pMAL9-26 is linked to MAL6. Since the MAL6 locus has previously been shown to contain a regulatory gene, the MAL6 locus must be a complex locus containing at least two of the factors needed for maltose fermentation: the structural gene for maltase and the maltase regulatory protein. The absence of other fragments which hybridize to the MAL6-derived probe shows that either MAL2 and MAL4 are not related to MAL6, or the DNA corresponding to these genes is absent from the MAL6 strain CB11.     

Chaperone-dependent gene expression of organic solvent-tolerant lipase from Pseudomonas aeruginosa strain S5

The gene coding for the intracellular organic solvent-tolerant lipase of Pseudomonas aeruginosa strain S5 was isolated from a genomic DNA library and cloned into pRSET. The cloned sequence included two open reading frames (ORF) of 1575 bp for the first ORF (ORF1), and 582 bp for the second ORF (ORF2). The ORF2, known as chaperone, plays an important role in the expression of the S5 gene. The ORF2 is located downstream of lipase gene, and functions as the act gene for ORF1. The conserved pentapeptide, Gly-X-Ser-X-Gly, is located in the ORF1. A sequence coding for a catalytic triad that resembles that of a serine protease, consisting of serine, histidine, and aspartic acid or glutamic acid residues, was present in the lipase gene. Expression of the S5 lipase gene in E. coli resulted in a 100-fold increase in enzyme activity 9 h after induction with 0.75 mM IPTG. The recombinant protein revealed a size of 60 kDa on SDS-PAGE. The Lip S5 gene was stable in the presence of 25% (v/v) n-dodecane and n-tetradecane after 2 h incubation at 37 °C.     

Cloning the amidase gene from Rhodococcus rhodochrous M8 and its expression in Escherichia coli

The amidase gene from Rhodococcus rhodochrous M8 was cloned by PCR amplification with primers developed by use of peptide amino acid sequences obtained after treating amidase with trypsin. Nucleotide sequence analysis of this gene revealed high homology with aliphatic amidases from R. erythropolis R312 and Pseudomonas aeruginosa. Considering the substrate specificity and the results of DNA analysis, amidase from R. rhodochrous M8 was assigned to the group of aliphatic amidases preferentially hydrolyzing short-chain aliphatic amides. The amidase gene was expressed in cells of Escherichia coli from the self promoter and from the lac promoter. To clone a fragment of R. rhodochrous M8 chromosome (approximately 9 kb), containing the entire structural gene and its flanking regions, plasmid pRY1 that can be integrated into the chromosome via homology regions was used. No sequences of the nitrile hydratase gene, the second key gene of nitrile degradation in strain R. rhodochrous M8, were detected. Thus, genes encoding amidase and nitrile hydratase in strain R. rhodochrous M8 are not organized into a single operon despite their common regulation.     

Genomic structure and sequence of the pufferfish (Fugu rubripes) growth hormone-encoding gene: a comparative analysis of teleost growth hormone genes

A nested polymerase chain reaction (PCR) technique for amplifying a fragment of the gene (GH) encoding teleost growth hormone has been developed. Using this technique, a fragment of the pufferfish, Fugu rubripes and Arothron maculatus; dwarf gourami, Colisa lalia; guppy, Poecilia reticulata; and goldfish, Carassius auratus GH genes were cloned. The Fugu rubripes (Fugu) gene fragment was used to isolate the GH gene from a Fugu genomic library. The complete nucleotide sequence of a 8.5-kb SacI genomic fragment containing the Fugu GH gene has been determined. The GH gene spans 2.5 kb from the first codon to polyadenylation signal, and contains six exons and five introns similar to the GH genes of salmonids, tilapia, barramundi, flounder and yellowtail. The GH introns contain microsatellite and satellite sequences. The microsatellites found in the fifth intron of the GH gene are also present in the corresponding introns of tilapia, barramundi and flounder GH genes. Southern analysis revealed that the GH gene is a single-copy gene in the Fugu. The promoter region of the Fugu GH gene contains conserved sequences that are likely to be involved in the pituitary-specific expression of the gene. A phylogenetic tree of nucleotide (nt) sequences of all known teleost GH genes has been inferred using the distance matrix method. The topology of this tree reflects the major phylogenetic groupings of teleosts. The intron patterns and repetitive sequences of GH genes can serve as useful natural markers for the classification and phylogenetic studies of teleosts.