Selection of new over-producing derivatives for the improvement of extracellular lipase production by the non-conventional yeast Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica produces an extracellular lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity were previously obtained either by chemical mutagenesis or genetic engineering. In this work, we used one of these mutants, named LgX64.81 to select new overproducing strains following by amplification of the LIP2 gene. We also developed a process for lipase production in bioreactors and compared lipase production levels in batch and fed-batch cultures. Batch culture led to a lipase production of 26450 U ml(-1) in a media containing olive oil and tryptone as carbon and nitrogen sources. Feeding of a combination of tryptone and olive oil at the end of the exponential growth phase yielded to lipase activity of 158246 U ml(-1) after 80 h of cultivation. In addition this production system developed for the extracellular lipase could also be applied for other heterologous protein production since we have demonstrated that LgX64.81 is an interesting alternative host strain.     

Isolation of bioactive peptides from tryptone that modulate lipase production in Yarrowia lipolytica

In this work the effect of several organic nitrogen sources on lipase production in Yarrowia lipolytica LgX64.81 overproducing mutant was studied. Among them, tryptone and peptone showed the most prominent stimulatory effect. Interestingly, only tryptic and peptic casein digest were found to highly induce lipase biosynthesis while lipase production was very limited in the presence of casein digest from papain and pronase-catalysed hydrolysis and absent in case of chymotryptic digest. It was also demonstrated that the stimulatory peptides should be present in the culture medium at specific proportions and molecular size to match the physiological requirement of Yarrowia lipolytica strain for lipase biosynthesis.     

Methyl Oleate Modulates <Emphasis Type="Italic">LIP2</Emphasis> Expression in the Lipolytic Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Methyl oleate was used as a primary carbon source and as an alternative inducer for the production of an extracellular lipase, Lip2, in Y. lipolytica strain LgX64.81 grown in a 20-l bioreactor. The lipase-encoding gene, LIP2, was investigated during culture on methyl oleate using a pLIP2–LacZ reporter fusion and we provide evidence for the involvement of methyl oleate in its regulation. Revisions requested 7 July 2005; Revisions received 30 August 2005     

A yeast mutation that stabilizes a plasmid bearing a mutated ARS1 element.

To identify the trans-acting factors involved in autonomously replicating sequence (ARS) function, we initiated a screen for Saccharomyces cerevisiae mutants capable of stabilizing a plasmid that contains a defective ARS element. The amm (altered minichromosome maintenance) mutations recovered in this screen defined at least four complementation groups. amm1, a mutation that has been studied in detail, gave rise to a 17-fold stabilization of one defective ARS1 plasmid over the level seen in wild-type cells. The mutation also affected the stability of at least one plasmid bearing a wild-type ARS element. amm1 is an allele of the previously identified TUP1 gene and exhibited the same pleiotropic phenotypes as other tup1 mutants. Plasmid maintenance was also affected in strains bearing a TUP1 gene disruption. Like the amm1 mutant, the tup1 disruption mutant exhibited ARS-specific plasmid stabilization; however, the ARS specificities of these two mutants differed. The recovery of second-site mutations that suppressed many of the tup1 phenotypes but not the increased plasmid maintenance demonstrates that the plasmid stability phenotype of tup1 mutants is not a consequence of the other defects caused by tup1.     

Isolation and Characterization of Mutants Which Show an Oversecretion Phenotype in Saccharomyces Cerevisiae

We have isolated mutants responsible for an oversecretion phenotype in Saccharomyces cerevisiae, using a promoter of SUC2 and the gene coding for alpha-amylase from mouse as a marker of secretion. These mutations defined two complementation groups, designated as ose1 (over secretion) and rgr1 (resistant to glucose repression). The ose1 mutant produced an oversecretion of amylase by 12- to 15-fold under derepressing conditions; however, the amylase mRNA was present at nearly the same amount as it was in the parent cells. No expression of the amylase gene was detected under repressing conditions. The rgr1 mutant oversecreted amylase by 11- to 13-fold under repressing conditions by 15- to 18-fold under derepressing conditions. The rgr1 mutant showed pleiotropic effects on the following cellular functions: (1) resistance to glucose repression, (2) temperature-sensitive lethality, (3) sporulation deficieny in homozygous diploid cells, and (4) abnormal cell morphology. The rgr1 mutation was not allelic with ssn6 and cyc9, and failed to suppress snf1.     

A trans-acting regulatory mutation that causes overproduction of phosphatidylserine synthase in Escherichia coli

We have isolated three mutants of Escherichia coli which have elevated levels of the phospholipid synthetic enzyme phosphatidylserine synthase. One of these strains carries a mutation, designated pssR1, which maps near minute 84 of the chromosome, distinct from the synthase structural gene (pss) at minute 56. The pssR1 mutation causes selective overproduction of phosphatidylserine synthase, since the levels of six other lipid synthetic enzymes are unaltered. The specific activity of the synthase in crude cell extracts of mutants harboring pssR1 is about five times greater than wild type. The synthase can also be overproduced 10-fold in wild type strains with hybrid ColE1 plasmids carrying the synthase structural gene (pss). A pssR1 mutant harboring such a pss plasmid overproduces the synthase about 50-fold. This multiplicative interaction of pssR1 and cloned pss demonstrates that pssR1 is trans-acting. The synthase has been purified in parallel from pssR1 and pssR+ strains. The pssR1 mutant yields more total synthase protein than pssR+, but the pure enzyme has the same specific activity in both cases. Therefore, pssR1 acts by increasing the amount of the normal protein, not by activating the enzyme. The discovery of pssR shows that there are regulatory loci which control the production of enzymes involved in membrane lipid synthesis.     

Mutations that increase the affinity of a translational repressor for RNA.

The coat protein of the RNA bacteriophage MS2 is a specific RNA binding protein that represses translation of the viral replicase gene during the infection cycle. As an approach to characterizing the RNA-binding site of coat protein we have isolated a series of coat mutants that suppress the effects of a mutation in the translational operator. Each of the mutants exhibits a super-repressor phenotype, more tightly repressing both the mutant and wild-type operators than does the wild-type protein. The variant coat proteins were purified and subjected to filter binding assays to determine their affinities for the mutant and wild-type operators. Each protein binds the operators from 3 to 7.5-fold more tightly than normal coat protein. The amino acid substitutions seem to extend the normal binding site by introducing new interactions with RNA.     

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae. Fungal Genetics and Biology 29, 28-37. Restriction enzyme-mediated integration (REMI) has been employed as a mutagen to generate two insertion libraries in an Aspergillus oryzae strain expressing a Thermomyces lanuginosus lipase. The REMI libraries were created using linearized plasmid containing the A. oryzae pyrG and either BamHI or EcoRI enzyme. The libraries were screened for lipase production, and mutants with increased production were isolated. The genomic DNA flanking the integration event was cloned from one of the mutants with increased lipase titers (DEBY10.3). Nucleotide sequence of the flanking DNA revealed similarity to the Aspergillus nidulans palB gene. Disruption of the palB gene in a strain producing lipase resulted in increased lipase expression. Additionally, complementation of the palB phenotype of DEBY10.3 led to a decrease in lipase production. These lines of evidence demonstrate that the increase in lipase yield in DEBY10.3 is linked to the palB phenotype generated by the integration of the pyrG gene into the palB gene. The results also demonstrated that tagged mutagenesis with REMI can be used to identify genes that influence expression of heterologous proteins.     

High-level production of extracellular lipase by Yarrowia lipolytica mutants from methyl oleate

The yeast Yarrowia lipolytica degrades efficiently low-cost hydrophobic substrates for the production of various added-value products such as lipases. To obtain yeast strains producing high levels of extracellular lipase, Y. lipolytica DSM3286 was subjected to mutation using ethyl methanesulfonate (EMS) and ultraviolet (UV) light. Twenty mutants were selected out of 1600 mutants of Y. lipolytica treated with EMS and UV based on lipase production ability on selective medium. A new industrial medium containing methyl oleate was optimized for lipase production. In the 20 L bioreactor containing new industrial medium, one UV mutant (U6) produced 356 U/mL of lipase after 24h, which is about 10.5-fold higher than that produced by the wild type strain. The properties of the mutant lipase were the same as those of the wild type: molecular weight 38 kDa, optimum temperature 37°C and optimum pH 7. Furthermore, the nucleotide sequences of extracellular lipase gene (LIP2) in wild type and mutant strains were determined. Only two silent substitutions at 362 and 385 positions were observed in the ORF region of LIP2. Two single substitutions and two duplications of the T nucleotide were also detected in the promoter region. LIP2 sequence comparison of the Y. lipolytica DSM3286 and U6 strains shows good targets to effective DNA recombinant for extracellular lipase of Y. lipolytica.     

The novel mutant scl of the medaka fish, Oryzias latipes, shows no secondary sex characters

A new mutant that has neither male nor female secondary sex characters was found in the medaka, Oryzias latipes. Both XX and XY mature mutants had gonads with many spermatozoa, but spawning did not occur when the mutants were paired with normal males or normal females. F1 progeny were successfully obtained by artificial insemination using unfertilized eggs from wild-type females and spermatozoa of the XY mutant. The mutant phenotype did not occur in the F1 progeny from this cross. Incrossing among the F1 progeny produced 17 mutant offspring out of 68 progeny (25%), demonstrating that the mutant phenotype is caused by a single recessive mutation. This mutant was named scl (sex character-less). Because papillary processes, a male secondary sex character, were induced in the XY mutants by androgen administration, it seems that the androgen receptor is functioning normally. We found a loss-of-function type mutation in the P450c17 gene of the mutant; this gene encodes a steroidogenic enzyme required for the production of estrogen and androgen. The scl phenotype was completely linked to the mutant genotype of P450c17, strongly suggesting that mutation at the P450c17 locus is responsible for the scl mutant phenotype.     

The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae.

The intracellular concentrations of the polypeptides encoded by the two enolase (ENO1 and ENO2) and three glyceraldehyde-3-phosphate dehydrogenase (TDH1, TDH2, and TDH3) genes were coordinately reduced more than 20-fold in a Saccharomyces cerevisiae strain carrying the gcr1-1 mutation. The steady-state concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA was shown to be approximately 50-fold reduced in the mutant strain. Overexpression of enolase and glyceraldehyde-3-phosphate dehydrogenase in strains carrying multiple copies of either ENO1 or TDH3 was reduced more than 50-fold in strains carrying the gcr1-1 mutation. These results demonstrated that the GCR1 gene encodes a trans-acting factor which is required for efficient and coordinate expression of these glycolytic gene families. The GCR1 gene and the gcr1-1 mutant allele were cloned and sequenced. GCR1 encodes a predicted 844-amino-acid polypeptide; the gcr1-1 allele contains a 1-base-pair insertion mutation at codon 304. A null mutant carrying a deletion of 90% of the GCR1 coding sequence and a URA3 gene insertion was constructed by gene replacement. The phenotype of a strain carrying this null mutation was identical to that of the gcr1-1 mutant strain.     

Specific Suppression of Mutations in Genes 46 and 47 by das, a New Class of Mutations in Bacteriophage T4D

Mutants in T4 genes 46 and 47 exhibit early cessation of deoxyribonucleic acid (DNA) synthesis ("DNA arrest") and decreased synthesis of late proteins and phage. In addition, mutants in genes 46 and 47 fail to degrade host DNA to acidsoluble products. It is shown here that this complex phenotype can be partially suppressed by mutation of a T4 gene external to genes 46 and 47 which has been named das for "DNA arrest suppressor." The das mutations were discovered as third-site mutations in spontaneous pseudorevertants of [46, 47] mutants; the pseudorevertants make small plaques on Escherichia coli B, whereas [46, 47] mutants make none. The [das, 46, 47] triple mutant exhibits increased DNA, late protein, and viable phage production compared to the double mutant [46, 47]. The [das, 46, 47] mutant also degrades more of the host DNA to acid-soluble products than does the [46, 47] mutant. The suppressor effect of the das mutation appears to be gene-specific: it suppresses both amber and temperature-sensitive mutations in genes 46 and 47 and does not suppress amber mutations in any of the other genes tested. The [das] single mutants make normal-sized plaques on E. coli B and exhibit nearly normal host DNA degradation, DNA synthesis, late protein synthesis, and viable phage production. The das mutations either define a new gene between genes 33 and 34 or are special mutations within gene 33.     

Selection and characterization of a glucokinase-deficient mutant of Tetrahymena thermophila.

We have isolated a mutant of Tetrahymena thermophila that is resistant to inhibition of growth by the glucose analog 2-deoxyglucose. The mutant exhibits a deficiency in a cytoplasmic glucokinase. This enzymatic defect and the attendant inability to convert 2-deoxyglucose to toxic phosphorylated derivatives is apparently the sole basis for the mutant phenotype since transport of glucose and 2-deoxyglucose is unimpaired; there is no elevation of glucose-6-phosphatase activity, which could decrease the level of toxic 2-deoxyglucose metabolites. Genetic analyses have shown that the mutant allele is recessive and inherited as a single Mendelian mutation. The glucokinase-deficient strain described here is useful for the selection of other mutants in this organism and for the investigation of various cellular processes initiated or modulated by glucose and its analogs. We have exploited the molecular defect in this strain to investigate the initial steps in the cyclic AMP-mediated repression of galactokinase gene expression which is caused by glucose.     

Use of reporter genes to identify recessive trans-acting mutations specifically involved in the regulation of Aspergillus nidulans penicillin biosynthesis genes.

Starting from three amino acid precursors, penicillin biosynthesis is catalyzed by three enzymes which are encoded by the following three genes: acvA (pcbAB), ipnA (pcbC), and aat (penDE). To identify trans-acting mutations which are specifically involved in the regulation of these secondary metabolism genes, a molecular approach was employed by using an Aspergillus nidulans strain (AXTII9) carrying acvA-uidA and ipnA-lacZ gene fusions integrated in double copies at the chromosomal argB gene. On minimal agar plates supplemented with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), colonies of such a strain stained blue, which is indicative of ipnA-lacZ expression. After mutagenesis with UV light, colonies were isolated on agar plates with lactose as the carbon source, which produced only a faint blue color or no color at all. Such mutants (named Prg for penicillin regulation) most likely were defective in trans-acting genes. Control experiments revealed that the mutants studied still carried the correct number of gene fusions. In a fermentation run, mutants Prg-1 and Prg-6 exhibited only 20 to 50% of the ipnA-lacZ expression of the wild-type strain and produced only 20 to 30% of the penicillin produced by the wild-type strain. Western blot (immunoblot) analysis showed that these mutants contained reduced amounts of ipnA gene product, i.e., isopenicillin N synthase. Both mutant Prg-1 and mutant Prg-6 also differed in acvA-uidA expression levels from the wild type. Segregation analysis indicated that for both mutants the Prg phenotype resulted from mutation of a single gene. Two different complementation groups, which were designated prgA1 and prgB1, were identified. However, the specific activity of the aat (penDE) gene product, i.e., acyl coenzyme A:6-aminopenicillanic acid acyltransferase, was essentially the same for the mutants as for the wild-type strain, implying that the last step of the penicillin biosynthetic pathway is not affected by the trans-acting mutations identified.     

Contrasting properties of gene-specific regulatory, coding, and copy number mutations in Saccharomyces cerevisiae: frequency, effects, and dominance

Genetic variation within and between species can be shaped by population-level processes and mutation; however, the relative impact of "survival of the fittest" and "arrival of the fittest" on phenotypic evolution remains unclear. Assessing the influence of mutation on evolution requires understanding the relative rates of different types of mutations and their genetic properties, yet little is known about the functional consequences of new mutations. Here, we examine the spectrum of mutations affecting a focal gene in Saccharomyces cerevisiae by characterizing 231 novel haploid genotypes with altered activity of a fluorescent reporter gene. 7% of these genotypes had a nonsynonymous mutation in the coding sequence for the fluorescent protein and were classified as "coding" mutants; 2% had a change in the S. cerevisiae TDH3 promoter sequence controlling expression of the fluorescent protein and were classified as "cis-regulatory" mutants; 10% contained two copies of the reporter gene and were classified as "copy number" mutants; and the remaining 81% showed altered fluorescence without a change in the reporter gene itself and were classified as "trans-acting" mutants. As a group, coding mutants had the strongest effect on reporter gene activity and always decreased it. By contrast, 50%-95% of the mutants in each of the other three classes increased gene activity, with mutants affecting copy number and cis-regulatory sequences having larger median effects on gene activity than trans-acting mutants. When made heterozygous in diploid cells, coding, cis-regulatory, and copy number mutant genotypes all had significant effects on gene activity, whereas 88% of the trans-acting mutants appeared to be recessive. These differences in the frequency, effects, and dominance among functional classes of mutations might help explain why some types of mutations are found to be segregating within or fixed between species more often than others.     

Strain improvement of Aspergillus niger for enhanced lipase production

The enhancement of lipase production from Aspergillus niger was attempted by ultraviolet (UV) and nitrous acid mutagenesis, and the mutants were selected on media containing bile salts. Nitrous acid mutants exhibited increased efficiency for lipase production when compared with UV mutants in submerged fermentation. The hyperproducing UV and nitrous acid mutants were further subjected to a second step of mutagenesis to devise an economical and ecofriendly technique for lipase production by the effective use of hydrocarbons. One percent kerosene was found to be optimal for lipase production, and one of the mutant strains NAII exhibited 2.53 times more increased lipase activity than the parental strain did. This investigation indicates a possible role for the A. niger mutant strains in the biodegradation of oil-polluted environments for the development of ecofriendly technologies.