Functional characterization of the Komagataella phaffii 1033 gene promoter and transcriptional terminator.

The methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is a widely used host for extracellularly producing heterologous proteins via an expression cassette integrated into the yeast genome. A strong promoter in the expression cassette is not always the most favorable choice for heterologous protein production, especially if the correct folding of the protein and/or post-translational processing is the limiting step.

Biochemical characterization of the β-glucosidase Glu1B from Coptotermes formosanus produced in Pichia pastoris.

β-glucosidases (E.C. 3.

Low specific growth rate and temperature in fed-batch cultures of a beta-propeller phytase producing Pichia pastoris strain under GAP promoter trigger increased KAR2 and PSA1-1 gene expression yielding enhanced extracellular productivity.

Previously, we showed that the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) could produce and secrete the beta-propeller phytase FTEII in an active form under the control of the AOX1 promoter and methanol as the inductor. In this work, we engineered P.

Sequence Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity.

Tannin acyl hydrolases or tannases (E.C.3.

Buried Kex2 Sites in Glargine Precursor Aggregates Prevent Its Intracellular Processing in Pichia pastoris Mut Strains and the Effect of Methanol-Feeding Strategy and Induction Temperature on Glargine Precursor Production Parameters.

Glargine is a long-acting insulin analog with less hypoglycemia risk. Like human insulin, glargine is a globular protein composed of two polypeptide chains linked by two disulfide bonds. Pichia pastoris KM71 Mut strains were engineered to produce and secrete insulin glargine through the cleavage of two Kex2 sites.

Identification and in silico structural and functional analysis of a trypsin-like protease from shrimp .

(Linnaeus, 1758) is a species of freshwater shrimp widely distributed from Florida southwards to southern Brazil, including southeast of Mexico. In the present work, we identified a putative trypsin-like protease cDNA fragment of 736 nucleotides from hepatopancreas tissue by the 3'RACE technique and compared the deduced amino acid sequence to other trypsin-related proteases to describe its structure and function relationship. The bioinformatics analyses showed that the deduced amino acid sequence likely corresponds to a trypsin-like protease closely related to brachyurins, which comprise a subset of serine proteases with collagenolytic activity found in crabs and other crustacea.

Biochemical characterization of recombinant Penaeus vannamei trypsinogen.

Trypsinogens are the inactive precursors of trypsins (EC 3.4.21.

Tannase sequence from a xerophilic Aspergillus niger Strain and production of the enzyme in Pichia pastoris.

Tannin acyl hydrolases, or tannases (EC 3.1.1.

Gene encoding a novel invertase from a xerophilic Aspergillus niger strain and production of the enzyme in Pichia pastoris.

β-Fructofuranosidases or invertases (EC 3.2.1.

Optimization of five environmental factors to increase beta-propeller phytase production in Pichia pastoris and impact on the physiological response of the host.

Recently, we engineered Pichia pastoris Mut(s) strains to produce several beta-propeller phytases, one from Bacillus subtilis and the others designed by a structure-guided consensus approach. Furthermore, we demonstrated the ability of P. pastoris to produce and secrete these phytases in an active form in shake-flask cultures.

Shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris bioreactor cultures.

Recently, we engineered a Pichia pastoris Mut(+) strain to produce and secrete recombinant Litopenaeus vannamei trypsinogen. Despite the observed toxicity of the recombinant shrimp trypsinogen to the P. pastoris cell host, when high density cell cultures in shake flasks with alanine in the induction medium were used recombinant shrimp trypsinogen could be produced.

Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris.

Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.

Expression of a Bacillus phytase C gene in Pichia pastoris and properties of the recombinant enzyme.

The cloning and expression of a native gene encoding a Bacillus subtilis phytase using Pichia pastoris as the host is described. In addition, the influence of N-glycosylation on the biochemical properties of the B. subtilis phytase, the influence of pH on the thermostability of the recombinant and native B.

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris.

Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time.

Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates.

A simplified amplified-fragment length polymorphism (AFLP) method was developed and applied to genotype 52 Mycobacterium tuberculosis isolates. This method can be carried out using only one restriction enzyme (XhoI), one double strand adapter, and one PCR primer. The amounts of DNA and DNA polymerase, and the concentrations of primer and Mg(2+) in the PCR step were optimized using the Basic Sequential Simplex method.

Detection and identification of mycobacteria by mycolic acid analysis of sputum specimens and young cultures.

Ziehl-Neelsen acid-fast staining and mycolic acid analysis of concentrated samples and Middlebrook 7H9 cultures were carried out on 127 sputum specimens to evaluate a rapid method for detecting and identifying mycobacteria by analyzing fluorescent derivatives of mycolic acids in concentrated sputum specimens and in Middlebrook 7H9 cultures and compare with mycobacterial detection using Lowenstein-Jensen (LJ) cultures. All samples were classified into five groups according to the number of acid-fast bacilli observed in the smear. The group of samples with 3+ acid-fast bacilli in the smear had the highest number of positive detections of mycolic acids in the concentrated samples and the Middlebrook 7H9 cultures (81.

Genotyping of recombinant Pichia pastoris strains.

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns.

Frequency of mutations in rpoB and codons 315 and 463 of katG in rifampin- and/or isoniazid-resistant Mycobacterium tuberculosis isolates from northeast Mexico.

A total of 48 isoniazid (INH)- and rifampin (RIF)-resistant Mycobacterium tuberculosis isolates, 19 INH-resistant isolates, and 9 RIF-resistant isolates were randomly selected and tested for detecting mutations at codons 315 and 463 of katG by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and/or for detecting mutations at a 69-bp region of the rpoB gene by the INNO-LiPA Rif TB assay. Of the 67 INH-resistant isolates tested, 36 (53.7%) showed the mutation at codon 315 of katG; however, none of them showed the mutation at codon 463.

Mycolic acid index susceptibility method for Mycobacterium tuberculosis.

A rapid drug susceptibility test to measure the susceptibility of Mycobacterium tuberculosis to isoniazid (INH) and rifampin (RIF) using clinical isolates and a newly defined mycolic acid index (MAI) was evaluated. A total of 200 clinical isolates of M. tuberculosis were tested for susceptibility or resistance to INH and RIF by the MAI susceptibility and indirect-proportion methods.

Drug Susceptibility of Mycobacterium tuberculosisThrough the Mycolic Acid Index.

The methods accepted to determine antimicrobial drug susceptibility of mycobacteria are based on the determination of the microorganisms' growth on solid or liquid medium containing a specified concentration of a single drug.

Identification of mycobacteria by mycolic acid pattern.

Background: Tuberculosis caused by Mycobacterium tuberculosis is a public health problem which has increased in importance during the last 12 years, due in part to the increasing number of cases caused by the association of acquired immunodeficiency syndrome (AIDS) and the appearance of multiple drug-resistant strains. Other mycobacteria which are often indistinguishable from tuberculosis have also increased.

Methods: Mycolic acid patterns were obtained from 53 clinical isolates of sputum, cerebrospinal fluid, bronchial washing, corneal ulcer, and bone marrow, as well as from 11 acid-fast stain smear-positive clinical specimens.

Determination of drug susceptibility of Mycobacterium tuberculosis through mycolic acid analysis.

In the present work a rapid method to determine the susceptibility of Mycobacterium tuberculosis to isoniazid and streptomycin by determining levels of mycolic acids by high-performance liquid chromatography (HPLC) was developed. Mycobacterial growth kinetics in the presence and absence of antituberculosis drugs was characterized by evaluating the total area corresponding to mycolic acid peaks (TAMA). Results show a linear relationship between the logarithm of CFU per milliliter and TAMA and show that it is possible to detect growth inhibition of M.

Natural anthracenone subcellular distribution and effects on NADPH-cytochrome P450 reductase microsomal activity.

Natural anthracenone subcellular distribution and effects on NADPH-cytochrome P450 reductase microsomal activity. Subcellular distribution study of a natural anthracenone (T-514) isolated from Karwinskia humboldtiana showed to be homogeneous on subcellular (nuclear, mitochondrial, peroxisomal and microsomal) fractions prepared from rat liver treated with an acute dose of T-514. These results indicate that T-514 can pass easily through subcellular compartment membranes and an absence of selectivity for some subcellular organelles.

[Pharmacokinetic design for the preclinical phase. Application to the study of an anthracenone].

T-514 is a toxic substance of Karwinskia humboldtiana which has been described as a possible anticancer agent. An animal model (New Zeland white rabbit) was selected for pharmacokinetic studies that would allow the performance of surgical techniques to catheterize central vessels in order to obtain blood samples at short time intervals. A nonlinear regression analysis method was used to fit the plasmatic concentration data to a multiexponential mathematical function; a computer program based on Marquant's iterating algorithm was used to fit the experimental curves.