Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion.

Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD-hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild-type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7-fold higher kcat /KM . Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high-producing strain, 8GM1, containing eight copies of AOX1 promoter-GAS1 signal peptide-MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co-expressing the trafficking components EES and SEC. 22, and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2-fold. In the 5-L bioreactor, conventional fed-batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3-fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high-producing strains for other industrial enzymes.

Synergistic Combination of Bioactive Hydroxyapatite Nanoparticles and the Chemotherapeutic Doxorubicin to Overcome Tumor Multidrug Resistance.

Multidrug resistance (MDR) is one of the biggest obstacles in cancer chemotherapy. Here, a remarkable reversal of MDR in breast cancer through the synergistic effects of bioactive hydroxyapatite nanoparticles (HAPNs) and doxorubicin (DOX) is shown. DOX loaded HAPNs (DHAPNs) exhibit a 150-fold reduction in IC50 compared with free DOX for human MDR breast cancer MCF-7/ADR cells, and lead to almost complete inhibition of tumor growth in vivo without obvious side effects of free DOX. This high efficacy and specificity could be attributed to multiple action mechanisms of HAPNs. In addition to acting as the conventional nanocarriers to facilitate the cellular uptake and retention of DOX in MCF-7/ADR cells, more importantly, drug-free HAPNs themselves are able to prevent drug being pumped out of MDR cells through targeting mitochondria to induce mitochondrial damage and inhibit ATP production and to trigger sustained mitochondrial calcium overload and apoptosis in MDR cancer cells while not affecting normal cells. The results demonstrate that this simple but versatile bioactive nanoparticle provides a practical approach to effectively overcome MDR.

Engineering Pichia pastoris to improve S-adenosyl- l-methionine production using systems metabolic strategies.

S-adenosyl-l-methionine (SAM) is a highly valued chemical that can be used as a dietary supplement and has been used to treat depression, osteoarthritis, and liver problems as well. We adopted systems metabolic engineering strategies to improve SAM production in a high-producing strain (GS115/DS56). First, the cystathionine ß-synthase gene CYS4 was downregulated using a weak promoter PG12 to reduce the removal of homocysteine from SAM cycle, thus leading to a 48.8% increase in the SAM titer (1.68 g/L) from the strain G12-CBS, while preventing cysteine auxotrophy induced by deletion of this essential gene. Subsequently, the SAM titer of G12-CBS was improved to 13.01 g/L in 15-L fed-batch fermentation using the optimal l-methionine feeding strategy. Finally, based on comparative transcriptomics, five genes were chosen and overexpressed for further enhancement of SAM production. Among them, GDH2 and ACS2 exhibited positive effects, and the additional overexpression of GDH2 led to a 52.3% increase of titer (2.71 g/L) in shake flask culture. Therefore, the engineered Pichia pastoris strains can be utilized in industrial production of SAM using a simple and cost-effective process, and these approaches could be employed for improving the production of other chemicals by P. pastoris.

Effect of the Polymorphism of Folylpolyglutamate Synthetase on Treatment of High-Dose Methotrexate in Pediatric Patients with Acute Lymphocytic Leukemia.

BACKGROUND The aim of this study was to investigate the association of the polymorphism of folylpolyglutamate synthetase (FPGS) with the dynamic plasma concentration of methotrexate (MTX) in pediatric patients with acute lymphocytic leukemia (ALL), as well as the prognosis. MATERIAL AND METHODS 57 ALL patients and 31 age and sex-matched children (control) were included in this study. Polymerase chain reaction-restriction fragment length polymorphism was performed for the analysis of the genotype of FPGS rs1544105 and high-performance liquid chromatography for measurement of MTX plasma concentration after 24-h and 44-h treatment. Overall survival was analyzed by Kaplan-Meier method. RESULTS No differences were observed between patients and controls regarding the distribution frequency of genotype and alleles of rs1544105. Patients carrying AA genotype had a significantly higher plasma concentration of MTX after 24 h than those carrying GG or GA (P<0.05) and no differences were found after 44 h. Kaplan-Meier survival analysis showed a longer median survival time in patients with AA than other genotypes with significant difference in overall survival. CONCLUSIONS Polymorphism of FPGS rs1544105 might be used as an effective approach for prediction of the treatment outcome of MTX.

Lipid-Encapsulated Engineered Bacterial Living Materials Inhibit Cyclooxygenase II to Enhance Doxorubicin Toxicity.

Recently, there has been increasing interest in the use of bacteria for cancer therapy due to their ability to selectively target tumor sites and inhibit tumor growth. However, the complexity of the interaction between bacteria and tumor cells evokes unpredictable therapeutic risk, which induces inflammation, stimulates the up-regulation of cyclooxygenase II (COX-2) protein, and stimulates downstream antiapoptotic gene expression in the tumor microenvironment to reduce the antitumor efficacy of chemotherapy and immunotherapy. In this study, we encapsulated celecoxib (CXB), a specific COX-2 inhibitor, in liposomes anchored to the surface of Escherichia coli Nissle 1917 (ECN) through electrostatic absorption (C@ECN) to suppress ECN-induced COX-2 up-regulation and enhance the synergistic antitumor effect of doxorubicin (DOX). C@ECN improved the antitumor effect of DOX by restraining COX-2 expression. In addition, local T lymphocyte infiltration was induced by the ECN to enhance immunotherapy efficacy in the tumor microenvironment. Considering the biosafety of C@ECN, a hypoxia-induced lysis circuit, pGEX-Pvhb-Lysis, was introduced into the ECN to limit the number of ECNs in vivo. Our results indicate that this system has the potential to enhance the synergistic effect of ECN with chemical drugs to inhibit tumor progression in medical oncology.

Progress in the research of S-adenosyl-L-methionine production.

This minireview mainly aims at the study of S-adenosyl-L-methionine (SAM) production by microbial fermentation. A brief introduction of the biological role and application of SAM was presented. In general, SAM production can be improved by breeding of the producing strain through the conventional mutation or genetic engineering approach in the molecular or cellular scale, by optimization of culture conditions in the cellular scale or bioreactor engineering scale, or by multiscale approach. The productivity of SAM fermentation has been improved greatly through the efforts of many researchers using the methods previously mentioned. The SAM-producing strains used extensively are Pichia pastoris and Saccharomyces cerevisiae. The effect of SAM on antibiotic production was also exemplified. The skill and scheme beneficial to the improvement of SAM production involves the enhancement of SAM synthetase (methionine adenosyltransferase) activity and selection of engineered constitutive promoters with appropriate strength; seeking for and eliminating the rate-limiting factors in SAM synthesis, namely, knocking off the genes that transform SAM and L-methionine (L-Met) to cysteine; release the feedback inhibition of SAM to methylenetetrahydrofolate reductase; blocking the transsulfuration pathway by interfering the responsible enzymes; enhancing ATP level through pulsed feeding of glycerol; and optimizing the L-Met feeding strategy. Precise control of gene expression and quantitative assessment of physiological parameters in engineered P. pastoris were highlighted. Finally, a discussion of the prospect of SAM production was presented.

Engineering of the hypoxia-induced Pichia stipitis ADH2 promoter to construct a promoter library for Pichia pastoris.

Hypoxia-inducible promoters of a wide range of activities are desirable for fine-tuning gene expression in response to oxygen limitation, especially for the Crabtree negative yeast Pichia pastoris (Komagataella phaffii) with a high oxygen consumption rate in large-scale fermentations. Here we constructed a hypoxia-inducible promoter library for P. pastoris through error-prone PCR of Pichia stipitis ADH2 promoter (PsADH2). The library of 30 selected promoters showing 0.4- to 5.5-fold of the PsADH2 activity was obtained through high-throughput screening in microplates using the reporter yeast-enhanced green fluorescent protein. Two strong promoters, AM23 and AM30, were further characterized in shake flask cultures at high and low dissolved oxygen levels. They responded more sensitively to the low dissolved oxygen level, achieving a 4.6-, 7.9-fold and 3.6-, 7.7-fold higher fluorescence intensity and transcript level, respectively, than the wild-type PsADH2. Their hypoxia-inducible properties were confirmed with two additional reporters: ß-galactosidase and Vitreoscilla hemoglobin, to demonstrate the broad applicability of the promoter library. During the typical fermentation process in shake flasks, the promoter AM30 showed strong expression with cell growth and decreased oxygen levels, without any additional chemical inducers or operations. Since the potent industrial host P. pastoris is recognized as an easy to scale-up system, it is reasonable to expect that the obtained hypoxia-inducible promoter library may have great potential to enable convenient regulation of gene expression under industrial fermentations which are usually run under oxygen limitation due to high cell density cultivations.

Hydroxyapatite nanoparticles induced calcium overload-initiated cancer cell-specific apoptosis through inhibition of PMCA and activation of calpain.

Hydroxyapatite nanoparticles (HAPNs) have been reported to specifically induce apoptosis and sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in cancer cells. However, it remains unclear whether calcium overload, the abnormal intracellular accumulation of Ca2+, is the intrinsic cause of cell apoptosis, how HAPNs specifically evoke calcium overload in cancer cells, and which potential pathways were involved in apoptosis initiation in response to calcium overload. In this study, using various cancer and normal cells, we observed a positive correlation between the degree of increased [Ca2+]i and the specific toxicity of HAPNs. Moreover, chelating intracellular Ca2+ with BAPTA-AM inhibited HAPN-induced calcium overload and apoptosis, thus demonstrating that calcium overload was the main cause of HAPN-induced cytotoxicity in cancer cells. Notably, the dissolution of particles outside the cells did not affect cell viability or [Ca2+]i. In contrast, internalized HAPNs dissolved more readily in cancer cells than in normal cells and inhibited the activity of plasma membrane calcium-ATPase solely in cancer cells to prevent extrusion of excessive Ca2+, hence leading to calcium overload in tumor cells. Upon exposure to HAPNs, the Ca2+-sensitive cysteine protease calpain was activated and then cleaved the BH3-only protein Bid. Consequently, cytochrome c was released, and caspase-9 and -3 were activated, leading to mitochondrial apoptosis. However, these effects were alleviated by the calpain inhibitor calpeptin, confirming the involvement of calpain in HANP-induced apoptosis. Therefore, our results demonstrated that calcium overload induced by HAPNs caused cancer cell-specific apoptosis by inhibiting PMCA and activating calpain in tumor cells and thus may contribute to a more comprehensive understanding of biological effects of this nanomaterial and facilitate the development of calcium overload cancer therapy.

Effect of size on the cellular endocytosis and controlled release of mesoporous silica nanoparticles for intracellular delivery.

Due to the unique physicochemical properties and membrane-permeable capacity, mesoporous silica nanoparticles (MSNs) are considered as an ideal carrier for intracellular delivery. Herein, we endeavored to address the size effect of MSNs on the cellular uptake, endosomal escape and controlled release, the key steps for the intracellular delivery. The well-ordered MSNs in the range from 55-nm to 440-nm with similar pore texture were prepared by modified base-catalyzed sol-gel method. With MC3T3-E1 model cell line, the in vitro results indicated that after 12 h cultivation, MSNs within 55 ~ 440 nm could all be internalized into the cells, and further escaped out of the endosomal compartment. The efficiency of the cellular uptake and endosomal escape strongly depended on the particle size, with the best efficiencies from 100-nm MSNs. Furthermore, the MTT results indicated that these MSNs materials were all biocompatible. The controlled release experiments with hydrophobic dexamethasone and hydrophilic vitamin C as models showed that for these small-molecular drugs, the loading amount all mainly determined by the surface area of the MSNs, and the subsequent release of the drug dramatically decreased with the increasing of the particle size. By contrast, the release rate of vitamin C was much quicker than that of the dexamethasone. These findings presented here could provide new means to tailor the size of MSNs and thus to guide the design of MSNs-based intracellular delivery system. Due to the good cell biocompatibility, high cellular uptake and endosomal escape, we conjectured that the 100-nm MSNs are more favorable for the intracellular delivery of drugs in live cells.

[Practical exploration of teaching microbiology in English to promote research and study].

Microbes are involved in every aspect of human life. Microbiology is a mandatory subject at the undergraduate level covering majors including life sciences, pharmacy, medicine, agriculture, forestry and food. Along with internationalization and development of the first-class disciplines, teaching microbiology courses in English is highly valued. Here we discuss how to conduct curriculum reform of microbiology teaching in English, and what are the advantages and challenges when teaching in English. The teaching system can be advanced by enhancing interdisciplinary communication so as to promote study and research for students and teachers. We take this practical exploration as an example to communicate with relevant teachers.

Fabrication and evaluation of a BMP-2/dexamethasone co-loaded gelatin sponge scaffold for rapid bone regeneration.

Improving the osteogenic activity of BMP-2 in vivo has significant clinical application value. In this research, we use a clinical gelatin sponge scaffold loaded with BMP-2 and dexamethasone (Dex) to evaluate the osteogenic activity of dual drugs via ectopic osteogenesis in vivo. We also investigate the mechanism of osteogenesis induced by BMP-2 and Dex with C2C12, a multipotent muscle-derived progenitor cell. The results show that the gelatin scaffold with Dex and BMP-2 can significantly accelerate osteogenesis in vivo. It is indicated that compared with the BMP-2 or Dex alone, 100 nM of Dex can dramatically enhance the BMP-2-induced alkaline phosphatase activity (ALP), ALP mRNA expression and mineralization. Further studies show that 100 nM of Dex can maintain the secondary structure of BMP-2 and facilitate recognition of BMP-2 with its receptors on the surface of C2C12 cells. We also find that in C2C12, Dex has no obvious effect on the BMP-2-induced Smad1/5/8 protein expression and the STAT3-dependent pathway, but Runx2-dependent pathway is involved in the Dex-stimulated osteoblast differentiation of BMP-2 both in vitro and in vivo. Based on these results, a potential mechanism model about the synergistic osteoinductive effect of Dex and BMP-2 in C2C12 cells via Runx2 activation is proposed. This may provide a theoretical basis for the pre-clinical application of Dex and BMP-2 for bone regeneration.

Toward improvement of erythromycin A production in an industrial Saccharopolyspora erythraea strain via facilitation of genetic manipulation with an artificial attB site for specific recombination.

Large-scale production of erythromycin A (Er-A) relies on the organism Saccharopolyspora erythraea, in which lack of a typical attB site largely impedes the application of phage ΦC31 integrase-mediated recombination into site-specific engineering. We herein report construction of an artificial attB site in an industrial S. erythraea strain, HL3168 E3, in an effort to break the bottleneck previously encountered during genetic manipulation mainly from homologous or unpredictable nonspecific integration. Replacement of a cryptic gene, nrps1-1, with a cassette containing eight attB DNA sequences did not affect the high Er-producing ability, setting the stage for precisely engineering the industrial Er-producing strain for foreign DNA introduction with a reliable conjugation frequency. Transfer of either exogenous or endogenous genes of importance to Er-A biosynthesis, including the S-adenosylmethionine synthetase gene for positive regulation, vhb for increasing the oxygen supply, and two tailoring genes, eryK and eryG, for optimizing the biotransformation at the late stage, was achieved by taking advantage of this facility, allowing systematic improvement of Er-A production as well as elimination of the by-products Er-B and Er-C in fermentation. The strategy developed here can generally be applicable to other strains that lack the attB site.

GAP promoter library for fine-tuning of gene expression in Pichia pastoris.

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (ß-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.

Cloning and characterization of Kluyveromyces marxianus Hog1 gene.

The mitogen-activated protein kinase Hog1 gene (Kmhog1) was isolated from Kluyveromyces marxianus strain NBRC 1777 by degenerate PCR and genome walking, and then disrupted to construct a mutant strain hog1∆. The mutant was now more sensitive to acetic acid and its growth was nearly completely inhibited by 0.5 M NaCl (97%) and 10 mM H(2)O(2) (93%) as compared with the wild-type cells. However, neither strain grew at 47°C. Kmhog1 may thus be required for adaptation to acetic acid, osmotic, and oxidative stress but is not involved in thermotolerance.

Advances in engineered Bacillus subtilis biofilms and spores, and their applications in bioremediation, biocatalysis, and biomaterials.

Bacillus subtilis is a commonly used commercial specie with broad applications in the fields of bioengineering and biotechnology. B. subtilis is capable of producing both biofilms and spores. Biofilms are matrix-encased multicellular communities that comprise various components including exopolysaccharides, proteins, extracellular DNA, and poly-γ-glutamic acid. These biofilms resist environmental conditions such as oxidative stress and hence have applications in bioremediation technologies. Furthermore, biofilms and spores can be engineered through biotechnological techniques for environmentally-friendly and safe production of bio-products such as enzymes. The ability to withstand with harsh conditions and producing spores makes Bacillus a suitable candidate for surface display technology. In recent years, the spores of such specie are widely used as it is generally regarded as safe to use. Advances in synthetic biology have enabled the reprogramming of biofilms to improve their functions and enhance the production of value-added products. Globally, there is increased interest in the production of engineered biosensors, biocatalysts, and biomaterials. The elastic modulus and gel properties of B. subtilis biofilms have been utilized to develop living materials. This review outlines the formation of B. subtilis biofilms and spores. Biotechnological engineering processes and their increasing application in bioremediation and biocatalysis, as well as the future directions of B. subtilis biofilm engineering, are discussed. Furthermore, the ability of B. subtilis biofilms and spores to fabricate functional living materials with self-regenerating, self-regulating and environmentally responsive characteristics has been summarized. This review aims to resume advances in biological engineering of B. subtilis biofilms and spores and their applications.

Real-time and on-line monitoring of ethanol fermentation process by viable cell sensor and electronic nose.

In this study, introduction of a viable cell sensor and electronic nose into ethanol fermentation was investigated, which could be used in real-time and on-line monitoring of the amount of living cells and product content, respectively. Compared to the conventional off-line biomass determination, the capacitance value exhibited a completely consistent trend with colony forming units, indicating that the capacitance value could reflect the living cells in the fermentation broth. On the other hand, in comparison to the results of off-line determination by high-performance liquid chromatography, the ethanol concentration measured by electronic nose presented an excellent consistency, so as to realize the on-line monitoring during the whole process. On this basis, a dynamic feeding strategy of glucose guided by the changes of living cells and ethanol content was developed. And consequently, the ethanol concentration, productivity and yield were enhanced by 15.4%, 15.9% and 9.0%, respectively. The advanced sensors adopted herein to monitor the key parameters of ethanol fermentation process could be readily extended to an industrial scale and other similar fermentation processes.

Significantly Improving the Bioefficacy for Rheumatoid Arthritis with Supramolecular Nanoformulations.

As a typical inflammatory disease with chronic pain syndromes, rheumatoid arthritis (RA) generally requires long-term treatment with frequent injection administration at 1-2 times per day, because common medications such as interleukin1 receptor antagonist (IL1ra) have poor bioavailability and very limited half-life residence. Here a novel strategy to fabricate nanotherapeutic formulations employing genetically engineered IL1ra protein complexes, yielding ultralong-lasting bioefficacy is developed rationally. Using rat models, it is shown that these nanotherapeutics significantly improved drug regimen to a single subcutaneous administration in a 14-day therapy, suggesting their extraordinary bioavailability and ultralong-acting pharmacokinetics. Specifically, the half-life and bioavailability of the nanoformulations are boosted to the level of 30 h and by 7 times, respectively, significantly greater than other systems. This new strategy thus holds great promise to potently improve patient compliance in RA therapy, and it can be adapted for other therapies that suffer similar drawbacks.

A novel strategy for tumor therapy: targeted, PAA-functionalized nano-hydroxyapatite nanomedicine.

The rapid development of nanotechnology has provided new strategies for the treatment of tumors. Nano-scale hydroxyapatite (HAP), as the main component of hard tissues in humans and vertebrates, have been found to specifically inhibit tumor cells. However, achieving controllable synthesis of HAP and endowing it with cancer cell-targeting properties remain enormous challenges. To solve this problem, we developed polyacrylic acid-coordinated hydroxyapatite nanoparticles (HAP-PAA) and further chemically grafted them with folic acid (HAP-PAA-FA) for cancer treatment in this study. The nucleation sites and steric hindrance provided by the PAA greatly inhibited the agglomeration of the nanoparticles, and at the same time, the excess functional groups further modified the surface of nanoparticles to achieve targeting efficiency. The spherical, low-crystallinity HAP-PAA nanoparticles exhibited good tumor cell lethality. After grafting the nanoparticles with folic acid for molecular targeting, their cellular uptake and specific killing ability of tumor cells were further enhanced. The HAP-PAA-FA nanoparticle system exerted a regulatory effect on the tumor microenvironment and had good biological safety. All the above results indicate that this research will broaden the application of hydroxyapatite in tumor treatment.

Optimization of L: -methionine feeding strategy for improving S-adenosyl-L: -methionine production by methionine adenosyltransferase overexpressed Pichia pastoris.

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-L: -methionine (SAM). Two L: -methionine (L: -Met) addition strategies were used to supply the precursor: the batch addition strategy (L: -Met was added separately at three time points) and the continuous feeding strategies (L: -Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l(-1) h(-1), respectively). SAM accumulation, L: -Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 +/- 0.31 g l(-1), 41.7 +/- 1.4%, and 0.18 +/- 0.01 g l(-1) h(-1) with the best continuous feeding strategy (0.2 g l(-1) h(-1)), respectively. The bottleneck for SAM production with the low L: -Met feeding rate (0.1 g L(-1) h(-1)) was the insufficient L: -Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing L: -Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the L: -Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the L: -Met feeding rate reached 0.5 g l(-1) h(-1).

A systematical investigation on the genetic stability of multi-copy Pichia pastoris strains.

A battery of Pichia pastoris transformants, G1, G6, A2, A3, C3, carrying 1, 6, 12, 18 and 29 copies of porcine insulin precursor (PIP) gene, were employed to investigate the genetic stability of these multi-copy P. pastoris strains. Both G6 and C3 maintained their original copy numbers in serial culture without methanol induction for 35 generations. With methanol as an inducer and carbon source, G1 and G6 remained stable but the average copy numbers (ACNs) of PIP gene in A2, A3, C3 were decreased to 10, 10 and 15 copies, respectively, after 96 h of induction in shake-flask culture. A PIP copy number distribution analysis of fermentation samples of C3 indicated that the majority of yeast cells have partially or completely lost their PIP genes. In 5-l fermentor culture, the ACNs of PIP gene in A2, A3, C3 were also decreased to 10, 15, 21 copies, respectively, after 72 h of methanol induction.