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The coordination of single or multiple microorganisms are required for the manufacture of traditional fermented foods, improving the flavour and nutrition of the food materials. However, both the additional economic benefits and safety concerns have been raised by microbiotas in fermented products. Among the fermented products, Lactobacillus and Saccharomyces cerevisiae are one of the stable microbiotas, suggesting their interaction is mediated by coexistence-relevant mechanisms and prevent to be excluded by other microbial species. Thus, aiming to guide the manufacture of fermented foods, this review will focus on interactions of coexistence-relevant mechanisms between Lactobacillus and S. cerevisiae, including metabolites communications, aggregation, and polymicrobial biofilm. Also, the molecular regulatory network of the coexistence-relevant mechanisms is discussed according to omics researches.

Objectives Pseudomonas aeruginosa is a medically important pathogen showing intrinsic low permeability to various antimicrobial agents and its potential to acquire multiple resistance mechanism. A longitudinal surveillance aimed to investigate the antimicrobial resistance and its determinants of Pseudomonas aeruginosa in Southern China. A total of 2163 P. aeruginosa isolates were obtained from patients in Southern China during 2004–2016. Methods The antimicrobial susceptibility of the isolates was performed by disk diffusion and Vitek 2 automated system and interpreted according to the Clinical and Laboratory Standard Institute (CLSI) 2015. Results A significant downtrend of resistant rate (>10.0%) was observed for tested antibiotic agents including ciprofloxacin (>30.0%), gentamicin (29.0%), tobramycin (24.2%) and ceftazidime (24.0%) except for aztreonam and amikacin. A total of 269 randomly selected isolates were further studied on the carriage of β-lactam resistance genes by using 7 groups of multiplex PCRs targeting on 20 genes. β-lactam resistance genes were rarely detected with a rate lower than 8%. Among all β-lactam resistance genes, blaSHV acquired the highest identification rate (18/269, 6.7%), followed by blaOXA-1-like (6/269, 2.2%) and blaPER (6/269, 2.2%). In addition, 8 different plasmid replicons were amplified using 8 groups of multiplex PCRs including 18 sets of primers. Only five plasmid replicons were identified in 5 different P. aeruginosa isolates. Insignificant clonal relatedness among the positive strains identified by regular PCR were further verified by randomly amplified polymorphic DNA (RAPD)-PCR. Conclusion This study has provided comprehensive knowledge on current antimicrobial resistance, β-lactam resistance genes and plasmid replicons carriage in a large scale of clinical P. aeruginosa isolates.

Pediococcus acidilactici may significantly reduce the pH-value, and thus has different influence, including serving as a probiotic in human microbiota but a spoilage in human food as it could change the flavor. Pediococcus acidilactici is also capable of entering into the viable but non-culturable (VBNC) state causing false negative results of standard culture-based detection method. Thus, development of detection method for VBNC state P. acidilactici is of great significance. In this study, propidium monoazide (PMA) combined with cross priming amplification (CPA) was developed to detect the VBNC cells of P. acidilactici and applied on the detection in different systems. With detection limit of 104 cells/ml, high sensitivity, and 100% specificity, PMA-CPA can successfully detect VBNC cells of P. acidilactici and be applied in with high robustness.

Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called “standards,” “guidelines,” or “gold standards” are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/μl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.

Salmonella enterica is a typical foodborne pathogen with multiple toxic effects, including invasiveness, endotoxins, and enterotoxins. Viable but nonculturable (VBNC) is a type of dormant form preserving the vitality of microorganisms, but it cannot be cultured by traditional laboratory techniques. The aim of this study is to develop a propidium monoazide-crossing priming amplification (PMA-CPA) method that can successfully detect S. enterica rapidly with high sensitivity and can identify VBNC cells in food samples. Five primers (4s, 5a, 2a/1s, 2a, and 3a) were specially designed for recognizing the specific invA gene. The specificity of the CPA assay was tested by 20 different bacterial strains, including 2 standard S. enterica and 18 non-S. enterica bacteria strains covering Gram-negative and Gram-positive isolates. Except for the two standard S. enterica ATCC14028 and ATCC29629, all strains showed negative results. Moreover, PMA-CPA can detect the VBNC cells both in pure culture and three types of food samples with significant color change. In conclusion, the PMA-CPA assay was successfully applied on detecting S. enterica in VBNC state from food samples.

Formation of viable but non-culturable (VBNC) status in methicillin-resistant Staphylococcus aureus (MRSA) has never been reported, and it poses a significant concern for food safety. Thus, this study aimed to firstly develop a rapid, cost-effective, and efficient testing method to detect and differentiate MRSA strains in the VBNC state and further apply this in real food samples. Two targets were selected for detection of MRSA and toxin, and rapid isothermal amplification detection assays were developed based on cross-priming amplification methodology. VBNC formation was performed for MRSA strain in both pure culture and in artificially contaminated samples, then propidium monoazide (PMA) treatment was further conducted. Development, optimization, and evaluation of PMA-crossing priming amplification (CPA) were further performed on detection of MRSA in the VBNC state. Finally, application of PMA-CPA was further applied for detection on MRSA in the VBNC state in contaminated food samples. As concluded in this study, formation of the VBNC state in MRSA strains has been verified, then two PMA-CPA assays have been developed and applied to detect MRSA in the VBNC state from pure culture and food samples.

Site-directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity, which is advantageous for many applications. Here, we report a facile, specific, and efficient strategy based on the SpyTag-SpyCatcher chemistry. Two SpyTag-fused model proteins, that is, the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl-7-aminocephalosporanic acid acylase, were easily immobilized onto a SpyCatcher-modified resin directly from cell lysates, with activity recoveries in the range of 85-91%. This strategy was further adapted to protein purification, which proceeded through the selective capture of the SpyCatcher-fused target proteins by a SpyTag-modified resin, with the aid of an intein to generate authentic N-termini. For two model proteins, that is, RFP and a variable domain of a heavy chain antibody, the yields were ∼3-7 mg/L culture with >90% purities. This approach could provide a versatile tool for producing high-performance immobilized protein devices and proteins for industrial and therapeutic uses.

Cronobacter sakazakii is an emerging opportunistic foodborne pathogen causing rare but severe infections in neonates. Furthermore, the formation of biofilm allows C. sakazakii to persist in different environments. We have demonstrated that the mutator phenotype ascribed to deficiency of the pmrA gene results in more biomass in the first 24 h but less during the post maturation stage (7–14 d) compared with BAA 894. The present study aimed to investigate the regulatory mechanism modulating biofilm formation due to pmrA mutation. The transcriptomic analyses of BAA 894 and s-3 were performed by RNA-sequencing on planktonic and biofilm cells collected at different time points. According to the results, when comparing biofilm to planktonic cells, expression of genes encoding outer membrane proteins, lysozyme, etc. were up-regulated, with LysR family transcriptional regulators, periplasmic proteins, etc. down-regulated. During biofilm formation, cellulose synthase operon genes, flagella-related genes, etc. played essential roles in different stages. Remarkably, pmrA varies the expression of a number of genes related to motility, biofilm formation, and antimicrobial resistance, including srfB, virK, mviM encoding virulence factor, flgF, fliN, etc. encoding flagellar assembly, and marA, ramA, etc. encoding AraC family transcriptional regulators in C. sakazakii. This study provides valuable insights into transcriptional regulation of C. sakazakii pmrA mutant during biofilm formation.

The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible λRed recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.

To deregulate the purine operon of the purine biosynthetic pathway and optimize energy generation of the respiratory chain to improve the yield of guanosine in Bacillus amyloliquefaciens XH7. The 5′-untranslated region of the purine operon, which contains the guanine-sensing riboswitch, was disrupted. The native promoter Pw in B. amyloliquefaciens XH7 was replaced by different strong promoters. Among the promoter replacement mutants, XH7purE::P41 gave the highest guanosine yield (16.3 g/l), with an increase of 23% compared with B. amyloliquefaciens XH7. The relative expression levels of the purine operon genes (purE, purF, and purD) in the XH7purE::P41 mutant were upregulated. The concentration of inosine monophosphate (IMP), the primary intermediate in the purine pathway, was also significantly increased in the XH7purE::P41 mutant. Combined modification of the low-coupling branched respiratory chains (cytochrome bd oxidase) improved guanosine production synergistically. The final guanosine yield in the XH7purE::P41△cyd mutant increased by 41% to 19 g/l compared with B. amyloliquefaciens XH7. The combined modification strategy used in this study is a novel approach to improve the production of guanosine in industrial bacterial strains.

To expand the repertoire of strong promoters for high level expression of proteins based on the transcriptome of Bacillus licheniformis. The transcriptome of B. licheniformis ATCC14580 grown to the early stationary phase was analyzed and the top 10 highly expressed genes/operons out of the 3959 genes and 1249 operons identified were chosen for study promoter activity. Using beta-galactosidase gene as a reporter, the candidate promoter pBL9 exhibited the strongest activity which was comparable to that of the widely used strong promoter p43. Furthermore, the pro-transglutaminase from Streptomyces mobaraensis (pro-MTG) was expressed under the control of promoter pBL9 and the activity of pro-MTG reached 82 U/ml after 36 h, which is 23% higher than that of promoter p43 (66.8 U/ml). In our analyses of the transcriptome of B. licheniformis, we have identified a strong promoter pBL9, which could be adapted for high level expression of proteins in the host Bacillus subtilis.

This study investigated the effect of the β2 receptor agonist terbutaline on the single channel activity of BKCa channel. The effects of racemate and two isomers of terbutaline were all assessed. β2 adrenoceptors were stably overexpressed on HEK293 cells by lentiviral transduction method and chicken BKCa channels were transiently expressed on normal HEK293 cell line or HEK293 cells overexpressing β2 receptors. Data showed that terbutaline significantly increased the single channel open probability of BKCa channel within 10min. The channel activating effects of terbutaline are stereoselective and mainly stay with the R-enantiomers. The opening probability of BKCa channel at 10min after drug application normalized to that just before drug application (Po10/Po0s) for R- and S-terbutaline were 7.85±3.20 and 1.06±0.45 respectively at 1μM concentration, corresponding to 28.37±9.96 and 2.68±1.09 at the higher concentration of 10μM. ICI 118551 blocked the effect of R- but not S-terbutaline (10μM), whereas atropine blocked the channel activating effects of S-terbutaline of higher concentration. In addition, the muscarinic receptor agonist carbachol increased the BKCa channel activity in an atropine-sensitive manner as an positive control experiment, which indicate the involvement of M receptor in the channel activating effect of S-terbutaline.

Tannin acyl hydrolase (tannase, EC3.1.1.20) catalyzes the hydrolysis of hydrolyzable tannins. It is used in the manufacture of instant tea and in the production of gallic acid. In this study, we reported that the overexpression, purification and characterization of an Aspergillus niger tannase. The tannase gene was cloned from A. niger SH-2 and expressed in the A. niger strain Bdel4 which is low-background of secreted proteins. The recombinant tannase was purified by desalting, followed by gel filtration for characterization. The tannase activity achieved 111.5 U/mL at 168 h, and the purity of the enzyme in the broth supernatant was estimated to be over 70%. The optimum temperature and pH of the recombinant tannase was ∼40 °C and 7.0, respectively. The tannase activity was inhibited by Mg2+, Ca2+, Cu2+, Ba2+, Ni2+ and EDTA, and was enhanced by Mn2+ and Co2+. Since A. niger is a GRAS microorganism, the recombinant tannase could be purification-free due to its high purity. The results of this study suggested that this recombinant strain could be subjected to large-scale production of A. niger tannase.

To construct a promoter probe vector, pBE-bgaB, to screen strong promoters from Bacillus amyloliquefaciens. 266 colonies containing active promoter elements from the genomic DNA of B. amyloliquefaciens were identified. Among these, promoter P41 exhibited the strongest β-Gal activity in Escherichia coli and B. amyloliquefaciens. Sequence analysis showed that promoter P41 contained P ykuN , a ykuN gene encoding flavodoxin. Optimization of the ribosome-binding site from P41 to P382 improved β-Gal activity by ~ 200%. A new strong promoter for protein expression and genetic engineering of Bacillus species.

Past studies have revealed major difficulties in applications of xylanase in the pulp and paper industry as enzymes isolated from many different species could not tolerate high temperatures or highly alkaline conditions. The thermostable xylanase A from Bacillus halodurans C-125 (C-125 xylanase A) was successfully cloned and expressed in Pichia pastoris with a yield as high as 3361 U/mL in a 2 L reactor. Its thermophilic and basophilic properties (optimal activity at 70 °C and pH 9.0), together with the fact it is cellulase-free, render this enzyme attractive for compatible applications in the pulp and paper industry. The pretreatment of wheat straw pulp with C-125 xylanase A at pH 9.0 and 70 °C for 90 min induced the release of both chromophores (Ab(237), Ab(254), Ab(280)) and hydrophobic compounds (Ab(465)) into the filtrate as well as sugar degradation. Moreover, the addition of 10 U xylanase to 1 g wheat straw pulp (dry weight) as pretreatment improved brightness by 5.2% ISO and decreased the kappa number by 5.0% when followed by hydrogen peroxide bleaching. In addition, compared with two commercial enzymes, Pulpzyme HC and AU-PE89, which are normally incorporated in ECF bleaching of wheat straw pulp, C-125 xylanase A proved to be more effective in enhancing brightness as well as preserving paper strength properties. When evaluating the physical properties of pulp samples, such as tensile index, tearing index, bursting index, and post-color (PC) number, the enzymes involved in pretreating pulps exhibited better or the same performances as chemical treatment. Compared with chemical bleaching, chlorine consumption can be significantly reduced by 10% for xylanase-pretreated wheat straw pulp while maintaining the brightness together with the kappa number at the same level. Scanning electron microscopy revealed significant surface modification of enzyme-pretreated pulp fibers with no marked fiber disruptions.

Compared to classical antibodies, camel heavy chain antibodies (HCAbs) are smaller in size due to lack of the light chain and the first constant domain of the heavy chain (CH1 region). The variable regions of HCAbs (VHHs) are more soluble and stable than that of conventional antibodies (VHs). Even with such simple structure, they are still functional in antigen binding. Although HCAbs have been extensively investigated over the past two decades, most efforts have been based upon low throughput sequence analysis, and there are only limited reports trying to analyze and describe the complete immune repertoire (IR) of camel HCAbs. Here we leveraged the high-throughput data generated by Next Generation Sequencing (NGS) of the variable domains of the antibody heavy chains from three Bactrian camels to conduct in-depth comparative analyses of the immunoglobulin repertoire. These include analyses of the complementary determining region 3 (CDR3) length and distribution, mutation rate, antibody characteristic amino acids, the distribution of the cysteine (Cys) codons, and the non-classical VHHs. We found that there is higher diversity in the CDR2 than in the other sub-regions, and there is a higher mutation rate in the VHHs than in the VHs (P < 0.05). In addition to substitutions at amino acid (AA) residue positions NO.49/50/52 between VH and VHH clones, we also observed other substitutions at the positions NO.40/54/57/96/101 that could lead to additional structural alterations. We also found that VH-derived VHH clones, referred to as non-classical VHH clones in this study, accounted for about 8% of all clones. Further, only 5%-10% clones had the Trp > Arg AA substitution at the first position of framework 4 for all types of clones. We present, for the first time, a relatively complete picture of the Bactrian camel antibody immune repertoire, including conventional antibody (Ab) and HCAbs, using PCR and in silico analysis based on high-throughput NGS data.

By predicting the potential signal peptides from proteins that are naturally secreted by Pichia pastoris, we identified three possible endogenous signal peptides: Scw, Dse and Exg. We compared their capability to mediate the secretion of enhanced green fluorescent protein (EGFP) and Candida antarctica lipase B (CALB) with that of the Saccharomyces cerevisiae α-factor prepro-signal. EGFP entered the secretory pathway of P. pastoris and was efficiently secreted into the culture medium by all three endogenous peptides. Further, these three putative endogenous signal peptides were also effective in secreting CALB. These endogenous signal peptides thus have the potential to mediate the efficient secretion of heterologous proteins in P. pastoris.

To develop a high efficiency Candida antarctica lipase B (CALB) yeast display system, we linked two CALB genes fused with Sacchromyces cerevisiae cell wall protein genes, the Sed1 and the 3'-terminal half of Sag1, separately by a 2A peptide of foot-and-mouth disease virus (FMDV) in a single open reading frame. The CALB copy number of recombinant strain KCSe2ACSa that harbored the ORF was identified, and the quantity of CALB displayed on the cell surface and the enzyme activity of the strain were measured. The results showed that the fusion of multiple genes linked by 2A peptide was translated into two independent proteins displayed on the cell surface of stain KCSe2ACSa. Judging from the data of immunolabeling assay, stain KCSe2ACSa displayed 94 % CALB-Sed1p compared with stain KCSe1 that harbored a single copy CALB-Sed1 and 64 % CALB-Sag1p compared with stain KCSa that harbored a single copy CALB-Sag1 on its surface. Besides, strain KCSe2ACSa possessed 170 % hydrolytic activity and 155 % synthetic activity compared with strain KCSe1 as well as 144 % hydrolytic activity and 121 % synthetic activity compared with strain KCSa. Strain KCSe2ACSa even owned 124 % hydrolytic activity compared with strain KCSe2 that harbored two copies CALB-Sed1. The heterogeneous glycosylphosphatidylinositol-anchored proteins co-displaying yeast system mediated by FMDV 2A peptide was shown to be an effective method for improving the efficiency of enzyme-displaying yeast biocatalysts.

Conventional lipase screening methods are mostly based on hydrolytic activity, which may not always be the best method to assess the enzyme activity, especially for evaluating synthetic activity. Here we developed a high throughput and visual method to screen clones with high synthetic activity and used it to assess lipases thermostability. All mutants’ lipase synthetic activity were identified through esterification of caprylic acid and ethanol with methyl red as the pH indicator adding in the substrates on according to the color change halo around the colony on culture plates since synthetic reaction was often accompanied with a rise in pH. After two rounds operation with the pH indicator screening method, we obtained a double mutant Asn120Lys/Lys131Phe from the Rhizomucor miehei lipase saturation mutated library based on amino acid residue B factors. The mutant's initial synthetic activity was a little higher than wild type and its thermostability in synthetic reaction was enhanced, which remained 63.1% residual activity after being heated at 70 °C for 5 h comparing to 51.0% of wild type. The double mutant with the two residue replacements balanced well between stability and activity. Yeast surface display technology and the pH indicator method, combined with colony screening were shown to facilitate high-throughput screening for lipase synthetic activity.

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.

Extensive alteration of gene expression and metabolic remodeling enable the budding yeast Saccharomyces cerevisiae to ensure cellular homeostasis and adaptation to heat shock. The response logic of the cells to heat shock is still not entirely clear. In this study, we combined the expression profiles with metabolic pathways to investigate the logical relations between heat shock response metabolic pathways. The results showed that the heat-stressed S. cerevisiae cell accumulated trehalose and glycogen, which protect cellular proteins against denaturation, and modulate its phospholipid structure to sustain stability of the cell wall. The TCA cycle was enhanced, and the heat shock-induced turnover of amino acids and nucleotides served to meet the extra energy requirement due to heat-induced protein metabolism and modification. The enhanced respiration led to oxidative stress, and subsequently induced the aldehyde detoxification system. These results indicated that new insight into the response logic of S. cerevisiae to heat shock can be gained by integrating expression profiles and the logical relations between heat shock response metabolic pathways.

Measuring the viability of the cells after chemical or physical stresses is a routine method in the determination of phenotypes in yeast. Numerous analytical techniques have been applied to the measurement of yeast cell viability, but few of them are suitable for real-time monitoring. Here we propose a microplate-based method for real-time monitoring of Saccharomyces cerevisiae cell viability. This method is also suitable for high-throughput measurement.

The methylotrophic yeast Pichia pastoris is an attractive platform for a plethora of recombinant proteins. There is growing evidence that host cells producing recombinant proteins are exposed to a variety of cellular stresses resulting in the induction of the unfolded protein response (UPR) pathway. At present, there is only limited information about the cellular reactions of the host cells at the level of the proteome, especially with regard to recombinant protein secretion. Here we monitored xylanase A secretion from Bacillus halodurans C-125 (xynA) in P. pastoris, using strains containing different copy numbers of the gene encoding xylanase A and co-overexpressing the gene encoding the UPR-regulating transcription factor HAC1 by applying a quantitative proteomics approach (iTRAQ-LC–MS/MS). Many important cellular processes, including carbon metabolism, stress response and protein folding are affected in the investigated conditions. Notably, the analysis revealed that strong over-expression of xynA can efficiently improve protein production but simultaneously cause an unfolded protein burden with a subsequent induction of the UPR. This limits the further improvement of protein production levels. Remarkably, constitutive expression of the gene encoding HAC1 lessens the unfolded protein burden by attenuating protein synthesis and increasing ER protein folding efficiency which is beneficial for protein secretion. Biological significance Pichia pastoris expression systems have been successfully used for over 20 years in basic research and in the biotechnology industry for the production and secretion of a wide range of recombinant proteins. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. It has become obvious that many protein products can lead to severe stress on the host cell when being over-expressed, thus limiting the potential yield. Detailed understanding of the physiological responses to such stresses gives rise to engineering of host cells that can better cope with the stress factors. Therefore, the regulatory mechanism of heterologous protein secretion by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavor in improving protein annotation. Many important cellular processes, including carbon and amino acid metabolism, stress response and protein folding are affected in the over-expression strains. This data represent a first step towards a systems wide approach to assess the response with recombinant protein induced stress in P. pastoris.

Background The methylotrophic yeast Pichia pastoris is widely used as a bioengineering platform for producing industrial and biopharmaceutical proteins, studying protein expression and secretion mechanisms, and analyzing metabolite synthesis and peroxisome biogenesis. With the development of DNA microarray and mRNA sequence technology, the P. pastoris transcriptome has become a research hotspot due to its powerful capability to identify the transcript structures and gain insights into the transcriptional regulation model of cells under protein production conditions. The study of the P. pastoris transcriptome helps to annotate the P. pastoris transcript structures and provide useful information for further improvement of the production of recombinant proteins. Results We used a massively parallel mRNA sequencing platform (RNA-Seq), based on next-generation sequencing technology, to map and quantify the dynamic transcriptome of P. pastoris at the genome scale under growth conditions with glycerol and methanol as substrates. The results describe the transcription landscape at the whole-genome level and provide annotated transcript structures, including untranslated regions (UTRs), alternative splicing (AS) events, novel transcripts, new exons, alternative upstream initiation codons (uATGs), and upstream open reading frames (uORFs). Internal ribosome entry sites (IRESes) were first identified within the UTRs of genes from P. pastoris, encoding kinases and the proteins involved in the control of growth. We also provide a transcriptional regulation model for P. pastoris grown on different carbon sources. Conclusions We suggest that the IRES-dependent translation initiation mechanism also exists in P. pastoris. Retained introns (RIs) are determined as the main AS event and are produced predominantly by an intron definition (ID) mechanism. Our results describe the metabolic characteristics of P. pastoris with heterologous protein production under methanol induction and provide rich information for further in-depth studies of P. pastoris protein expression and secretion mechanisms.

The imitation of translation, as mediated by internal ribosome entry sites, has not yet been reported in Pichia pastoris. An IRES element from Saccharomyces cerevisiae was demonstrated to direct the translation of a dicistronic mRNA in P. pastoris. The 5′-untranslated region of GPR1 mRNA, termed GPR, was cloned into a dual reporter construct containing an upstream Rhizomucor miehei lipase (RML) and a downstream β-galactosidase gene (lacZ) from Escherichia coli BL21. After being transformed into P. pastoris, the RML gene and lacZ were simultaneously expressed. The possibility of DNA rearrangement, spurious splicing, or cryptic promoter in the GPR sequence were eliminated, indicating that expression of a second ORF was IRES-dependent. These findings strongly suggested that the IRES-dependent translation initiation mechanism is conserved in P. pastoris and provides a useful means to express multiple genes simultaneously.