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Propanol had been widely used as a precursor for erythromycin synthesis in industrial production. However, the knowledge on the exact metabolic fate of propanol was still unclear. In the present study, the metabolic fate of propanol in industrial erythromycin-producing strain Saccharopolyspora erythraea E3 was explored via 13C labeling experiments. An unexpected pathway in which propanol was channeled into tricarboxylic acid cycle was uncovered, resulting in uneconomic catabolism of propanol. By deleting the sucC gene, which encodes succinyl-CoA synthetase that catalyse a reaction in the unexpected propanol utilization pathway, a novel strain E3-ΔsucC was constructed. The strain E3-ΔsucC showed a significant enhancement in erythromycin production in the chemically defined medium compared to E3 (786.61 vs 392.94 mg/L). Isotopically nonstationary 13C metabolic flux analysis were employed to characterize the metabolic differences between Saccharopolyspora erythraea E3 and E3-ΔsucC. The results showed that compared with the starting strain E3, the fluxes of pentose phosphate pathway in E3-△sucC increased by almost 200%. The flux of the metabolic reaction catalyzed by succinyl-CoA synthetase in E3-ΔsucC was almost zero, while the glyoxylate bypass flux significantly increased. These new insights into the precursor utilization of antibiotic biosynthesis by rational metabolic engineering in Saccharopolyspora erythraea provided the new vision in increasing industrial production of secondary metabolites.

Porcine circovirus Type 2 (PCV2) is a primary etiological pathogen of post-weaning multi-systemic wasting syndrome (PMWS). The capsid protein of PCV2 is the crucial immunogenic protein which can induce antibody generation and immune responses. However, there is still a lack of efficient PCV2 vaccines with high immunogenicity. In the current study, we developed a novel engineered PCV2 capsid (∆1-41aa)-pFc fusion protein (PCFP), which comprised a truncated capsid protein of PCV2 and a porcine IgG Fc fragment, fused to the capsid protein of PCV2 at the C-terminus. We found that this novel fusion protein could auto-assemble into virus-like nanoparticles with an estimated mean diameter of 22.6 nm, characterized by transmission electron microscopy. Immunization of BALB/c mice with this fusion protein significantly increased the production levels of anti-PCV2-capsid protein antibody in serum. Besides, the virus-like nanoparticles, PCFP was demonstrated to induce efficient cellular immune responses in mice, as evident by the high specific T cell reactivity to the PCFP fusion protein and the high production of the immune cytokines IFN-γ and IL-10 in an ex vivo re-stimulation system. Collectively, these findings demonstrate that the PCV2 truncated capsid subunit Fc-fusion protein can induce both cellular and humoral immune responses, and it displays great application potential.

The selection of informative variables in partial least squares (PLS) is important in process analytical technology (PAT) applications in the pharmaceutical industry, for example, the calibration of spectrometers. In the past, numerous approaches have been proposed to select the variables in partial least squares. In this work, a new variable selection method for PLS with the weighted variable contribution (PLS-WVC) to the first singular value of the covariance matrix for each PLS component is proposed. Several variants of PLS-WVC with different weighting factors are proposed. One variant of PLS-WVC is equivalent to the PLS with variable importance in projection (PLS-VIP). However, the variants with the correlation between X γ w γ and Y γ q γ as the weighting factor are preferred based on the results of the simulation cases studies. The proposed PLS-WVCs are integrated with interval PLS (iPLS) further to select the informative wavelength intervals for spectroscopic modelling. The utility of the proposed WVC based variable selection methods in PLS is demonstrated with the real spectral data sets.

Propanol had been widely used as a precursor for erythromycin synthesis in industrial production. However, the knowledge on the exact metabolic fate of propanol was still unclear. In the present study, the metabolic fate of propanol in industrial erythromycin-producing strain Saccharopolyspora erythraea E3 was explored via

Embryonic Sertoli cells (eSCs) possess multiple supporting functions and research value in gonadal development and sex determination. However, the limitation of acquiring quality eSCs had hindered the further application. Herein, we successfully derived non-genetically modified (non-GM)-induced embryonic Sertoli-like cells (eSLCs) from mouse embryonic stem cells (ESCs) with a TM4 cell-derived conditioned medium containing recombinant endogenous protein factorsSry,Sox9,Sf1,Wt1,Gata4, andDmrt1. These eSLCs were determined through morphology; transcriptional expression levels of stage-specific, epithelial, and mesenchymal marker genes; flow cytometry, immunofluorescence; and immunocytochemistry and functionally determined by coculture with spermatogonia stem cells. Results indicated that these eSLCs performed similarly to eSCs in specific biomarkers and expression of marker genes and supported the maturation of spermatogonia. The study induced eSLCs from mouse ESCs by defined protein factors. However, the inducing efficiency of the non-GM method was still lower than that of the lentiviral transduction method. Thus, this work established a foundation for future production of non-GM eSLCs for clinical applications and fundamental theory research.

In this study, we prepared a series of edible blend films of xanthan and curdlan by mixing different ratios of these two biopolymers. Characterization techniques like FTIR, XRD, TGA and SEM analysis were applied to investigate the newly formed films. Moreover, mechanical properties, moisture absorbance properties and water solubility of these films were also determined. The obtained results demonstrated that the strong intermolecular hydrogen bonding was observed between xanthan and curdlan at pH 5. At this pH, the xanthan-curdlan hydrogel retained the original structure of xanthan and sustained self-aggregation of xanthan chains via hydrogen bonding, which led to strong intermolecular bonding between xanthan and curdlan. Furthermore, the 5:5 and 4:6 ratios of xanthan and curdlan showed greater interaction in the blend films that resulted in their excellent miscibility. Moreover, highest tensile strength of 28.13 and 26.45 MPa were also found in the same rational of XG/CG blend films. In addition, it was observed that the curdlan incorporation improved the water solubility properties of XG/CG blend films. Conclusively, this xanthan/curdlan nexus with excellent mechanical and moisture barrier properties confirm its potential application and prospective use as food packaging material.

Figure S1. Five constructed plasmids. FUW-lightO-GAVPO is made to produce element GAVPO for lightOn expression system. These plasmids are made for lentiviral transduction. FUW-lightO-Wt1 was constructed from FUW-TetOn-GFP which was applicated to express gene Wt1. The rest plasmids in this paper were done in the same manner.

BackgroundEmbryonic Sertoli cells (eSCs) have been known for playing important roles in male reproductive development system. In current studies, eSCs were mainly generated from induced intermediate mesoderm. The deriving mechanism of eSCs has been unclear so far. Therefore, this work was aimed to reveal the molecular pathways during derivation of eSCs.MethodsIn this scenario, a differentiation model from mouse embryonic stem cells (mESCs) to eSCs was established through spatiotemporal control of 5 key factors, Wilms tumor 1 homolog (Wt1), GATA binding protein 4 (Gata4), nuclear receptor subfamily 5, group A, member 1 (Nr5a1, i.e.,Sf1), SRY (sex determining region Y)-box 9 (Sox9), doublesex, and mab-3 related transcription factor 1 (Dmrt1). To investigate the molecular mechanism, these key factors were respectively manipulated through a light-switchable (light-on) system, tetracycline-switchable (Tet-on) system, and CRISPR/Cas9 knock out (KO) system.ResultsVia the established approach, some embryonic Sertoli-like cells (eSLCs) were induced from mESCs and formed ring-like or tubular-like structures. The key factors were respectively manipulated and revealed their roles in the derivation of these eSLCs. Based on these results, some molecular pathways were mapped during the development of coelomic epithelial somatic cells to eSCs.ConclusionsThis differentiation model provided a high controllability of some key factors and brought a novel insight into the deriving mechanism of Sertoli cells.

To further understand fermentation kinetics of sodium gluconate (SG) production by Aspergillus niger with different inlet oxygen concentrations, logistic model for cell growth and two-step models for SG production and glucose consumption were established. The results demonstrated that the maximum specific growth rate (µm) presented exponential relationship with inlet oxygen concentration and the maximum biomass (Xm) exhibited linear increase. In terms of SG production, two-step model with Luedeking-Piret equation during growth phase and oxygen-dependent equation during stationary phase could well fit the experimental data. Notably, high inlet oxygen concentration exponentially improved SG yield (YP/S), whereas biomass yield to glucose (YX/S) and cell maintenance coefficient (m) were almost independent on inlet oxygen concentration, indicating that high oxygen supply enhancing SG synthesis not only functioning as a substrate directly, but also regulating glucose metabolism towards SG formation. Finally, the applicability and predictability of the proposed models were further validated by additional experiments.

Background Compared with shake flask, bioreactor offers a relatively stable and controllable extracellular environment for cell growth and metabolism. Meanwhile, parallel micro-bioreactor system could well meet the screening flux requirement in the process of high-throughput strain screening. In this study, a self-developed 8-parallel micro-bioreactor system with non-invasive optical biosensors was introduced to substitute the traditional shake flask. Results Optical pH and DO biosensors could be well applied for the process monitoring and controlling in the cultures of commonly used microorganisms through maintaining constant temperature and bioreactor shading treatment. Subsequently, 8-parallel micro-bioreactor system was adopted in the rescreening procedure of high-throughput screening process, and it significantly increased the feasibility of scaling up a cultivating system without any sacrifice on the screening flux. As a result, a designated mutant strain Lactobacillus paracasei S4 was obtained, which presented an improvement of 18.9% on titer value of l-lactic acid. Moreover, the yield also increased from 0.903 ± 0.005 to 0.932 ± 0.013 g/g. Conclusion In this study, the micro-bioreactor system proved to be applicable and effective in the rescreening procedure of high-throughput screening process. The adopted approach provided a robust tool for screening the strain with high l-lactic acid producing performance.

Background In general, high-quality seed is the prerequisite of an efficient bioprocess. However, in terms of sodium gluconate production by Aspergillus niger, reports have seldom focused on seed culture with rational optimization by process analysis technology, especially for carbon source effects. In this study, based on the online physiological parameter of oxygen uptake rate (OUR), and intracellular metabolite profiling, as well as cell morphology analysis, the effects of different initial glucose concentrations on seed culture by A. niger were investigated. Results The optimum initial glucose concentration was 300 g/L, corresponding to 1900 mOsm/kg, with OUR level about 70% higher than other conditions. Besides, the cells from optimized seed culture accumulated more osmoprotectants of alanine and glutamate. Interestingly, high glucose concentration could induce glucose oxidase (GOD) activity possibly by affecting the synthesis of histidine, one key component of active site of GOD. Prominently, the fermentation yield using the optimized seed culture was up to 1.198 g/g, 99% of the theoretical value, which was the best in literature. Conclusion The initial glucose concentration appropriately 300 g/L in seed cultivation was determined to be the most optimal. Further, this study would be helpful for guiding sodium gluconate production on industrial scale.

In the current study, the effect of different available nitrogen sources on erythromycin fermentation by Saccharopolyspora erythraea No. 8 is evaluated. Three different combinations of corn steep liquor and yeast powder were developed to investigate their impacts on erythromycin production. The results indicate that the optimal combination of available nitrogen sources was 10.0 g/L corn steep liquor and 4.0 g/L yeast power, generating a maximum yield of erythromycin of 13672 U/mL. To explore the effects of nitrogen perturbations on cell metabolism, metabolic flux analyses were performed and compared under different conditions. A high flux pentose phosphate pathway provided more NADPH for erythromycin synthesis via nitrogen optimization. Moreover, high n-propanol specific consumption rate enhanced erythromycin synthesis and n-propanol flowed into the central carbon metabolism by methylmalonyl-CoA node. These results indicate that the selection of an appropriate organic nitrogen source is essential for cell metabolism and erythromycin synthesis, and this is the first report of the successful application of available nitrogen source combinations in industrial erythromycin production.

Background Adipose-derived mesenchymal stem cells (ADMSCs) are considered an efficient and important candidate for male infertility treatment because they contain pluripotent stem cells, which can differentiate into all cells from three germ layers. However, the efficient generation of male germ-like cell (MGLCs) is one of the key issues, and little is known about the mechanisms underlying generation of MGLCs. Herein, we attempt to improve the efficient generation of MGLCs derived during co-culturing of rat ADMSCs with SCs under retinoic acid (RA) and testosterone (T) treatment. Methods ADMSCs isolated from male SD rat were induced into generation of MGLCs by using respective methods in vitro. Transwell insert system was used for co-culturing. Busulfan-induced non-obstructive azoospermia rat mode was used to evaluate spermatogenic recovery ability of treated ADMSCs. Besides, the relative gene expression level was detected by reverse transcription PCR, quantitative RT-PCR. The relative protein expression level was detected by western blot (WB) and immunostaining analysis. Results The results showed that ADMSCs co-cultured with TM4 cells under RA and T induction enhanced the formation of bigger and tightly packed MGLCs feature colonies in vitro. Moreover, the expression of male germ cell-related markers (Oct4, Stella, Ddx4, Dazl, PGP9.5, Stra8, and ITGα6) is significantly upregulated in TM4 cell-co-cultured ADMSCs in vitro and in busulfan-treated rat testis after injecting TM4 cell-treated ADMSCs for 2 months. Comparatively, the ADMSCs treated by TM4 cell with RA and T exhibited the highest expression of male germ cell-related markers. RA- and T-treated TM4 cell showed fewer dead cells and higher cytokine secretion than untreated groups. The protein expression level of TGFβ-SMAD2/3, JAK2-STAT3, and AKT pathways in ADMSCs co-cultured with TM4 cells under RA and T was higher than others. Whereas, downregulation of male germ cell-related marker expression subsequently inhibited the phosphorylation of SMAD2/3, JAK2, STAT3, and AKT. Conclusion These results suggested that TM4 cells could efficiently stimulate in vitro generation of MGLCs during co-culturing of ADMSCs under RA and T treatment. Conclusively, the ADMSCs co-cultured with TM4 cell under RA and T induction stimulate the efficient generation of MGLCs in vitro through activating TGFβ-SMAD2/3, JAK2-STAT3, and AKT pathways. Among them, JAK2-STAT3 and AKT pathways are being first reported to show involvement of in vitro generation of MGLCs during ADMSC co-culturing with SCs. Electronic supplementary material The online version of this article (10.1186/s13287-019-1181-5) contains supplementary material, which is available to authorized users.

Background Embryonic Sertoli cells (eSCs) play an important role in sex determination and in male gonad development which makes them a very useful cell type for therapeutic applications. However, the deriving mechanism of Sertoli cells has been unclear and challenging to create a large number of quality eSCs. Therefore, this study aimed to create the eSCs induced from mouse embryonic stem (mES) cells by regulating defined factors and to explore the relevant regulatory mechanism. Methods Six inducing factors, Sry, Sox9, SF1, WT1, GATA4, and Dmrt1, were respectively transduced into mES cells by lentiviral infection according to the experimental design. The test groups were identified by development stage-specific markers, AMH, Emx2, SF1, and FasL, using flow cytometry. Induced eSCs were determined by FasL and AMH biomarkers under immunofluorescence, immunocytochemistry, and flow cytometry. Moreover, the pluripotency markers, gonad development-related markers, epithelial markers and mesenchymal markers in test groups were transcriptionally determined by qPCR. Results In this study, the co-overexpression of all the six factors effectively produced a large population of eSCs from mES cells in 35 days of culturing. These eSCs were capable of forming tubular-like and ring-like structures with functional performance. The results of flow cytometry indicated that the upregulation of GATA4 and WT1 contributed to the growth of somatic cells in the coelomic epithelium regarded as the main progenitor cells of eSCs. Whereas, SF1 facilitated the development of eSC precursor cells, and Sry and Sox9 promoted the determination of male development. Moreover, the overexpression of Dmrt1 was essential for the maintenance of eSCs and some of their specific surface biomarkers such as FasL. The cellular morphology, biomarker identification, and transcriptomic analysis aided in exploring the regulatory mechanism of deriving eSCs from mES cells. Conclusion Conclusively, we have elucidated a differentiation roadmap of eSCs derived from mES cells with a relevant regulatory mechanism. Through co-overexpression of all these six factors, a large population of eSCs was successfully induced occupying 24% of the whole cell population (1 × 105 cells/cm2). By adopting this approach, a mass of embryonic Sertoli cells can be generated for the purpose of co-culture technique, organ transplantation, gonadal developmental and sex determination researches. Electronic supplementary material The online version of this article (10.1186/s13287-019-1180-6) contains supplementary material, which is available to authorized users.

Detailed materials and methods. (DOCX 8979 kb)

Experimental methods. (DOCX 4120 kb)

The main indicators for industrial production of high-quality lactic acid at elevated temperatures are high titer, productivity, yield, and optical purity. However, no such strains have been reported to meet all these requirements simultaneously. In this study, a high optical purity L-lactic acid producing strain is developed through the CRISPR-Cas9 gene editing platform. Further, adaptive evolution was used to breed and select a high-performance strain (NCBIO01-M2-ldhL1-HT) that could efficiently produce L-lactic acid at a high temperature of 45℃. This strain produced 221.0 g/L of L-lactic acid in open fermentation with high initial glucose concentration. Also, L-lactic acid productivity and yield was above 7.5 g/L/h and 0.96 g/g respectively, as well as the optical purity of L-lactic acid in the fermentation broth exceeded 99.1%. In short, this breeding strain possess high potential to be considered for the commercial production of polymer-grade L-lactic acid.

In this study we have established a rational and high performance high-throughput screening system to select for a sophorolipids (SLs) high-producing strain of Candida bombicola. Introduction of mutagen combination, relaxation culture and multi-stress significantly improved both mutation and positive mutation rates. A high-performing strain, Ncbio 5, was selected out of 6212 mutants. Final SLs titer, productivity and yield of this strain in 5 L bioreactors were 26.9%, 27.0% and 35.0% higher than in the original strain, respectively. The improved fermentation performance in Ncbio 5 is contributed by the longer production period, higher SLs productivity and more efficient oil utilization. The strategy adopted herein to optimize the high-throughput screening system should be readily extendable to other similar systems.

This study presents an engineered approach for sustainable biosynthesis of curdlan by Alcaligenes faecalis using orange peels. To confirm the substrate suitability a four step study was organized. Firstly, drying of substrate was carried within temperature range of 60–120 °C, along with the application of moisture diffusion control model. Secondly, fermentation medium was obtained via saccharification and detoxification, releasing highest sugar at 72.34 g/L with phenolics removal of 95–98%. Thirdly, curdlan fermentation was conducted in detoxified orange peel hydrolysate followed by optimization of batch culture fermentation via kinetic modeling using Logistic and Luedeking-Piret equations. In 5 L bioreactor, highest specific growth rate (μm = 0.233/h), highest curdlan production (Pm = 23.24 g/L) and growth associated rate constant (α = 3.403) were achieved. Moreover, the total sugar consumption and conversion rates were 83.27% and 53.20%. Lastly, characterization techniques such as FTIR, NMR, XRD, TGA, HPGPC and EDS were applied to biosynthesized curdlan for qualitative validation.

Curdlan has wide-ranging benefits in food and pharmaceutical industries for its unique rheological and thermal gelling properties. To analyze the cell growth and curdlan biosynthesis kinetics of Alcaligenes faecalis, the kinetic properties of the curdlan fermentation under different carbon sources conditions (maltose, sucrose, glucose and fructose) were investigated using Logistic and Luedeking-Piret equations. The results demonstrated that curdlan fermentation is partial growth-associated process. With maltose as the sole carbon source, the highest curdlan production (Pm = 39.3 g/L), the maximum specific growth rate (μm = 0.44/h) and the growth-associated rate constant (α = 2.05 g curdlan/g cell) were achieved. In contrast, the fructose was the less desired carbon source in both the cell growth and curdlan production. Further, the results demonstrated that slow-releasing glucose from maltose boosted cell growth and curdlan production.

Background Riboflavin, an intermediate of primary metabolism, is one kind of important food additive with high economic value. The microbial cell factory Bacillus subtilis has already been proven to possess significant importance for the food industry and have become one of the most widely used riboflavin-producing strains. In the practical fermentation processes, a sharp decrease in riboflavin production is encountered along with a decrease in the dissolved oxygen (DO) tension. Influence of this oxygen availability on riboflavin biosynthesis through carbon central metabolic pathways in B. subtilis is unknown so far. Therefore the unveiled effective metabolic pathways were still an unaccomplished task till present research work. Results In this paper, the microscopic regulation mechanisms of B. subtilis grown under different dissolved oxygen tensions were studied by integrating 13C metabolic flux analysis, metabolomics and transcriptomics. It was revealed that the glucose metabolic flux through pentose phosphate (PP) pathway was lower as being confirmed by smaller pool sizes of metabolites in PP pathway and lower expression amount of ykgB at transcriptional level. The latter encodes 6-phosphogluconolactonase (6-PGL) under low DO tension. In response to low DO tension in broth, the glucose metabolic flux through Embden–Meyerhof–Parnas (EMP) pathway was higher and the gene, alsS, encoding for acetolactate synthase was significantly activated that may result due to lower ATP concentration and higher NADH/NAD+ ratio. Moreover, ResE, a membrane-anchored protein that is capable of oxygen regulated phosphorylase activity, and ResD, a regulatory protein that can be phosphorylated and dephosphorylated by ResE, were considered as DO tension sensor and transcriptional regulator respectively. Conclusions This study shows that integration of transcriptomics, 13C metabolic flux analysis and metabolomics analysis provides a comprehensive understanding of biosynthesized riboflavin’s regulatory mechanisms in B. subtilis grown under different dissolved oxygen tension conditions. The two-component system, ResD–ResE, was considered as the signal receiver of DO tension and gene regulator that led to differences between biomass and riboflavin production after triggering the shifts in gene expression, metabolic flux distributions and metabolite pool sizes. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0764-z) contains supplementary material, which is available to authorized users.

Additional file 1. Mass isotopomer distributions (MID) of intracellular free amino acids.

The physiology of feeding ammonium sulphate in erythromycin biosynthesis phase of Saccharopolyspora erythraea on the regulation of erythromycin A (Er-A) biosynthesis was investigated in 50 L fermenter. At an optimal feeding ammonium sulphate rate of 0.03 g/L per h, the maximal Er-A production was 8281 U/mL at 174 h of growth, which was increased by 26.3% in comparison with the control (6557 U/mL at 173 h). Changes in cell metabolic response of actinomycete were observed, i.e. there was a drastic increase in the level of carbon dioxide evolution rate and oxygen consumption. Assays of the key enzyme activities and organic acids of S. erythraea and amino acids in culture broth revealed that cell metabolism was enhanced by ammonium assimilation, which might depend on the glutamate transamination pathway. The enhancement of cell metabolism induced an increase of the pool of TCA cycle and the metabolic flux of erythromycin biosynthesis. In general, ammonium assimilation in the erythromycin biosynthesis phase of S. erythraea exerted a significant impact on the carbon metabolism and formation of precursors of the process for dramatic regulation of secondary metabolites biosynthesis.

Heterologous protein expression using Pichia pastoris causes metabolic stress on the physiology of host cells, which may compromise the yields of secreted foreign proteins. Thus, understanding these metabolic stresses during secretory expression allows us to circumvent these undesirable effects. We investigated the effect of co-feeding two alternative carbon resources, sorbitol and yeast extract (YE), on the physiology of A3, a P. pastoris strain carrying 18 copies of the porcine insulin precursor (PIP) gene. Comparative transcriptional analysis was performed on 13 selected genes involved in important cellular processes. Results showed that co-feeding of either sorbitol or YE along with methanol improved the performance of A3. The co-feeding of YE enhanced the specific growth rate of A3 and the specific PIP productivity. However, the oxidative stress in the yeast cells increased. The co-feeding of methanol and sorbitol increased the specific growth rate of A3 but did not affect the specific PIP productivity. The transcriptional results suggested that sorbitol may have repressed the expression of foreign proteins. These observations will not only guide the application of mixed feeding strategies but also give a deeper understanding of the metabolic burden in the secretory expression of foreign proteins.

Effects of feeding different available nitrogen sources from 80 h in erythromycin biosynthesis phase on the erythromycin A (Er-A) production were investigated in 50 l fermenter. Feeding corn steep liquor and yeast extract, the Er-A production was enhanced, while the biotransformation from erythromycin C (Er-C) to Er-A had no increase. When ammonium sulphate was fed at high feeding rate, the maximal Er-A production and ratio of Er-A to Er-C were 7953 U/ml and 98.18:1 at 184 h, respectively, which were higher than that of the control (6742 U/ml and 5.47:1). The feeding ammonium sulphate process was successfully scaled up from 50 l to 25 m(3) fermenter. The maximal Er-A production reached 7938 U/ml at 203 h, which was enhanced by 22.1% compared with the control (6501 U/ml at 192 h). The ratio of Er-A to Er-C was 24.05:1, which was higher than that of the control (4.77:1).

The methylotrophic yeast Pichia pastoris has become a popular host for the production of recombinant protein under the tight control of methanol-induced alcohol oxidase promoter (P AOX1 ), which is, however, strongly repressed by glucose at the transcription level. A real-time parameter-based controlled glucose feed strategy (rpcGFS) is developed here to realize Pichia high-cell density culture. It allows for the high-level production of recombinant phytase by P. pastoris YY113 using glucose as the growth substrate instead of glycerol. As a result, high phytase production (2200 FTU phytase ml −1 ) and yield (0.25 FTU ml −1 [phytase] g −1 [methanol]) are achieved successfully, respectively, after 100 h methanol induction by this novel simple fermentation strategy.

Based on the fact that Pichia cell growth follows a Monod equation under the condition of methanol con- centration limitation, a kinetics model of recombinant methylotrophic yeast Pichia pastoris expressing porcine insulin precursor (PIP) was developed in the quasi-steady state in the induction phase. The model revealed that the relationship between specific growth rate (µ) and substrate methanol concentration was in accord with the Monod equation. The fermentation kinetic parameters maximum specific growth rate (µmax), saturation constant (Ks) and maintenance coeffi- cient (M) were estimated to be 0.101 h −1 , 0.252 g l −1 , and 0.011 g MeOH g −1 DCW h −1 , respectively. The unstructured model was validated in methanol induction phase with different initial cell densities. Results showed that the maximum specific protein production rate (qp.max) of 0.098 mg g −1 DCW h −1 was achieved when µ was kept at 0.016 h −1 , and the maximum yield of PIP reached 0.97 g l −1 , which was 1.5-fold as that of the control. Therefore, the simple Monod model proposed has proven to be a robust control system for recombinant porcine insulin precursor production by P. pastoris on pilot scale, which would be further applied on production scale.

Recombinant phytase expressed in Pichia pastoris FPHY34 is a typical glycoprotein, whose gene possesses 10 potential N-glycosylation sites without O-glycosylation. Effects of glycosylation on phytase's biochemical characterization were investigated. Secreted phytase's micro-heterogenious glycosylation phenomenon was observed and verified by SDS–PAGE, N-terminal amino acid sequencing and mass spectrum analyses. Deglycosylation of recombinant phytase reduced the molecular weight from 83 to 63 kDa, which indicates the presence of total carbohydrate content of approximately 24.1%. According to image analysis of 2-dimension gel electrophoresis, the range of isoeclectric point of phytase covers nearly 1 pH value rather than a specific point, which might be due to heavy and complex glycosylation. The phytase expressed by P. pastoris still retained 40% and 30% activities at 80 and 90 °C for 10 min, respectively. However, upon deglycosylation in vitro by peptide-N-glycohydrolase F (PNGase F), thermostability of deglycosylated phytase significantly declined after 10 min treatment at 40 and 50 °C. Optimum pH of phytase tested was shifted from pH 5.0 to 2.5 by deglycosylation, although there exhibit bi-humps pH optima at both before and after PNGase F digestion cases.

Human papillomavirus virus-like particles (HPV VLPs) have been shown to be effective in preventing cervical cancer when used as a prophylactic vaccine. However, little attention has been paid to the process of L1 expression and self-assembly of VLPs in vivo. In this study, the methanol concentration is optimized during fermentation to optimize L1 expression and self-assembly into VLPs. HPV 16 L1 was expressed in Pichia pastoris GS115, and successfully self-assembled into HPV 16 VLPs. The results demonstrate that there is a strong correlation between methanol concentration and the expression of HPV L1 as well as VLP formation. During the methanol induction phase, low concentrations of residual methanol (0.1-0.3%) promoted the expression of free L1 and increased the total content of L1, whereas a higher residual methanol concentration (0.32-0.6%) was conducive for the self-assembly of LI into VLPs. To conclude, if the residual methanol concentration is too low or too high, the levels of L1 can easily decrease during the fermentation process.

Effects of different nitrogen sources on the erythromycin production were investigated in 50 l fermenter with multi-parameter monitoring system firstly. With the increase of soybean flour concentration from 27 g/l to 37 g/l to the culture medium, the erythromycin production had no obvious increase. Whereas adding corn steep liquor 15 g/l in the medium was beneficial for the production of erythromycin, the maximum erythromycin production was 22.2% higher than that of the control. It was found that corn steep liquor can regulate and enhance the oxygen uptake rate (OUR) which characterizes the activity of the microbial metabolism by inter-scale observation and data association. Both Intracellular and extracellular organic acids of central metabolism were analyzed, and it was found that the whole levels of lactic acid, pyruvic acid, citric acid, and propionic acid were higher than those of control before 64th h. The consumption amount of amino acids, which could be transformed into the precursors for erythromycin synthesis (i.e. threonine, serine, alanine, glycine and phenylalanine), were elevated compared with the control in erythromycin biosynthesis phase. The results indicated that corn steep liquor can regulate OUR to certain level in the early phase of fermentation, and enhance the metabolic flux of erythromycin biosynthesis. Erythromycin production was successfully scaled up from a laboratory scale (50 l fermenter) to an industrial scale (132 m(3) and 372 m(3)) using OUR as the scale-up parameter. Erythromycin production on industrial scale was similar to that at laboratory scale.