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The stability and high productivity of heterogeneous terpenoid production in Escherichia coli expression system is one of the most key issues for its large scale industrialization. In the current study on taking lycopene biosynthesis as an example, an integrated Escherichia coli system has been generated successfully, which resulted into stable and high lycopene production. In this process, two modules of mevalonate (MVA) pathway and one module of lycopene expression pathway were completely integrated in the chromosome. Firstly, the copy number and integrated position of three modules of heterologous pathways were rationally optimized. Later, a strain DH416 equipped with heterogeneous expression pathways through chromosomal integration was efficiently derived from parental strain DH411. The evolving DH416 strain efficiently produced the lycopene level of 1.22 g/L (49.9 mg/g DCW) in a 5 L fermenter with mean productivity of 61.0 mg/L/h. Additionally, the integrated strain showed more genetic stability than the plasmid systems after successive 21st passage.

Abstract Background Embryonic 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. Methods In 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. Results Via 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. Conclusions This differentiation model provided a high controllability of some key factors and brought a novel insight into the deriving mechanism of Sertoli cells.

Abstract 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.

Abstract 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.

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 factors Sry, Sox9, Sf1, Wt1, Gata4, and Dmrt1. 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.

Abstract 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.

Abstract 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.

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.

Abstract 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.