Ali Mohsin

Optimized biosynthesis of xanthan via effective valorization of orange peels using response surface methodology: A kinetic model approach

Herein, an enhanced green production of xanthan gum has been achieved by utilizing orange peels. Response surface methodology and kinetic modeling were adapted for the process optimization and its influence on scale up production respectively. Optimal conditions for the maximum xanthan production were 1.62% acid hydrolysis, 85% carbon source of orange peel hydrolysate and 30.4°C temperature. Furthermore, the optimized treatment was conducted in the batch culture fermentor to observe the associated variations during scale up process. In bio-fermentor, to the first time ever, xanthan production along with reducing sugar conversion and utilization rates reached 30.19g/L, 69.29% and 99.99%, respectively. Employed characterization techniques of FTIR, XRD and HPLC confirmed the fermented product as xanthan gum and obtained an average molecular weight of 1.01×106g/mol. This work on account of optimized process parameters presented maximum xanthan production from a waste material.

Application of 8-parallel micro-bioreactor system with non-invasive optical pH and DO biosensor in high-throughput screening of l-lactic acid producing strain

BackgroundCompared 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.ResultsOptical 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 0.903u2009±u20090.005 to 0.932u2009±u20090.013xa0g/g.ConclusionIn 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.

Enhanced production of biosynthesized lycopene via heterogenous MVA pathway based on chromosomal multiple position integration strategy plus plasmid systems in Escherichia coli

The multiple plasmid system, mostly relied, for heterogeneous gene expression, results in genetic instability and low mean productivity. To address this, an integration method was employed for investigating expression of heterogenous pathway in E. coli cells; where mevalonate upper pathway was found efficiently expressed. Subsequently, to improve lycopene production, chromosomal multiple position integration strategy was used to strengthen mevalonate upper pathway. Meanwhile, the plasmid system was employed for mevalonate lower pathway and lycopene pathway expression to finally generate the mutant D711 strain. Comparatively, highest level of 68.5u202fmg/L lycopene was produced by D711 outperforming its maximum average productivity of 2.85u202fmg/L/h with over 2-folds enhancement. In addition, lycopene level was almost 224u202fmg/L after optimization of induction time, which was 3.3-fold higher than standard control condition. Finally, expression Performance Parameter was developed for scoring mutants and evaluating these two strategies, indicating chromosomal multiple position integration strategy as more efficient approach.

An electroporation-free method based on Red recombineering for markerless deletion and genomic replacement in the Escherichia coli DH1 genome

The λ-Red recombination system is a popular method for gene editing. However, its applications are limited due to restricted electroporation of DNA fragments. Here, we present an electroporation-free λ-Red recombination method in which target DNA fragments are excised by I-CreI endonuclease in vivo from the landing pad plasmid. Subsequently, the I-SceI endonuclease-cutting chromosome and DNA double-strand break repair were required. Markerless deletion and genomic replacement were successfully accomplished by this novel approach. Eight nonessential regions of 2.4–104.4 kb in the Escherichia coli DH1 genome were deleted separately with selection efficiencies of 5.3–100%. Additionally, the recombination efficiencies were 2.5–45%, representing an order of magnitude improvement over the electroporation method. For example, for genomic replacement, lycopene expression flux (3.5 kb) was efficiently and precisely integrated into the chromosome, accompanied by replacement of nonessential regions separately into four differently oriented loci. The lycopene production level varied approximately by 5- and 10-fold, corresponding to the integrated position and expression direction, respectively, in the E. coli chromosome.

Exploring cellular fatty acid composition and intracellular metabolites of osmotic-tolerant mutant Lactobacillus paracasei NCBIO-M2 for highly efficient lactic acid production with high initial glucose concentration

High titer, productivity and yield are the main pre-requisites of an efficient lactic acid production process. However, the hyperosmotic stress inhibits cell metabolism in the later phase of fermentation. In this study, an osmotic-tolerant mutant named Lactobacillus paracasei NCBIO01-M2 was obtained through a high-throughput screening technology, which exhibited a higher tolerance to osmotic stress due to its more flexible regulation of the unsaturated fatty acid proportion along with the intracellular compatible solute pools. The mutant successfully consumed all 248u2009g/L initial glucose and produced 223.7u2009g/L lactic acid with a productivity of 5.53u2009g/L/h in a single batch fermentation by the neutralizing agent strategy. Moreover, similar fermentation performances were also achieved in the open fermentation mode without sterilization by the mutant, which suggested that the mutant would be a potential for cost-effective commercial lactic acid production.

Co-culture with TM4 cells enhances the proliferation and migration of rat adipose-derived mesenchymal stem cells with high stemness

The proliferation and migration of mesenchymal stem cells (MSCs) are the efficiency determinants in MSCs transplant therapy. Sertoli cells considered as “nurse cell” possesses the ability to enhance the proliferation and migration of umbilical cord mesenchymal stem cells (UCMSCs). However, no reports about TM4 cells effect on the proliferation and migration of adipose tissue-derived mesenchymal stem cells (ADSCs) have been found until at present research work. Therefore, this study investigates the effect of TM4 cells on the proliferation and migration of ADSCs. We found that the performance of proliferation and migration of ADSCs were improved significantly while maintaining their stemness and reducing their apoptosis rate. After co-culturing with TM4 cells, the co-cultured ADSCs demonstrated higher proportion of synthetic phase (S) cells and colony-forming units-fibroblastic (CFU-F) number, lower proportion of sub-G1 phase cells and enhanced osteogenic and adipogenic differentiation ability. Moreover, results confirmed the higher multiple proteins involved in cell proliferation and migration including expression of the phospho-Akt, mdm2, pho-CDC2, cyclin D1 CXCR4, MMP-2, as well as phospho-p44 MAPK and phospho-p38 MAPK in co-cultured ADSCs. Furthermore, the process of TM4 cells promoting the proliferation of ADSCs was significantly inhibited by the administration of the PI3K/AKT inhibitor LY294002. Obtained results indicated that TM4 cells through MAPK/ERK1/2, MAPK/p-38 and PI3K/Akt pathways influence the proliferation and migration of ADSCs. These findings indicated that TM4 cells were found effective in promoting stemness and migration of ADSCs, that proves adopted co-culturing technique as an efficient approach to obtain ADSCs in transplantation therapy.

Sustainable biosynthesis of curdlan from orange waste by using Alcaligenes faecalis: A systematically modeled approach

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 60-120u2009°C, along with the application of moisture diffusion control model. Secondly, fermentation medium was obtained via saccharification and detoxification, releasing highest sugar at 72.34u2009g/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 5u2009L bioreactor, highest specific growth rate (μmu2009=u20090.233/h), highest curdlan production (Pm = 23.24u2009g/L) and growth associated rate constant (αu2009=u20093.403) 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.

Effective utilization of wastewater for valuable validamycin A biosynthesis by Streptomyces hygroscopicus K2509 in plant-scale bioreactor

BackgroundThe continuous escalation in wastewater production with declining dependency on conventional resources as a result of rapid urbanization, increasing global water scarcity and growing population have initiated many researchers to look out for efficient means of utilizing wastewater. In this regard, an effectual process economizing approach has been achieved, upon utilizing the discharged wastewater from validamycin A recovery process in fermentation medium as a replacement of costly tap water.ResultsIn this study, wastewater was successfully used as a fermentation medium for the production of validamycin A in plant-scale bioreactor. Moreover, a new strain Streptomyces hygroscopicus K2509 was screened showing a good production capability in this low-cost culture environment and showed maximum validamycin A production and productivity of 21.23xa0g/L and 0.29xa0g/Lxa0h, respectively. Execution of this study has managed the effective utilization of wastewater by reducing 12.42% production cost per kilogram of validamycin A in an environmental friendly way.ConclusionThe novel successful approach for using process wastewater even on being executed in plant-scale bioreactor was proved cost effective. In short, the presented effective way of utilizing wastewater in study will definitely serve as potentially cheap fermentation medium upon replacing tremendously used expensive tap water.

Mixomics analysis of Bacillus subtilis: effect of oxygen availability on riboflavin production

BackgroundRiboflavin, 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.ResultsIn 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.ConclusionsThis 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.

Enhanced protein production by sorbitol co-feeding with methanol in recombinant Pichia pastoris strains

Pichia pastoris strains carrying 1, 6, 12, and 18 copies of the porcine insulin precursor (PIP) gene, were employed to investigate the effects of sorbitol co-feeding with methanol on the physiology of the strains. Multicopy clones of the methylotrophic yeast were generated to vary the PIP gene dosage and recombinant proteins. Elevated gene dosage increased levels of the recombinant PIP protein when methanol served as the sole carbon and energy source i.e., an increase of 1.9% for a strain carrying 1 copy, 42.6% for a strain carrying 6 copies, 34.7% for a strain carrying 12 copies and 80.9% for a strain carrying 18 copies, respectively (using sorbitol co-feeding with methanol during the induction phase). However, it had no significant influence on a lower gene dosage strain (1 copy), but this approach affirmed enhancement in cell growth and PIP production for higher gene dosage strain (6, 12, and 18 copies) via using sorbitol co-feeding with methanol. Additionally, the co-feeding strategy could hold vital importance for recombinant protein production by a multi-copy P. pastoris system.