Investigation of the influence of Candida tropicalis on bioethanol production using sugarcane bagasse: stochastic and in silico analysis.

This study investigated the impact of Candida tropicalis NITCSK13 on sugarcane bagasse (SCB) consolidated bioprocessing (CSB) using various parameters, such as pH, steam explosion (STEX) pretreatment, and temperature (at two different temperatures, cellulose hydrolysis and ethanol fermentation). The backpropagation neural network (BPNN) method simulated the optimal CSB conditions, achieving a maximum ethanol yield of 44 ± 0.32 g/L (0.443 g of ethanol/g of SCB) from STEX pretreated SCB within 48 h at 55 °C for cellulose hydrolysis and 33 °C for ethanol fermentation and pH 3.5. The simulated conditions were experimentally validated and showed an R2 value of 0.998 and absolute average deviation (AAD) of 1.23%. The strain NITCSK13 also exhibited a high ethanol tolerance of 16% (v/v). The interactions between the inhibitors, cellobiose, furfural, and thermocellulase were assessed through molecular docking. The results revealed a maximum inhibitory constant of 3.7 mM for furfural against the endoglucanase (EnG) of Humicola insolens (2ENG) at 50 °C. Acremonium chrysogenum endoglucanase (5M2D) exhibited a maximum of 88.7 µM for cellobiose at 50 °C. The SWISS homology model of EnG from Candida viswanathii exhibited inhibitory effects similar to those of EnG from Thermoascus and Thermotoga, indicating that the moderately thermophilic yeast Candida sp. cellulase may be capable of efficiently tolerating inhibitors and could be a promising candidate for consolidated bioprocessing of cellulosic ethanol.

Valorization of sugarcane bagasse for high-yield production of laccase through Aspergillus terreus for effective azo dye decolourization.

Synthetic dyes such as azo dyes are significant pollutants in the wastewater released from various textile industries. The low biodegradability and production from synthetic sources with high shelf life make azo dyes a challenging material for degradation. This study used chemically mutated Aspergillus terrus in the laccase production under solid-state fermentation using sugarcane bagasse. Initially, the wild-type strain produced a laccase activity of 4.12 U/mL. Later, the alkaline pretreatment of sugarcane bagasse showed a significant increase in laccase activity by 38.9%. Further, random mutagenesis treatment with 100 mM EMS generated a hyper laccase-producing strain with a 2.3-fold increment in laccase activity compared to the wild-type strain. The enzyme displayed optimal activity at pH 6.5 and 35 °C. The metal ions such as Fe3+ (29.4 U/mL), Fe2+ (20.8 U/mL) and Cu2+ (18.05 U/mL) showed positive effects on laccase activity. The crude laccase was used to bioremediate Congo red, a prominent azo dye used in textile and pharmaceutical industries. The preliminary studies with a crude enzyme displayed 68.86% dye decolourization after 24 h of incubation. Additionally, with Taguchi orthogonal array optimization experiments, the maximal dye decolorization of 78.24% was achieved by maintaining crude enzyme concentration (20 U), dye concentration (25 mg/L) and pH 4.5.

Sustainable biorefinery approach by utilizing xylose fraction of lignocellulosic biomass.

Lignocellulosic biomass (LCB) has been a lucrative feedstock for developing biochemical products due to its rich organic content, low carbon footprint and abundant accessibility. The recalcitrant nature of this feedstock is a foremost bottleneck. It needs suitable pretreatment techniques to achieve a high yield of sugar fractions such as glucose and xylose with low inhibitory components. Cellulosic sugars are commonly used for the bio-manufacturing process, and the xylose sugar, which is predominant in the hemicellulosic fraction, is rejected as most cell factories lack the five­carbon metabolic pathways. In the present review, more emphasis was placed on the efficient pretreatment techniques developed for disintegrating LCB and enhancing xylose sugars. Further, the transformation of the xylose to value-added products through chemo-catalytic routes was highlighted. In addition, the review also recapitulates the sustainable production of biochemicals by native xylose assimilating microbes and engineering the metabolic pathway to ameliorate biomanufacturing using xylose as the sole carbon source. Overall, this review will give an edge on the bioprocessing of microbial metabolism for the efficient utilization of xylose in the LCB.

Mixed food waste valorization using a thermostable glucoamylase enzyme produced by a newly isolated filamentous fungus: A sustainable biorefinery approach.

Food waste is a lucrative source of complex nutrients, which can be transformed into a multitude of bioproducts by the aid of microbial cell factories. The current study emphasizes isolating Glucoamylase enzyme (GA) producing strains that can effectively break down mixed food waste (MW), which serves as a substrate for biomanufacturing. The screening procedure relied heavily on the growth of isolated fungi on starch agar media, to specifically identify the microbes with the highest starch hydrolysis potential. A strain displayed the highest GA activity of 2.9 ± 0.14 U/ml which was selected and identified as Aspergillus fumigatus via molecular methods of identification. Exposure of the A. fumigatus with 200 mM Ethyl methanesulphonate (EMS) led to a 23.79% increase compared to the wild-type GA. The growth conditions like cultivation temperature or the number of spores in the inoculum were investigated. Further, maximum GA activity was exhibited at pH 5, 55 °C, and at 5 mM Ca2+ concentration. The GA showed thermostability, retaining activity even after long periods of exposure to temperatures as high as 95 °C. The improvement of hydrolysis of MW was achieved by Taguchi design where a maximum yield of 0.57 g g-1 glucose was obtained in the hydrolysate. This study puts forth the possibility that mixed food waste, despite containing spices and other microbial growth-inhibitory substances, can be efficiently hydrolyzed to release glucose units, by robust fungal cell factories. The glucose released can then be utilized as a carbon source for the production of value-added products.

Story of Pore-Forming Proteins from Deadly Disease-Causing Agents to Modern Applications with Evolutionary Significance.

Animal venoms are a complex mixture of highly specialized toxic molecules. Among them, pore-forming proteins (PFPs) or toxins (PFTs) are one of the major disease-causing toxic elements. The ability of the PFPs in defense and toxicity through pore formation on the host cell surface makes them unique among the toxin proteins. These features made them attractive for academic and research purposes for years in the areas of microbiology as well as structural biology. All the PFPs share a common mechanism of action for the attack of host cells and pore formation in which the selected pore-forming motifs of the host cell membrane-bound protein molecules drive to the lipid bilayer of the cell membrane and eventually produces water-filled pores. But surprisingly their sequence similarity is very poor. Their existence can be seen both in a soluble state and also in transmembrane complexes in the cell membrane. PFPs are prevalent toxic factors that are predominately produced by all kingdoms of life such as virulence bacteria, nematodes, fungi, protozoan parasites, frogs, plants, and also from higher organisms. Nowadays, multiple approaches to applications of PFPs have been conducted by researchers both in basic as well as applied biological research. Although PFPs are very devastating for human health nowadays researchers have been successful in making these toxic proteins into therapeutics through the preparation of immunotoxins. We have discussed the structural, and functional mechanism of action, evolutionary significance through dendrogram, domain organization, and practical applications for various approaches. This review aims to emphasize the PFTs to summarize toxic proteins together for basic knowledge as well as to highlight the current challenges, and literature gap along with the perspective of promising biotechnological applications for their future research.

Integrated biorefineries for repurposing of food wastes into value-added products.

Food waste (FW) generated through various scenarios from farm to fork causes serious environmental problems when either incinerated or disposed inappropriately. The presence of significant amounts of carbohydrates, proteins, and lipids enable FW to serve as sustainable and renewable feedstock for the biorefineries. Implementation of multiple substrates and product biorefinery as a platform could pursue an immense potential of reducing costs for bio-based process and improving its commercial viability. The review focuses on conversion of surplus FW into range of value-added products including biosurfactants, biopolymers, diols, and bioenergy. The review includes in-depth description of various types of FW, their chemical and nutrient compositions, current valorization techniques and regulations. Further, it describes limitations of FW as feedstock for biorefineries. In the end, review discuss future scope to provide a clear path for sustainable and net-zero carbon biorefineries.

Development of Hypertolerant Strain of Yarrowia lipolytica Accumulating Succinic Acid Using High Levels of Acetate.

Acetate is emerging as a promising feedstock for biorefineries as it can serve as an alternate carbon source for microbial cell factories. In this study, we expressed acetyl-CoA synthase in Yarrowia lipolytica PSA02004PP, and the recombinant strain grew on acetate as the sole carbon source and accumulated succinic acid or succinate (SA). Unlike traditional feedstocks, acetate is a toxic substrate for microorganisms; therefore, the recombinant strain was further subjected to adaptive laboratory evolution to alleviate toxicity and improve tolerance against acetate. At high acetate concentrations, the adapted strain Y. lipolytica ACS 5.0 grew rapidly and accumulated lipids and SA. Bioreactor cultivation of ACS 5.0 with 22.5 g/L acetate in a batch mode resulted in a maximum cell OD600 of 9.2, with lipid and SA accumulation being 0.84 and 5.1 g/L, respectively. However, its fed-batch cultivation yielded a cell OD600 of 23.5, SA titer of 6.5 g/L, and lipid production of 1.5 g/L with an acetate uptake rate of 0.2 g/L h, about 2.86 times higher than the parent strain. Cofermentation of acetate and glucose significantly enhanced the SA titer and lipid accumulation to 12.2 and 1.8 g/L, respectively, with marginal increment in cell growth (OD600: 26.7). Furthermore, metabolic flux analysis has drawn insights into utilizing acetate for the production of metabolites that are downstream to acetyl-CoA. To the best of our knowledge, this is the first report on SA production from acetate by Y. lipolytica and demonstrates a path for direct valorization of sugar-rich biomass hydrolysates with elevated acetate levels to SA.

Prospects on bio-based 2,3-butanediol and acetoin production: Recent progress and advances.

The bio-based platform chemicals 2,3-butanediol (BDO) and acetoin have various applications in chemical, cosmetics, food, agriculture, and pharmaceutical industries, whereas the derivatives of BDO could be used as fuel additives, polymer and synthetic rubber production. This review summarizes the novel technological developments in adapting genetic and metabolic engineering strategies for selection and construction of chassis strains for BDO and acetoin production. The valorization of renewable feedstocks and bioprocess development for the upstream and downstream stages of bio-based BDO and acetoin production are discussed. The techno-economic aspects evaluating the viability and industrial potential of bio-based BDO production are presented. The commercialization of bio-based BDO and acetoin production requires the utilization of crude renewable resources, the chassis strains with high fermentation production efficiencies and development of sustainable purification or conversion technologies.

Microbial itaconic acid production from starchy food waste by newly isolated thermotolerant Aspergillus terreus strain.

In the present study, we have explored the potential of newly isolated Aspergillus terreus BD strain, which can accumulate itaconic acid (IA) at higher temperature. The shake flask cultivation of thermotolerant strain with medium optimized using Box-Behnken Design at 45 °C resulted in IA accumulation of 28.9 g/L with yield of 0.27 g/g. The enzymatic saccharification of the synthetic food waste (SFW) consisting of potatoes, rice & noodles were optimized using Taguchi method of orthogonal array to maximize the release of fermentable sugar. The maximum glucose release of 0.60 g/g was achieved with 10% biomass loading, 5% enzyme concentration, pH 5.5 and temperature 60 0C. The sugars obtained from SFW was integrated with IA production and maximum IA titer achieved with SFW hydrolysate during bioreactor cultivation was 41.1 g/L with conversion yield of 0.27 g/g while with pure glucose IA titer and yield were 44.7 g/L and 0.30 g/g, respectively.

Enhanced xylitol production using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse by newly isolated Pichia fermentans.

BACKGROUND: Integrated management of hemicellulosic fraction and its economical transformation to value-added products is the key driver towards sustainable lignocellulosic biorefineries. In this aspect, microbial cell factories are harnessed for the sustainable production of commercially viable biochemicals by valorising C5 and C6 sugars generated from agro-industrial waste. However, in the terrestrial ecosystem, microbial systems can efficiently consume glucose. On the contrary, pentose sugars are less preferred carbon source as most of the microbes lack metabolic pathway for their utilization. The effective utilization of both pentose and hexose sugars is key for economical biorefinery. RESULTS: Bioprospecting the food waste and selective enrichment on xylose-rich medium led to screening and isolation of yeast which was phylogenetically identified as Pichia fermentans. The newly isolated xylose assimilating yeast was explored for xylitol production. The wild type strain robustly grew on xylose and produced xylitol with > 40% conversion yield. Chemical mutagenesis of isolated yeast with ethyl methanesulphonate (EMS) yielded seven mutants. The mutant obtained after 15 min EMS exposure, exhibited best xylose bioconversion efficiency. This mutant under shake flask conditions produced maximum xylitol titer and yield of 34.0 g/L and 0.68 g/g, respectively. However, under the same conditions, the control wild type strain accumulated 27.0 g/L xylitol with a conversion yield of 0.45 g/g. Improved performance of the mutant was attributed to 34.6% activity enhancement in xylose reductase with simultaneous reduction of xylitol dehydrogenase activity by 22.9%. Later, the culture medium was optimized using statistical design and validated at shake flask and bioreactor level. Bioreactor studies affirmed the competence of the mutant for xylitol accumulation. The xylitol titer and yield obtained with pure xylose were 98.9 g/L and 0.67 g/g, respectively. In comparison, xylitol produced using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse was 79.0 g/L with an overall yield of 0.54 g/g. CONCLUSION: This study demonstrates the potential of newly isolated P. fermentans in successfully valorising the hemicellulosic fraction for the sustainable xylitol production.

Bioproduction of succinic acid from xylose by engineered Yarrowia lipolytica without pH control.

BACKGROUND: Xylose is the most prevalent sugar available in hemicellulose fraction of lignocellulosic biomass (LCB) and of great interest for the green economy. Unfortunately, most of the cell factories cannot inherently metabolize xylose as sole carbon source. Yarrowia lipolytica is a non-conventional yeast that produces industrially important metabolites. The yeast is able to metabolize a large variety of substrates including both hydrophilic and hydrophobic carbon sources. However, Y. lipolytica lacks effective metabolic pathway for xylose uptake and only scarce information is available on utilization of xylose. For the economica feasibility of LCB-based biorefineries, effective utilization of both pentose and hexose sugars is obligatory. RESULTS: In the present study, succinic acid (SA) production from xylose by Y. lipolytica was examined. To this end, Y. lipolytica PSA02004 strain was engineered by overexpressing pentose pathway cassette comprising xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulose kinase (XK) gene. The recombinant strain exhibited a robust growth on xylose as sole carbon source and produced substantial amount of SA. The inhibition of cell growth and SA formation was observed above 60 g/L xylose concentration. The batch cultivation of the recombinant strain in a bioreactor resulted in a maximum biomass concentration of 7.3 g/L and SA titer of 11.2 g/L with the yield of 0.19 g/g. Similar results in terms of cell growth and SA production were obtained with xylose-rich hydrolysate derived from sugarcane bagasse. The fed-batch fermentation yielded biomass concentration of 11.8 g/L (OD600: 56.1) and SA titer of 22.3 g/L with a gradual decrease in pH below 4.0. Acetic acid was obtained as a main by-product in all the fermentations. CONCLUSION: The recombinant strain displayed potential for bioconversion of xylose to SA. Further, this study provided a new insight on conversion of lignocellulosic biomass into value-added products. To the best of our knowledge, this is the first study on SA production by Y. lipolytica using xylose as a sole carbon source.

Biovalorisation of crude glycerol and xylose into xylitol by oleaginous yeast Yarrowia lipolytica.

BACKGROUND: Xylitol is a commercially important chemical with multiple applications in the food and pharmaceutical industries. According to the US Department of Energy, xylitol is one of the top twelve platform chemicals that can be produced from biomass. The chemical method for xylitol synthesis is however, expensive and energy intensive. In contrast, the biological route using microbial cell factories offers a potential cost-effective alternative process. The bioprocess occurs under ambient conditions and makes use of biocatalysts and biomass which can be sourced from renewable carbon originating from a variety of cheap waste feedstocks. RESULT: In this study, biotransformation of xylose to xylitol was investigated using Yarrowia lipolytica, an oleaginous yeast which was firstly grown on a glycerol/glucose for screening of co-substrate, followed by media optimisation in shake flask, scale up in bioreactor and downstream processing of xylitol. A two-step medium optimization was employed using central composite design and artificial neural network coupled with genetic algorithm. The yeast amassed a concentration of 53.2 g/L xylitol using pure glycerol (PG) and xylose with a bioconversion yield of 0.97 g/g. Similar results were obtained when PG was substituted with crude glycerol (CG) from the biodiesel industry (titer: 50.5 g/L; yield: 0.92 g/g). Even when xylose from sugarcane bagasse hydrolysate was used as opposed to pure xylose, a xylitol yield of 0.54 g/g was achieved. Xylitol was successfully crystallized from PG/xylose and CG/xylose fermentation broths with a recovery of 39.5 and 35.3%, respectively. CONCLUSION: To the best of the author's knowledge, this study demonstrates for the first time the potential of using Y. lipolytica as a microbial cell factory for xylitol synthesis from inexpensive feedstocks. The results obtained are competitive with other xylitol producing organisms.

Glucose-methanol-based fed-batch fermentation for the production of recombinant human interferon gamma (rhIFN-γ) and evaluation of its antitumor potential.

Squamous cell carcinoma (SCC) is nonmelanoma skin cancer, which is very common in patients having T-cell immunosuppressant drugs. Anticancerous agents such as cytokines showed effective response on SCC. Human interferon-gamma (hIFN-γ), a type II cytokines, are having potent antiproliferative and immunomodulatory effects. In the current study, the fed-batch cultivation of recombinant Pichia pastoris was carried out, and its effect on cell biomass production, recombinant human interferon-gamma (rhIFN-γ) production, and the overflow metabolites was estimated. P. pastoris GS115 strain coexpressed with 6-phosphogluconolactonase (SOL3) and ribulose-phosphate 3-epimerase (RPE1) gene (GS115/rhIFN-γ/SR) resulted in 60 mg L-1 of rhIFN-γ production, which was twofold higher as compared with the production from GS115/rhIFN-γ strain. The antiproliferative potential of rhIFN-γ was examined on the human squamous carcinoma (A431) cell lines. Cells treated with 80 ng mL-1 of rhIFN-γ exhibited 50% growth inhibition by enhancing the production of intracellular reactive oxygen species levels and disrupting membrane integrity. Our findings highlight a state of art process development strategy for the high-level production of rhIFN-γ and its potential application as a therapeutic drug in SCC therapy.

Cationic reverse micellar based purification of recombinant glutaminase free L-asparaginase II of Bacillus subtilis WB800N from fermentation media.

Reverse micellar extraction (RME), a liquid-liquid based separation is a versatile tool for protein purification. A statistical approach was employed for the purification of recombinant glutaminase free anti-cancerous enzyme viz., l-asparaginase II to evaluate the effects of RME in current study. The cationic system (CTAB/iso-octane/hexanol/butanol) was used in RME to optimize both forward and backward protein extraction efficiency. By adapting Taguchi's orthogonal array (OA), maximum forward extraction efficiency (FEE) of 86.98% with 84.82% enzyme activity recovery and 1.04 times purification fold achieved with the optimized parameters. Under the optimal levels, the back extraction efficiency (BEE) was observed to be 96.97% with 93.07% enzyme activity recovery and 1.38 times purification fold. Further, mass transfer kinetic studies of RME indicated the mass transfer coefficients of forward and backward extraction to be 0.049 min-1 and 0.036 min-1 respectively.

Metabolic engineering of Pichia pastoris GS115 for enhanced pentose phosphate pathway (PPP) flux toward recombinant human interferon gamma (hIFN-γ) production.

In the present study, the effects of individual as well as multiple genes of pentose phosphate pathway (PPP) on human interferon gamma (hIFN-γ) production were analyzed. With overexpression of 6-phosphogluconate dehydrogenase (GND2), 1.9-fold increase in hIFN-γ was achieved, while synergetic effect of 6-phosphogluconolactonase (SOL3) and D-ribulose-5-phosphate 3-epimerase (RPE1) resulted in 2.56-fold increase in hIFN-γ as compared to control. Fed batch fermentation using mixed feeding of gluconate and methanol (carbon source) was carried out, resulting in 80 and 123 mg L-1 of hIFN-γ enhancement in recombinant Pichia GS115 strain encoding codon optimized hIFN-γ (GS115/hIFN-γ) and Pichia GS115 strain encoding codon optimized hIFN-γ with co-expressed 6-phosphogluconolactonase(SOL3) and D-ribulose-5-phosphate 3-epimerase (RPE1) (GS115/hIFN-γ/SR) respectively. To get more insight of the flux distribution towards hIFN-γ, studies were carried out by applying flux balance analysis during methanol fed batch phase for both strains. In both strains (GS115/hIFN-γ and GS115/hIFN-γ/SR) more than 95% of formaldehyde flux is directed towards assimilatory pathway. The analysis revealed that with the overexpression of SOL3 and RPE1 the flux towards PPP triggering the alleviation in hIFN-γ production.

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis.

The present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box-Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80 g/L lactose, 10.353 g/L yeast extract, and 15 mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881 µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1 mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.

A novel reverse micellar purification strategy for histidine tagged human interferon gamma (hIFN-γ) protein from Pichia pastoris.

In the present study, we have demonstrated the process development of human interferon gamma (hIFN-γ) (upstream to downstream). The codon optimized hIFN-γ gene was cloned in Pichia pastoris X-33 and the expression was evaluated in batch reactor study. The purification was carried out with modified nickel chelated reverse micellar system and compared with the existing Nickle- Nitrilotriacetic acid (NI-NTA) method. The parameter optimization for forward extraction demonstrated a significant enhancement of 72% in forward extraction efficiency (FEE). Furthermore, the factors governing back extraction efficiency (BEE) were also optimized with sequential optimization involving Taguchi orthogonal array and Artificial Neural Network linked Simulated Annealing Algorithm (ANN-SA). The optimization resulted in 91.2% back extraction efficiency of recombinant human interferon gamma (rhIFN-γ). The development of this purification system with optimized parameters led to an efficient recovery of 67.3% and improved purity of 79.54%. Alongside, the anti-proliferative activity in MCF-7 cell lines were also investigated and it demonstrated that at 60ngmL-1 concentration of rhIFN-γ more that 25%.

Dual-substrate inhibition kinetic studies for recombinant human interferon gamma producing Pichia pastoris.

Pichia pastoris is considered as one of the prominent host extensively used as a platform for heterologous protein production. In the present study, the growth inhibition kinetics of recombinant P. pastoris expressing human interferon gamma was studied under different initial substrate concentrations of gluconate (10-100 g L-1) and methanol (2-50 g L-1) in modified FM22 medium. The highest specific growth rate of 0.0206 and 0.019 hr-1 was observed at 60 g L-1 of gluconate and 10 g L-1 of methanol, respectively. Various three- and four-parametric Monod-variant models were chosen to analyze the inhibition kinetics. The model parameters as well as goodness of fit were estimated using nonlinear regression analysis. The three-parameter Haldane model was found to be best fit for both gluconate (R2 = 0.95) and methanol substrate (R2 = 0.96). The parameter sensitivity analysis revealed that µmax, Ki, and Ks are the most sensitive parameters for both methanol and gluconate. Different substrate inhibition models were fitted to the growth kinetic data and the additive form of double Webb model was found to be the best to explain the growth kinetics of recombinant P. pastoris.

Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris.

BACKGROUND: Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. RESULT AND CONCLUSION: In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized.

Batch and fed-batch bioreactor studies for the enhanced production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428.

The effect of dissolved oxygen (DO) level and pH (controlled/uncontrolled) was first studied to enhance the production of novel glutaminase-free L-asparaginase by Pectobacterium carotovorum MTCC 1428 in a batch bioreactor. The optimum level of DO was found to be 20%. The production of L-asparaginase was found to be maximum when pH of the medium was maintained at 8.5 after 12 h of fermentation. Under these conditions, P. carotovorum produced 17.97 U/mL of L-asparaginase corresponding to the productivity of 1497.50 U/L/h. The production of L-asparaginase was studied in fed-batch bioreactor by feeding L-asparagine (essential substrate for production) and/or glucose (carbon source for growth) at the end of the reaction period of 12 h. The initial medium containing both L-asparagine and glucose in the batch mode and L-asparagine in the feeding stream was found to be the best combination for enhanced production of glutaminase-free L-asparaginase. Under this condition, the L-asparaginase production was increased to 38.8 U/mL, which corresponded to a productivity of 1615.8 U/L/h. The production and productivity were increased by 115.8% and 7.9%, respectively, both of which are higher than those obtained in the batch bioreactor experiments.