Co-feeding Strategy Research Articles

High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins

Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of ROL gene cassettes, which were then transformed into P. pastoris GS115 to generate a strain with specific copy numbers of ROL. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of ROL was screened, revealing production of the highest activity (2700 U/mL), which was 8-fold higher than that of the strain harboring one copy. The activity of GS115/pAOα-5ROL 11# was then enhanced to 3080 U/mL in a shaking flask under optimized culture conditions. Subsequently, the endoplasmic reticulum-associated protein-degradation-related genes Ubc1 or/and Hrd1 were co-expressed with ROL to further increase ROL expression. The activities of the recombinant strains, GS115/5ROL-Ubc1 22#, -Hrd1 15#, and -Hrd1-Ubc1 1#, were 4000 U/mL, 4200 U/mL, and 4750 U/mL, which was 29.9%, 36.4%, and 54.2% higher, respectively, than that observed in GS115/pAOα-5ROL 11#. Using the combined strategy, ROL expression was improved 15.8-fold, with maximum GS115/5ROL-Hrd1-Ubc1 1# activity reaching 33,900 U/mL via a sorbitol/methanol co-feeding strategy in a 3-L fermenter and resulting in a 1.65-, 1.26-, and 1.14-fold enhancement relative to the activities observed in strains GS115/pAOα-5ROL 11#, GS115/5ROL-Ubc1 22#, and GS115/5ROL-Hrd1 15#, respectively. These results indicated that heterologous overexpression of ROL in P. pastoris using this combined strategy is feasible for large-scale industrialization.

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.

Chemical looping as a reactor concept for the oxidative coupling of methane over the MnxOy-Na2WO4/SiO2 catalyst, benefits and limitation

The chemical looping concept (CLC) has been implemented to suppress the formation of COX in an oxidative coupling of methane (OCM) reactor under a wide range of conditions. In comparison to the normal co-feeding strategy, this technique resulted in an enhanced C2 selectivity at the same methane conversions. Nevertheless, the obtained yield never exceeded 25% which is still lower than the minimum value needed for industrializing OCM. The CLC was applied in mechanistic studies to investigate the consecutive reaction of the main products of OCM, i.e. ethane and ethene. The performance of the reaction of C2 components at 750°C in the chemical looping reactor was compared with that obtained in the co-feeding experiments. The effect of the surface adsorbed oxygen species on the reaction of both ethane and ethene was investigated. The results of these experiments reveal that some of the mechanistic assumptions about the OCM reaction are not compatible with the nature of MnxOy-Na2WO4/SiO2, one of the most stable and best performing catalysts known for this reaction. The yield limitation is shown to be inherent to the catalyst. However, this limitation should be solvable through the modified process concept aiming at the production of ethane in a separated first step.

Transcriptional analysis for carbon metabolism and kinetic modeling for heterologous proteins productions by Pichia pastoris in induction process with methanol/sorbitol co-feeding

Abstract It is difficult to control concentrations of methanol/dissolved oxygen at high levels simultaneously in heterologous proteins productions by Pichia pastoris during induction phase. Two strains, a methanol utilization slow (MutS) type and a plus (Mut+) type were used with methanol/sorbitol co-feeding strategy to induce porcine interferon-α and human serum albumin-human granulocyte colony stimulating factor respectively, under the conditions of “methanol sufficient-oxygen limited (MS-OL)” and “methanol limited-oxygen sufficient (ML-OS)”. For the MutS/Mut+ strains, the target proteins titers under “MS-OL” were 6-fold/19.2% of those under “ML-OS”. The key genes in methanol metabolism of the MutS strain were up-regulated under “MS-OL”, but they were not differently expressed in the Mut+ strain. Methanol utilization rate (rMeOH) of the MutS strain reduced when decreasing methanol concentration, but rMeOH of the Mut+ strain unchanged unless methanol concentration decreased to a low-limit of 0.6 g/L. Finally, kinetic models were designed to describe the methanol/sorbitol co-feeding process.

High-level extracellular production of glucose oxidase by recombinant Pichia pastoris using a combined strategy.

In this work, a combined strategy was developed to improve the production of glucose oxidase (GOD) (EC 1.1.3.4) in Pichia pastoris. One of the main challenges facing protein production by the high-density fermentation of P. pastoris is the high demand for oxygen. Another challenge is how to balance a reduction in oxygen consumption and its effects on protein production. Herein, a combined strategy involving mannitol co-feeding, two-stage methanol induction, and the co-expression of the transcriptional activator general control non-derepressible 4 (GCN4) from P. pastoris was used. A two-stage, co-feeding strategy, based on a mannitol/methanol mixture in a 3-L fermentor was used to enhance cell viability and protein production. This resulted in an increased GOD yield of 1208.2 U/mL compared with a control strain (427.6 U/mL). An increase in the copy number of the GCN4 gene enhanced the GOD yield (1634.7 U/mL) by 2.8-fold and the protein concentration (19.55 g/L) by 1.58-fold compared with the control (7.59 g/L). This strategy illustrates a way to overcome the high oxygen requirement during high-density fermentation of P. pastoris and balances the reduction of oxygen consumption and protein production. Moreover, the series of strategies presented in this work provide valuable and novel information for the industrial production of GOD.

Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies.

This study attempted to enhance the expression level of Thermomyces lanuginosus lipase (TLL) in Pichia pastoris using a series of strategies. The tll gene was first inserted into the expression vector pPIC9K and transformed into P. pastoris strain GS115. The maximum hydrolytic activity of TLL reached 4,350U/mL under the optimal culture conditions of a 500mL shaking flask containing 20mL culture medium with the addition of 1.2% (w/v) methanol, cultivation for 144h at pH 7.0 and 27°C. To further increase the TLL expression and copy number, strains containing two plasmids were obtained by sequential electroporation into GS115/9k-TLL #3 with a second vector, either pGAPZαA-TLL, pFZα-TLL, or pPICZαA-TLL. The maximum activity of the resultant strains GS115/9KTLL-ZαATLL #40, GS115/9KTLL-FZαATLL #46 and GS115/9KTLL-GAPTLL #45 was 6,600U/mL, 6,000U/mL and 4,800U/mL, respectively. The tll copy number in these strains, as assessed by real-time quantitative PCR, was demonstrated to be seven, five, and three, respectively, versus two copies in GS115/9k-TLL #3. When a co-feeding strategy of sorbitol/methanol was adopted in a 3-L fermenter, the maximum TLL activity of GS115/9k-TLL #3 increased to 27,000U/mL after 130h of fed-batch fermentation, whereas, the maximum TLL activity was 19,500U/mL after 145h incubation when methanol was used as the sole carbon source.

English

Process techniques of the methylotrophic yeast Pichia pastoris for the production and recovery of heterologous phytase proteins has developed in last 20 years. High expression levels using methanol as induction have been made in the quality of recombinant proteins in the fermenter culture and in the quality of the protein product. This allowed rapidly P. pastoris to become the system of choice for the expression of recombinant proteins in yeast. The experimental designs, the methanol/L-alanine co-feeding strategy and optimization of phytase production by P. pastoris supported by the optimum levels of variables and lower temperature expression produced high level of phytase activity which could be scaled up to produce phytase for food additives at industrial level.  An overall phytase activity was 8632 U/ml, this means 332 fold increase compare to the wild type of phytase. This work demonstrates not only the impact of α-factor prepro secretion signal and efficiency of methanol/L-alanine co-induction strategy for phytase production by recombinant P. pastoris Mut+ strains, but also shows new insights for the expression of bioproduct at lower temperature.   Key words: Phytase, gene expression, Pichia pastoris, process optimization, co-feeding strategy, Deglycosylation.

Efficient production of l-lactic acid using co-feeding strategy based on cane molasses/glucose carbon sources

L-Lactic acid is an important platform chemical, which ought to be produced under cost control to meet its huge demand. Cane molasses, a waste from sugar manufacturing processes, is hopeful to be utilized as a cheap carbon source for L-lactic acid fermentation. Considering that cane molasses contains nutrients and hazardous substances, efficient production of L-lactic acid was developed by using a co-feeding strategy based on the utilization of cane molasses/glucose carbon sources. Based on the medium optimization with response surface method, 168.3g/L L-lactic acid was obtained by a Bacillus coagulans strain H-1 after 78h fed-batch fermentation, with a productivity of 2.1g/Lh and a yield of 0.88g/g. Since cane molasses is a feasible carbon source, the co-feeding fermentation might be a promising alternative for the economical production of L-lactic acid.

Optimizing the co-feeding strategy of Persian sturgeon (Acipenser persicus) larvae using Artemia nauplii and formulated diet

High mortality and labor costs are associated with first-feeding sturgeon culture, particularly during the period of dietary transition from live to formulated feed. Therefore we investigated the effects of various feeding treatments on the survival and growth of the Persian sturgeon ( Acipenser persicus ) larvae during a 20-day culture period. Three replicate groups (250 fish/replicate) of first-feeding larvae were fed according to four main feeding regimes: (1) live food (live nauplii of brine shrimp Artemia urmiana ); (2) indirect transition (5 days live food followed by gradual transition to formulated diet); (3) direct transition (using different combinations of live and formulated diet from the start feeding onwards); (4) formulated feed (FD) from the start feeding. Results indicated that growth and survival were higher in the indirect transition feeding regime than in other regimes. Based on our study, co-feeding of A. persicus should start five days after prior feeding with live food.

A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut+/pAOX1-lacZ strain

BackgroundOne of the main challenges for heterologous protein production by the methylotrophic yeast Pichia pastoris at large-scale is related to its high oxygen demand. A promising solution is a co-feeding strategy based on a methanol/sorbitol mixture during the induction phase. Nonetheless, a deep understanding of the cellular physiology and the regulation of the AOX1 promoter, used to govern heterologous protein production, during this co-feeding strategy is still scarce.ResultsTransient continuous cultures with a dilution rate of 0.023 h-1 at 25°C were performed to quantitatively assess the benefits of a methanol/sorbitol co-feeding process with a Mut+ strain in which the pAOX1-lacZ construct served as a reporter gene. Cell growth and metabolism, including O2 consumption together with CO2 and heat production were analyzed with regard to a linear change of methanol fraction in the mixed feeding media. In addition, the regulation of the promoter AOX1 was investigated by means of β-galactosidase measurements. Our results demonstrated that the cell-specific oxygen consumption (qO2) could be reduced by decreasing the methanol fraction in the feeding media. More interestingly, maximal β-galactosidase cell-specific activity (>7500 Miller unit) and thus, optimal pAOX1 induction, was achieved and maintained in the range of 0.45 ~ 0.75 C-mol/C-mol of methanol fraction. In addition, the qO2 was reduced by 30% at most in those conditions. Based on a simplified metabolic network, metabolic flux analysis (MFA) was performed to quantify intracellular metabolic flux distributions during the transient continuous cultures, which further shed light on the advantages of methanol/sorbitol co-feeding process. Finally, our observations were further validated in fed-batch cultures.ConclusionThis study brings quantitative insight into the co-feeding process, which provides valuable data for the control of methanol/sorbitol co-feeding, aiming at enhancing biomass and heterologous protein productivities under given oxygen supply. According to our results, β-galactosidase productivity could be improved about 40% using the optimally mixed feed.

Effect of Feeding Enriched Formulated Diet and Live Feed on Growth, Survival and Fatty Acid Profile of Deccan Mahseer, Tor Khudree (Sykes) First Feeding Fry

Deccan mahseer (Tor khudree) has recently been included in the IUCN list of threatened species due to its declining population. Intervention of nutritional strategies for better growth and survival of early stages for grow out and ranching is essential. The present investigation intended to study the effect of various dietary combinations containing live and inert feed enriched with cod liver oil on growth and survival of Tor khudree first feeding fry, while effects on whole body fatty acid composition of fry were also studied. Cod liver oil as a source of essential fatty acids was used for enriching both live and inert formulated feed. The experimental design consist of four treatments viz. T0 (inert diet without enrichment), T1 (inert diet enriched with cod liver oil), T2 (inert diet enriched with cod liver oil+unenriched Artemia) and T3 (unenriched inert diet+enriched Artemia) fed to four distinct experimental groups for a period of 60 days. At the end of the experiment growth, survival and fatty acid profile of the fry were examined. Fry fed combination of unenriched inert diet and enriched Artemia (T3) had the highest growth and feed efficiency in terms of percentage weight gain (PWG), specific growth rate (SGR), average daily growth (ADG) and feed conversion ratio (FCR). Survival percentage was also significant in T3 compared to other groups. Enrichment of dietary components had a direct effect on the whole body fatty acid profile of mahseer fry with significant (P<0.05) changes in the eicosapentaenoic acid (EPA 20:5 n-3) and docosahexaenoic acid (DHA 22:6 n-3) composition. This study provides avenues for improving the growth and survival of mahseer fry through enrichment and concurrent co-feeding strategies with dietary formulated diet which can further reduce feed cost during fry rearing.

Burbot, Lota lota (Linnaeus, 1758), an Opportunity for Diversification of European Inland Aquaculture

SUMMARY While marine aquaculture has experienced a signific ant increase over the last decade, freshwater production has declined. And as land-loc ked countries do not have access to the sea, they cannot benefit from the improvements mari ne culture followed. But one of the possible strategies to enhance inland production co uld be the diversification of the selected species, trying to aim for high value species, whic h would make bigger investment possible with an increased production as outcome. Being at the origin of a famous traditional Flemish dish, as well as being highly appreciated for its quality meat in Poland, there i s no doubt on the culinary attraction of the burbot ( Lota lota ) as even its eggs and liver are found a delicacy i n respectively Finland and Austria. The current status on the aquaculture prod uction of burbot shows a limited production based on pond culture with little guidel ines known regarding the culture methods. But the potential fast growth of this cold-water sp ecies and its market value makes the burbot an interesting candidate for inland aquaculture (Za rski et al., 2009). For this reason, current research at the AQUA-ERF, launched by the Flemish University College KaHo Sint-Lieven in the context of the European Interreg project “AQ UAVLAN“, is focused on the development of the optimal culture methods of the b urbot. Although at the beginning research was centered on the burbot grow out in recirculation systems, the uncertainty on the avail ability of weaned fingerlings has pushed Aqua-ERF to run experiments concerning larviculture of which the results are presented in this poster. As a preliminary trial had shown indication of a po sitive weaning result, a new trial was set up comparing a co-feeding versus a non-co-f eeding protocol. After a one month weaning period, equal survival rate, rate of cannib alism and SGR were found for both protocols, but a bigger size variation between the larvae of the co-feeding strategy was noticed. Because of the more uniform batch and the higher fraction of larger fry found for the non-co-feeding protocol, the latter would be prefer red in practical fingerling production. Allthemore the application of direct feeding (non-c o-feeding) would be recommended in terms of economics as it reduces the cost and labou r for artemia production.

Enhancement of cell viability and alkaline polygalacturonate lyase production by sorbitol co-feeding with methanol in Pichia pastoris fermentation

Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL −1, which was enhanced 1.85-fold compared to the control (863 U mL −1) cultured with sorbitol added at a constant rate of 3.6 g h −1 L −1 after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL.

Co-Feeding of Pacu, Piaractus mesopotamicus Holmberg (1887), Larvae with Artemia Nauplii and a Microencapsulated Diet

Aiming at a precocious substitution of live prey by artificial diet, a 20-day experiment with pacu, Piaractus mesopotamicuslarvae using co-feeding and abrupt weaning strategies was set up. At the end of the experiment, larvae fed Artemia showed the best results (P < 0.05) in weight, total length and biomass, compared with other treatments. Larvae fed exclusively a microencapsulated diet never ingested the diet. Diet ingestion in co-fed and abrupt-weaned larvae was low, but did increase during the experiment; however, Artemia influenced diet ingestion on co-fed larvae. Careful considerations should be given to diet processing and formulation to ensure survival and growth of larvae fed exclusively on prepared diets.

Reducing Artemia use in the culture of Atlantic cod ( Gadus morhua)

The acquisition and production of Artemia for the feeding of larval marine fish can constitute a significant portion of total fry culturing costs. The availability and price of Artemia can vary widely from year to year and, recently, Artemia cysts attained record high prices. This experiment was conducted to examine early weaning of cod ( Gadus morhua) larvae followed by supplementing microparticulate diet (MPD) with reduced Artemia rations. The objective was to determine whether Artemia use could be minimized while maintaining high growth and survival through supplementation of the MPD diet. Eggs were volumetrically counted to achieve a stocking density of 75 larvae l −1. The experiment consisted of a control group (100% A), which was fed live food during the experimental period, and three experimental treatments. All treatments were run in triplicate. The three experimental treatments received MPD and 50% of the amount Artemia in the 100% A treatment (50% A), MPD and 25% of the amount Artemia in the 100% A treatment (25% A), and MPD only (MPD). Standard lengths and dry weights were collected at hatch, 8, 22, 36, 50 and 64 days post-hatch (dph). Survival was calculated by counting all remaining fish at the conclusion of the experiment. The results indicate that the 100% A, 50% A and 25% A treatments did not differ significantly in standard length, dry weight, specific growth rate or survival. The MPD treatment had lower growth and survival than any of the treatments receiving Artemia. This study supports that, when Artemia prices are high (>US$50/kg), significant savings are possible for marine larval fish culture by using co-feeding strategies similar to those presented here.