Exobiopolymer production of Ophiocordyceps dipterigena BCC 2073: optimization, production in bioreactor and characterization

<p>Abstract</p> <p>Background</p> <p>Biopolymers have various applications in medicine, food and petroleum industries. The ascomycetous fungus <it>Ophiocordyceps dipterigena </it>BCC 2073 produces an exobiopolymer, a (1→3)-β-<it>D</it>-glucan, in low quantity under screening conditions. Optimization of <it>O. dipterigena </it>BCC 2073 exobiopolymer production using experimental designs, a scale-up in 5 liter bioreactor, analysis of molecular weight at different cultivation times, and levels of induction of interleukin-8 synthesis are described in this study.</p> <p>Results</p> <p>In order to improve and certify the productivity of this strain, a sequential approach of 4 steps was followed. The first step was the qualitative selection of the most appropriate carbon and nitrogen sources (general factorial design) and the second step was quantitative optimization of 5 physiological factors (fractional factorial design). The best carbon and nitrogen source was glucose and malt extract respectively. From an initial production of 2.53 g·L^-1, over 13 g·L^-1could be obtained in flasks under the improved conditions (5-fold increase). The third step was cultivation in a 5 L bioreactor, which produced a specific growth rate, biomass yield, exobiopolymer yield and exobiopolymer production rate of 0.014 h^-1, 0.32 g·g^-1glucose, 2.95 g·g biomass^-1(1.31 g·g^-1sugar), and 0.65 g.(L·d)^-1, respectively. A maximum yield of 41.2 g·L^-1was obtained after 377 h, a dramatic improvement in comparison to the initial production. In the last step, the basic characteristics of the biopolymer were determined. The molecular weight of the polymer was in the range of 6.3 × 10⁵- 7.7 × 10⁵Da. The exobiopolymer, at 50 and 100. μg·mL^-1, induced synthesis in normal dermal human fibroblasts of 2227 and 3363 pg·mL^-1interleukin-8 respectively.</p> <p>Conclusions</p> <p>High exobiopolymer yield produced by <it>O. dipterigena </it>BCC 2073 after optimization by qualitative and quantitative methods is attractive for various applications. It induced high IL-8 production by normal dermal fibroblasts, which makes it promising for application as wound healing material. However, there are still other possible applications for this biopolymer, such as an alternative source of biopolymer substitute for hyaluronic acid, which is costly, as a thickening agent in the cosmetic industry due to its high viscosity property, as a moisturizer, and in encapsulation.</p

Characterization of recombinant Bacillus halodurans CM1 xylanase produced by Pichia pastoris KM71 and its potential application in bleaching process of bagasse pulp

Thermoalkalophilic xylanases promise potential application in pulp biobleaching to reduce the use of toxic chlorinated chemical agents, which are harmful to the environment. In this study, a thermoalkalophilic endoxylanase gene (bhxyn3) originating from Indonesian indigenous Bacillus halodurans CM1 was cloned into yeast expression vector pPICZα A and expressed in Pichia pastoris KM71 under the control of AOX1 promoter. Recombinant P. pastoris expressed the highest final level of xylanase (146 U/mL) on BMGY medium after five days of cultivation. Optimization of xylanase production on a small scale was carried out by varying the methanol concentrations and the optimal xylanase production by the recombinant P. pastoris was observed in the culture with 2% (v/v) methanol after four days of the induction phase. The recombinant xylanase (BHxyn3E) was thermotolerant and alkalophilic, with an optimal temperature at around 55‐65 °C and under pH 8.0. The enzyme activity was slightly induced by K+, Fe2+, and MoO42‐. Enzymatic bleaching of bagasse pulp with no prior pH adjustment (pH 9) using BHxyn3E at 200 U/g oven dried pulp increased the lightness index (L*) and changed substantially the color a index (a*); however, the treatments did not change the whiteness index in a significant way. Therefore, further optimization and assessment such as adjustment of incubation temperature and pH in biobleaching were needed to reduce the use of harmful chemical agents in industrial applications

Evaluation of Methylotrophic Yeast Ogataea thermomethanolica TBRC 656 as a Heterologous Host for Production of an Animal Vaccine Candidate

Multiple yeast strains have been developed into versatile heterologous protein expression platforms. Earlier works showed that Ogataea thermomethanolica TBRC 656 (OT), a thermotolerant methylotrophic yeast, can efficiently produce several industrial enzymes. In this work, we demonstrated the potential of this platform for biopharmaceutical manufacturing. Using a swine vaccine candidate as a model, we showed that OT can be optimized to express and secrete the antigen based on porcine circovirus type 2d capsid protein at a respectable yield. Crucial steps for yield improvement include codon optimization and reduction of OT protease activities. The antigen produced in this system could be purified efficiently and induce robust antibody response in test animals. Improvements in this platform, especially more efficient secretion and reduced extracellular proteases, would extend its potential as a competitive platform for biopharmaceutical industries

Metabolic engineering of Saccharomyces cerevisiae for polyhydroxybutyrate production

Establishing industrial biotechnology for the production of chemical compounds from the biosynthetic pathway has received a significant boost with the implementation of metabolic engineering. At present, metabolic engineering in Saccharomyces cerevisiae gains significant advantages of integration of knowledge acquired through a long history of use and data acquisition from novel –omics technologies hence enabling the development of a tailor-made S. cerevisiae with desired features for various industrial applications.With regard to environmentally friendly (eco-friendly) materials, engineering of biodegradable polyhydroxybutyrate (PHB) producing microbes has been studied as a potential alternative to petroleum-based thermoplastics. Heterologous expression of the bacterial PHB biosynthesis pathway in S. cerevisiae involves the utilization of acetyl-CoA, an intermediate of the central carbon metabolism, as precursor and NADPH, a redox cofactor used during anabolic metabolism, as a required cofactor for the catalyzing enzymes in the PHB biosynthesis pathway. Provision of acetyl-CoA and NADPH by alteration of the endogenous pathways and/or implementation of a heterologous gene/pathway was investigated with the aim to improve PHB production in S. cerevisiae. Since the specific growth rate and the type of carbon source (fermentable/non-fermentable) influence cell physiology and affect the growth of S. cerevisiae, PHB production was examined at different specific growth rates on different carbon sources. Overexpression of genes in the native ethanol degradation pathway and heterologous expression of a phosphoketolase pathway from Aspergillus nidulans aiming to increase the production of cytosolic acetyl-coA and chromosomal integration of gapN from Streptococcus mutans to enhance the availability of NADPH were evaluated for their possibility to promote PHB production in S. cerevisiae. The enhancement of acetyl-CoA and NADPH either by the combined strategies of the ethanol degradation pathway and gapN or utilization of the phosphoketolase pathway resulted in the improved PHB content from 4 mg/gDW in the reference strain to approximately 28 mg/gDW. It is difficult for S. cerevisiae to compete with other natural PHB producers like Ralstonia eutropha which benefit from native enzymes for the biosynthesis or with the engineered E. coli since the metabolism in S. cerevisiae is more complex and involves compartmentalization and shuttle systems for precursor and redox balancing. However, the strategies employed in this study involve both engineering of the central carbon and redox metabolism and it demonstrated that it is possible to substantially improve PHB production. Furthermore, the applied strategies may well be suitable also for improving the production of other chemicals, derived from acetyl-CoA and requires NADPH for its biosynthesis

Specific growth rate and substrate dependent polyhydroxybutyrate production in Saccharomyces cerevisiae

Production of the biopolymer polyhydroxybutyrate (PHB) in Saccharomyces cerevisiae starts at the end of exponential phase particularly when the specific growth rate is decreased due to the depletion of glucose in the medium. The specific growth rate and the type of carbon source (fermentable/non-fermentable) have been known to influence the cell physiology and hence affect the fermentability of S. cerevisiae. The production of PHB utilizes cytosolic acetyl-CoA as a precursor and the S. cerevisiae employed in this study is therefore a strain with the enhanced cytosolic acetyl-CoA supply. Growth and PHB production at different specific growth rates were evaluated on glucose, ethanol and a mixture of glucose and ethanol as carbon source. Ethanol as carbon source yielded a higher PHB production compared to glucose since it can be directly used for cytosolic acetyl-CoA production and hence serves as a precursor for PHB production. However, this carbon source results in lower biomass yield and hence it was found that to ensure both biomass formation and PHB production a mixture of glucose and ethanol was optimal, and this resulted in the highest volumetric productivity of PHB, 8.23 mg/L \ub7 h-1, at a dilution rate of 0.1 h-1

Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway

The metabolic pathways of the central carbon metabolism in Saccharomyces cerevisiae are well studied and consequently S. cerevisiae has been widely evaluated as a cell factory for many industrial biological products. In this study, we investigated the effect of engineering the supply of precursor, acetyl-CoA, and cofactor, NADPH, on the biosynthesis of the bacterial biopolymer polyhydroxybutyrate (PHB), in S. cerevisiae. Supply of acetyl-CoA was engineered by over-expression of genes from the ethanol degradation pathway or by heterologous expression of the phophoketolase pathway from Aspergillus nidulans. Both strategies improved the production of PHB. Integration of gapN encoding NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans into the genome enabled an increased supply of NADPH resulting in a decrease in glycerol production and increased production of PHB. The strategy that resulted in the highest PHB production after 100h was with a strain harboring the phosphoketolase pathway to supply acetyl-CoA without the need of increased NADPH production by gapN integration. The results from this study imply that during the exponential growth on glucose, the biosynthesis of PHB in S. cerevisiae is likely to be limited by the supply of NADPH whereas supply of acetyl-CoA as precursor plays a more important role in the improvement of PHB production during growth on ethanol

Toward Design-based Engineering of Industrial Microbes

Engineering industrial microbes has been hampered by incomplete knowledge of cell biology. Thus an iterative engineering cycle of modeling, implementation, and analysis has been used to increase knowledge of the underlying biology while achieving engineering goals. Recent advances in Systems Biology technologies have drastically improved the amount of information that can be collected in each iteration. As well, Synthetic Biology tools are melding modeling and molecular implementation. These advances promise to move microbial engineering from the iterative approach to a design-oriented paradigm, similar to electrical circuits and architectural design. Genome-scale metabolic models, new tools for controlling expression, and integrated -omics analysis are described as key contributors in moving the field toward Design-based Engineering. \ua9 2010 Elsevier Ltd. All rights reserved

Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae

Through metabolic engineering microorganisms can be engineered to produce new products and further produce these with higher yield and productivities. Here, we expressed the bacterial polyhydroxybutyrate (PHB) pathway in the yeast Saccharomyces cerevisiae and we further evaluated the effect of engineering the formation of acetyl coenzyme A (acetyl-CoA), an intermediate of the central carbon metabolism and precursor of the PHB pathway, on heterologous PHB production by yeast. We engineered the acetyl-CoA metabolism by co-transformation of a plasmid containing genes for native S. cerevisiae alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA acetyltransferase (ERG10) and a Salmonella enterica acetyl-CoA synthetase variant (acs(L641P)), resulting in acetoacetyl-CoA overproduction, together with a plasmid containing the PHB pathway genes coding for acetyl-CoA acetyltransferase (phaA), NADPH-linked acetoacetyl-CoA reductase (phaB) and poly(3-hydroxybutyrate) polymerase (phaC) from Ralstonia eutropha H16. Introduction of the acetyl-CoA plasmid together with the PHB plasmid, improved the productivity of PHB more than 16 times compared to the reference strain used in this study, as well as it reduced the specific product formation of side products