Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD₆₀₀ compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.     

Techno‐economic evaluation of a two‐step biological process for hydrogen production

An integrated biological process for the production of hydrogen based on thermophilic and photo‐heterotrophic fermentation was evaluated from a technical and economic standpoint. Besides the two fermentation steps the process also includes pretreatment of the raw material (potato steam peels) and purification of hydrogen using amine absorption. The study aimed neither at determining the absolute cost of biohydrogen nor at an economic optimization of the production process, but rather at studying the effects of different parameters on the production costs of biohydrogen as a guideline for future improvements. The effect of the key parameters, hydrogen productivity and yield and substrate concentration in the two fermentations on the cost of the hydrogen produced was studied. The selection of the process conditions was based mainly on laboratory data. The process was simulated by use of the software Aspen Plus and the capital costs were estimated using the program Aspen Icarus Process Evaluator. The study shows that the photo‐fermentation is the main contributor to the hydrogen production cost mainly because of the cost of plastic tubing, for the photo‐fermentors, which represents 40.5% of the hydrogen production cost. The costs of the capital investment and chemicals were also notable contributors to the hydrogen production cost. Major economic improvements could be achieved by increasing the productivity of the two fermentation steps on a medium‐term to long‐term scale. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large‐scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale‐up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha‐1‐antitrypsin (rAAT) by co‐infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD₆₀₀ of 0.5 and a 0.25‐min vacuum infiltration, and multiple 1‐min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post‐induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Recovery of biologically produced 3‐hydroxypropionaldehyde and its dehydrated product acrolein

3‐Hydroxypropionaldehyde (3‐HPA), which can be derived from biomass, is an important precursor for low‐cost, large‐volume acrolein‐based chemicals like acrylic acid and acrylamide with a wide range of applications. In order to find an efficient process for isolating 3‐HPA from fermentation broth, we comparatively investigated several separation methods including precipitation with hydrazides, immobilization with amines, reactive extraction with thiols, extraction with hydrophilic solvents, and reactive distillation as acrolein. It turned out that the reactive distillation is the most efficient method for in situ recovery of 3‐HPA as acrolein. In a reactive distillation process at 37°C and Hammett acidity H₀ = –1, the aldehyde concentration was reduced to 6 ± 1 mM in the transformation medium and increased to 1866 ± 146 mM in the distillate. The yield was 96 ± 8%. These experimental results are close to the calculated ideal equilibrium results assuming total dehydration of 3‐HPA to acrolein. The main advantages of the reactive distillation process are that the recovery, purification, and concentration of acrolein are carried out in one step and the process is well suited for large‐scale production at low costs.     

Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs

The transient expression of recombinant biopharmaceutical proteins in plants can suffer inter‐batch variation, which is considered a major drawback under the strict regulatory demands imposed by current good manufacturing practice (cGMP). However, we have achieved transient expression of the monoclonal antibody 2G12 and the fluorescent marker protein DsRed in tobacco leaves with ～15% intra‐batch coefficients of variation, which is within the range reported for transgenic plants. We developed models for the transient expression of both proteins that predicted quantitative expression levels based on five parameters: The OD_600nm of Agrobacterium tumefaciens (from 0.13 to 2.00), post‐inoculation incubation temperature (15–30°C), plant age (harvest at 40 or 47 days after seeding), leaf age, and position within the leaf. The expression models were combined with a model of plant biomass distribution and extraction, generating a yield model for each target protein that could predict the amount of protein in specific leaf parts, individual leaves, groups of leaves, and whole plants. When the yield model was combined with a cost function for the production process, we were able to perform calculations to optimize process time, yield, or downstream costs. We illustrate this procedure by transferring the cost function from a production process using transgenic plants to a hypothetical process for the transient expression of 2G12. Our models allow the economic evaluation of new plant‐based production processes and provide greater insight into the parameters that affect transient protein expression in plants. Biotechnol. Bioeng. 2012; 109: 2575–2588. © 2012 Wiley Periodicals, Inc.     

Cell wall biochemical alterations during Agrobacterium‐mediated expression of haemagglutinin‐based influenza virus‐like vaccine particles in tobacco

Influenza virus‐like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant‐based biotechnology allows for the large‐scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium‐mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post‐Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG‐I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin‐based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing.     

Spaceflight‐induced enhancement of 2‐keto‐L‐gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis

Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l^?1 h^?1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m³ fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.

Significance and Impact of the Study

KB2194‐320, a combination of two bacterial strains bred by spaceflight mutation, exhibited significantly improved 2‐KLG productivity and hence could potentially increase the efficiency and reduce the cost of vitamin C production by the two‐step fermentation process. In addition, a new pH indicator method was applied for rational screening of K2, which dramatically improved the efficiency of screening.     

Improved one‐step steam pretreatment of SO2‐Impregnated softwood with time‐dependent temperature profile for ethanol production

In the production of ethanol from lignocellulosic material, pretreatment of the raw material before enzymatic hydrolysis and fermentation is essential to obtain high overall yields of sugar and ethanol. Two‐step steam pretreatment results in higher ethanol yields from softwood than the standard one‐step pretreatment process. However, the difficulty with separation and washing of the material at high pressure between the two pretreatment steps is a major drawback. In this study, a new one‐step pretreatment procedure was investigated, in which the time‐temperature profile was varied during pretreatment. The efficiency of pretreatment was assessed by performing simultaneous saccharification and fermentation on the pretreated slurries. Pretreatment of SO₂‐impregnated softwood performed by varying the temperature (190–226°C), the residence time (5–10 min), and the mode of temperature increase (linear or stepwise), resulted in recovery of about 90% of the mannose and glucose present in the raw material. The highest ethanol yield, 75% of theoretical based on the glucan and mannan content of the raw material, was obtained at pretreatment conditions of 190°C for 12 min. Similar ethanol yields were achieved when running the pretreatment as one‐step (190–200°C), two levels of temperature, at shorter residence time (7 min), which results in lower capital costs for the process. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Design of experiments reveals critical parameters for pilot‐scale freeze‐and‐thaw processing of L‐lactic dehydrogenase

Freezing constitutes an important unit operation of biotechnological protein production. Effects of freeze‐and‐thaw (F/T) process parameters on stability and other quality attributes of the protein product are usually not well understood. Here a design of experiments (DoE) approach was used to characterize the F/T behavior of L‐lactic dehydrogenase (LDH) in a 700‐mL pilot‐scale freeze container equipped with internal temperature and pH probes. In 24‐hour experiments, target temperature between –10 and –38°C most strongly affected LDH stability whereby enzyme activity was retained best at the highest temperature of –10°C. Cooling profile and liquid fill volume also had significant effects on LDH stability and affected the protein aggregation significantly. Parameters of the thawing phase had a comparably small effect on LDH stability. Experiments in which the standard sodium phosphate buffer was exchanged by Tris‐HCl and the non‐ionic surfactant Tween 80 was added to the protein solution showed that pH shift during freezing and protein surface exposure were the main factors responsible for LDH instability at the lower freeze temperatures. Collectively, evidence is presented that supports the use of DoE‐based systematic analysis at pilot scale in the identification of F/T process parameters critical for protein stability and in the development of suitable process control strategies.     

Isolation and characterization of new poly(3HB)‐accumulating star‐shaped cell‐aggregates‐forming thermophilic bacteria

Aims: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3‐hydroxybutyrate), poly(3HB), at elevated temperatures. Methods and Results: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram‐negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star‐shaped cell‐aggregates (SSCAs) during growth; only strain MW10 consisted of free‐living rod‐shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii , Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8 g l^?l (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. Conclusions: New PHA‐accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. Significance and Impact of the Study: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA‐accumulating bacteria and also by thermophilic bacteria. PHA‐producing thermophiles can significantly reduce the costs of fermentative PHA production.     

Techno‐economic analysis for incorporating a liquid–liquid extraction system to remove acetic acid into a proposed commercial scale biorefinery

Mitigating the effect of fermentation inhibitors in bioethanol plants can have a great positive impact on the economy of this industry. Liquid–liquid extraction (LLE) using ethyl acetate is able to remove fermentation inhibitors—chiefly, acetic acid—from an aqueous solution used to produce bioethanol. The fermentation broth resulting from LLE has higher performance for ethanol yield and its production rate. Previous techno‐economic analyses focused on second‐generation biofuel production did not address the impact of removing the fermentation inhibitors on the economic performance of the biorefinery. A comprehensive analysis of applying a separation system to mitigate the fermentation inhibition effect and to provide an analysis on the economic impact of removal of acetic acid from corn stover hydrolysate on the overall revenue of the biorefinery is necessary. This study examines the pros and cons associated with implementing LLE column along with the solvent recovery system into a commercial scale bioethanol plant. Using details from the NREL‐developed model of corn stover biorefinery, the capital costs associated with the equipment and the operating cost for the use of solvent were estimated and the results were compared with the profit gain due to higher ethanol production. Results indicate that the additional capital will add 1% to the total capital and manufacturing cost will increase by 5.9%. The benefit arises from the higher ethanol production rate and yield as a consequence of inhibitor extraction and results in a $0.35 per gallon reduction in the minimum ethanol selling price (MESP). © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:971–977, 2016     

Production of paramylon,a β‐1,3‐glucan,by heterotrophic growth of Euglena gracilis on potato liquor in fed‐batch and repeated‐batch mode of cultivation

Recently, it had been shown that Euglena gracilis was able to grow heterotrophically not only on synthetic media, but also on media based on potato liquor. Supplementation with glucose in both cases led to the accumulation of paramylon, a β‐1,3‐glucan. Thus, such a process may yield a valuable product accompanied by the revaluation of an otherwise annoying waste stream of the potato‐starch industry. Actually, process strategies have been evaluated in order to optimise the concentration of paramylon obtained at the end of the cultivation process. Therefore, cultivation processes based on fed‐batch and in particular repeated‐batch strategies have been studied. It is shown that repeated‐batch operation maybe particularly suited for such a process since E. gracilis seems to adapt gradually to the cultivation medium so that the concentration of media components may be increased step by step. Repeated‐batch cultivation of E. gracilis leads to biomass concentrations in access of 20 g/L with a consistent paramylon mass fraction of about 75%. Cultivations have been carried out at an operating temperature of 27.5°C. As had been found earlier already, pH control is not required during cultivation. On the basis of these results it is clear that repeated‐batch cultivation represent a simple and economic way for the production of paramylon by heterotrophic cultivation of E. gracilis.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Integrated two‐liquid phase bioconversion and product‐recovery processes for the oxidation of alkanes: Process design and economic evaluation

Pseudomonas oleovorans and recombinant strains containing the alkane oxidation genes can produce alkane oxidation products in two‐liquid phase bioreactor systems. In these bioprocesses the cells, which grow in the aqueous phase, oxidize apolar, non‐water soluble substrates. The apolar products typically accumulate in the emulsified apolar phase. We have studied both the bioconversion systems and several downstream processing systems to separate and purify alkanols from these two‐liquid phase media. Based on the information generated in these studies, we have now designed bioconversion and downstream processing systems for the production of 1‐alkanols from n‐alkanes on a 10 kiloton/yr scale, taking the conversion of n‐octane to 1‐octanol as a model system. Here, we describe overall designs of fed‐batch and continuous‐fermentation processes for the oxidation of octane to 1‐octanol by Pseudomonas oleovorans, and we discuss the economics of these processes. In both systems the two‐liquid phase system consists of an apolar phase with hexadecene as the apolar carrier solvent into which n‐octane is dissolved, while the cells are present in the aqueous phase. In one system, multiple‐batch fermentations are followed by continuous processing of the product from the separated apolar phase. The second system is based on alkane oxidation by continuously growing cultures, again followed by continuous processing of the product. Fewer fermentors were required and a higher space‐time‐yield was possible for production of 1‐octanol in a continuous process. The overall performance of each of these two systems has been modeled with Aspen software. Investment and operating costs were estimated with input from equipment manufacturers and bulk‐material suppliers. Based on this study, the production cost of 1‐octanol is about 7 US$kg⁻¹ when produced in the fed‐batch process, and 8 US$kg⁻¹ when produced continuously. The comparison of upstream and downstream capital costs and production costs showed significantly higher upstream costs for the fed‐batch process and slightly higher upstream costs for continuous fermentation. The largest cost contribution was due to variable production costs, mainly resulting from media costs. The organisms used in these systems are P. putida alk+ recombinants which oxidize alkanes, but cannot oxidize the resulting alkanols further. Hence, such cells need a second carbon source, which in these systems is glucose. Although the continuous process is about 10% more expensive than the fed‐batch process, improvements to reduce overall cost can be achieved more easily for continuous than for fed‐batch fermentation by decreasing the dilution rate while maintaining near constant productivity. Improvements relevant to both processes can be achieved by increasing the biocatalyst performance, which results in improved overall efficiency, decreased capital investment, and hence, decreased production cost. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 84: 459–477, 1999.     

Three‐phase succession of autochthonous lactic acid bacteria to reach a stable ecosystem within 7 days of natural bamboo shoot fermentation as revealed by different molecular approaches

Microbial community structure and population dynamics during spontaneous bamboo shoot fermentation for production of ‘soidon’ (indigenous fermented food) in North‐east India were studied using cultivation‐dependent and cultivation‐independent molecular approaches. Cultivation‐dependent analyses (PCR‐amplified ribosomal DNA restriction analysis and rRNA gene sequencing) and cultivation‐independent analyses (PCR‐DGGE, qPCR and Illumina amplicon sequencing) were conducted on the time series samples collected from three independent indigenous soidon fermentation batches. The current findings revealed three‐phase succession of autochthonous lactic acid bacteria to attain a stable ecosystem within 7 days natural fermentation of bamboo shoots. Weissella spp. (Weissella cibaria, uncultured Weissella ghanensis) and Lactococcus lactis subsp. cremoris predominated the early phase (1–2 days) which was joined by Leuconostoc citreum during the mid‐phase (3 days), while Lactobacillus brevis and Lactobacillus plantarum emerged and became dominant in the late phase (5–7 days) with concurrent disappearance of W. cibaria and L. lactis subsp. cremoris. Lactococcus lactis subsp. lactis and uncultured Lactobacillus acetotolerans were predominantly present throughout the fermentation with no visible dynamics. The above identified dominant bacterial species along with their dynamics can be effectively utilized for designing a starter culture for industrialization of soidon production. Our results showed that a more realistic view on the microbial ecology of soidon fermentation could be obtained by cultivation‐dependent studies complemented with cultivation‐independent molecular approaches. Moreover, the critical issues to be considered for reducing methodological biases while studying the microbial ecology of traditional food fermentation were also highlighted with this soidon fermentation model.     

Pilot production of Clonostachys rosea conidia in a solid‐state fermentor optimized using response surface methodology

The nonpathogenic, saprophytic fungus Clonostachys rosea is one of the most powerful fungal biological control agents (BCAs). However, the production of fungal BCAs is still a major constraint for their large‐scale use and commercialization. Here, we developed a novel solid‐fermentation reactor that is light transparent and ventilated both at the top and the bottom, and optimized C. rosea cultivation conditions in solid‐state fermentation using response surface methodology. The growth area of spores provided by the novel fermentor was two times that of the traditional one. A quadratic polynomial model was developed, which indicated the effects of variables on the conidia yield. The greatest spore production of 3.50 × 10¹⁰ spores/g‐dry‐matter was obtained after 11 days at the initial moisture content of 69.2% w/w, the medium thickness of 3.84 cm, and the porosity of 0.37%. The optimized spore yield was increased by one order of magnitude. The fermentation time was shortened from 15 to 11 days. With the novel solid‐fermentation reactor, increase in C. rosea spores production and decrease in fermentation time were achieved. Current data imply that both the novel solid‐fermentation reactor designed and the optimized fermentation conditions are suitable for industrial‐scale C. rosea spore production.     

High yield and cost‐effective production of poly(γ‐glutamic acid) with Bacillus subtilis

Poly(γ‐glutamic acid) (γ‐PGA) is a promising biopolymer with many potential industrial and pharmaceutical applications. To reduce the production costs, the effects of yeast extract and L ‐glutamate in the substrate for γ‐PGA production were investigated systematically at shake flask scale. The results showed that lower concentrations of yeast extract (40 g/L) and L ‐glutamate (30 g/L) were beneficial for the cost‐effective production of γ‐PGA in the formulated medium. By maintaining the glucose concentration in the range of 3–10 g/L via a fed‐batch strategy in a 10‐L fermentor, the production of γ‐PGA was greatly improved with the highest γ‐PGA concentration of 101.1 g/L, a productivity of 2.19 g/L·h and a yield of 0.57 g/g total substrate, which is about 1.4‐ to 3.2‐fold higher than those in the batch fermentation. Finally, this high‐density fermentation process was successfully scaled up in a 100‐L fermentor. The present work provides a powerful approach to produce this biopolymer as a bulk chemical in large scale.     

Attachment of Agrobacterium tumefaciens to leaf tissue in response to infiltration conditions

Transient expression of recombinant proteins in plant tissues following Agrobacterium‐mediated gene transfer is a promising technique for rapid protein production. However, transformation rates and transient expression levels can be sub‐optimal depending on process conditions. Attachment of Agrobacterium tumefaciens to plant cells is an early, critical step in the gene transfer pathway. Bacterial attachment levels and patterns may influence transformation and, by extension, transient expression. In this study, attachment of A. tumefaciens to lettuce leaf tissue was investigated in response to varying infiltration conditions, including bacterial density, surfactant concentration, and applied vacuum level. Bacterial density was found to most influence attachment levels for the levels tested (10⁸, 10⁹, and 10¹⁰ CFU/mL), with the relationship between bacterial density and attachment levels following a saturation trend. Surfactant levels tested (Break‐Thru S240: 1, 10, 100, and 1,000 µL/L) also had a significant positive effect on bacterial attachment while vacuum level (5, 25, and 45 kPa) did not significantly affect attachment in areas exposed to bacteria. In planta transgene transient expression levels were measured following infiltration with 10⁸, 10⁹, and 10¹⁰ CFU/mL bacterial suspension. Notably, the highest attachment level tested led to a decrease in transient expression, suggesting a potential link between bacterial attachment levels and downstream phenomena that may induce gene silencing. These results illustrate that attachment can be controlled by adjusting infiltration conditions and that attachment levels can impact transgene transient expression in leaf tissue. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1137–1144, 2014     

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Porcine circovirus type 2 (PCV‐2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus‐associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV‐2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV‐2 capsid protein (CP) from plants is an essential first step towards the goal of a plant‐produced PCV‐2 vaccine candidate. In this study, the PCV‐2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV‐2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self‐assembled into virus‐like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant‐produced PCV‐2 VLPs elicited specific antibody responses to PCV‐2 CP. This is the first report describing the expression of PCV‐2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.     

Mature‐stem expression of a silencing‐resistant sucrose isomerase gene drives isomaltulose accumulation to high levels in sugarcane

Isomaltulose (IM) is a natural isomer of sucrose. It is widely approved as a food with properties including slower digestion, lower glycaemic index and low cariogenicity, which can benefit consumers. Availability is currently limited by the cost of fermentative conversion from sucrose. Transgenic sugarcane plants with developmentally‐controlled expression of a silencing‐resistant gene encoding a vacuole‐targeted IM synthase were tested under field conditions typical of commercial sugarcane cultivation. High yields of IM were obtained, up to 483 mm or 81% of total sugars in whole‐cane juice from plants aged 13 months. Using promoters from sugarcane to drive expression preferentially in the sugarcane stem, IM levels were consistent between stalks and stools within a transgenic line and across consecutive vegetative field generations of tested high‐isomer lines. Germination and early growth of plants from setts were unaffected by IM accumulation, up to the tested level around 500 mm in flanking stem internodes. These are the highest yields ever achieved of value‐added materials through plant metabolic engineering. The sugarcane stem promoters are promising for strategies to achieve even higher IM levels and for other applications in sugarcane molecular improvement. Silencing‐resistant transgenes are critical to deliver the potential of these promoters in practical sugarcane improvement. At the IM levels now achieved in field‐grown sugarcane, direct production of IM in plants is feasible at a cost approaching that of sucrose, which should make the benefits of IM affordable on a much wider scale.