Cell Engineering and Molecular Pharming for Biopharmaceuticals

Biopharmaceuticals are often produced by recombinant E. coli or mammalian cell lines. This is usually achieved by the introduction of a gene or cDNA coding for the protein of interest into a well-characterized strain of producer cells. Naturally, each recombinant production system has its own unique advantages and disadvantages. This paper examines the current practices, developments, and future trends in the production of biopharmaceuticals. Platform technologies for rapid screening and analyses of biosystems are reviewed. Strategies to improve productivity via metabolic and integrated engineering are also highlighted

Degradation of cellulosic biomass and its subsequent utilization for the production of chemical feedstocks. Progress report, June 1, 1977--August 31, 1977

Studies on the microbial degradation of cellulose biomass continues to be centered around Clostridium thermocellum. The effect of surfactants on growth and cellulase production by C. thermocellum was investigated. The effect of pH on growth and reducing sugar accumulation rate of Clostridium thermocellum on solka floc was evaluated. Activity of extracellular cellulase of Clostridium thermocellum ATCC 27405 was examined using TNP--CMC and Avicel as substrates. The pH optima are 5 and 4.5, respectively. Hydrolysis of either substrate is not inhibited by cellobiose, xylose, or glucose. The enzyme appears to be quite stable under reaction conditions at 60/sup 0/C. Thus far, regulation studies indicate that CMCase formation is not repressed by cellobiose. The search for plasmids in C. thermocellum was continued. The presence of plasmids was confirmed by cesium chloride ethidium bromide gradient centrifugation and electron microscopy. Two plasmids were detected, one with an approximate molecular weight of 1 x 10/sup 6/ daltons. Studies on the fermentation of lactic acid to propionic acid showed the pathway in C. propionicum to be simpler than in M. elsdenii and hence more amenable to manipulation for acrylate production. Using Lactobacillius delbrueckii, it was possible to convert glucose, cellobiose, and cellulose hydrolysates to lactic acid rapidly and quantitatively. Fermentations of C. acetobutylicum growing in soluble media were performed. Detailed studies of Clostridium thermoaceticum have shown that pH is the primary limiting factor in the production of acetic acid. pH-controlled fermentations indicated accumulations of over 30 gm/l of acetic acid

Degradation of cellulosic biomass and its subsequent utilization for the production of chemical feedstocks. Progress report, March 1, 1977--May 31, 1977

The degradation of cellulosic biomass continues to focus on the anaerobic thermophile Clostridium thermocellum. When grown on crystalline cellulose (MN300) in batch culture, there is an initial rapid accumulation of reducing sugars but the sugars are rapidly metabolized in later times during the fermentation. When grown on Solka floc with periodic addition of the substrate, there is a continual accumulation of reducing sugars (xylose, glucose, and cellobiose) as well as ethanol and acetic acid during the entire course of the fermentation. In the presence of surfactant in the growth medium, there is an increased appearance of extracellular cellulases. A chemically defined medium is being developed for growth Cl. thermocellum in order to study the enzyme regulations. Lastly, a trinitrophenyl-carboxylmethyl cellulose substrate for determining cellulose activity appears to be a promising and rapid assay. Progress in the genetic manipulations has been cautious but promising. Preliminary evidence leads to optimistic projection on the presence of plasmids and bacteriophage in Cl. thermocellum. The production of chemical feedstocks continues to focus on acrylic acid, acetone/butanol and acetic acid. Studies with cell free extracts of Clostridium propionicum have shown the production and accumulation of acrylic acid from lactic acid. The use of electron acceptor in cell-free systems has shown effective prevention on the reduction of acrylic acid to propionic acid. Medium development and strain selection using available acetone/butanol producing Cl. acetobutylicum have been initiated. There is every indication that these strains are capable to produce mixed solvents close to the theoretical maximum yield. An accurate and rapid method for quantifying acetic acid was developed. This technique is being used to examine the pertinent parameters on the production of acetic acid by Clostridium thermoaceticum

Degradation of cellulosic biomass and its subsequent utilization for the production of chemical feedstocks. Progress report, June 1-August 31, 1978

Studies concerning the cellobiose properties of Clostridium thermocellum were started to determine if the cellulose degradation end products can be enhanced for glucose (with a subsequent decrease in cellobiose). Implications of preliminary studies indicate that the cells or the enzyme(s) responsible for converting cellobiose to glucose can be manipulated environmentally and genetically to increase the final yield of glucose. The second area of effort is to the production of chemical feedstocks. Three fermentations have been identified for exploration. Preliminary reports on acrylic acid acetone/butanol, and acetic acid production by C. propionicum, C. acetobutylicum, and C. thermoaceticum, respectively, are included. (DMC

Degradation of cellulosic biomass and its subsequent utilization for the production of chemical feedstocks. Progress report, December 1, 1978-February 28, 1979

The ongoing progress of a coordinated research program aimed at optimizing the biodegradation of cellulosic biomass to ethanol and chemical feedstocks is summarized. Growth requirements and genetic manipulations of clostridium thermocellum for selection of high cellulose producers are reported. The enzymatic activity of the cellulase produced by these organisms was studied. The soluble sugars produced from hydrolysis were analyzed. Increasing the tolerance of C. thermocellum to ethanol during liquid fuel production, increasing the rate of product formation, and directing the catabolism to selectively achieve high ethanol concentrations with respect to other products were studied. Alternative substrates for C. thermocellum were evaluated. Studies on the utilization of xylose were performed. Single stage fermentation of cellulose using mixed cultures of C. thermocellum and C. thermosaccharolyticum were studied. The study of the production of chemical feedstocks focused on acrylic acid, acetone/butanol, acetic acid, and lactic acid

Degradation of cellulosic biomass and its subsequent utilization for the production of chemical feedstocks

Progress in studies on the production of reducing sugars and other products by Clostridium thermocellum on cellulosic biomass is reported. The rate of reducing sugar production using corn residue was found to be equal if not greater than on solka floc. Current work is being devoted towards elucidating discrepancies between reducing sugar analysis and high pressure liquid chromatography sugar analysis in order to permit accurate material balances to be completed. Studies are reported in further characterizing the plasmics of C. thermocellum and in the development of protoplasts of the same microorganism. A process and economic analysis for the production of 200 x 10/sup 6/ pounds (90 x 10/sup 6/ kilograms) per year of soluble reducing sugars from corn stover cellulose, using enzymes derived from Clostridium thermocellum was designed. Acrylic acid was produced in resting cell preparation of Clostridium propionicum from both ..beta..-alanine and from propionic acid. Results from the conversion of corn stover hydrolyzates to lactic acid, a precursor to acrylic acid, show that up to 70% of the sugars produced are converted to lactic acid. Efforts are proceeding to improve the conversion yield and carry out the overall conversion of corn stover to acrylic acid in the same fermentor. Results on the production of acetone and butanol by Clostridium acetobutylicum demonstrated the capability of the strain to produce mixed solvents in concentration and conversion similar to that achieved in industrial processes. Various studies on the production of acetic acid by Clostridium thermoaceticum are also reported