Bacteroides succinogenes S-85 grows readily in media containing 0.2% (w/v) filter paper cellulose or microcrystalline cellulose as the carbohydrate source. During growth, the cells appear to adhere to the cellulose. Cell-free culture supernates and cell extracts from cellulose-grown cultures had very low hydrolytic activity against either filter paper or crystalline cellulose (Avicel) as substrate, although H3PO4-swollen cellulose, carboxmethylcellulose, and cellobiose were readily hydrolyzed. Cells grown on either cellobiose or glucose exhibited cell-bound carboxymethylcellulase (CMCase) and cellobiase activities. Cultures grown on cellulose had seven to eight times more CMCase activity than either cellobiose- or glucose-grown cultures. Seventy percent of the CMCase activity was present in the supernate, of which 50--60% was associated with sedimentable membranous fragments. the cellobiase, which was largely cell associated, appeared to be constitutive, and the only product detected on enzymic hydrolysis of cellobiose was glucose. The cellobiase activity was strongly inhibited by 0.02 M tris(hydroxymethyl)-aminomethane (Tris), pH 7.1, but this was partially relieved by phosphate ions. These data indicate that B. succinogenes S-85 contains high endo-beta-1,4-glucanase and beta-1,4-glucanase and beta-1,4-glucosidase-like activities.

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Agarose is degraded by a beta-agarase from Pseudomonas atlantica to neoagarooligosaccharides of degree of polymerization (DP), 4, 6, 8, and 10. A beta-neoagarotetraose hydrolase cleaves the central beta-linkage in neoagarotetraose and the beta-linkage near the nonreducing end in neoagarohexaose and -octaose to yield neoagarobiose. The beta-neoagarotetraose hydrolase was localized on or outside the cytoplasmic membrane, in the cell wall region. The enzyme was activated by NaCl, KCl, CaCl2, MnCl2, and MgSO4, has a Km of 3.4 X 10(-3) M for neoagarotetraose, was free from beta-agarase and alpha-neoagarobiose hydrolase activity, and showed no transglycosidic activity.

A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the Pseudomonas-based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive. The characteristics and potential of the expression system were determined. Using flow cytometry and fed-batch fermentations, we have shown that, with the newly developed cumate-regulated system, (i) higher recombinant product yields can be obtained than with the pET (isopropyl-␤-D-thiogalactopyranoside [IPTG])-induced expression system, (ii) expression is tightly regulated, (iii) addition of cumate quickly results in a fully induced and homogenous protein-expressing population in contrast to the bimodal expression profile of an IPTGinduced population, (iv) expression can be modulated by varying the cumate concentration, and (v) the cumate-induced population remains induced and fully expressing even at 8 h following induction, resulting in high yields of the target protein Furthermore, the cumate gene switch described in this article is applicable to a wide range of E. coli strains.A variety of gene expression systems exist for the production of recombinant proteins and as a tool in metabolic engineering. These systems differ in the hosts, plasmids, and promoters being utilized. The variety of existing expression systems reflects the diversity, complexity, and toxicity of the proteins being produced, requiring in certain instances that the product be expressed at various concentrations (14) or at different phases of cell growth (14) and be soluble (4), secreted (13) or compartmentalized (40,45). In an attempt to meet these challenges, the search for or the development of new hosts and expression vectors is ongoing.Gene transcription can be enhanced by replacing a promoter sequence naturally associated with that gene with a sequence of a stronger promoter (17, 42). The nature of the bioprocess will dictate the promoter of choice. A process that requires high-level expression will necessitate a strong constitutive or regulated promoter. A process requiring the expression of a product with toxicity issues for the cell will benefit from a regulated expression system that does not possess basal expression under repressed conditions (26, 37). The promoters P lac , P trp , P tac , P L , P T7 , P BAD , and P lacUV5 are commonly utilized for the construction of expression vectors (1, 18). Among these, P lacUV5 , P tac , and the combined system of P T7 with P lacUV5 are widely used, be...

The green fluorescent protein (GFP) was used as a model protein to study the recombinant protein production by the strain Methylobacterium extorquens ATCC 55366. Scale-up from shake flasks to 20 l fed-batch fermentation was achieved using methanol as a sole carbon and energy source and a completely minimal culture medium. Two different expression vectors were used to express GFP. Clone PCM-GFP containing the vector pCM110 with native promoter of the methanol dehydrogenase P mxaF produced approximately 100-fold more GFP than the clone PRK-GFP containing the vector pRK310 with the heterogeneous promoter P lac . Several fed-batch fermentations with and without selective pressure (tetracycline) were run in a 20 l stirred tank fermenter using the two different clones of M. extorquens. The methanol concentration was monitored with an on-line semiconductor gas sensor in the culture broth. It was maintained at a non-toxic level of 1.4 g l 31 with an adaptative control which regulates the methanol feed rate. The same growth profile was achieved in all fermentations. The maximum growth rate (W max ) was 0.18 h 31 with an overall yield (Y X =S ) of 0.3 g g 31 methanol. With this high cell density fermentation process, we obtained high levels (up to 4 g l 31 ) of GFP with the clone PCM-GFP. The maximum specific GFP production (Y GFP=X ) with this clone was 80 mg g 31 representing approximately 16% of the total cell protein. Additional feeding of pure oxygen to the fermenter permitted a longer phase of exponential growth but had no effect on the total yields of biomass and GFP. The specific GFP production of clone PCM-GFP remained unaffected in the presence or absence of selective pressure (tetracycline), within the initial 50 h of the fermentation culture. These results suggest that M. extorquens ATCC 55366 could be an interesting candidate for overexpression of recombinant proteins.

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