Den Haan et al. [49] calculated that a 20- to 120-fold improvement in CBH ex­pression, as well as simultaneous high-level expression of other cellulase com­ponents, will be necessary for slow growth on crystalline cellulose. This calcu­lation assumes a strain that can grow at 0.02 h-1 has a typical anaerobic yield of 0.1 gbiomass/g substrate or an aerobic yield of 0.45 gbiomass/gsubstrate, that the expressed cellulase has a specific activity which is the same as that of crude T. reesei cellulase on avicel (0.6 U/mg), and that CBH1 would make up the same fraction of total cellulase protein as in the T. reesei system [9]. While techniques for rational design of cellulases for improvement in ex­pression level and potentially specific activity will be important to achieving this goal, techniques involving random natural and/or induced mutation will also play an important role. The well-established success of directed evolu­tion techniques for enzymes and enzyme systems (e. g., see reviews in [132, 133]) can be transferred to engineering organisms for CBP, although this application does present unique challenges due to the lack of a good high — throughput screening technique for activity on insoluble cellulosic substrates. On the other hand, the natural connection between cellulase expression and growth on cellulose for CBP organisms makes whole cell selection-based strategies for improving cellulase production a powerful way to screen very large libraries of candidate cells, mimicking the evolutionary process found in nature.

An assumption for any selection-based improvement for CBP organisms is that mutations can result in increased cellulase activity expression. For total cellulase activity such mutations would increase either the per cell expression level (g cellulase/g cell) or the cellulase specific activity (U/mg cellulase). Mu­tagenesis and screening techniques have allowed researchers to isolate strains of S. cerevisiae with “super-secreting” phenotypes [134-136], and similar techniques for the expression of individual cellulase components have been successful [137]. Also, random mutation has been used to change the prop­erties of cellulase enzymes (e. g., [138-140]; a further review can be found in [141]), although to our knowledge enhanced overall specific activity of cellulase on insoluble substrates has not been demonstrated via directed evo­lution. However, the specific activity of a mixture of cellulases also depends on the relative amounts of cellulase components to achieve the highest degree of enzyme-enzyme synergy [142], as well as other parameters (as yet not elu­cidated) that determine enzyme-microbe synergy [6]. These features could be impacted by mutation and therefore lead to enhanced specific activity of cellulase systems expressed by recombinant cellulolytic CBP organisms.

Earlier in this review (Sect. 3) the relationship between cellulase activity and growth rate was examined from a whole-population perspective, using parameters that are averages for many cells. The relationship between growth rate and cellulase expression for an individual cell, especially a cell harbor­ing mutations affecting cellulase expression, as compared to other cells in the population depends on the diffusion of the soluble reaction products from the point they are created at the cellulose surface to the point they are taken up by a particular cell. When a connection between growth rate and en­zyme production can be established, selection in liquid culture—particularly continuous culture—has the potential to screen many cells. For example, if a continuous reactor had a cell concentration of 1010 cells/L and was operat­ing at a dilution rate of 0.02 h-1, then 108 cells/(L*h) would be screened, and a 100-h continuous culture would screen 1010 cells.

The power of this system has been recognized previously (see [143-145] for reviews) and demonstrated in many examples where the enzyme of in­terest is located intracellularly [146-153], including some cases where the limiting enzyme made up 25% of the total cellular protein after selection, an approximately fourfold increase in expression in both cases [154,155]. In a very recent study, the authors were able to create a strain of S. cerevisiae capable of utilizing xylose as the sole carbon source with a 6-h doubling time without using recombinant genetic techniques—only using selection on xylose minimal media from a strain that could grow only very poorly ini­tially [156]. For secreted enzymes (both cell-associated or extracellularly), far fewer studies have shown improvements via selection in liquid culture. Francis and Hansche [157] were able to isolate a mutant of S. cerevisiae in a well-mixed chemostat with 1.7-fold improvement in acid phosphatase activ­ity, and Naki et al. [158] were able to isolate mutants of Bacillus subtilis with about fivefold increased secretion of protease by growing the cells in a hollow fiber apparatus, which physically separated cells, with bovine serum albumin as the limiting nitrogen source. Therefore, understanding the physical char­acteristics of the cell/enzyme/substrate system and the resulting magnitude of differences in growth rate between mutants is critical to applying selection to this system.

When cells are grown on solid media, with significant space between ini­tial cell colonies, those cells that produce more or better cellulase will retain the products of their hydrolytic reactions, and will form larger colonies. This technique—selection by people judging the size of colonies—has the ad­vantage of maintaining separation between cells. It has the disadvantage of limiting the number of cells that can be screened. It is hard to imagine how more than 109 cells (103 colonies/plate* 106 plates) can be screened in a rea­sonable amount of time, even utilizing high-throughput approaches.

When cells are grown in well-mixed liquid culture, the situation is much different because the products of hydrolysis are free to diffuse. A schematic representation of some of the liquid culture cases relevant to recombinant cellulolytic CBP organisms is presented in Fig. 4. In case A, where cellulase enzymes are secreted away from the cell, cellulases with cellulose binding do­mains will diffuse to cellulose, bind to it, and release soluble hydrolysis prod­ucts. In the final step of the overall hydrolysis reaction secreted в-glucosidase converts soluble glucose oligomers into glucose (an overview of fungal cellu­lase systems can be found in [1]). Lelieveld [159] predicted that in cases such as this, the limiting enzyme will form a gradient in the diffusion boundary layer around the cell, creating a gradient of the limiting nutrient as well. Such a gradient would provide a link between mutations conferring increased en­zymatic activity and supply of the limiting nutrient to the cell. With respect to selecting CBP organisms, when a cell secretes a growth limiting cellulase that binds cellulose, it will not necessarily take up the products of the reaction preferentially compared to another cell in the population. Thus, increased activity of that cellulase cannot be selected for. The remaining question is whether the postulated gradient of в-glucosidase exists, and if so what is the effect of the glucose gradient (ДА) on a mutant’s growth rate compared to a parent strain producing less в-glucosidase. Fan et al. [160] used a 2-D reac — tion/diffusion model to predict that the differences in growth rates between mutants and parents in this case are too small to allow the mutant to outgrow the parent in a reasonable length of time.

Recombinant xylanases and cellulases can also be expressed as tethered enzymes [59,119,130] (Fig. 4, cases B and C). In the case where a cell does not bind to the cellulose substrate (case B) (e. g., cellulases with cellulose bind­ing domains are not tethered to the cell surface), the limiting enzyme reaction is once again в-glucosidase conversion of cello-oligomers to glucose. The в — glucosidase enzyme is concentrated at the cell surface, setting up a larger gradient (ДВ) than in case A. However, in this case Fan et al. [160] found that unless the Monod constant (kS) for the substrate was very low, this gradi­ent would not be large enough to allow mutants to outgrow parents in liquid culture.

Case C presents the situation when cellulases are tethered to the surface and the cell binds to a substrate particle. In this case, the particle acts to trap the hydrolysis products, creating a substantial difference between the glucose

concentration in this gap and the substrate and the bulk fluid. When this cell/enzyme/substrate relationship is operative, Fan et al. [160] predict that differences in enzyme expression level will lead to differences in growth rates between mutant and parent cells, and that this will allow selection-based pop­ulation changes to occur in a reasonable amount of time. To date, the promise of selection for improving cellulase production by recombinant cellulolytic microorganisms has not been realized, and knowledge of the local concen­tration of glucose around such cells is limited to prediction. However, it is known that cellulose hydrolysis by naturally occurring cellulolytic microor­ganisms occurs much faster when mediated by cells adhering to the substrate as compared to nonadherent mutants [161]. It has been suggested that adher­ence confers a competitive advantage associated with first access to hydrolysis products.

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