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ELSEVIER FEMS Microbiology Reviews 16 (1995) 271-276

MICROBIOLOGY REVIEWS

Critical limitations in biological production of chemicals: process or genetic solutions?

James E. Ba i l ey *

Institute of Biotechnology, ETH Hi~nggerberg, CH-8093 Ziirich, Switzerland

Abstract

Production of bulk chemicals by biological processes is presently limited by failure of contemporary biological and bioreactor technology to deliver high product concentrations in high space-time yields in fluids of sufficiently low water content for subsequent down-stream processing operations. Limitations in the bioreactor portion of the process can arise due to failure to process sufficient substrate, substrate inhibition, inadequate rates or yields, and product inhibition. Various process approaches for addressing many of these limitations have been demonstrated or conceptualized. Less developed but potentially effective are genetic strategies addressing these process limitations. Ideally, the most effective combination of genetic and process approaches should be integrated in a synergistic fashion to maximize the economic potential of biological production of chemicals.

Keywords: Biological production; Bulk chemicals

I . Introduct ion

Biological routes to chemicals offer the advan- tages of renewable feedstocks, energy-efficient pro- cessing, and biodegradable wastes. However, such processes are usually not employed at present rela- tive to their petroleum-based alternatives, in part because of a current political end corresponding economic framework which supports extremely low oil prices and does not charge producers fully for the environmental costs of their effluents. Although fu- ture changes in public opinion and political influ- ences on the economics of the chemical industry can be expected to shift the decision-making framework over time, it is extremely important in the meantime to improve the technical efficiency of the biological

* Corresponding author. Tel: (+41-1) 633 3170; Fax: (+41-1) 371 0658

technologies available for chemicals production. This will lead to eventual intersection of bioprocess capa- bility with a correspondingly favorable economic context for an increasing array of chemical products.

Comparison of a relatively low-cost petrochemi- cal production process with a higher-cost biological reaction technology suggests several basic differ- ences which give rise to the cost structures of these two cases. Examining these differences reveals sev- eral general types of process limitations in biopro- cess-based routes. Although these limitations are not mutually exclusive, each can arise in a somewhat independent fashion, and for each there are different types of approaches available to attempt to surmount or minimize the limitations. The particular bioreactor limitations which will be considered here are the following: (i) Incompatibility with large-volume, low-cost sub- strates;

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272 J.E. Bailey / FEMS Microbiology Reviews 16 (1995) 271-276

(ii) Inadequate supply of a rate- or yield-limiting substrate; (iii) Inhibition by excess substrate (can be caused directly by the substrate itself or indirectly by a catabolic by-product); (iv) Inhibition by product, resulting in low effluent product concentration; (v) Inadequate rate of formation or yield of the desired product. This survey of limitations applies to both enzyme- and whole cell-based bioreaction processes. How- ever, the central focus of the remaining discussion will be on cellular processes.

Each of these limitations can be attacked by both process approaches (this term is used here as a shorthand for bioprocess engineering) and also by genetic approaches. Because high priority for ethanol production technology development existed before genetic engineering technology, a repertoire of pro- cess concepts for addressing these limitations is rela- tively well-known. Of course further process innova- tions and improvements to relieve these basic limita- tions can be foreseen. However, because the oppor- tunity for genetic approaches to address these limita- tions is relatively new and because the implementa- tion of these approaches is relatively less well devel- oped, these will be emphasized primarily in the following commentary.

Some general comments about opportunities pro- vided by 'metabolic engineering' (which means the use of recombinant DNA technology for improving the metabolic capabilities of industrial organisms [1]) are appropriate before exploring more specific appli- cations. Genetic engineering provides the enabling technology for transfer of one or more functional enzymes, transport proteins, or regulatory proteins from one organism into another. This capability re- moves many restrictions which exist if only natural organisms or their mutants are considered. It is in fact highly unlikely that organisms which have evolved in nature will possess precisely the combina- tion of substrate-utilizing and uptake proteins, metabolic pathways, export systems, and general environmental tolerance which are optimized or even reasonably effective for a particular bioprocess appli- cation. Through the use of genetic engineering, de- sirable attributes obtained from different organisms can be combined in a single system in order to

provide enhanced capabilities or, as will be discussed briefly in the concluding section, to enable synthesis of entirely new chemicals. Furthermore, protein en- gineering can be used to alter the activity, stability, and regulatory properties of enzymes and transport proteins, further expanding the potential benefits available through metabolic engineering.

Bioreactor processes are typically designed to support the requirements and also to tolerate the constraints imposed by use of a particular organism. Therefore, the capability of creating a large class of new hybrid organisms by metabolic engineering gives rise to corresponding opportunities and challenges in bioprocess engineering to accomplish the best reac- tor design and operation consistent with the newly expanded and more critically matched potential of the production organism. The most successful devel- opment of biological routes to chemicals will inte- grate genetic constructions of more productive or- ganisms with process engineering conception and development.

In the following sections, each of the limitations listed above will be briefly discussed, with indica- tions of process and genetic approaches available for each. In both process and genetic arenas, the indi- cated points are not intended to be exhaustive, but instead are provided to show the existence of com- plementary strategies and also the opportunities which exist for connecting process and genetic im- provements.

2. Incompatibility with large-volume, low-cost substrates

Most of the microorganisms which have the enzy- matic capability to synthesize interesting chemicals cannot use the most widely available and cheapest carbon feedstocks-namely petroleum and its frac- tions. Also recalcitrant are most of the available renewable, biological substrates which typically are agricultural or food-processing wastes. Process ap- proaches would provide prior conversion of these wastes to substrates which could be used. Genetic level approaches, which offer great potential in this very important domain of bioprocess limitations, would incorporate in the chemical-producing organ- ism the degradation, uptake, and dissimilatory en-

.I.E. Bailey/FEMS Microbiology Reviews 16 (1995) 271-276 273

zymes needed for utilization of carbon sources avail- able in abundance at relatively low cost. The con- struction of ethanol-producing organisms capable of using C5 sugars in the laboratory of Prof. L.O. Ingram, described elsewhere in this volume, is an excellent illustration of the effectiveness of this ap- proach.

In connection with this work and as a general comment touching several of the limitations ad- dressed here, the importance of engineering transport of substrates into the cell and products leaving the cell is a critical one which is often overlooked or inadequately emphasized in formulating metabolic engineering strategies. The involvement of particular types of uptake systems in repression of utilization of co-substrates, which occurs, for example, in the action of the phosphotransferase system of glucose uptake in Escherichia coli and other bacteria, is another potential target for genetic modification to enable a desirable process outcome.

3. Inadequate supply of a rate- or yield-limiting substrate

Certain substrates have relatively low solubility or may exist as a heterogeneous phase in the bioreactor. Process approaches involve improved mixing, novel multiphase contacting schemes and high-efficiency dispersion systems to increase the rate of substrate transport, and modifications of the medium or use of multiphase systems with solvents.

Genetic approaches would seek to install a system more efficient in utilizing the substrate for the de- sired metabolic activities and might also involve installation of a higher affinity uptake system or an uptake system which is free of controls which limit the rate of substrate entry.

A necessary substrate in the production of organic acids and amino acids is oxygen, a substance diffi- cult to deliver to cells at high rates because of its low solubility in aqueous media. A genetic approach to improve cellular activity and product synthesis rates in oxygen-limited aerobic biological processes in- volves expression of a heterologous hemoglobin ex- pressed in nature in the bacterium Vitreoscilla [2]. The positive effect of this hemoglobin on the effi- ciency of growth, oxygen-limited growth rates, and

protein synthesis in E. coli have been well docu- mented [3], as have increases in specific production rates of actinorhodin by Streptomyces coelicolor [4], of oxytetracycline by Streptomyces rimosus, of cephalosporin C by Acremonium chrysogenum, and lysine by Corynebacterium glutamicum. The most detailed physiological studies of this system have been conducted in E. coli and show an increased efficiency of proton pumping, a higher ATP produc- tion rate, and higher ATP contents in oxygen-limited E. coli when VHb is expressed [5]. These observa- tions are all consistent with a hypothesis for VHb action which postulates a higher available oxygen level inside cells due to the presence of oxygenated V ~ [5].

4. Inhibition by excess substrate

Simple stoichiometric considerations indicate that achieving high product concentration implies conver- sion of a correspondingly large amount of substrate. If substrate is present in high concentration initially, the cells may exhibit very low activity either due to direct inhibition by substrate of key metabolic func- tions or to rapid accumulation of inhibitory by-prod- ucts in the presence of high concentrations of sub- strate. Accumulation of acetate, ethanol, and lactate in cultures of E. coli, Saccharomyces cerevisiae, and CHO cells, respectively, in media with high glucose concentration illustrate the latter phenomenon.

Process strategies based upon fed-batch opera- tions which provide an ongoing feed of substrate, in either continuous or intermittent dosage fashion, and which maintain a low residual concentration of sub- strate in the bioreactor have proven very successful for alleviating these problems in a number of appli- cations. Operational strategies are aided by on-line process analysis and associated computer control systems. In many applications, off-gas monitoring combined with material-balancing calculations have proven sufficient for excellent fed-batch perfor- mance.

Another process strategy effective in addressing this problem is use of continuous bioreactors which retain a low residual level of substrate. In the case of highly inhibitory substrates such as methanol, it is

274 J.E. Bailey/FEMS Microbiology Reviews 16 (1995) 271-276

important that substrate be effectively dispersed in the bioreaction fluid in order to avoid locally high concentrations.

Genetic modification of the organisms can also alleviate inhibition associated with high substrate concentrations. One approach involves altering the distribution of metabolic fluxes genetically in order to reroute catabolic intermediates away from particu- larly inhibitory products and into either more benign end-products or into storage compounds. The former strategy has been demonstrated in the laboratory of Prof. L.O. Ingram. E. coli carrying heterologous genes encoding the pyruvate decarboxylase and alco- hol dehydrogenase of Zymomonas mobilis synthe- size greatly elevated levels of ethanol and minimal levels of weak organic acid metabolites, with the associated consequence of culture growth to higher cell densities. Ability to achieve higher cell densities has also been demonstrated in E. coli which has been engineered to overproduce glycogen [6]. As a result, this construct accumulates far lower maxi- mum concentrations of pyruvate than the unmodified control, which is a mutant lacking the genes for acetate production from acetyl CoA.

Both of these approaches also provide possible opportunities for improving management of carbon flow into desired end-products. In the former con- struct, ethanol can be produced at sufficiently high concentrations to be interesting for large-scale pro- duction. In the second case, the carbon which is sequestered as glycogen is potentially available for subsequent routing into any metabolic products of interest.

On a longer-term basis, it may prove possible by protein engineering to alleviate direct inhibition by substrate. In cases in which substrate has a deleteri- ous effect on the cell membrane, modification of the membrane genetically may provide some relief from substrate or from product inhibition. However, the goal of membrane modification, like several other long-term genetic possibilities, will likely entail a complicated, multigene strategy which is signifi- cantly more sophisticated than those so far demon- strated. However, such major changes in phenotype by interchange or transfer of multiple genes respon- sible for a given structure or cellular function are certainly available in principle. The primary chal- lenge is effective design of the regulation of such a

multiple gene modification at both the genetic and protein levels in a fashion which enables the inserted genes to function in a fashion well integrated with the operation and regulation of the remaining host metabolism.

5. Inhibition by product, resulting in low effluent product concentration

Many commercially desirable chemical products of microbial metabolism are inhibitory to the produc- ing organisms. Several process approaches have been studied and demonstrated to address this problem, particularly in connection with ethanol production. These involve continuous or intermittent selective removal of the product from the fermentation medium. This can be in a stream being withdrawn and, after treatment, reintroduced into the bioreactor. Also approaches involving in situ product absorption or extraction have been proposed and demonstrated. Bioreactors employing a two-liquid phase medium have potential advantages in such applications.

Genetic approaches to this problem are relatively difficult in view of the poorly understood and likely complex mechanism by which different products in- hibit cell metabolism. These mechanisms appear to involve disruption of membrane fluidity or mem- brane structure by organic end-products and also reduction in the proton motive force across the cyto- plasmic membrane by weak acids. It is possible that, by utilization of host organisms which are naturally adapted to acidic environments or to growth in the presence of polar organic molecules, these problems can be alleviated genetically. The biosynthesis path- way of interest could be transferred into such an organism, or the genes coding for the important structural or functional features resistance to product inhibition could be transferred to the producing or- ganism. In other cases protein engineering of key enzymes or transport systems might alleviate diffi- culties with direct product inhibition. Also, it is likely that in some cases product inhibition arises because products accumulate at higher concentra- tions intracellularly and could be reduced to lower levels by installing genetically a more active system for product export from the cytoplasm.

J.E. Bailey / FEMS Microbiology Reviews 16 (1995) 271-276 275

6. Inadequate rate of formation or yield of the desired product

Often the product does not appear sufficiently rapidly, necessitating a large capital investment for the bioreactor. In other cases the product is not produced efficiently as a fraction of the substrate utilized. This results in higher feedstock operating costs which can also be a crucial limitation in the production of chemicals by biological routes.

Process approaches can have a significant effect on volumetric production rates by increasing cell density in the bioreactor. This is most commonly achieved with fed-batch operations or with use of cell concentration and recycle in continuous or semi-continuous processes. This approach generally has no benefits with respect to yield, however. In order to alter yield through process manipulations, one must explore alternative cultivation conditions on a trial-and-error basis, seeking the cellular envi- ronment which results in the most favorable distribu- tion of metabolic activity towards production of the desired chemical.

Such process manipulations are obviously acting at the level of the pathway activities involved, and genetic intervention provides a much more direct vehicle for influencing and controlling flux distribu- tion in the production organism. Genetic approaches enable modification of the relative levels of enzymes involved and also of regulation of these activities. The effectiveness of such genetic manipulations is well known from many prior examples in amino acid production, in which both intelligent mutagenesis/ selection and genetic engineering have had major impacts in redirecting fluxes in favor of the desired product.

Genetic manipulations should also be useful in increasing the specific formation rate of product (that is, the amount of product produced by the cells per unit time per unit mass of cells).

The challenge for redirecting and maximizing fluxes include identification of which enzyme levels should be changed and of what regulatory features should be modified. Local guidance for the most effective changes in enzyme levels can be obtained through sensitivity analysis. In particular, given suf- ficient information on intracellular metabolite con- centrations and metabolic fluxes, and perhaps also on

changes in these quantities in response to certain perturbations in operation of the pathways, an analy- sis can be achieved, for example by using the formal- ism of so-called Metabolic Control Theory, aug- mented by consideration of the metabolic context in the cell of the particular pathway of interest [7].

Another approach for accomplishing sensitivity analysis is formulation and validation of a mathemat- ical kinetic model for the pathway which is subse- quently employed for sensitivity calculations at both local and global levels. This approach has been demonstrated in a simplified context of non-growing Saccharomyces cerevisiae converting glucose to ethanol [8]. This study showed that the sensitivity of the rate of ethanol production to small changes in the levels of enzymes in the pathway were not localized in a single rate-controlling step under many circum- stances, but instead that several steps have a signifi- cant influence on the flux to ethanol. Moreover, simulation studies for large changes in the involved activities show when the activity of one step is increased significantly, other steps rapidly assume control of the ethanol production flux. This simple example illustrates the likely general principle that achieving major changes in the flux of a particular pathway with relatively little branching will require the simultaneous amplification of several enzymes and transport activities in the pathway. This may explain why prior random mutagenesis and genetic engineering approaches which have sought to in- crease fluxes in primary metabolite and secondary metabolite pathways have generally yielded disap- pointingly small effects.

7. Discussion of current opportunities

Based upon experience to date, concepts demon- strated in laboratory or small-scale studies but not yet proven at large-scale, and also currently available scientific information and concepts, it appears that process strategies are currently the best hopes for increasing the final product concentration and sub- strate throughput. On the other hand, genetic strate- gies are most useful for improving yields and chang- ing the flux distribution. Both process and genetic approaches should be used together to increase cell density and to improve the overall rate of product formation.

276 .I.E. Bai ley /FEMS Microbiology Reviews 16 (1995) 271-276

In closing, the unique capabilities of genetic ap- proaches to make possible entirely new process routes to particular products should be recognized. For example, no amount of process manipulation can enable an E. coli bacterium to produce a human protein. Extending this same concept into the domain of chemicals production, the possibilities have al- ready been demonstrated that genetic methods offer the enabling technology to combine genes, and thereby the corresponding enzymatic activities, from different organisms to create new catabolic or syn- thetic pathway s . This makes possible new biosyn- thetic routes to current chemicals such as the dye indigo and biosynthesis of entirely new chemicals such as novel polyketides synthesized by genetically engineered strains of Streptomyces (see [1]). The opportunity to create new molecules by construction of new multienzyme catalytic pathways is particu- larly important in view of the primary role of new products and new processes to expansion of the biochemical and biotechnological industry.

Acknowledgement

The author's research on metabolic engineering is supported in part by the Swiss Priority Program in Biotechnology.

References

[1] Bailey, J.E. (1991) Toward a science of metabolic engineer- ing. Science 252, 1668-1675.

[2] Khosla, C. and Bailey J.E. (1988) Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant E. coli. Nature 331, 633-634.

[3] Khosla, C., Curtis, J., DeModena, J., Rinas, U. and Bailey, J.E. (1990) Expression of intraceUular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli. Bio/ Technology 8, 849-853.

[4] Magnolo, S., Leenutaphong, D., DeModena, J., Curtis J., Bailey, J., Galazzo, J. and Hughes, D. (1991) Actinorhodin production by Streptomyces coelicolor and growth of Strepto- myces lividans are improved by the expression of a bacterial hemoglobin. Bio/Technology 9, 473-476.

[5] Kallio, P.T., Kim, D.J., Tsai P.S., and Bailey, J.E. (1994) Intracellular expression of VitreosciUa hemoglobin alters Es- cherichia coli energy metabolism under oxygen-limited condi- tions. Eur. J. Biochem., in press.

[6] Dedhia, N., Hottiger, Th., Chen, W. and Bailey, J.E. (1992) Genetic manipulation of central carbon metabolism in Es- cherichia coli. In: Harnessing Biotechnology for the 21st Century (M.R. Ladisch and A. Bose, Eds.), pp. 59-62. Ameri- can Chemical Society, New York, NY.

[7] Schlosser, P.M. and Bailey, J.E. (1990) An integrated mod- elling-experimental strategy for the analysis of metabolic path- ways. Math. Biosci. 100, 87-114.

[8] Galazzo, J.L. and Bailey, J.E. (1990) Fermentation pathway kinetics and metabolic flux control in suspended and immobi- lized Saccharomyces cerevisiae. Enz. Microb. Technol. 12, 162-172.