Microb Ecol (1986) 12:3-13
MICROBIAL ECOLOGY ~) 1986 Springer-Verlag
Genetic Engineering of Microorganisms for Biotechnology Edmund J. Stellwag* and Jean E. Brenchleyt Genex Corporation, 16020 IndustrialDrive, Gaithersburg,Maryland20877
introduction Investigations of a variety of microorganisms have demonstrated the existence of a considerable biochemical diversity. Different microbes have evolved the capabilities to survive in environmental extremes of nutrient supply, temperature, pH, pressure, minerals, and toxic chemicals. Although attempts have been made to understand the physiology of many different microorganisms, the study of microbial genetics has centered on a few bacteria, primarily Escherichia coli and related enterics. This focused approach has been highly successful for the development of the detailed genetic linkage map of E. coli, the elaborate understanding of regulatory mechanisms, the elucidation of macromolecular synthesis, and, most importantly, for the purposes of this article, the establishment of recombinant DNA procedures for genetic engineering. The ability to form recombinant DNA molecules in vitro that can be stably replicated in a recipient host has had a major impact on the biological community and on society. One aspect of this impact has been the formation of biotechnology companies. Biotechnology, when broadly defined as the application of biological materials to industrial processes, is not new. Microorganisms have traditionally produced such useful compounds as alcohol and antibiotics. More recently, considerable attention has been directed toward exploitation of diverse microbial species for enzyme production as well as for the synthesis and degradation of chemicals. Unfortunately, the application of microorganisms and their enzymes has been limited by several problems, including low product yield, poor growth, or pathogenicity. A major advantage of the recombinant DNA techniques is that they provide the means to circumvent these barriers and have greatly extended the potential for using the existing microbial diversity. It is ironic that it has been this breakthrough in molecular biology that has greatly revitalized the interest and excitement in the study of general microbiology for industrial applications. This article will examine areas where the use of recombinant DNA procedures with microorganisms other than E. coli are important to biotechnology. Because our goal is to illustrate how recombinant DNA methods can be applied to a wide variety of industrially important microbes, the article will present the
~address:
Departmentof Microbiologyand Immunology,EastCarolinaUniversity,Green-
ville, NC 27834.
t Presentaddress:DepartmentofMicrobiology,Molecular,and CellBiology,PennStateUniversity, Collegeof Science, UniversityPark, PA 16802.
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historical development and current status of a few microorganisms rather than a detailed review of all publications in the field. Extensive discussions of certain microorganisms that are described elsewhere in this volume were omitted to avoid duplication. In addition, we outline some of the potential that recombinant DNA procedures provide as tools for greatly expanding our understanding and use of many other diverse microorganisms.
Discussion
Development o f Recombinant DNA Procedures in Bacillus and Streptomyces Bacillus subtilis is perhaps foremost among the many industrially significant microbes to have received increasing attention as a host for recombinant DNA experiments [29]. B. subtilis, unlike E. coli, is proficient at the secretion of proteins into the extracellular medium [35]. The property of extracellular protein secretion is important in industry not only because of the potential for high product yields, but because many extracellular proteins are purified more readily than intracellular proteins [44]. The inability orE. coli to secrete proteins encoded by foreign genes results in the formation of intracellular protein aggregate structures termed "inclusion bodies" [8]. The proteins assembled in these inclusion bodies are frequently denatured making the recovery of the active product difficult [8]. One solution to this problem is to clone these genes into an organism, such as B. subtilis, capable of both synthesis and extracellular secretion of active proteins. A prerequisite to the development of gene-cloning in any organism is the identification of a suitable vector for the introduction and stable maintenance of cloned DNA in the host. The search for a suitable vector in B. subtilis yielded only cryptic plasmids [30, 41] and was therefore of limited significance for the establishment of a molecular genetic system. A major breakthrough occurred when it was discovered that plasmids carrying antibiotic-resistance determinants in Staphylococcus aureus could be transformed into and stably maintained in B. subtilis [13, 14, 18]. Two of the plasmids, pC194 and pUB110, have served as the cornerstone for the development of gene-cloning in B. subtilis. A second major hurdle in the establishment of a gene-cloning system is the ability to introduce, with high efficiency, molecules of the vector DNA into the host organism [9]. While transformation of competent B. subtilis by chromosomal DNA is a highly efficient process in which up to 10% of the recipient ceils undergo genetic alteration [1 ], the frequency of transformation by plasmid DNA is several orders of magnitude lower [13, 18]. Despite the efficient competence system in B. subtilus, it was of little use for recombinant DNA-cloning experiments. This difficulty has been remedied by the development of a highly efficient procedure involving the polyethylene glycol (PEG)-induced uptake of DNA by B. subtilis protoplasts [9]. This procedure is so efficient that it is suitable even for the introduction of phenotypically cryptic plasmids [9]. High efficiency transformation has permitted the cloning and expression in
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B. subtilis of a diverse list of genes, including those encoding antibiotic resistance markers from B. pumilis, a thermostable a-amylase for B. licheniformis, and the mouse dihydrofolate reductase [21, 33, 37]. Perhaps more important than the actual number and types of genes cloned in B. subtilis is the application of the PEG transformation technique to other Bacillus species. PEG-mediated D N A uptake has been reported in a succession of Bacillus species, including thermophiles [24] and strains that secrete prodigious amounts of protein [40]. The genetic flexibility afforded the bacilli by the protoplast transformation technique has permitted the successful heterologous gene cloning among a limited number of species [17, 34, 36]. However, considering the possibilities that exist, the actual number of reports concerning the cloning and expression of homologous or heterologous genes a r e few. Before the recombinant D N A tools can be used to full advantage, a better understanding of fundamental physiology and metabolism of this diverse group is needed. Numerous opportunities exist for studying the effects of different cloned genes in a wide range of host backgrounds. The utility of the PEG-mediated D N A uptake system has not been restricted to the bacilli. In fact, the high frequency uptake of D N A by PEG-treated protoplasts has contributed significantly to the success of D N A cloning efforts in the genus Streptornyces [2]. The Streptomyces have historically received considerable attention from industrial researchers primarily because of their important position to antibiotic synthesis [42, 45]. As in B. subtilis, the advent of molecular genetic manipulation in Streptomyces was preceded by discovery of a high-efficiency, interspecific gene-transfer system [23]. In the case of Streptomyces, this was mediated by a conjugal plasmid capable of transferring either plasmid or chromosomal D N A [23]. Unfortunately, the difficulty in isolating this large plasmid precluded its use as a cloning vector [22]. The first useful Streptomyces vector was constructed by cloning the neomycin-resistance gene from Streptomycesfradiae and the thiostrepton-resistance genes from Streptomyces azureus onto a plasmid, SLP 1.2, which had been discovered in Streptomyces lividans 66 only after mating with Streptornyces coelicolor [22]. Although this vector has several unique restriction s~tes in the gene encoding neomycin resistance, the copy number of the plasmid is low (4-5 copies/chromosome equivalent). Consequently, this vector, pIJ61, is useful for cloning via insertional inactivation of the neomycin resistance marker, but it is of limited utility for expressing large quantities of the gene product. Although plasmids with high copy number have been isolated from Streptornyces, the recent cloning of the complete biosynthetic pathway for the synthesis of the antibiotic actinorhodin from S. coelicolor was attributed to the use of a low-copy number plasmid pIJ922 as the vector [31]. In this instance, the use of high-copy number plasmids was not effective; the authors suggest the high-level production of a "physiologically active" gene product may have lethal effects in the recipient strain resulting in a failure to isolate the appropriate clone. This observation places a different perspective on the potential strategies for cloning genes which specify products effecting normal host metabolism. There may be situations where low levels of expression are required for cell viability. In such instances, the production of large quantities of a compound may require the conditional regulation of gene expression.
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In addition to the plasmids described above, other sophisticated vectors based on both high-copy number plasmids [28] and bacteriophage [20] are presently employed in the analysis ofgene expression in Streptomyces. Although these gene-cloning tools are available, other obstacles have prevented the rapid full-scale industrial exploitation of the recent developments in molecular biology. The cloning of genes coding for the enzymes involved in the biosynthesis of antibiotics in Streptomyces provides an example of the limitations of conventional gene-cloning techniques for directly increasing product yield. Because the biosynthetic pathways for antibiotics and other complex molecules are highly regulated and involve many enzymatic steps, the cloning of genes encoding one or a few enzymes in the pathway generally fails to increase product yield. Thus, several genes may be needed before the total pathway and product yield are affected. The matter is made more difficult because the cloning of genes coding for enzymes in biosynthetic pathways frequently confer no detectable phenotype on the host. Consequently, cloning genes encoding biosynthetic enzymes often relies on complementation of mutants blocked at specific steps in the biosynthetic pathway. The "complementation cloning'" technique was used in the isolation o f a gene involved in the biosynthesis o f the antibiotic undecylprodigiosin [15]. A S. coelicolor mutant defective in antibiotic biosynthesis was used as the recipient strain for wild-type S. coelicolor DNA cloned into a plasmid. A single antibiotic-producing colony was found, and DNA from this clone restored antibiotic production in two of the five classes of S. coelicolor antibiotic-biosynthetic mutants. The cloning of genes encoding undecylprodiglosin production was possible because of the availability of mutants blocked in antibiotic production. The need to isolate mutants in each step of a biosynthetic pathway can significantly delay cloning efforts, particularly for organisms that are poorly characterized genetically. 9. An alternate method termed "mutational cloning" circumvents the need for generating mutants prior to cloning the wild-type allele [10]. The mutational cloning system is dependent on the availability of a lysogenic phage-derived cloning vector containing a selectable marker, a suitable cloning site, and a defect in the normal phage chromosomal integration mechanism. Generally lysogenic phage integrate into the host genome by site specific recombination between a unique phage DNA sequence and a nonhomologous host DNA sequence [6]. A defect in this normal phage-specified integration mechanism destroys the ability of the phage to insert into its specific host attachment site. The defective integration mechanism can be circumvented by cloning host DNA into the phage genome. Integration of the phage genome carrying host DNA can now proceed via homologous recombination between the DNA cloned into the phage genome and the homologous DNA of the host. The integration of the phage genome in this matter interrupts the normal gene coding sequence, resulting in mutations. Each mutant generated by integration of phage DNA will contain not only the phage genome but also the cloned DNA homologous to the gene sequence disrupted by the integration event, i.e., the mutant gene. The DNA obtained from phage released from such a lysogenic mutant strain contains cloned DNA and can be used for further molecular biological manipulation. Mutational cloning procedure was first demonstrated in the isolation of genes involved in methylenomycin A biosynthesis from S. coelicolor [10].
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The above presentation of gene-cloning in Bacillus and Streptomyces has neglected the importance of understanding the regulation of gene expression. The fact that these two genera must coordinate expression of both vegetative and developmentally transcribed genes suggests that regulation may be quite sophisticated. Interest in developmental controls in Bacillus and Streptomyces led to the construction of promotor-probe vectors for each genus [3, 47]. Promoter-probe vectors contain a cloning site 5' distal to a marker gene that possesses no promotor. Expression of the marker gene following introduction of foreign D N A at the cloning site indicates promotor activity on the cloned D N A fragment. Streptomyces D N A possessing promotor activity has been isolated using promotor-probe vectors functional in Streptomyces species [3]. Streptomyces D N A sequences possessing promotor activity showed no sequence relationship to well-characterized promotor sequences from other genera [3]. In addition, Streptomyces D N A fragments showing promotor activity in the homologous Streptomyces host do not demonstrate detectable promotor activity in E. coli [3]. In contrast, some promotors from E. coli, Serratia, and Bacillus are functional in Streptomyces [3]. These results suggest that Streptomyces promotors are unrelated to those from other genera, but that Streptomyces R N A polymerase and/or ribosomes are promiscuous with regard to recognition of foreign promotors and translation initiation sequences. These observations may have important implications for the potential application of Streptomyces as a host for foreign DNA.
Application of Genetic Engineering to Other Industrially Important Microorganisms The presentation of the Bacillus and Streptomyces work focused on the critical aspects in the development of DNA-cloning systems. But it should not be concluded that significant strides in recombinant DNA-mediated strain manipulation are dependent on years of vector construction. The following example will demonstrate that recombinant D N A tools developed in well-characterized systems can be employed in the manipulation of strains lacking an established genetic system. The obligate methylotroph Methylophilus methylotrophus is able to efficiently convert methanol to single-cell protein [49], a process of major importance to a variety of industries [ l 2]. Interestingly, instead of using the one-step glutamate dehydrogenase catalyzed conversion of ammonia and a-ketoglutarate to glutamate, this organism uses a two-step pathway catalyzed by glutamine synthetase and glutamate synthase [49]. The two-step pathway is less energy efficient and therefore reduces the ~r of single-cell protein from methanol grown cultures. Researchers at International Cell Products, Inc. used chromosomal D N A from E. coli and vectors from E. coli and Pseudomonas to construct a strain ofM. methylotrophus that assimilated ammonia via the energetically favorable glutamate dehydrogenase (GDH) pathway. The G D H gene was obtained by complementation cloning of wild-type E. coli D N A into a G D H deficient strain
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o f E. coli [49]. Since the vector used to clone the E. coli G D H gene could not be transferred directly to M. methylotrophus, the G D H gene was subcloned onto broad host range, Pseudomonas-derived vectors capable o f transfer to M. rnethylotrophus by conjugation. Transconjugants were selected for the antibiotic resistance encoded by the vector, and the G D H enzyme activity measured to confirm the presence o f the G D H gene. The strain constructed by these manipulations was able to convert methanol to single-cell protein more efficiently than the original parent strain. As the authors point out in their discussion, "the alteration made in M. methylotrophus could not have been achieved either by prolonged selection or by conventional mutagenesis, and therefore represents the planned adaptation of a microorganism to a man-made environment" [49]. Specific examples o f recombinant D N A manipulations such as described above are rare; however, they provide insight into the utility of these procedures for future strain improvement. A second example o f strain improvement by recombinant DNA manipulation o f a metabolic pathway is presented below. In this example DNA obtained from a procaryote is used to mediate an alteration in an eucaryotic metabolic pathway. During the wine-making process, a secondary fermentation convering L-malate to L-lactate is performed by various species o f lactic acid bacteria. This process reduces the acidity o f the wine and makes it more stable for storage. Unfortunately, the lactic acid bacteria normally do not grow rapidly in wine, and starter cultures are often needed. To circumvent this step, Williams et al. [48] cloned the gene for the malolactic enzyme from Lactobacillus delbreuchii into the plasmid pBR322, transformed E. coli, and selected for transformants that converted malate to lactate. Since pBR322 cannot replicate in Saccharomyces cerevisiae, the D N A restriction fragment carrying the gene for the malolactic enzyme was purified and recombined into an E. coli-yeast shuttle vector (i.e., a vector capable o f replicating in both E. coli and S. cerevisiae). S. cerevisiae cells containing the shuttle vector carrying the L. delbreuchii gene for the malolactic enzyme produced about 3.5 times more L-lactate during nonaerobic growth than the control strains [48]. These strains and their derivatives are being tested for their utility in the wine-making process. The interest in industrial ethanol fermentation extends beyond the winemaking industry. A considerable amount of attention has been directed toward the production o f ethanol as an energy source. Because traditional fermentation for alcohol production has used highly selected strains o f the yeast, S. cerevisiae, there has been substantial development o f yeast genetic systems. Vectors for S. cerevisiae were developed carrying origins of replication, such as the 2u circle or autonomously replicating sequences (ARS elements), and genes for biosynthetic enzymes that could be used for complementation [4, 11, 19, 39]. These genes, the trp 1 and leu2, were important because the natural resistance of S. cerevisiae to many commonly used antibiotics prevented the use of antibiotic resistance as a direct selection. The different yeast plasmids are being used successfully to clone and express a variety of eucaryotic genes, and S. cerevisiae may become a useful production organism for the synthesis o f medically important proteins. Another group o f microorganisms o f long-standing interest to industry are the coryneform bacteria. The c o m m o n feature of reproduction by mycelial
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fragmentation or snapping fission results in the variability o f the cell shape characteristic of these microbes. Despite this similarity in mode of cell division the different genera in these groups have diverse metabolic properties, occur in a wide variety of habitats, and, in many cases, are not closely related [ 16]. The group generally termed the coryneforms has as representative genera, Corynebacteriurn, Arthrobacter, Brevibacterium, and Microbacterium. Despite the importance ofcoryneforms for industrial amino acid production, no genetic transfer systems have been developed for these bacteria. Recently, however, several research groups have reported the development of recombinant DNA procedures for coryneforms. Patent applications from Ajinomoto Co., Inc. [32, 43] and Kyowa Hakko Kogyo Company, LTD [26, 27] describe transformation methods and the construction o f plasmids that can be used as vec!ors in Corynebacterium and Brevibacteriurn. One plasmid (pCG4) was obtained from C. glutamicum and h a s a gene conferring resistance to streptomycin and/or spectinomycin as a selectable marker [26, 27]. This plasmid was used to transform C. glutamicum, C. herculis, B. flavum, and B. lactofermenturn, demonstratit~g that it could replicate in these distinct strains and suggesting that it will be generally useful as a coryneform vector. Critical to this successful development of coryneform vectors has been protoplast transformation. However, because coryneform bacteria are extremely resistant to the lysozyme treatment used to obtain protoplasts, mutants that are substantially more sensitive to lysozyrne were isolated to facilitate protoplast transformation [25]. This approach could be useful for other microorganisms where protoplast formation or regeneration has been difficult. A different strategy was used by Dr. A. Sinsky and colleagues to develop cloning vectors from existing cryptic plasmids isolated from coryneform strains (38, and personal communication). Plasmids purified from C. glutamicum or B. lactofermentum were combined in vitro with a B. subtilis plasmid carrying a kanamycin resistance gene. These plasmid constructions were transformed into 17. subtilis selecting for kanamycin resistance and screening for shuttle vectors carrying D N A from both the coryneform and Bacillus plasmids. The plasmids were purified and used to test for transformation into coryneform recipients. The few kanamycin-resistant transformants that were obtained mere used to prepare quantities of the shuttle vector for use in retransformation and cloning experiments. As we pointed out in the discussion of gene cloning in Streptomyces, phage can be employed as recombinant D N A vectors in lieu ofplasmids. The phage, pEC, was isolated for its ability to infect Rhodococcus erythropolis, an organism related to the industrially important coryneforms [5]. The phage has a broad host range and can be propagated in several species of Rhodococcus and Nocardia. A map identifying sites recognized by restriction endonucleases was constructed and several deletion mutants isolated to determine the regions of the phage containing nonessential DNA. Phage deletion mutants can be used as cloning vectors whereby foreign D N A can be recombined into the phage in vitro and then introduced into cell protoplasts by transfection. Since the phage DNA can integrate, the new genes can be stably maintained. Temperate phage can provide the necessary components for recombinant D N A experiments in organisms lacking plasmids carrying selectable markers.
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The ability to use genetic engineering principles to construct microorganisms that can use alternate growth substrates could be extremely important to the development of better production strains and to using new raw materials as growth substrates. Preliminary efforts in this regard have involved the transfer of a plasmid carrying antibiotic resistance markers and a lactose transposon to various different strains by conjugation. Using this strategy, the lactose genes were transferred to Z y m o m o n a s mobilis [7] and Xanthomonas carnpestris [46] to enable these strains to use whey as a raw material for ethanol and xanthan gum production, respectively.
Conclusions and Future Prospects The application of recombinant DNA procedures to microorganisms other than E. coli is rapidly increasing. The construction of new recombinant DNA vectors and the elucidation of transformation techniques for previously untransformable microorganisms has tremendous potential for applications in both basic and applied research. These developments in genetic engineering of diverse microbial species have the potential for generating genetic heterogeneity previously unknown in the natural environment. This article has described a few situations where recombinant DNA techniques are being used to improve industrial processes. The examples presented provide the basis for outlining general conclusions regarding the development of gene-cloning systems: 1. An efficient gene transfer system, for example, protoplast transformation, is essential. 2. An elaborate genetic characterization of either the donor or host is not always necessary for gene cloning. 3. Vectors can be constructed from either plasmids or phage, depending on their availability and the eventual use. 4. Different types of vectors, such as ones with either low or high copy number, are required for different kinds of experiments. 5. Cryptic plasmids are potential vectors if appropriate markers that confer a selectable phenotype on the recipient can be incorporated in vitro. 6. Vectors using plasmids or genes from organisms other than the final host can often be useful. 7. The presence of selectable markers on vectors is essential for selecting transformants. 8. Almost any eucaryotic or procaryotic gene can be cloned into a new host if appropriate vectors and screening techniques are available. 9. A persistent investigation of strains of any particular genus is likely to provide suitable materials for the development of a gene cloning system. Because recombinant DNA procedures can be rapidly developed for widely diverse microorganisms, it is relevant to consider how these new approaches will impact future industrial applications. In the past, enhanced production of compounds was obtained by optimizing growth or culture conditions and by repeated strain selection and improvement programs. These approaches remain important, but they can be supplemented by gene cloning. For example, the
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production o f a m i n o acids or antibiotics can be increased by cloning genes for biosynthetic enzymes which remain rate limiting even in highly i m p r o v e d production strains. P r o d u c t i o n can also be i m p r o v e d by transferring genes that enable the strain to utilize alternate carbon sources that m a y be present as secondary sugars in the fermentation broth. T h e transfer o f the genes for lactose degradation to strains normally unable to use lactose is an example of increasing the range o f carbon source utilization. In situations where a protein is itself the p r i m a r y product, gene cloning can be used directly to increase the copy number, a n d generally, the p r o d u c t yield. More importantly, r e c o m b i n a n t D N A procedures m a k e it possible to transfer the gene o f interest to strains better suited for p r o d u c t i o n than the original host. Changes in the biochemical properties o f the protein can be m a d e by in vitro localized mutagenesis o f the cloned gene and subsequent analysis o f transformants. T h e ability to alter e n z y m e activity and greatly increase the cellular concentration o f enzymes is i m p o r t a n t not only for the direct c o m m e r c i a l production o f enzymes, but also for the d e v e l o p m e n t o f new industrial processes using immobilized cells and enzymes. T h e use o f enzymes as catalysts in manufacturing will b e c o m e increasingly i m p o r t a n t as the m e t h o d s for gene cloning and immobilization improve. T h e above applications only superficially illustrate the potential for genetic engineering in biotechnology. T h e future possibilities are virtually limitless as the methods are applied to a greater diversity o f microorganisms. It will b e feasible to construct entirely novel pathways by combining genes f r o m several different organisms into one host. It m a y be possible to enrich for previously undescribed phenotypes by r a n d o m l y cloning D N A from one strain into a different host and screening for a desired property. As i m p o r t a n t as the current progress has been, it is likely to be rapidly o v e r s h a d o w e d as these approaches are extended to thermophils and psychrophiles, acidophiles and alkalophiles, anaerobes, autotrophs, fungi, photosynthetic microorganisms, and m a n y others. Gene-cloning should not be expected to be a panacea, rather it should be viewed as a d e v e l o p m e n t in technology which will aid in the exploration and exploitation o f the vast diversity o f biochemical reactions in microorganisms.
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9. Chang S, Cohen SN (1979) High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168:111-115 10. Chater KF, Bruton CJ (1983) Mutational cloning in Streptomyces and the isolation of antibiotic production genes. Gene 26:67-78 11. Clarke L, Carbon J (1980) Isolation of a yeast centromere and construction of functional small chromosomes. Nature 287:504-509 12. Cooney CL (1975) Microbial growth on C~--compounds ! 83-197. Society of Fermentation Technology, Tokyo 13. Ehrlich SD (1977) Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc Natl Acad Sci USA 74:1680-1682 14. Ehrlich SD (1978) DNA cloning in Bacillus subtilis. Proc Natl Acad Sci USA 75:1433-1436 15. Feitelson TS, Hopwood DA (1983) Cloning o f a Streptornyces gene for an o-methyltransferase involved in antibiotic biosynthesis. Mol Gen Genet 190:394-398 16. Goodfellow M, Williams ST (1983) Ecology of actinomycetes. Ann Rev Microbiol 37:189226 17. Gray O, Chang S (1981) Molecular cloning and expression of Bacillus licheniformis/~-lactamase gene in E. coli and Bacillus subtilis. J Bacteriol 145:422-428 18. Gryczan TJ, ContenteS, Dubnau D (1978) Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol 134:318-329 19. Gunge N (1983) Yeast DNA plasmids. Ann Rev Microbiol 37:253-276 20. Harris JE, Chater KF, Bruton CJ, Piret JM (1983) The restriction mapping o f c gene deletions in Streptomyces bacteriophage ~ C 31 and their use in cloning vector development. Gene 22: 167-174 21. Harwood CR, Williams DM, L o v e r PS (1983) Nucleotide sequence o f a Bacilluspumilis gene specifying chloramphenicol acetyltransferase. Gene 24:163-169 22. Hopwood DA, Bibb MJ, Bruton CJ, Chater KF, Feitelson JS, Gil JA (1983) Cloning Streptomyces genes for antibiotic production. Trends Biotechnol 1:43-48 23. Hopwood DA, Chater KF, Dowding JE, Vivian A (1973) Advances in Streptomyces coelicolor genetics. Bacteriol Rev 37:371-401 24. Irnanaka J, Fujii M, Aramori I, Aiba S (1982) Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between B. stearothermophilus and B. subtilis. J Bacteriol 149:824-830 25. Katsumata R, Oka T, Furuya A (1982) Novel lysozyme sensitive microorganism. European Patent Application 92103677.9 26. Katsumata R, Oka T, Furuya A (1982) Novel plasmids. European Patent Application 92103222.4 27. Katsumata R, Oka T, Furuya A (1982) Transformation method of a microorganism. European Patent Application 92103223.2 28. Kiesen T, Hopwood DA, Wright HM, Thompson CJ (1972) plJ 107, a multicopy broad hostrange Streptomyces plasmid: functional analysis and development of DNA cloning vectors. Mol Gen Genet 185:223-238 29. Levin MA, Zaugg RH, Kidd GH, Swarz JR (1983) Applied genetic engineering 37-40, Noyes Publications, Park Ridge, New Jersey 30. Lovett PS, Duvall EJ, Keggins KM (1976) Bacillus pumilus plasmid pPLI0: properties and insertion into Bacillus subtilis 168 by transformation. 31. Malpartida F, Hopwood DA (1984) Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heteroiogous host. Nature 309:462-464 32. Miwa K, Terabe M, Ito K, Ishida M, Matsui K. Makamori S, Sano K (1983) Composite plasmid. European Patent Application 93302478.9 33. Ortlepp SA. Ollington .IF, McConnell DJ (1983) Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase. Gen 23:267-276 34. Palva I (1982) Molecular cloning of a-amylase gene from Bacillus amyloliquefaciens and its expression in B. subtilis. Gene 19:81-87 35. Priest FG (1977) Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev 41:711753
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MICROBIAL ECOLOGY ~) 1986 Springer-Verlag
Genetic Engineering of Microorganisms for Biotechnology Edmund J. Stellwag* and Jean E. Brenchleyt Genex Corporation, 16020 IndustrialDrive, Gaithersburg,Maryland20877
introduction Investigations of a variety of microorganisms have demonstrated the existence of a considerable biochemical diversity. Different microbes have evolved the capabilities to survive in environmental extremes of nutrient supply, temperature, pH, pressure, minerals, and toxic chemicals. Although attempts have been made to understand the physiology of many different microorganisms, the study of microbial genetics has centered on a few bacteria, primarily Escherichia coli and related enterics. This focused approach has been highly successful for the development of the detailed genetic linkage map of E. coli, the elaborate understanding of regulatory mechanisms, the elucidation of macromolecular synthesis, and, most importantly, for the purposes of this article, the establishment of recombinant DNA procedures for genetic engineering. The ability to form recombinant DNA molecules in vitro that can be stably replicated in a recipient host has had a major impact on the biological community and on society. One aspect of this impact has been the formation of biotechnology companies. Biotechnology, when broadly defined as the application of biological materials to industrial processes, is not new. Microorganisms have traditionally produced such useful compounds as alcohol and antibiotics. More recently, considerable attention has been directed toward exploitation of diverse microbial species for enzyme production as well as for the synthesis and degradation of chemicals. Unfortunately, the application of microorganisms and their enzymes has been limited by several problems, including low product yield, poor growth, or pathogenicity. A major advantage of the recombinant DNA techniques is that they provide the means to circumvent these barriers and have greatly extended the potential for using the existing microbial diversity. It is ironic that it has been this breakthrough in molecular biology that has greatly revitalized the interest and excitement in the study of general microbiology for industrial applications. This article will examine areas where the use of recombinant DNA procedures with microorganisms other than E. coli are important to biotechnology. Because our goal is to illustrate how recombinant DNA methods can be applied to a wide variety of industrially important microbes, the article will present the
~address:
Departmentof Microbiologyand Immunology,EastCarolinaUniversity,Green-
ville, NC 27834.
t Presentaddress:DepartmentofMicrobiology,Molecular,and CellBiology,PennStateUniversity, Collegeof Science, UniversityPark, PA 16802.
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historical development and current status of a few microorganisms rather than a detailed review of all publications in the field. Extensive discussions of certain microorganisms that are described elsewhere in this volume were omitted to avoid duplication. In addition, we outline some of the potential that recombinant DNA procedures provide as tools for greatly expanding our understanding and use of many other diverse microorganisms.
Discussion
Development o f Recombinant DNA Procedures in Bacillus and Streptomyces Bacillus subtilis is perhaps foremost among the many industrially significant microbes to have received increasing attention as a host for recombinant DNA experiments [29]. B. subtilis, unlike E. coli, is proficient at the secretion of proteins into the extracellular medium [35]. The property of extracellular protein secretion is important in industry not only because of the potential for high product yields, but because many extracellular proteins are purified more readily than intracellular proteins [44]. The inability orE. coli to secrete proteins encoded by foreign genes results in the formation of intracellular protein aggregate structures termed "inclusion bodies" [8]. The proteins assembled in these inclusion bodies are frequently denatured making the recovery of the active product difficult [8]. One solution to this problem is to clone these genes into an organism, such as B. subtilis, capable of both synthesis and extracellular secretion of active proteins. A prerequisite to the development of gene-cloning in any organism is the identification of a suitable vector for the introduction and stable maintenance of cloned DNA in the host. The search for a suitable vector in B. subtilis yielded only cryptic plasmids [30, 41] and was therefore of limited significance for the establishment of a molecular genetic system. A major breakthrough occurred when it was discovered that plasmids carrying antibiotic-resistance determinants in Staphylococcus aureus could be transformed into and stably maintained in B. subtilis [13, 14, 18]. Two of the plasmids, pC194 and pUB110, have served as the cornerstone for the development of gene-cloning in B. subtilis. A second major hurdle in the establishment of a gene-cloning system is the ability to introduce, with high efficiency, molecules of the vector DNA into the host organism [9]. While transformation of competent B. subtilis by chromosomal DNA is a highly efficient process in which up to 10% of the recipient ceils undergo genetic alteration [1 ], the frequency of transformation by plasmid DNA is several orders of magnitude lower [13, 18]. Despite the efficient competence system in B. subtilus, it was of little use for recombinant DNA-cloning experiments. This difficulty has been remedied by the development of a highly efficient procedure involving the polyethylene glycol (PEG)-induced uptake of DNA by B. subtilis protoplasts [9]. This procedure is so efficient that it is suitable even for the introduction of phenotypically cryptic plasmids [9]. High efficiency transformation has permitted the cloning and expression in
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B. subtilis of a diverse list of genes, including those encoding antibiotic resistance markers from B. pumilis, a thermostable a-amylase for B. licheniformis, and the mouse dihydrofolate reductase [21, 33, 37]. Perhaps more important than the actual number and types of genes cloned in B. subtilis is the application of the PEG transformation technique to other Bacillus species. PEG-mediated D N A uptake has been reported in a succession of Bacillus species, including thermophiles [24] and strains that secrete prodigious amounts of protein [40]. The genetic flexibility afforded the bacilli by the protoplast transformation technique has permitted the successful heterologous gene cloning among a limited number of species [17, 34, 36]. However, considering the possibilities that exist, the actual number of reports concerning the cloning and expression of homologous or heterologous genes a r e few. Before the recombinant D N A tools can be used to full advantage, a better understanding of fundamental physiology and metabolism of this diverse group is needed. Numerous opportunities exist for studying the effects of different cloned genes in a wide range of host backgrounds. The utility of the PEG-mediated D N A uptake system has not been restricted to the bacilli. In fact, the high frequency uptake of D N A by PEG-treated protoplasts has contributed significantly to the success of D N A cloning efforts in the genus Streptornyces [2]. The Streptomyces have historically received considerable attention from industrial researchers primarily because of their important position to antibiotic synthesis [42, 45]. As in B. subtilis, the advent of molecular genetic manipulation in Streptomyces was preceded by discovery of a high-efficiency, interspecific gene-transfer system [23]. In the case of Streptomyces, this was mediated by a conjugal plasmid capable of transferring either plasmid or chromosomal D N A [23]. Unfortunately, the difficulty in isolating this large plasmid precluded its use as a cloning vector [22]. The first useful Streptomyces vector was constructed by cloning the neomycin-resistance gene from Streptomycesfradiae and the thiostrepton-resistance genes from Streptomyces azureus onto a plasmid, SLP 1.2, which had been discovered in Streptomyces lividans 66 only after mating with Streptornyces coelicolor [22]. Although this vector has several unique restriction s~tes in the gene encoding neomycin resistance, the copy number of the plasmid is low (4-5 copies/chromosome equivalent). Consequently, this vector, pIJ61, is useful for cloning via insertional inactivation of the neomycin resistance marker, but it is of limited utility for expressing large quantities of the gene product. Although plasmids with high copy number have been isolated from Streptornyces, the recent cloning of the complete biosynthetic pathway for the synthesis of the antibiotic actinorhodin from S. coelicolor was attributed to the use of a low-copy number plasmid pIJ922 as the vector [31]. In this instance, the use of high-copy number plasmids was not effective; the authors suggest the high-level production of a "physiologically active" gene product may have lethal effects in the recipient strain resulting in a failure to isolate the appropriate clone. This observation places a different perspective on the potential strategies for cloning genes which specify products effecting normal host metabolism. There may be situations where low levels of expression are required for cell viability. In such instances, the production of large quantities of a compound may require the conditional regulation of gene expression.
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In addition to the plasmids described above, other sophisticated vectors based on both high-copy number plasmids [28] and bacteriophage [20] are presently employed in the analysis ofgene expression in Streptomyces. Although these gene-cloning tools are available, other obstacles have prevented the rapid full-scale industrial exploitation of the recent developments in molecular biology. The cloning of genes coding for the enzymes involved in the biosynthesis of antibiotics in Streptomyces provides an example of the limitations of conventional gene-cloning techniques for directly increasing product yield. Because the biosynthetic pathways for antibiotics and other complex molecules are highly regulated and involve many enzymatic steps, the cloning of genes encoding one or a few enzymes in the pathway generally fails to increase product yield. Thus, several genes may be needed before the total pathway and product yield are affected. The matter is made more difficult because the cloning of genes coding for enzymes in biosynthetic pathways frequently confer no detectable phenotype on the host. Consequently, cloning genes encoding biosynthetic enzymes often relies on complementation of mutants blocked at specific steps in the biosynthetic pathway. The "complementation cloning'" technique was used in the isolation o f a gene involved in the biosynthesis o f the antibiotic undecylprodigiosin [15]. A S. coelicolor mutant defective in antibiotic biosynthesis was used as the recipient strain for wild-type S. coelicolor DNA cloned into a plasmid. A single antibiotic-producing colony was found, and DNA from this clone restored antibiotic production in two of the five classes of S. coelicolor antibiotic-biosynthetic mutants. The cloning of genes encoding undecylprodiglosin production was possible because of the availability of mutants blocked in antibiotic production. The need to isolate mutants in each step of a biosynthetic pathway can significantly delay cloning efforts, particularly for organisms that are poorly characterized genetically. 9. An alternate method termed "mutational cloning" circumvents the need for generating mutants prior to cloning the wild-type allele [10]. The mutational cloning system is dependent on the availability of a lysogenic phage-derived cloning vector containing a selectable marker, a suitable cloning site, and a defect in the normal phage chromosomal integration mechanism. Generally lysogenic phage integrate into the host genome by site specific recombination between a unique phage DNA sequence and a nonhomologous host DNA sequence [6]. A defect in this normal phage-specified integration mechanism destroys the ability of the phage to insert into its specific host attachment site. The defective integration mechanism can be circumvented by cloning host DNA into the phage genome. Integration of the phage genome carrying host DNA can now proceed via homologous recombination between the DNA cloned into the phage genome and the homologous DNA of the host. The integration of the phage genome in this matter interrupts the normal gene coding sequence, resulting in mutations. Each mutant generated by integration of phage DNA will contain not only the phage genome but also the cloned DNA homologous to the gene sequence disrupted by the integration event, i.e., the mutant gene. The DNA obtained from phage released from such a lysogenic mutant strain contains cloned DNA and can be used for further molecular biological manipulation. Mutational cloning procedure was first demonstrated in the isolation of genes involved in methylenomycin A biosynthesis from S. coelicolor [10].
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The above presentation of gene-cloning in Bacillus and Streptomyces has neglected the importance of understanding the regulation of gene expression. The fact that these two genera must coordinate expression of both vegetative and developmentally transcribed genes suggests that regulation may be quite sophisticated. Interest in developmental controls in Bacillus and Streptomyces led to the construction of promotor-probe vectors for each genus [3, 47]. Promoter-probe vectors contain a cloning site 5' distal to a marker gene that possesses no promotor. Expression of the marker gene following introduction of foreign D N A at the cloning site indicates promotor activity on the cloned D N A fragment. Streptomyces D N A possessing promotor activity has been isolated using promotor-probe vectors functional in Streptomyces species [3]. Streptomyces D N A sequences possessing promotor activity showed no sequence relationship to well-characterized promotor sequences from other genera [3]. In addition, Streptomyces D N A fragments showing promotor activity in the homologous Streptomyces host do not demonstrate detectable promotor activity in E. coli [3]. In contrast, some promotors from E. coli, Serratia, and Bacillus are functional in Streptomyces [3]. These results suggest that Streptomyces promotors are unrelated to those from other genera, but that Streptomyces R N A polymerase and/or ribosomes are promiscuous with regard to recognition of foreign promotors and translation initiation sequences. These observations may have important implications for the potential application of Streptomyces as a host for foreign DNA.
Application of Genetic Engineering to Other Industrially Important Microorganisms The presentation of the Bacillus and Streptomyces work focused on the critical aspects in the development of DNA-cloning systems. But it should not be concluded that significant strides in recombinant DNA-mediated strain manipulation are dependent on years of vector construction. The following example will demonstrate that recombinant D N A tools developed in well-characterized systems can be employed in the manipulation of strains lacking an established genetic system. The obligate methylotroph Methylophilus methylotrophus is able to efficiently convert methanol to single-cell protein [49], a process of major importance to a variety of industries [ l 2]. Interestingly, instead of using the one-step glutamate dehydrogenase catalyzed conversion of ammonia and a-ketoglutarate to glutamate, this organism uses a two-step pathway catalyzed by glutamine synthetase and glutamate synthase [49]. The two-step pathway is less energy efficient and therefore reduces the ~r of single-cell protein from methanol grown cultures. Researchers at International Cell Products, Inc. used chromosomal D N A from E. coli and vectors from E. coli and Pseudomonas to construct a strain ofM. methylotrophus that assimilated ammonia via the energetically favorable glutamate dehydrogenase (GDH) pathway. The G D H gene was obtained by complementation cloning of wild-type E. coli D N A into a G D H deficient strain
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o f E. coli [49]. Since the vector used to clone the E. coli G D H gene could not be transferred directly to M. methylotrophus, the G D H gene was subcloned onto broad host range, Pseudomonas-derived vectors capable o f transfer to M. rnethylotrophus by conjugation. Transconjugants were selected for the antibiotic resistance encoded by the vector, and the G D H enzyme activity measured to confirm the presence o f the G D H gene. The strain constructed by these manipulations was able to convert methanol to single-cell protein more efficiently than the original parent strain. As the authors point out in their discussion, "the alteration made in M. methylotrophus could not have been achieved either by prolonged selection or by conventional mutagenesis, and therefore represents the planned adaptation of a microorganism to a man-made environment" [49]. Specific examples o f recombinant D N A manipulations such as described above are rare; however, they provide insight into the utility of these procedures for future strain improvement. A second example o f strain improvement by recombinant DNA manipulation o f a metabolic pathway is presented below. In this example DNA obtained from a procaryote is used to mediate an alteration in an eucaryotic metabolic pathway. During the wine-making process, a secondary fermentation convering L-malate to L-lactate is performed by various species o f lactic acid bacteria. This process reduces the acidity o f the wine and makes it more stable for storage. Unfortunately, the lactic acid bacteria normally do not grow rapidly in wine, and starter cultures are often needed. To circumvent this step, Williams et al. [48] cloned the gene for the malolactic enzyme from Lactobacillus delbreuchii into the plasmid pBR322, transformed E. coli, and selected for transformants that converted malate to lactate. Since pBR322 cannot replicate in Saccharomyces cerevisiae, the D N A restriction fragment carrying the gene for the malolactic enzyme was purified and recombined into an E. coli-yeast shuttle vector (i.e., a vector capable o f replicating in both E. coli and S. cerevisiae). S. cerevisiae cells containing the shuttle vector carrying the L. delbreuchii gene for the malolactic enzyme produced about 3.5 times more L-lactate during nonaerobic growth than the control strains [48]. These strains and their derivatives are being tested for their utility in the wine-making process. The interest in industrial ethanol fermentation extends beyond the winemaking industry. A considerable amount of attention has been directed toward the production o f ethanol as an energy source. Because traditional fermentation for alcohol production has used highly selected strains o f the yeast, S. cerevisiae, there has been substantial development o f yeast genetic systems. Vectors for S. cerevisiae were developed carrying origins of replication, such as the 2u circle or autonomously replicating sequences (ARS elements), and genes for biosynthetic enzymes that could be used for complementation [4, 11, 19, 39]. These genes, the trp 1 and leu2, were important because the natural resistance of S. cerevisiae to many commonly used antibiotics prevented the use of antibiotic resistance as a direct selection. The different yeast plasmids are being used successfully to clone and express a variety of eucaryotic genes, and S. cerevisiae may become a useful production organism for the synthesis o f medically important proteins. Another group o f microorganisms o f long-standing interest to industry are the coryneform bacteria. The c o m m o n feature of reproduction by mycelial
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fragmentation or snapping fission results in the variability o f the cell shape characteristic of these microbes. Despite this similarity in mode of cell division the different genera in these groups have diverse metabolic properties, occur in a wide variety of habitats, and, in many cases, are not closely related [ 16]. The group generally termed the coryneforms has as representative genera, Corynebacteriurn, Arthrobacter, Brevibacterium, and Microbacterium. Despite the importance ofcoryneforms for industrial amino acid production, no genetic transfer systems have been developed for these bacteria. Recently, however, several research groups have reported the development of recombinant DNA procedures for coryneforms. Patent applications from Ajinomoto Co., Inc. [32, 43] and Kyowa Hakko Kogyo Company, LTD [26, 27] describe transformation methods and the construction o f plasmids that can be used as vec!ors in Corynebacterium and Brevibacteriurn. One plasmid (pCG4) was obtained from C. glutamicum and h a s a gene conferring resistance to streptomycin and/or spectinomycin as a selectable marker [26, 27]. This plasmid was used to transform C. glutamicum, C. herculis, B. flavum, and B. lactofermenturn, demonstratit~g that it could replicate in these distinct strains and suggesting that it will be generally useful as a coryneform vector. Critical to this successful development of coryneform vectors has been protoplast transformation. However, because coryneform bacteria are extremely resistant to the lysozyme treatment used to obtain protoplasts, mutants that are substantially more sensitive to lysozyrne were isolated to facilitate protoplast transformation [25]. This approach could be useful for other microorganisms where protoplast formation or regeneration has been difficult. A different strategy was used by Dr. A. Sinsky and colleagues to develop cloning vectors from existing cryptic plasmids isolated from coryneform strains (38, and personal communication). Plasmids purified from C. glutamicum or B. lactofermentum were combined in vitro with a B. subtilis plasmid carrying a kanamycin resistance gene. These plasmid constructions were transformed into 17. subtilis selecting for kanamycin resistance and screening for shuttle vectors carrying D N A from both the coryneform and Bacillus plasmids. The plasmids were purified and used to test for transformation into coryneform recipients. The few kanamycin-resistant transformants that were obtained mere used to prepare quantities of the shuttle vector for use in retransformation and cloning experiments. As we pointed out in the discussion of gene cloning in Streptomyces, phage can be employed as recombinant D N A vectors in lieu ofplasmids. The phage, pEC, was isolated for its ability to infect Rhodococcus erythropolis, an organism related to the industrially important coryneforms [5]. The phage has a broad host range and can be propagated in several species of Rhodococcus and Nocardia. A map identifying sites recognized by restriction endonucleases was constructed and several deletion mutants isolated to determine the regions of the phage containing nonessential DNA. Phage deletion mutants can be used as cloning vectors whereby foreign D N A can be recombined into the phage in vitro and then introduced into cell protoplasts by transfection. Since the phage DNA can integrate, the new genes can be stably maintained. Temperate phage can provide the necessary components for recombinant D N A experiments in organisms lacking plasmids carrying selectable markers.
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The ability to use genetic engineering principles to construct microorganisms that can use alternate growth substrates could be extremely important to the development of better production strains and to using new raw materials as growth substrates. Preliminary efforts in this regard have involved the transfer of a plasmid carrying antibiotic resistance markers and a lactose transposon to various different strains by conjugation. Using this strategy, the lactose genes were transferred to Z y m o m o n a s mobilis [7] and Xanthomonas carnpestris [46] to enable these strains to use whey as a raw material for ethanol and xanthan gum production, respectively.
Conclusions and Future Prospects The application of recombinant DNA procedures to microorganisms other than E. coli is rapidly increasing. The construction of new recombinant DNA vectors and the elucidation of transformation techniques for previously untransformable microorganisms has tremendous potential for applications in both basic and applied research. These developments in genetic engineering of diverse microbial species have the potential for generating genetic heterogeneity previously unknown in the natural environment. This article has described a few situations where recombinant DNA techniques are being used to improve industrial processes. The examples presented provide the basis for outlining general conclusions regarding the development of gene-cloning systems: 1. An efficient gene transfer system, for example, protoplast transformation, is essential. 2. An elaborate genetic characterization of either the donor or host is not always necessary for gene cloning. 3. Vectors can be constructed from either plasmids or phage, depending on their availability and the eventual use. 4. Different types of vectors, such as ones with either low or high copy number, are required for different kinds of experiments. 5. Cryptic plasmids are potential vectors if appropriate markers that confer a selectable phenotype on the recipient can be incorporated in vitro. 6. Vectors using plasmids or genes from organisms other than the final host can often be useful. 7. The presence of selectable markers on vectors is essential for selecting transformants. 8. Almost any eucaryotic or procaryotic gene can be cloned into a new host if appropriate vectors and screening techniques are available. 9. A persistent investigation of strains of any particular genus is likely to provide suitable materials for the development of a gene cloning system. Because recombinant DNA procedures can be rapidly developed for widely diverse microorganisms, it is relevant to consider how these new approaches will impact future industrial applications. In the past, enhanced production of compounds was obtained by optimizing growth or culture conditions and by repeated strain selection and improvement programs. These approaches remain important, but they can be supplemented by gene cloning. For example, the
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production o f a m i n o acids or antibiotics can be increased by cloning genes for biosynthetic enzymes which remain rate limiting even in highly i m p r o v e d production strains. P r o d u c t i o n can also be i m p r o v e d by transferring genes that enable the strain to utilize alternate carbon sources that m a y be present as secondary sugars in the fermentation broth. T h e transfer o f the genes for lactose degradation to strains normally unable to use lactose is an example of increasing the range o f carbon source utilization. In situations where a protein is itself the p r i m a r y product, gene cloning can be used directly to increase the copy number, a n d generally, the p r o d u c t yield. More importantly, r e c o m b i n a n t D N A procedures m a k e it possible to transfer the gene o f interest to strains better suited for p r o d u c t i o n than the original host. Changes in the biochemical properties o f the protein can be m a d e by in vitro localized mutagenesis o f the cloned gene and subsequent analysis o f transformants. T h e ability to alter e n z y m e activity and greatly increase the cellular concentration o f enzymes is i m p o r t a n t not only for the direct c o m m e r c i a l production o f enzymes, but also for the d e v e l o p m e n t o f new industrial processes using immobilized cells and enzymes. T h e use o f enzymes as catalysts in manufacturing will b e c o m e increasingly i m p o r t a n t as the m e t h o d s for gene cloning and immobilization improve. T h e above applications only superficially illustrate the potential for genetic engineering in biotechnology. T h e future possibilities are virtually limitless as the methods are applied to a greater diversity o f microorganisms. It will b e feasible to construct entirely novel pathways by combining genes f r o m several different organisms into one host. It m a y be possible to enrich for previously undescribed phenotypes by r a n d o m l y cloning D N A from one strain into a different host and screening for a desired property. As i m p o r t a n t as the current progress has been, it is likely to be rapidly o v e r s h a d o w e d as these approaches are extended to thermophils and psychrophiles, acidophiles and alkalophiles, anaerobes, autotrophs, fungi, photosynthetic microorganisms, and m a n y others. Gene-cloning should not be expected to be a panacea, rather it should be viewed as a d e v e l o p m e n t in technology which will aid in the exploration and exploitation o f the vast diversity o f biochemical reactions in microorganisms.
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