Recombinant organisms for production of industrial products

Abstract

A revolution in industrial microbiology was sparked by the discoveries of ther double-sttranded structure of DNA and the development of trecombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding techniques and their modifications are contributing greatly to the development of improved industrial processes. In addition, functional genomics, proteomics and metabolomics are being exploited for the discovery of novel valuable small molecules for medicine as well as enzymes for catalysis. The sequencing of industrial microbal genomes is beuing carried out which bodes well for future process improvement and discovery of new industrial products.

Genetic Recombination in Industrial Microorganisms

In contrast to the extensive use of mutation in industry, employment of genetic recombination was meager at first. This lack of interest was prevalent despite early claims of success,1,2 mainly due to the absence or the extremely low frequency of genetic recombination in industrial microorganisms (in streptomycetes, it was usually 10⁻⁶ or even less). Recombination was erroneously looked upon as an alternative to mutation instead of a method which would complement mutagenesis programs. The most balanced and efficient strain development strategy would not emphasize one to the exclusion of the other; it would contain both mutagenesis-screening and recombination-screening components. In such a program, strains at different stages of a mutational line, or from lines developed from different ancestors, would be recombined. Such strains would no doubt differ in many genes and by crossing them, genotypes could be generated which would never occur as strictly mutational descendants of either parent. Recombination was also of importance in the mapping of production genes. The model for such investigations was the genetic map of Streptomyces coelicolor3 which was found to be very similar to those of other Streptomyces species, such as Streptomyces bikiniensis, Streptomyces olivaceous, Streptomyces glaucescens and Streptomyces rimosus.

Recombination in microorganisms occurs through three parasexual processes: conjugation, transduction, and transformation. Internal genetic rearrangements can also occur via translocatable DNA segments (insertion sequences or transposons). Conjugation involves transfer of DNA via cell-to-cell contact. Transduction occurs from host cell to recipient cell via mediation by bacteriophage. Transformation involves uptake and expression of naked DNA by competent cells. Competence occurs naturally but can also be induced by changes in the physical and chemical environment. In the laboratory, it can be induced by cold calcium chloride treatment, protoplasting, electroporation and heat shock.

As mentioned above, genetic recombination was virtually ignored in industry, mainly due to the low frequency of recombination. However, use of protoplast fusion changed the situation markedly. After 1980, there was a heightened interest in the application of genetic recombination to the production of important microbial products such as antibiotics. Today, frequencies of recombination have increased to even greater than 10^-1 in some cases,4 and strain improvement programs routinely include protoplast fusion between different mutant lines. The power of recombination was demonstrated by Yoneda5 who recombined individual mutations, each of which increased α-amylase production by two- to seven-fold in Bacillus subtilis. A strain constructed by genetic transformation, which contained all five mutations, produced 250-fold more α-amylase than the starting strain.

Primary Metabolites

Primary metabolites are microbial products made during the exponential phase of growth whose synthesis is an integral part of the normal growth process. They include intermediates and end products of anabolic metabolism, which are used by the cell as building blocks for essential macromolecules (e.g., amino acids, nucleotides) or are converted to coenzymes (e.g., vitamins). Other primary metabolites (e.g., citric acid, acetic acid and ethanol) result from catabolic metabolism; they are not used for building cellular constituents but their production, which is related to energy production and substrate utilization, is essential for growth. Industrially, the most important primary metabolites are amino acids, nucleotides, vitamins, solvents and organic acids.

Recent approaches utilize the techniques of modern genetic engineering to develop strains overproducing primary metabolites. This rationale for strain construction aims at assembling the appropriate characteristics by means of in vitro recombinant DNA techniques. This is particularly valuable in organisms with complex regulatory systems, where deregulation would involve many genetic alterations.

Production of a particular primary metabolite by deregulated organisms may inevitably be limited by the inherent capacity of the particular organism to make the appropriate biosynthetic enzymes, i.e., even in the absence of repressive mechanisms, there may not be enough of the enzyme made to obtain high productivity. There are some ways to overcome this: (1) to increase the number of copies of structural genes coding for these enzymes by genetic engineering, and (2) to increase the frequency of transcription, which is related to the frequency of binding of RNA polymerase to the promoter region. Increasing gene copies can be achieved by incorporating the biosynthetic genes in vitro into a plasmid which, when placed in a cell, will replicate into multiple copies. Increasing the frequency of transcription involves constructing a hybrid plasmid, which contains the structural genes of the biosynthetic enzymes but lacks the regulatory sequences (promoter and operator) normally associated with them. The ideal plasmid for metabolite synthesis would contain a regulatory region with a constitutive phenotype, preferably not subject to nutritional repression.

One of the major problems in using strains in which the desired characteristics are encoded by a plasmid is the difficulty in maintaining the plasmids during fermentation. One solution is to use antibiotic pressure during fermentation so that only organisms resistant to the antibiotic (due to the presence of a plasmid-borne resistance gene) can survive. Combinations of deregulation and plasmid amplification can yield a synergistic effect.

Novel genetic technologies are important for the development of effective overproducers. Those which have proven to be very useful for increasing production of primary metabolites are shown in Table 1. Ongoing sequencing projects involving hundreds of genomes, the availability of sequences corresponding to model organisms, new DNA microarray and proteomic tools, as well as the new techniques for mutagenesis and recombination described above, will no doubt accelerate strain improvement programs.

Genome-based strain reconstruction achieves the construction of a superior strain which only contains mutations crucial to hyperproduction, but not other unknown mutations which accumulate by brute-force mutagenesis and screening.6

The directed improvement of product formation or cellular properties via modification of specific biochemical reactions or introduction of new ones with the use of recombinant DNA technology is known as metabolic engineering.7,8 Analytical methods are combined to quantify fluxes and to control them with molecular biological techniques in order to implement suggested genetic modifications. Different means of analyzing flux are (1) kinetic based models, (2) control theories, (3) tracer experiments, (4) NMR magnetization transfer, (5) metabolite balancing, (6) enzyme analysis and (7) genetic analysis.9 The overall flux through a metabolic pathway depends on several steps, not just a single rate-limiting reaction. Amino acid production is one of the fields with many examples of this approach.10 Other processes improved by this technique include vitamins, organic acids, ethanol, 1,3-propanediol and carotenoids.

Reverse (inverse) metabolic engineering is another approach that involves (1) choosing a strain which has a favorable cellular phenotype, (2) evaluating and determining genetic and/or environmental factors that confer that phenotype, and (3) transferring that phenotype to a second strain via direct modifications of the identified genetic and/or environmental factors.11,12

Molecular breeding techniques come closer to mimicking natural recombination by allowing in vitro homologous recombination.13 DNA shuffling not only recombines DNA fragments but also introduces point mutations at a very low controlled rate.14 Unlike site directed mutagenesis, this method of pooling and recombining parts of similar genes from different species or strains has yielded remarkable improvements in a very short amount of time.15 Whole genome shuffling is a novel technique for strain improvement combining the advantage of multi-parental crossing allowed by DNA shuffling with the recombination of entire genomes.16,17

Amino acids.

Genetic engineering has made an impact on the production of amino acids by using the following strategies: (1) amplification of a rate-limiting enzyme of pathway; (2) amplification of the first enzyme after a branchpoint; (3) cloning of a gene encoding an enzyme with more or less feedback regulation; (4) introduction of a gene encoding an enzyme with a functional or energetic advantage as replacement for the normal enzyme; and (5) amplification of the first enzyme leading from central metabolism to increase carbon flow into the pathway followed by sequential removal of bottlenecks caused by accumulation of intermediates.

Transport mutations have also become useful, i.e., a mutation decreasing amino acid uptake allows for improved excretion and lower intracellular feedback control. This has been especially useful in production of tryptophan and threonine. In cases where excretion is carrier-mediated, increase in activity of these carrier enzymes increases production of the amino acid.

Among the amino acids, L-glutamate (MSG) and L-lysine, mostly used as feed and food additives, respectively, represent the largest products in this category. Produced by fermentation are 1.5 million tons of L-glutamate and 850,000 tons of L-lysine-HCl. The total amino acid market was about 4.5 billion dollars in 2004.18 Top fermentation titers reported in the literature are shown in Table 2.

Strains of Corynebacterium, Brevibacterium and Serratia19 have been routinely used for the commercial production of amino acids. Plasmid vector systems for cloning in Corynebacterium glutamicum were established and amino acid production by C. glutamicum and related strains has been improved by gene cloning.20 Extensive research has been done on sequencing the genome of C. glutamicum by Kyowa Hakko scientists, by scientists at the Ajinomoto Co., of Japan, and also by a collaboration of German workers.21,22 The genome of the closely related glutamate-overproducing species, Corynebacterium efficiens, has also been sequenced.23

L-Glutamate. Glutamate was the first amino acid to be produced by fermentation, because of its use as MSG in flavoring. High titers have been obtained over the years. Introduction of the Vitreoscilla hemoglobin gene vgb into C. glutamicum strains increased cell growth and glutamic acid and glutamine production via increased oxygen uptake.24 Workers at Ajinomoto Co., Inc., increased glutamate production from glucose by 9% by suppressing CO₂ liberation in the pyruvate dehydrogenase reaction.25 They did this by cloning the xfp gene encoding phosphoketolase from Bifidobacterium animalis into C. glutamicum and overexpressing it.

L-Lysine. Metabolic engineering has been used in C. glutamicum to improve L-lysine production.10 Metabolic flux studies of wild-type C. glutamicum and four improved lysine-producing mutants available from the ATCC showed that yield increased from 1.2% to 24.9% relative to the glucose flux. Lysine production by C. glutamicum mutants has been increased by several different approaches including:

1. deletion of PEP carboxykinase, deletion of glucose-6-phosphate dehydrogenase, disruption of malate quinone oxidoreductase, and lowering of citrate synthase activity by mutation,26

2. improving the availability of the precursor pyruvate by eliminating pyruvate dehydrogenase activity,27

3. overexpression of pyruvate carboxylase or DAP dehydrogenase genes,

4. overexpression of gene NCg10855 encoding a methyltransferase or the amtA-ocd-soxA operon encoding an ammonium uptake system, a putative ornithine cyclodeaminase and an uncharacterized protein.28

L-Threonine. Overproduction of L-threonine has been achieved with the use of several microorganisms. In Serratia marcescens, construction of a high threonine producer was achieved by transductional crosses which combined several feedback control mutations in a single organism.29 It produced 25 g l^-1 of threonine. Another six regulatory mutations derived by resistance to amino acid analogues were combined into a single strain of S. marcescens by transduction. These mutations led to desensitization and derepression of the key enzymes in threonine synthesis. The resultant transductant produced 40 g l^-1 of threonine.30 These S. marcescens overproducing strains were further improved by recombinant DNA technology. A mutant overproducing PEP carboxylase made 63 g l^-1 threonine, a 21% increase.31 An Escherichia coli strain was developed via mutation and genetic engineering and optimized by inactivation of threonine dehydratase (TD) resulting in a process yielding 100 g l^-1 of L-threonine in 36 hours of fermentation.32

In C. glutamicum ssp. lactofermentum, L-threonine production reached 58 g l⁻¹ when a strain producing both threonine and lysine (an isoleucine auxotroph resistant to thialysine, α-amino-β-hydroxyvaleric acid and S-methylcysteine sulfoxide) was transformed with a recombinant plasmid carrying its own hom (encoding homoserine dehydrogenase), thrB (encoding homoserine kinase) and thrC (encoding L-threonine synthase) genes.33 A recombinant strain of E. coli (made by mutating to isoleucine auxotrophy, cloning in extra copies of the thrABC operon, inactivating the threonine-degrading gene tdh, and mutating to resistance to high concentrations of L-threonine and L-homoserine) produced 80 g l⁻¹ L-threonine in 1.5 days at a yield of 50%.34 Cloning extra copies of threonine export genes into E. coli led to increased threonine production.35

L-Valine. Production by mutant strain VAL1 of C. glutamicum amounted to 105 g l⁻¹.36 The mutant was constructed by overexpressing biosynthetic enzymes via a plasmid, eliminating ilvA encoding threonine dehydratase, and deleting two genes encoding enzymes of pantothenate biosynthesis. The culture was grown with limitation of isoleucine and pantothenate.

L-Isoleucine. Feedback regulation in C. glutamicum was eliminated37 by replacing the native threonine dehydratase gene ilvA with the feedback resistant gene from E. coli. By introducting additional copies of genes encoding branched amino and biosynthetic enzymes, lysine- or threonine-producing strains were converted into L-isoleucine producers with improved titers.38,39 Amplification of the wild-type threonine dehydratase gene ilvA in a threonine-producing strain of Corynebacterium lactofermentum led to isoleucine production.40

L-Alanine. Lee et al.41 introduced into an E. coli double mutant [lacking genes encoding a protein of the pyruvate dehydrogenase complex (aceF) and lactate dehydrogenase (ldhA)] a plasmid containing the Bacilus sphaericus alanine dehydrogenase gene (alaD). The strain produced L-alanine in 27 hours with a yield on glucose of 0.63 g g⁻¹ and a maximum productivity of 2 g l⁻¹h⁻¹.

L-Proline. A mutant of S. marcescens resistant to 3,4-dehydroproline, thiazolidine-4-carboxylate and azetidine-2-carboxylate and unable to utilize proline produced 50 to 55 g l⁻¹ L-proline.42 Cloning of a gene bearing the dehydroproline-resistance locus on a plasmid yielded a recombinant strain of S. marcescens producing 75 g 1⁻¹.43 Further development work increased production to over 100 g l⁻¹.44

L-Hydroxyproline. Introduction of the proline 4-hydroxylase gene from Dactylosporangium sp. into recombinant E. coli producing L-proline at 1.2 g l⁻¹ led to a new strain producing 25 g l⁻¹ of hydroxyproline (trans-4-hydroxy-L-proline).45 When proline was added, hydroxyproline reached 41 g l⁻¹, with a yield of 87% from proline.

Aromatic amino acids. Overproduction of aromatic amino acids and derivatives has also been achieved by metabolic engineering.46 An engineered strain of C. glutamicum producing 50 g l⁻¹ of L-tryptophan was further modified by cloning in additional copies of its own transketolase gene in order to increase the level of the erythrose-4-phosphate precursor of aromatic biosynthesis.47 A low copy number plasmid increased production to 58 g l⁻¹, whereas a high copy number plasmid decreased production. Early use of a 6-fluorotryptophan- and 8-azaguanine-resistant mutant of a recombinant E. coli strain yielded more than 50 g l⁻¹ tryptophan from added anthranilic acid and glucose. Work on C. glutamicum in which the first enzyme (3-deoxy D-arabino-heptulosonate 7-phosphate synthase) entering the aromatic pathway was amplified, yielded 50 g l⁻¹ of L-tryptophan without adding anthranilic acid.48 By further engineering central metabolism to raise the availability of PEP and E4P (via a PEP-carboxylase-deficient mutant with only 25% remaining activity and amplification of tkt-encoding transketolase), C. glutamicum produced 58 g l⁻¹ L-tryptophan in a fed batch sucrose process. Using a plasmid containing tryptophan synthase plus induction with 3-indole acrylate, recombinant E. coli was able to carry out a bioconversion producing 180 g l⁻¹ of L-tryptophan from indole plus L-serine in 8 hours.49

Production of L-phenylalanine amounted to 28 g l⁻¹ when a plasmid was cloned into E. coli containing a feedback inhibition- resistant version of the CM-prephenate dehydratase (PD) gene, a feedback inhibition-resistant DAHPS and the O_RP_R and O_LP_L operator-promoter system of lambda phage. Control of plasmid expression was by temperature manipulation.50 Further process development of genetically engineered E. coli strains brought phenylalanine titers up to 46 g l⁻¹.51 Independently, genetic engineering based on cloning aroF and feedback resistant pheA genes created an E. coli strain producing 50 g l⁻¹.52

L-Tyrosine overproduction was obtained by amplification of aro II (encoding desensitized type II-DS) and csm in a tryptophan-producer of C. glutamicum. The manipulation shifted production from 18 g l⁻¹ L-tryptophan to 26 g l⁻¹ L-tyrosine.53

Vitamins.

Biotin. Biotin has been made traditionally by chemical synthesis but recombinant microbes have approached a competitive economic position. Cloning of a biotin operon (bioABFCD) on a multicopy plasmid allowed E. coli to produce 10,000 times more biotin than did the wild type strain.54 Sequential mutation of S. marcescens to resistance to the biotin antimetabolite acidomycin (=actithiazic acid) led to mutant strain SB412 which produced 20 mg l⁻¹ of biotin.55 Further improvements were made by mutating selected strains to ethionine-resistance (strain ET2, 25 mg l⁻¹), then mutating ET2 to S-2-aminoethylcysteine resistance (strain ETA23, 33 mg l⁻¹) and finally cloning in the resistant bio operon yielding a strain able to produce 500 mg l⁻¹ in a fed-batch fermentor along with 600 mg/l of biotin vitamers. Later advances led to production by recombinant S. marcescens of 600 mg l⁻¹ of biotin.56

Riboflavin. A process for riboflavin production by Corynebacterium ammoniagenes (previously Brevibacterium ammoniagenes) was developed by cloning and overexpressing the organism's own riboflavin biosynthesis genes57 and its own promoter sequences. The resulting culture produced 15 g l⁻¹ riboflavin in three days. Genetic engineering of a B. subtilis strain already containing purine analog-resistance mutations led to improved production of riboflavin.58 The industrial strain of B. subtilis was produced by inserting multiple copies of the rib operon at two different sites in the chromosome, making purine analog-resistance mutations to increase guanosine triphosphate (GTP; a precursor) production and a riboflavin analog (roseflavin)-resistance mutation in ribC that deregulated the entire pathway.59 Resulting production was over 25 g l⁻¹. A genome-wide transcript expression analysis called massive parallel signature sequencing60 was successfully used to discover new targets for futher improvement of riboflavin production by the fungus A. gossypii.61 The authors identified 53 genes of known function, some of which could clearly be related to riboflavin production. This approach also allowed the finding of sites within the genome with high transcriptional activity during riboflavin biosynthesis that are suitable integration loci for the target genes found.

Vitamin C. Vitamin C (ascorbic acid) has traditionally been made in a five-step chemical process by first converting glucose to 2-keto-L-gulonic acid (2-KGA) with a yield of 50% and then converting the 2-KGA by acid or base to ascorbic acid. A novel process for vitamin C synthesis involved the use of a genetically engineered Erwinia herbicola strain containing a gene from Corynebacterium sp. The engineered organism converted glucose into 1 g l⁻¹ of 2-KGA.62 A better process was devised independently which converted 40 g l⁻¹ glucose into 20 g l⁻¹ of 2-KGA.63 This process involved cloning and expressing the gene encoding 2,5-diketo-D-gluconate reductase from Corynebacterium sp. into Erwinia citreus. Another process used a recombinant strain of Gluconobacter oxydans containing genes encoding L-sorbose dehydrogenase and L-sorbosone dehydrogenase from G. oxydans T-100. The new strain was an improved producer of 2-KGA.64 Further mutation to suppress the L-idonate pathway and to improve the promoter led to production of 130 g l⁻¹ of 2-KGA from 150 g l⁻¹ sorbitol.

Organic acids.

Microbial production of organic acids is an excellent approach for obtaining building-block chemicals from renewable carbon sources.65 Production of some organic acids started decades ago and titers have been improved by classical mutation and screening/selection techniques as well as by metabolic engineering.66 Metabolic engineering of E. coli and C. glutamicum has yielded high levels of acetate, pyruvate, succinate and lactate.67

Acetic acid. Titers of acetic acid reached 53 g l⁻¹ with genetically engineered E. coli68 and 83 g l⁻¹ with a Clostridium thermoaceticum mutant.69 Cloning of the aldehyde dehydrogenase gene from Acetobacter polyoxogenes on a plasmid vector into Acetobacter aceti subsp. xylinum increased the rate of acetic acid production by over 100% (1.8 g l⁻¹h⁻¹ to 4 g l⁻¹h⁻¹) and titer by 40% (68 g l⁻¹ to 97 g l⁻¹).70

Lactic acid. A transgenic wine yeast genetically engineered to contain six copies of the bovine L-lactate dehydrogenase gene produced L-(+)-lactate at 122 g l⁻¹.71 Whole genome shuffling was used to improve the acid-tolerance of a commercial lactic acid-producing Lactobacillus sp.72 Further approaches using the recursive protoplast fusion technique yielded strains of Lactobacillus rhamnosus ATCC 11443 with improved glucose tolerance (above 160–200 g l⁻¹ glucose as initial concentration), while simultaneously enhancing L-lactic acid production by 71% as compared to the wild type. Shuffling of a mutant strain of Lactobacillus delbrueckii NCIM 2025 and the amylase-producing non-fastidious strain of Bacillus amyloliquefaciens ATCC 23842 produced a fusant that could utilize liquefied cassava bagasse starch directly with minimal nutrient supplementation for lactic acid production. A titer of 40 g l⁻¹ of lactic acid was achieved which amounted to a 96% conversion of starch to lactic acid.73

A recombinant E. coli strain was constructed that produced optically active pure D-lactic acid from glucose at virtually the theoretical maximum yield, e.g., two molecules from one molecule of glucose.74 The organism was engineered by eliminating genes of competing pathways encoding fumarate reductase, alcohol/aldehyde dehydrogenase and pyruvate formate lyase, and by a mutation in the acetate kinase gene. D-Lactic acid has also been produced at 61 g l⁻¹ by a recombinant strain of S. cerevisiae containing the D-lactic dehydrogenase gene from Leuconostoc mesenteroides.75

Whereas lactobacilli make more lactic acid than Rhizopus oryzae, they require yeast extract and produce mixed isomers. However, R. oryzae produces L-(+) lactic acid exclusively. The fungus yields lactic acid at 60–80% of added glucose, the remainder going to ethanol. By increasing lactic dehydrogenase levels via plasmid transformation with ldhA, more lactate could be made from pyruvate.76 Production was increased to 78 g l⁻¹ whereas undesirable coproduct ethanol was reduced from 10.6 to 8.7 g l⁻¹ and fumaric acid from 0.2 to 0.05 g l⁻¹. Recombinant Saccharomyces cerevisiae containing six copies of bovine L-lactate dehydrogenase produced 122 g l⁻¹ from cane sugar with optical purity of 99.9% or higher.77 Kluyveromyces lactis, also with the bovine enzyme and a deleted pyruvate decarboxylase gene, produced 109 g l⁻¹.78

Succinic acid. Metabolic engineering of Mannheimia succiniciproducens led to a strain producing 52 g l⁻¹ of succinic acid at a yield of 1.16 mol mol⁻¹ glucose and a productivity of 1.8 g l⁻¹h⁻¹ in fed-batch culture.79 Reduced formation of acetic, formic and lactic acids was accomplished by disruption of genes ldhA, pflB, pta and ackA. A metabolically engineered succinate-producing strain of E. coli with inactivated genes encoding succinate dehydrogenase, pyruvate oxidase, acetate kinase-phosphotransacetylase, glucose phosphotransferase and the repressor of the aceBAK operon, and overexpression of the gene encoding PEP carboxylase, yielded 58 g l⁻¹ succnate in a 59 hour fed-batch fermentation under aerobic conditions.80 The average succinate yield was 0.94 mol mol⁻¹ of glucose, the average productivity was 1.08 g l⁻¹h⁻¹; and the average specific activity was 90 mg g⁻¹h⁻¹. Sanchez et al.81 used metabolic engineering to create an E. coli strain which had three deactivated genes of the central metabolic pathway, i.e., adhE, ldhA and act-pta, and an inactivated iclR gene which resulted in activation of the glyoxylate pathway. The strain produced 40 g l⁻¹ of succinate. A titer of 99 g l⁻¹ has been reached with recombinant E. coli with a productivity of 1.3 g l⁻¹h⁻¹.82

Shikimic acid. Shikimic acid is the starting point for chemical synthesis of Tamiflu, an antiviral agent. Metabolic engineering of E. coli yielded an overproducer making 84 g l⁻¹ of shikimic acid with a 0.33 molar yield from glucose.83 50–90 g l⁻¹ of shikimic acid was produced in 30% (mol mol⁻¹) yield from glucose with an E. coli aroL and aroK mutant which overexpressed aroF, aroB and aroE, and tktA and ppsA of central metabolism.83,84

Other acids. Metabolic engineering of Clostridium tyrobutyricum created a fermentation strain yielding 80 g l⁻¹ butyric acid and a yield on glucose of 0.45 g g⁻¹.85

Whereas S. cerevisiae nomally produces 2 g l⁻¹ of malic acid from fumaric acid, a recombinant strain containing a cloned fumarase gene was able to produce 125 g l⁻¹ with a yield of almost 90%.86

An oxidative bioconversion of saturated and unsaturated linear aliphatic 12–22 carbon substrates to their terminal dicarboxylic acids was developed by gene disruption and gene amplification.87 Product concentrations reached 200 g l⁻¹ and problematic side-reactions such as unsaturation, hydroxylation and chain-shortening did not occur.

Alcohols. Ethanol. E. coli was converted into a good ethanol producer (43 g l⁻¹) by recombinant DNA technology.88 Alcohol dehydrogenase II and pyruvate decarboxylase genes from Zymomonas mobilis were inserted in E. coli and became the dominant system for NAD regeneration. Ethanol represented over 95% of the fermentation products in the genetically-engineered strain. By cloning and expressing the same two genes in Klebsiella oxytoca, the recombinant was able to convert crystalline cellulose to ethanol in high yield when fungal cellulase was added.89 Maximum theoretical yield was 81–86% and titers as high as 47 g l⁻¹ of ethanol were produced from 100 g l⁻¹ of cellulose.

Most recombinant strains of E. coli, Zymomonas and Saccharomyces convert corn fiber hydrolysate to 21–35 g l⁻¹ with yields of 0.41–0.50 ethanol per g of sugar consumed.90 For a recombinant E. coli strain making 35 g l⁻¹, time was 55 hours and yield was 0.46 g ethanol per g of available sugar, which is 90% of the attainable maximum.

A S. cerevisiae fusant library obtained by genome shuffling was screened for growth at 35, 40, 45, 50 and 55°C on agar plates containing different concentrations of ethanol.91 After three rounds of genome shuffling, a strain (F34) was obtained which was able to grow on plate culture up to 55°C, completely utilized 20% (w/v) glucose at 45–48°C, produced 99 g l⁻¹ ethanol and tolerated 25% (v/v) ethanol stress.

Three rounds of shuffling allowed isolation of a strain with a substantial improvement in multiple stress tolerance to ethanol, glucose, and heat. Its cycle of fermentation was not only shortened, but ethanol yield was increased by up to 11% as compared with the control in very-high-gravity (VHG) fermentations.92 Tolerance of Candida krusei GL560 to acetic acid was also improved by genome shuffling.93 A mutant, S4-3, which was isolated and selected after four rounds, had a higher viability in different media containing acetic acid than did the parent strain GL560. The mutant also improved its multiple stress tolerance to ethanol, H₂O₂, heat and freeze-thawing.

1,3-propanediol. A strain of Clostridium butyricum converts glycerol to 1,3-propanediol (PDO) at a yield of 0.55 g g⁻¹ glycerol consumed.94 In a two-stage continuous fermentation, a titer of 41 to 46 g l⁻¹ was achieved with a maximum productivity of 3.4 g l⁻¹ h⁻¹. Recent metabolic engineering triumphs have included the development of an E. coli culture that grows on glucose and produces PDO at 135 g l⁻¹, with a yield of 51% and a rate of 3.5 g l⁻¹ h⁻¹.95 To do this, eight new genes were introduced to convert dihydroxyacetone phosphate (DHAP) into PDO. These included yeast genes converting dihydroxyacetone to glycerol and Klebsiella pneumoniae genes converting glycerol to PDO. Production was further improved in the recombinant by modifying eighteen E. coli genes, including regulatory genes.95 PDO is the monomer used to chemically synthesize industrial polymers such as polyurethanes and the polyester fiber Sorono™ by DuPont. This new bioplastic is polytrimethylene terephthalate (3GT polyester) made by reacting terephthalic acid with PDO.96 PDO is also used as a polyglycol-like lubricant and as a solvent.

D-Mannitol. D-Mannitol is a naturally occurring polyol, widely used in the food, chemical and pharmaceutical industries. About 40,000 tons are produced annually. It is considered to be a low-calorie sweetener. It is produced mainly by catalytic hydrogenation of glucose/fructose mixtures but 75% of the product is sorbitol, not mannitol. For this reason, microbial processes are being considered. A whole cell bioconversion of D-fructose to D-mannitol was developed by metabolic engineering of E. coli.97 The mdh gene encoding mannitol dehydrogenase from Leuconostoc pseudomesenteroides and the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae were co-expressed in E. coli along with the glf gene encoding the glucose facilitator protein of Z. mobilis. The process yielded 75–91 g l⁻¹ of D-mannitol, a specific productivity of 3.1–4.1 g g⁻¹h⁻¹ and a molar yield of 84–92% with no byproducts. An improved bioconversion process was developed with a recombinant E. coli strain in the presence of added glucose isomerase yielding 145 g l⁻¹ of D-mannitol from 180 g l⁻¹ glucose.98

Sorbitol. Sorbitol, also called D-glucitol, is 60% as sweet as sucrose and has use in the food, pharmaceutical and other industries. Its worldwide production is estimated to be higher than 500,000 tons per year and it is made chemically by catalytic hydrogenation of D-glucose. Metabolic engineering of Lactobacillus plantarum for high sorbitol production was successfully achieved by a simple two-step strategy overexpressing the two sorbitol-6-phosphate dehydrogenase genes (srlD1 and srlD2) identified in the genome sequence.99

Other compounds.

Nucleosides. Genetic engineering of the inosine monophosphate (IMP) dehydrogenase gene in a B. subtilis strain producing 7 g l⁻¹ of the desirable guanosine and 19 g l⁻¹ of the undesirable inosine changed production to that of 20 g l⁻¹ guanosine and 5 g l⁻¹ inosine.100 Guanosine is used to produce guanosine monophosphate (GMP) a potent flavoring agent.

Carotenoids. Carotenoids were overproduced by introducing carotenoid gene clusters from Erwinia uredovora into E. coli and overexpressing the E. coli deoxyxylulose phosphate synthase, the key enzyme of the non-mevalonate isoprenoid biosynthetic pathway.101 Lycopene accumulated to 1.3 mg g⁻¹ dry cell weight and zeaxanthin to 0.6 mg g⁻¹.

Glucosamine. Glucosamine is used for osteoarthritis and is made by acid hydrolysis of chitin from shellfish waste. Since many patients have shellfish allergies, a microbial source is desirable. Metabolic engineering of E. coli yielded a process producing 17 g l⁻¹ of glucosamine.102 In this strain, glucosamine synthase was overexpressed, glucosamine degradative genes were inactivated, and the inhibition of glucosamine synthase by glucosamine was decreased by mutational modification of the enzyme via error-prone PCR. Overexpression of a heterologous glucosamine-6-P-N acetyltransferase yielded a strain making 110 g l⁻¹ of N-acetylglucosamine which is easily converted to glucosamine by mild acid hydrolysis.

Solvents. Cloning of its ace (acetone) operon genes adc (encoding acetoacetate decarboxylase), and ctfA and ctfB (two genes encoding coenzyme A transferase) on a plasmid containing the adc promoter into Clostridium acetobutylicum resulted in a 95% increased production. The increases were 95% in production of acetone, 37% for butanol, 90% for ethanol, 50% for solvent yield from glucose and a 22-fold lower production of undesirable acids.103 Introduction of the acetone operon from C. acetobutylicum into E. coli led to high acetone production by the latter.104

Secondary Metabolites

These compounds have a major effect on the health, nutrition and economics of our society. The best-known are the antibiotics. This remarkable group of compounds form a heterogeneous assemblage of biologically active molecules with different structures and modes of action. They attack virtually every type of microbial activity such as DNA, RNA, and protein synthesis, membrane function, electron transport, sporulation, germination and many others. Other secondary metabolites are pesticides, pigments, toxins, effectors of ecological competition and symbiosis, pheromones, enzyme inhibitors, immunomodulating agents, receptor antagonists and agonists, pesticides, antitumor agents, immunosuppressives, cholesterol-lowering agents, plant protectants and growth promotants of animals and plants. To obtain/develop overproducing strains, the technologies already described for the production of primary metabolites (see section 2) have been applied.

Antibiotics.

Antibiotics have tremendous economic importance. More than 350 agents have reached the world market as antimicrobials.

β-lactams. Of great importance in chemotherapy of bacterial infections are the β-lactam antibiotics, namely penicillin G, cephalosporin C, cephamycin C and the semi-synthetic penicillins and cephalosporins.

Penicillin G. Protoplast fusion was used to modify the characteristics of an improved penicillin-producing strain of Penicillium chrysogenum which showed poor sporulation and poor seed growth. Backcrossing with a low-producing strain yielded a higher-producing strain with better sporulation and better growth in seed medium.105

Metabolic engineering of the penicillin-producing P. chrysogenum strains showed that increases could result by introducing extra copies of biosynthetic genes and by increasing copy number and high transcription levels of the whole cluster. Penicillin production was increased by overexpressing the gene encoding phenylacetic acid-activating CoA ligase from Pseudomonas putida. Overexpression of gene acvA in A. nidulans, by replacing the normal promoter with the ethanol dehydrogenase promoter,106 increased penicillin production up to 30-fold.

Cephalosporin C. Protoplast fusion was carried out with strains of Acremonium chrysogenum (formerly Cephalosporium acremonium) obtained from a commercial strain improvement program. A low-titer, rapidly-growing, spore-forming strain which required methionine to optimally produce cephalosporin C was crossed with a high-titer, slow-growing, asporogenous strain which could use the less expensive inorganic sulfate. The progeny included a recombinant which grew rapidly, sporulated, produced cephalosporin C from sulfate and made 40% more antibiotic than the parent.107

Cloning multiple copies of cyclase into A. chrysogenum yielded an improved cephalosporin C-producing strain.108 When an industrial production strain of A. chrysogenum 394-4 was transformed with a plasmid containing the pcbC and the cefEF gene from an early strain of the mutant line, a transformant producing 50% more cephalosporin C than the production strain, as well as less penicillin N, was obtained. Production in pilot plant (150 liter) fermentors was further improved by 15%.109 One copy of the cefEF had been integrated into chromosome III whereas the native gene is on chromosome II. Cephalosporin C production by A. chrysogenum was further improved by introducing extra copies of the the acetyl-transferase gene (cefG) along with additional copies of the expandase/hydroxylase gene (cefEF).

Cephamycin C. Another application of protoplast fusion is the recombination of improved producers from a single mutagenesis treatment. By recombination, one can combine the yield-increase mutations and obtain an even more superior producer before carrying out further mutagenesis. Two improved cephamycin C-producing strains from Nocardia lactamdurans were fused and among the recombinants were two cultures which produced 10–15% more antibiotic than the best parent.110 Overexpression of ccaR, a positive regulatory gene in Streptomyces clavuligerus111 led to a two- to three-fold increase in antibiotic production-Cephamycin C production by Nocardia lactamdurans has also been improved by overxpression of lat, encoding lysine-aminotransferase.112

Semisynthetic cephalosporins. Semisynthetic cephalosporins are made from 7-aminocephalosporanic acid (7-ACA) or 7-aminodeacetoxycepalosporanic acid (7-ADCA). Chemical methods had traditionally been used to produce 7-ACA and 7-ADCA using expensive and toxic reagents and yielding sideproducts harmful to the environment. These processes are being replaced by safer microbiological processes. Transformation of P. chrysogenum with the Streptomyces lipmanii cefD and S. clavuligerus cefE genes allowed production of the intermediate deacetoxycephalosporin C (DAOC)113 at titers of 2.5 g l⁻¹, along with penicillin V. DAOC is another valuable intermediate in the commercial production of semi-synthetic cephalosporins. Also, cloning of cefE from S. clavuligerus or cefEF and cefG from A. chrysogenum into P. chrysogenum grown with adipic acid as side-chain precursor114 resulted in formation of adipyl-6-aminopenicillanic acid (adipyl-6-APA) and adipyl-7-ADCA in the case of cefE, and adipyl-6-APA, adipyl-7ADCA, adipyl-7-DAC, and 7-ACA in the case of cefEF and cefG. Feeding adipic acid as side-chain precursor and then enzymatically removing the adipoyl side chain leads to production of 7-ADCA.

Disruption and one-step replacement of the cefEF gene of an industrial cephalosporin C production strain of A. chrysogenum yielded strains accumulating up to 20 g l⁻¹ of penicillin N. Cloning and expression of the cefE gene from S. clavuligerus into those high producing strains yielded recombinant strains producing high titers of DAOC.115 Production levels were nearly equivalent (80%) to the total β-lactams biosynthesized by the parental strain. Luo et al.116 constructed a strain of E. coli containing the D-amino acid oxidase gene from Trigonopsis variabilis and the glutaryl-7-aminocephalosporanic acid acylase gene from Pseudomonas sp. The recombinant E. coli was able to convert cephalosporin C directly to 7-ACA.

Clavulanic acid. Although clavulanic acid is a β-lactam compound, it has only low antibacterial activity and instead, it is used widely as an inhibitor of β-lactamase, an enzyme strongly incvolved in antibiotic resistance. Conventional strain improvement increased clavulanate production 10-fold over the wild-type S. clavuligerus NRRL 3585. Then, protoplast fusion of arginine and cysteine auxotrophs yielded a fusant (CKD 1386) producing 30-fold more clavulanic acid than the wild-type.117

Clavulanic acid biosynthesis begins with a condensation of L-arginine and D-glyceraldehyde-3-phosphate (G3P). The supply of G3P is limited, so inactivation of two G-3-P dehydrogenases, encoded by gap1 and gap2 by targeted gene disruption, doubled clavulanic acid production.118 Also, increased dosage of biosynthetic genes ceas and cs2119 or overexpression of positive regulatory genes increased production two- to three-fold.120,121

Non-β-lactam antibiotics. Non-β-lactam antibiotics have also been genetically engineered. Daptomycin. Some Streptomyces roseosporus Tn 5099 transposition mutants produce 57–66% more daptomycin than the parent.122

Transposition increased the rate-limiting step of tylosin biosynthesis in Streptomyces fradiae, i.e., the conversion of macrocin to tylosin. Transposing a second copy of tylF into a neutral site on the S. fradiae chromosome increased its gene product, macrocin O-methyltransferase, and tylosin production up to 60%, while decreasing the concentration of the final intermediate (macrocin).123

Need for new antibiotics. Despite the success of the antibiotic field, new antibiotics are sorely needed. About 30 years ago, the difficulty and high cost of isolating novel antibiotic structures and agents with new modes of action for such uses became apparent and the field entered a phase of decline. However, efforts must continue to discover and develop new antibiotics, in order to protect the health of the world's population. This is coming about by the combination of complementary technologies such as natural product discovery via high throughput screening with new genetic technologies. The most promising genetic techniques are shown in Table 3.

Genetic recombination allows the discovery of new antibiotics by fusing producers of different or even the same antibiotics. Protoplast fusion between non-producing mutants of a streptomycin producer (Streptomyces griseus) and an istamycin producer (Streptomyces tenjimariensis) led to a hybrid strain producing a new antibiotic.124 Another new antibiotic, indolizomycin, was produced by protoplast fusion between non-antibiotic producing mutants of S. griseus and S. tenjimariensis.125

A recombinant obtained from two different rifamycin-producing strains of Nocardia mediterranei produced two new rifamycins (16,17-dihydrorifamycin S and 16,17-dihydro-17-hydroxy-rifamycin S).126 Interspecific protoplast fusion between S. griseus and five other species (Streptomyces cyaneus, Streptomyce exfoliatus, Streptomyces griseoruber, Streptomyces purpureus and Streptomyces rochei) yielded recombinants of which 60% produced no antibiotics and 24% produced antibiotics different from the parent strains.127

The production of antibiotics in heterologous hosts via combinatorial biosynthesis is becoming very popular in antibiotic production and discovery.128 New derivatives of antibiotics have been obtained after the biosynthetic paths were elucidated and the biosynthetic genes isolated.129 Techniques used are (1) targeted gene disruption in which single genes are inactivated; (2) tailoring by introducing a single gene or a few genes from another pathway; and (3) by combination of (1) and (2) above. Over 200 new polyketides have been made by combinatorial biosynthesis.130,131

Mutation in the genes encoding synthases of erythromycin biosynthesis in Saccharopolyspora erythrea or introducing heterologous DNA in a blocked mutant has led to production of new erythromycins. These products are the 2-norerythromycins,132 6-deoxyerythromycins,133 Δ6,7-anhydroerythromycin C134 and 5-6-dideoxy-3α-mycarosyl-5-oxyerythronolide B.135 Two amino acid substitutions were made in the NAD(P)H binding motif of the enoyl reductase domain encoded by eryAII. By substituting the acyltransferase (AT) and β-carbon processing domains of 6-deoxyerythronolide B synthase [DEBS; the polyketide synthase (PKS) of the erythromycin macrolide ring] with counterparts from the rapamycin PKS, a library of over 60 new macrolide aglycones was produced.136

Novel erythromycins with fatty acids attached to C-13 were produced using a recombinant S. erythraea culture with the loading domain of the erythromycin PKS replaced by the one from the avermectin producer, Streptomyces avermitilis.137 One such compound containing a cycopentyl group at C13 instead of an ethyl group was much more active against Pasteurella multocida and E. coli, although less active against S. aureus than erythromycins A and B.

A three plasmid system for heterologous expression of 6-deoxyerythronolide B synthase was used for combinatorial biosynthesis.138 A library of modified polyketide lactones was produced by using 14 modified versions of each plasmid and inserting them in combinations into Streptomyces lividans. The modified versions were produced by replacing distinct gene segments in the erythromycin PKS with those from the rapamycin producer. Triple transformants able to produce 43 different polyketides were obtained.

Combinatorial biosynthesis has also been used to construct macrolides with new sugar moieties.139,140 Methymycin and pikromycin, produced by a gene cluster of Streptomyces venezuelae and normally containing the sugar desosamine, were modified by cloning of a gene from the calicheamicin producer, Micromonospora echinospora subsp. calichensis. The gene encodes TDP-glycero-hexulose aminotransferase. Transfer of a 12.6 kb DNA fragment from the tetracenomycin C-producing S. glaucescens to S. lividans resulted in tetracenomycin C production by the latter. The fragment contains 12 genes of biosynthesis and resistance. Novel hybrid tetracenomycins were produced by introducing a 25 kb cosmid from the elloramycin biosynthetic pathway of S. olivaceus into the polyketide synthase (PKS)-deleted mutant of the urdamycin producer, S. fradiae, and into the mithramycin producer, Streptomyces argillaceus. The cosmid contains a glycosyltransferase gene whose enzyme has broad substrate specificity and thus produces hybrid products containing different D-and -L sugars.

3-0-Acetyl-4″-0-isovaleryltylosin (AIV) is useful in veterinary medicine against tylosin-resistant Staphylococcus aureus. It is made by first producing tylosin with S. fradiae and then using Streptomyces thermotolerans (producer of carbomycin) to bioconvert tylosin into AIV. A novel direct fermentation organism was constructed by transforming S. fradiae with S. thermotolerans plasmids containing acyl transferase genes.141

Introduction of a gene encoding a modifying enzyme has been used to generate analogs of the macrolide spiramycin. Thus, spiramycin-4″-O-isovaleryl ester was produced by cloning the carE gene from S. thermotolerans (carbomycin producer) into Streptomyces ambofaciens (spiramycin producer).142 These are both 16-membered macrolides but one of the two sugars in carbomycin is isovalerylmycarose and the corresponding sugar in spiramycin is mycarose. Furthermore, spiramycin was converted into its 3-O-acetyl- and 3-O-propionyl esters by S. lividans transformed with the mdmB gene from Streptomyces mycorofaciens, the producer of midecamycin.143

Cloning of the actI, actIV and actVII genes from S. coelicolor into the 2-hydroxyaklavinone producer, Streptomyces galilaeus 31671, yielded novel hybrid metabolites, desoxyerythrolaccin and 1-O-methyl-desoxyerythrolaccin.144 Cloning of these genes into other streptomycetes resulted in the production of the novel metabolite aloesaponarin II.145

New peptide antibiotics (modified surfactins) were produced by combinatorial biosynthesis.146 Genes encoding activating domains for phenylalanine, ornthine and leucine from the gramicidin S producer Bacillus brevis and for cysteine and valine from the ACV and penicillin producer P. chrysogenum were transferred into a mutant of the surfactin producer B. subtilis which had a disrupted leu7 gene. The five new surfactins were slightly less hemolytic than surfactin.

The use of microbial genomics for improving the discovery of new antibiotics has been reviewed by Van Lanen and Shen.147 The efforts include mining of whole genome sequences, genome scanning, heterologous expression and discovery of novel chemistry. 294 microbes had been sequenced and annotated at that time. The number of genes expected to be part of secondary metabolic sequences is much greater than the number of known secondary metabolites.148

Genomics will provide a huge group of new targets against which natural products can be screened.149 The human genome has 30,000–35,000 genes, less than 50% having a putative function.150 These genes have the potential to produce over 100,000 proteins. Estimates of the number of proteins acting as useful targets range from 600–10,000.

Genome mining utilizes DNA sequencing and bioinformatics to rapidly identify clusters of genes in antibiotic-producing cultures encoding biosynthesis of new bioactive products and to predict structure based on gene sequences.151 More than 450 natural product clusters were identified in this 2003 study.

Bacterial pathogens contain about 2,700 genes of which current antibiotics target less than 25.152 A genomic comparison of the pathogenic Haemophilus influenzae with a nonpathogenic E. coli revealed 40 potential drug targets in the former.153 Similarly, a comparison of genomes of Helicobacter pylori with E. coli and H. influenzae revealed 594 H. pylori-specific genes of which 196 were known, 123 of the known genes being involved in known host-pathogen interactions and 73 targets of novel potential.154

Antitumor agents.

In their review on the use of microbes to prescreen potential antitumor compounds, Newman and Shapiro155 concluded that microorganisms have played a cucial role in identifying compounds with therapeutic benefit against cancer. Most of the important compounds used for chemotherapy of tumors are microbially-produced antibiotics. Approved antitumor agents from microorganisms are actinomycin D (dactinomycin), anthracyclines [including daunorubicin, doxorubicin (adriamycin), epirubicin, pirirubicin, idarubicin, valrubicin, amrubicin], glycopeptolides (bleomycin, phleomycin), the mitosane mitomycin C, anthracenones (mithramycin, streptozotocin, pentostatin) and the enediyne calcheamycin attached to a monoclonal antibody (Mylotarg®).

Anthracyclines. A new anthracycline, 11-hydroxyaclacino mycin A, was produced by cloning the doxorubicin resistance gene and the aklavinone 11-hydroxylase gene dnrF from the doxorubicin producer, Streptomyces peucetius subsp. caesius, into the aclacinomycin A producer. The hybrid molecule showed greater activity against leukemia and melanoma than aclacinomycin A. Additional new anthracyclines have been made by (1) introducing DNA from Streptomyces purpurascens into Streptomyces galilaeus, both of which normally produce known anthracyclines, (2) cloning DNA from the nogalomycin producer, Streptomyces nogalater, into S. lividans and into an aclacinomycin-negative mutant of S. galilaeus, (3) cloning of the actI, actIV and actVII genes from S. coelicolor into the 2-hydroxyaklavinone producer, S. galilaeus 31671 yielding the novel hybrid metabolites, desoxyerythrolaccin and 1-O-methyl-desoxyerythrolaccin, and (4) blocking deoxysugar biosynthesis in a S. peucetius strain and cloning of the avrE gene of the avermectin-producing S. avermitilis or the eryBIV genes from the erythromycin producer, Streptomyces erythraea.

Epothilones. Epothilones are highly promising prospective anticancer agents that are produced by the myxobacterium, Sorangium cellulosum. Using the classical UV-mutation method plus selection pressure, the production capacity was increased 2.5 times that of the starting strain.156 The mutants with higher production and different phenotypes were further subjected to recursive protoplast fusion and the fusants products were screened under multi-selection pressure. Production of epothilone B by one fusant was increased about 130 times compared to the starting strain.

Cholesterol-lowering agents.

Integrating transcriptional and metabolite profiles from 21 strains of Aspergillus terreus producing different levels of lovastatin and another 19 strains with altered (+)-geodin levels led to an improvement in lovastatin production of over 50%.157 This approach, named association analysis, served to reduce the complexity of profiling data sets to identify those genes whose expression was most tightly linked to metabolite production. Improvement of lovastatin titers was achieved by increasing dosage of lovastatin biosynthetic genes and of regulatory genes for secondary metabolism, i.e., lovF, creA, fadA, ganA, gnaI, gna3 and gpa1. Of these, gene lovF was found to be the important one. They fused its promoter to the ble gene encoding resistance to phleomycin. The lovF::ble transcriptional fusion protein was used as a reporter-based system to select improved mutants.

Antihelminthics.

A S. avermitilis culture, isolated by Omura and coworkers at the Kitasato Institute in Japan, was found by Merck scientists to produce a family of secondary metabolites having both antihelminthic and insecticidal activities. These killed the intestinal nematode, Nematosporoides dubius, in mice and were named avermectins. They are disaccharide derivatives of macrocyclic lactones with exceptional activity against parasites, i.e., at least ten times higher than any synthetic antihelminthic agent known. Despite their macrolide structure, avermectins lack antibiotic activity, do not inhibit protein synthesis nor are they ionophores; instead they interfere with neurotransmission in many invertebrates. They have activity against both nematode and arthropod parasites in sheep, cattle, dogs, horses and swine.

S. avermitilis has gene afsR2 which was originally discovered as a global regulatory gene in S. lividans with positive control action on actinorhodin and undecylprodigiosin formation. Incorporation of multiple copies of afsR2 from S. lividans into wildtype S. avermitilis increased avermectin production by 2.3-fold.158 Another regulatory gene appears to be an 8 kb DNA fragment of S. avermitilis which stimulates actinorhodin and undecylprodigiosin formation in S. lividans. It also stimulated avermectin production in wild-type S. avermitilis by 2.5-fold, in an improved strain by 1-fold, and by 0.4-fold in a semi-industrial strain.159 Transposon mutagenesis eliminated production of the troublesome toxic oligomycin by the avermectin-producing S. avermitilis.160

A semi-synthetic derivative, 22,23-dihydroavermectin B1 (Ivermectin) is 1,000 times more active than thiobenzole and is a commercial veterinary product. Ivermectin is made by hydrogenation at C22–C23 of avermectin B1a and B1b with rhodium chloride acting as catalyst. By genetic engineering of S. avermitilis in which certain PKS genes were replaced by genes from the PKS of S. venezuelae (the pikromycin producer), ivermectin could be made directly by fermentation, thus avoiding semisynthesis.161

Biopharmaceuticals

Recombinant proteins.

By means of genetic engineering, desired proteins have been produced in great quantities to meet the copious demands of industry.162 Hence, most biopharmaceuticals produced today are recombinant. Protein quality, functionality, production speed and yield are the most important factors to consider when choosing the right expression system for recombinant protein production. Top yields for different systems are shown in Table 4.

Between 100 and 200 therapeutic proteins have been approved in Europe and the USA. Non-glycosylated proteins are usually made in E. coli or yeasts and they constitute 55% of the therapeutic protein market (39% by E. coli, 1% by other bacteria and 15% by the yeasts).163 N-glycosylated proteins are usually made in mammalian cells which mimic human glycosylation. Chinese hamster ovary (CHO) cells provide about 35% of the therapeutic protein market but the process is very expensive and the glycoproteins made are not exactly the human type, and in some cases, they must be modified. Ten percent of the market is supplied by other mammalian systems such as NSO (mouse myeloma) cells. Insect cells are also used. Although yeasts, molds and insect cells are generally unable to provide mammalian glycosylation, the popular methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce a human type of glycosylation.164

Bacteria. Bacterial systems are used to make somatostatin, insulin, bovine growth hormone for veterinary applications, α-1 antitrypsin, interleukin-2, tumor necrosis factor, β-interferon, and γ-interferon. Avecia Biologics has achieved a titer of 14 g l⁻¹ of recombinant protein using E. coli.165 Bacilli have yields as high as 3 g l⁻¹. An improved Gram-negative host for recombinant protein production has been developed using Ralstonia eutropha.166 The system appears superior to E. coli with respect to inclusion body formation. Organophosphohydrolase, a protein prone to inclusion body formation with a production of less than 100 mg l⁻¹ in E. coli, was produced at 10 g l⁻¹ in R. eutropha. Staphylococcus carnosus can produce 2 g l⁻¹ of secreted mammalian protein.

Yeasts. High recombinant protein yields can be obtained in the methylotrophic yeast, P. pastoris, e.g., as high as 15 g l⁻¹.167 Claims have been made that P. pastoris can make 20–30 g l⁻¹ of recombinant proteins.168 Some other proteins were made at or over 10 g l⁻¹ by P. pastoris, e.g., tumor necrosis factor, gelatin, intracellular tetanus toxin fragment C and serum albumin. Heterologous gene expression in another methylotroph Hansenula polymorpha yielded 13.5 g l⁻¹ of phytase. Another utilized yeast is S. cerevisiae. Glucose oxidase from Aspergillus niger is produced by S. cerevisiae at 9 g l⁻¹. Recombinant products on the market which are made in S. cerevisiae are insulin, hepatitis B surface antigen, urate oxidase, glucagons, granulocyte macrophage colony stimulating factor (GM-CSF), hirudin, and platelet-derived growth factor.

Mammalian cells. CHO cells constitute the preferred system for producing monoclonal antibodies and some other recombinant proteins. Other cell types include (1) various mouse myelomas such as NSO murine myeloma cells,169 (2) baby hamster kidney (BHK) cells for production of cattle foot-and-mouth disease vaccine, (3) green monkey kidney cells for polio vaccine170 and (iv) human cell lines such as human embryonic kidney (HEK) cells. NSO is a non-secreting subclone of the NS-1 mouse melanoma cell line.

Monoclonal antibody production in NSO animal cells reached over 2.5 g l⁻¹ in fed-batch processes.171 Animal-free, protein-free and even chemically-defined media with good support of production have been developed.172 Protein production by CHO cells went from 5–50 mg l⁻¹ in 1985 to 50–500 mg l⁻¹ in 1995, to 5 g l⁻¹ in 2005.173 A number of mammalian processes are producing 3–5 g l⁻¹ of recombinant protein174 and, in some cases, protein titers have reached 10 g l⁻¹ in industry175 including antibodies.176 A rather new system is that of a human cell line known as PER. C6 of Crucell Holland BV, which, in cooperation with DSM Biologics, was reported to produce 15 g l⁻¹,177 and then later, 27 g l⁻¹ of a monoclonal antibody.178 Protein production of over 20 g l⁻¹ has been achieved in serum-free medium but production of 2–3 g l⁻¹ in such media is more usual.

Insect cells. Insect cells are able to carry out more complex post-translational modifications than can be accomplished with fungi. They also have the best machinery for the folding of mammalian proteins and are therefore quite suitable for making soluble protein of mammalian origin.179 The most commonly used vector system for recombinant protein expression in insects is the baculovirus, especially the nuclear polyhedrosis virus (Autographa californica) which contains circular double-stranded DNA, is naturally pathogenic for lepidopteran cells, and can be grown easily in vitro. The usual host is the fall armyworm (Spodoptera frugiperda) in suspension culture. A larval culture can be used which is much cheaper than mammalian cell culture. Recombinant insect cell cultures have yielded over 200 proteins encoded by genes from viruses, bacteria, fungi, plants and animals.180 Current thought is that the baculovirus insect cell system can produce 11 g l⁻¹ of recombinant protein.

Enzymes

Genes encoding many microbial enzymes have been cloned and the enzymes expressed at levels hundreds of times higher than those naturally produced. Over 60% of the enzymes used in the detergent, food and starch processing industry are recombinant proteins.181 Recombinant DNA technology has been used beneficially in the enzyme industry in the following ways:182,183

1. to produce in industrial organisms enzymes obtained from microbes which are difficult to grow or modify genetically;

2. to increase enzyme productivity by use of multiple gene copies, strong promoters, and efficient signal sequences;

3. to produce in a safe host useful enzymes obtained from a pathogenic or toxin-producing microorganism; and

4. to improve the stability, activity or specificity of an enzyme by protein engineering.

The industrial enzyme business adopted rDNA methods eagerly to increase production levels and to produce enzymes from industrially-unknown microorganisms in industrial organisms such as Aspergillus spp., Trichoderma spp., K. lactis, S. cerevisiae, Yarrowia lipolytica and Bacillus licheniformis.

Filamentous fungi can produce recombinant proteins at levels as high as 4.6 g l⁻¹. They include Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Chrysosporium lucknowense and A. chrysogenum. Recombinant molds are one of the main sources of enzymes for industrial applications.

There are three fungal recombinant lipases currently used in the food industry. They are from Rhizomucor miehi, Thermomyces lanuginosus and Fusarium oxysporum, and are produced in A. oryzae. Lipases are employed in leather processing where they are combined with proteases, peroxidases and oxidases. In order to be effective in detergents, lipases need to remain functional under harsh conditions such as the presence of surfactants and oxidants, temperatures above 45°C, pH values of about 10, and should be alkalophilic. Washing powders have been improved in activity and low temperature operation by application of recombinant DNA technology and site-directed mutagenesis to proteases and lipases.183,184 In 1994, Novo Nordisk introduced Lipolase, the first commercial recombinant lipase for use in a detergent by cloning the Humicola lanuginose lipase gene into the A. oryzae genome. Such lipases are used for laundry cleaning, interesterification of lipids, and esterification of glucosides producing glycolipids which have applications as biodegradable non-ionic surfactants for detergents, skin care products, contact lenses and as food emulsifiers.

The α-amylase gene from B. amyloliquefaciens was cloned using multicopy plasmid pUB110 in B. subtilis.185 Production was 2,500-fold greater then that in wild-type B. subtilis and five-fold that of the B. amyloliquefaciens donor. An exoglucanase from the cellulolytic Cellulomonas fimi was overproduced after cloning in E. coli to a level of over 20% of cell protein.186 The endo-β-glucanase components of the cellulase complexes from Thermomonospora and Clostridium thermocellum were cloned in E. coli as was the cellobiohydrolase I gene of Trichoderma reesei.187 P. pastoris, the methanol-utilizing yeast, was engineered to produce S. cerevisiae invertase and to excrete it into the medium at 100 mg l⁻¹.188 Self-cloning of the xylanase gene in S. lividans resulted in six-fold overproduction of the enzyme.189

Many enzymes are made by filamentous organisms which are slow-growing and difficult to handle in fermentors. Transfer of these genes to rapidly-growing unicellular bacteria provide an economical move achieving rapid growth and more reproducible production. Other advantages are more rapid nutrient uptake due to a greater surface/volume ratio, better oxygen transfer, better mixing and thus more reliable control of pO2, pCO2 and pH, and a better organism for mutagenesis.

Aspartase production was increased 30-fold by cloning in E. coli.190 Captopril esterase of P. putida, used in preparing the chiral captopril sidechain, was cloned in E. coli with a 38-fold increase in activity.191 A 1,000-fold increase in phytase production was achieved in A. niger by use of recombinant technology.192 Cloning of the benzylpenicillin acylase gene of E. coli on multicopy50 plasmids resulted in a 45-fold increase as compared to uninduced wild-type production. Interestingly, the cloned enzyme is constitutive.193

The properties of many enzymes have been altered by genetic means. “Brute force” mutagenesis and random screening of microorganisms over the years led to changes in pH optimum, thermostability, feedback inhibition, carbon source inhibition, substrate specificity, Vmax, K_m and K_i. This information was later exploited by the more rational techniques of protein engineering. Single changes in amino acid sequences have yielded similar types of changes in a large variety of enzymes. Today, it is no longer necessary to settle for an enzyme's natural properties; these can be altered to suit the needs of the investigator or the process. For example, a protease from Bacillus stearothermophilus was increased in heat tolerance from 86°C to 100°C, being made resistant to boiling. The enzyme was developed by site directed mutagenesis.194 Only eight amino acids had to be modified. Temperature stability at 100°C was increased 340-fold without a decrease in activity at lower temperatures. All eight mutations were far from the enzyme's active site.

Molecular breeding techniques, e.g., DNA shuffling, are being currently used to generate enzymes with improved properties such as: activity and stability at different pH values and temperatures,195 increased or modified enantioselectivity,196 altered substrate specificity,197 stability in organic solvents,198 novel substrate specificity and activity,199 increased biological activity of protein pharmaceuticals and biological molecules200 as well as novel vaccines.201,202 Proteins from directed evolution work were already on the market by 2000.203 These were green fluorescent protein of Clontech204 and Novo Nordisk's LipoPrime® lipase.

Other Compounds

Rapamycin.

Rapamycin, a 31-member ring macrolide immuno suppressant produced by Streptomyces hygroscopicus, has many applications in clinical medicine. One round of genome shuffling using seven mutants of S. hygroscopicus with different features and rapamycin productivities, generated an improved rapamycin-producing strain.205 An interspecies fusion of protoplasts of S. hygroscopicus D7-804 and S. erythreus ZJU325 also yielded an improved strain.

Artemisinin.

A leading antimalarial today is artemisinin obtained from extracts of the Wormwood tree, Artemisia annua.206 Artemisinin is a sesquiterpene lactone, active against both drug-resistant and cerebral malaria-causing strains. It still is isolated from this plant but it is a very expensive process and there is an extreme shortage. Martin et al.207 used metabolic engineering of E. coli to produce 100 mg l⁻¹ in 12 hours. The genes came from a truncated mevalonic acid pathway in S. cerevisiae. A genetically-engineered strain of S. cerevisiae can produce its precursor artemisinic acid.208 Using this yeast in a fed-batch process in chemically-defined medium with galactose as carbon source, addition of methionine and controlled aeration and agitation speed, 2.5 g l⁻¹ of artemisinic acid was produced.209

Spinosad.

Spinosad (spinosyns A and D) is a mixture of secondary metabolites produced by Saccharopolyspora spinosa which is used in agriculture as a potent insect control agent with exceptional safety with respect to non-target organisms. After four rounds of genome shuffling with 10 mutant strains, generated by nitrosoguanidine and ultraviolet irradiation, a markedly improved strain was isolated.210

Xanthan gum.

Microbially-produced xanthan gum is not only an acceptable food-thickener but is one of the most promising agents for enhanced oil recovery in the petroleum industry. Recombinant DNA manipulation of Xanthomonas campestris increased titers of xanthan by two-fold and increased pyruvate content by over 45%.211,212 Formation amounted to 0.6 g g⁻¹ of sucrose utilized.213 Ten-twenty thousand tons of xanthan are produced annually for use in the oil, pharmaceutical, cosmetic, paper, paint and textile industries.214

Closing Remarks

Discovery of new bioactive compounds, as well as the development of new and more efficient bioprocesses, is coming about by the use of recombinant strains. The combination of complementary technologies such as mutation and genetic recombination have led to remarkable improvements in the productivity of many primary and secondary metabolites as well as protein biopharmaceuticals and enzymes. New genetic approaches for the development of overproducing strains are continuously emerging. Among those that have proven to be very successful are metabolic engineering, combinatorial biosynthesis and molecular breeding techniques.

More exciting advances are still to come as the complete sequencing of industrially important microbial genomes takes place. Functional genomics, proteomics and metabolomics are now being exploited for the discovery of novel valuable small molecules for medicine and enzymes for catalysis.

Figures and Tables

Table 1 Novel genetic technologies successfully applied to improvement of primary metabolite production

Genetic technologies	Metabolites
Genome-based strain reconstruction	Amino acids, vitamins, organic acids, alcohols, carotenoids.
Metabolic engineering including reverse (inverse) metabolic engineering	Amino acids, vitamins, organic acids, ethanol, 1,3-propanediol, carotenoids, 5′-inosinic acid.
Genome-wide transcript expression analysis	Riboflavin
Molecular breeding (whole genome shuffling)	Lactic acid, ethanol

Table 2 Top titers of amino acid fermentations

Amino Acid	Titer (g l^−1)
L-alanine	114
L-arginine	96
L-glutamic acid	130
L-glutamine	49
L-histidine	42
L-hydroxyproline	41
L-isoleucine	40
L-leucine	34
L-lysine HCl	170
L-phenylalanine	51
L-proline	100
L-serine	65
L-threonine	100
L-tryptophan	60
L-tyrosine	55
L-valine	105

Table 3 Genetic techniques used to increase secondary metabolite production

Genetic techniques	Metabolites
Protoplast fusion	Penicillin G, cephalosporin C, cephamycin C, clavulanic acid, indolizomycin, rifamycins
Metabolic engineering	Antibiotics (penicillin G, cephalosporin C, cephamycin C, clavulanic acid, semisynthetic cephalosporins), antitumor agents (anthracyclines, glycopeptolides, anthracenones), avermectins, xanthan gum, artemisinin
Transposition	Daptomycin, tylosin
Association analysis	Lovastatin
Combinatorial biosynthesis	Erythromycins, tetracenomycins, tylosin, spiramycins, surfactins
Whole genome shuffling	Epothilones, spinosad
Genome mining	Echinosporamicin-type antibiotics, antifungal compounds (ECO-02301) and others

Table 4 Top levels of recombinant proteins made by different biological systems*

System	Protein (g/L)
Bacteria	14
Yeasts	30
Mammalian cells	27
Insect cells	11