In: Barredo J (Ed) Microbial Carotenoids from Fungi: Methods and Protocols.

Humana Press/Springer,
New York, 2018.
En Prensa
Chapter 1
Advancement of Biotechnology by Genetic Modifications

Arnold L. Demaina* and Sergio Sanchezb

Abstract
One of the greatest sources of metabolic and enzymatic diversity are microorrganisms. In recent
years, emerging recombinant DNA and genomic techniques have facilitated the development of
new efficient expression systems, modification of biosynthetic pathways leading to new
metabolites by metabolic engineering, and enhancement of catalytic properties of enzymes by
directed evolution. Complete sequencing of industrially important microbial genomes is taking
place very rapidly and there are already hundreds of genomes sequenced. Functional genomics
and proteomics are major tools used in the search for new molecules and development of
higher-producing strains.

Key words: Agriculture, Bioconversions, Biopharmaceuticals, Enzymes, Genetic engineering,

Hosts, Metabolic engineering, Polymers, Primary metabolites, Organic acids, Alcohols,
Secondary metabolites, Bioinsecticides, Recombinant proteins

1. Introduction
Advantages of microorganisms in the production of compounds, as compared to isolation from
plants and animals or synthesis by chemists, include (a) rapid uptake of nutrients that supports
high rates of metabolism and biosynthesis, (b) capability of carrying out a wide variety of
reactions, (c) facility to adapt to a large array of different environments, (d) ease of genetic
manipulation, both in vivo and in vitro, to increase production, to modify structures and activities,
and to make entirely new products, (e) simplicity of screening procedures, and (f) a wide
diversity.
Products from microbes are very diverse, ranging from very large molecules, such as
proteins, nucleic acids, carbohydrate polymers, or even cells, to small molecules that are
usually divided into primary metabolites, i.e., those essential for vegetative growth, and
secondary metabolites, i.e., those nonessential for growth.

1.1. Primary Metabolites

Synthesis of microbial products during the exponential phase of growth is an integral part of the
normal growth process. They are intermediates or end products of the pathways, are building
blocks for essential macromolecules (e.g., amino acids and nucleotides), or are converted into
coenzymes (e.g., vitamins). Other primary metabolites (e.g., organic acids 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, alcohols, and organic acids.
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*Corresponding author: Arnold L. Demain, RISE Institute, Drew University, 36 Madison Avenue,
Madison, New Jersey, 07940, USA; ademain@drew.edu
Sergio Sánchez Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de
México, Ciudad de Mexico, 04510, CDMX, Mexico.

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 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. Recent approaches utilize the techniques of modern genetic engineering to correct
such deficiencies and develop strains overproducing primary metabolites. There are two ways
to accomplish after this (a) to increase the number of copies of structural genes coding for these
enzymes and (b) to increase the frequency of transcription.
Novel genetic technologies are important for the development of overproducers. Ongoing
genome-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 below, 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 (1).
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 (2, 3). Analytical methods are combined to
quantify fluxes and to control them with molecular biological techniques to implement suggested
genetic modifications. 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 (4). 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 choosing a
strain which has a favorable cellular phenotype, evaluating and determining the genetic and/or
environmental factors that confer that phenotype, and finally transferring that phenotype to a
second strain via direct modifications of the identified genetic and/or environmental factors (5,
6).
Molecular breeding techniques are based on mimicking natural recombination by in vitro
homologous recombination (7). DNA shuffling not only recombines DNA fragments but also
introduces point mutations at a very low controlled rate (8). 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 (9). Whole genome
shuffling is another technique that combines the advantage of multiparental crossing allowed by
DNA shuffling with the recombination of entire genomes through recursive protoplast fusion (10,
11).
Systems biology is an integrated, systemic approach to the analysis and optimization of
cellular processes by introducing a variety of perturbations and measuring the system response
(12). Altered phenotypes are created by molecular biological techniques or by altering
environments. Further characterization of the phenotype leading to maximal product formation is
analyzed and quantified through the use of genome-wide high-throughput omics data and
genome-scale computational analysis.

1.2. Amino Acids

Many of the techniques mentioned above have made a great impact on the production of amino
acids. Different strategies including (a) amplification of a rate-limiting enzyme of pathway, (b)
amplification of the first enzyme after a branchpoint, (c) cloning of a gene encoding an enzyme
with more or less feedback regulation, (d) introduction of a gene encoding an enzyme with a
functional or energetic advantage as a replacement for the normal enzyme, (e) amplification of
the first enzyme leading from central metabolism to increase carbon flow into the pathway, and
(f) isolation of a transport mutant decreasing amino acid uptake and intracellular feedback
control while improving excretion.

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 Among the amino acids, L-glutamate and L-lysine, mostly used as feed and food additives,
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 (13). High titers of amino acids are shown in Table 1.

Table 1
High amino acids levels produced by fermentation
Amino acid Titer (g L-1)
L-Alanine 120
L-Arginine 96
L-Glutamate 150
L-Glutamine 49
L-Histidine 42
L-Hydroxyproline 41
L-Isoleucine 40
L-Leucine 34
L-Lysine HCl 170
L-Methionine 25
L-Phenylalanine 51
L-Proline 108
L-Serine 65
L-Threonine 124
L-Tryptophan 60
L-Tyrosine 55
L-Valine 51

1.2.1. L –Glutamate
Glutamate was the first amino acid to be produced by fermentation because of its use as a
flavoring agent (monosodium glutamate, MSG). The process employs various species of the
genera Corynebacterium and Brevibacterium. Molar yields of glutamate from sugar are 50–60%
and broth concentrations have reached 150 g L-1. Glutamic acid overproduction is feedback
regulated. However, mutant strains with modifications in the cell membrane are able to pump
glutamate out of the cell, thus allowing its biosynthesis to proceed unabated. Introduction of the
Vitreoscilla hemoglobin gene vgb into Corynebacterium glutamicum increased cell growth and
glutamic acid and glutamine production via increased oxygen uptake (14). Workers at Ajinomoto
Co., Inc. increased glutamate production from glucose by 9% by suppressing CO2 liberation in
the pyruvate dehydrogenase reaction (15). They did this by cloning the xfp gene-encoding
phosphoketolase from Bifidobacterium animalis into C. glutamicum and overexpressing it.

1.2.2. L-Lysine
Since the bulk of the cereals consumed in the world are deficient in L-lysine, this essential
amino acid became an important industrial product. Although the lysine biosynthetic pathway is
controlled very tightly in an organism such as Escherichia coli and in lysine-producing
organisms (e.g., mutants of C. glutamicum and its relatives), there is only a single aspartate
kinase (AK), which is regulated via concerted feedback inhibition by threonine plus lysine.
Metabolic engineering has been used in C. glutamicum to improve L–lysine production (4). A
chimeric AK, composed of the N-terminal catalytic region from Bacillus subtilis AKII and the C-
terminal region from Thermus thermophilus, was evolved through random mutagenesis and
then screened using a high-throughput synthetic RNA device which comprises an L-lysine-
sensing riboswitch and a selection module. Of three evolved aspartate kinases, the best mutant
(BT3) showed 160 % increased in vitro activity compared to the wild-type enzyme (16).

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 Comparative genome analysis between a wild-type strain and an L-lysine-producing C.
glutamicum strain identified three mutations that increased L-lysine production when introduced
into the wild-type strain. Introduction of the 6-phosphogluconate-dehydrogenase gnd mutation
increased yield by 15% (17). Further improvement was achieved by introducing the mqo
mutation (malate: quinone oxidoreductase) resulting in an increased titer of 95 g L-1 in a fed-
batch culture (18). Transcriptome analysis revealed that strain B-6, as compared to the wild
type, is upregulated in both the pentose phosphate pathway genes and the amino acid
biosynthetic genes and downregulated in TCA cycle genes. Lysine HCl titers have reached 170
g L-1 (19). Metabolic flux studies of wild-type C. glutamicum and improved lysine-producing
mutants showed that yield increased from 1.2% to 24.9% relative to the glucose flux. Different
approaches have been used to increase lysine production by C. glutamicum mutants including
(1) deletion of different genes from the glycolytic pathway (20), (2) improving the availability of
pyruvate by eliminating pyruvate dehydrogenase activity (21), (3) overexpression of pyruvate
carboxylase or DAP dehydrogenase genes, and (4) overexpression of gene NCg10855
encoding a methyltransferase or the amtA-ocd-soxA operon (22). A genetically defined C.
glutamicum L-lysine overproducer strain was developed by system metabolic engineering of the
wild type. Implementation of only 12 defined genome-based changes in genes encoding central
metabolic enzymes redirected major carbon fluxes as desired towards the optimal pathway
usage predicted by in silico modeling. The final engineered C. glutamicum strain was able to
produce lysine with a high yield of 0.55 g per gram of glucose, a titer of 120 g L-1 of lysine and a
productivity of 4.0 g L-1 h-1 in fed-batch culture (23).

1.2.3. L –Threonine
Overproduction of L-threonine has been achieved in Serratia marcescens by transductional
crosses, which combined several feedback control mutations in a single organism. This resulted
in titers up to 25 g L-1 (24). Combination in a single strain of another six regulatory mutations
derived by resistance to amino acid analogs led to desensitization and derepression of the key
enzymes of threonine synthesis. The resultant transductant produced 40 g L-1 of threonine (25).
The use of recombinant DNA technology led to strains that made 63 g L-1 threonine (26). An E.
coli strain was developed via mutation and genetic engineering and optimized by the
inactivation of threonine dehydratase (TD) resulting in a process yielding 100 g L-1 of L-
threonine in 36 h of fermentation (27).
In C. glutamicum ssp. lactofermentum, L-threonine production reached 58 g L-1 when a
strain producing both threonine and lysine was transformed with a plasmid carrying its own
hom, thrB, and thrC genes (28). A recombinant isoleucine auxotrophic strain of E. coli (carrying
extra copies of the thrABC operon, an inactivated tdh gene and mutated to resist high
concentrations of L–threonine and L-homoserine) produced 80 g L-1 L-threonine in 1.5 days
with a yield of 50% (29). Cloning extra copies of threonine export genes into E. coli also
increased threonine production (30).
Systems metabolic engineering was used to develop an L- threonine1-overproducing E. coli
strain (31). Feedback inhibition of aspartokinase I and III (encoded by thrA and lysC,
respectively) and transcriptional attenuation regulation (thrL) were removed. Deletion of tdh and
mutation of ilvA avoid threonine degradation. The metA and lysA genes were deleted to make
more precursors available for threonine biosynthesis. Further target genes to be engineered
were identified by transcriptome profiling combined with in silico flux response analysis. The
final engineered E. coli strain was able to reach a high yield of 0.393 g of threonine per gram of
glucose and 82.4 g L-1 in a fed-batch culture. With the use of an L-isoleucine leaky (ILEL) and α-
amino-β-hydroxyvaleric acid resistant (AHVr) E. coli TRFC strain, carrying a pTHR101 plasmid
containing a Thr operon, a threonine production of 118 g L-1 was achieved after 38 h with a
productivity of 3.1 g L-1 h-1 (46% conversion ratio from glucose to threonine) (32Chen et al.,
2009). A further increase in amino acid production to 124.57 g L-1 was obtained in TRFC strain

4
by decreasing acetic acid production using combined feeding strategies (33). Acetic acid is an
inhibitor of growth and threonine production.

1.2.4. L-Valine
Combined rational modification, transcriptome profiling, and systems-level in silico analysis was
used to develop an E. coli strain for the production of this amino acid (34). The ilvA, leuA, and
panB genes were deleted to make more precursors available for L–valine biosynthesis. This
engineered strain, harboring a plasmid overexpressing the ilvBN genes, produced 1.3 g L-1 L-
valine. Overexpression of the lrp and ygaZH genes (encoding a global regulator Lrp and L-
valine exporter, respectively), and amplification of the lrp, ygaZH, and lrp-ygaZH genes
enhanced production of L-valine by 21.6, 47.1, and 113%, respectively. Further improvement
was achieved by using in silico gene knockout simulation, which identified the aceF, mdh, and
pfkA genes as knockout targets. The VAMF strain (Val aceF mdh pfkA) was able to
produce 7.5 g L-1 L–valine from 20 g L-1 glucose in batch culture, resulting in a high yield of
0.378 g of L-valine per gram of glucose. Production by mutant strain VAL1 of C. glutamicum
amounted to 31 g L-1 (35). 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 limiting
concentrations of isoleucine and pantothenate. By applying metabolic engineering, a C.
glutamicum strain (WCC003 harboring pJYW-4-ilvBNC1-lrp1-brnFE) deleted in the genes aceE,
alaT and ilvA and overexpressing the ilvB, ilvN, ilvC, lrp1, brnF and brnE genes, produced 51 g
L-1 L-valine in fed-batch fermentation, with almost no detectable amino-acid by-products such as
L-alanine and L-isoleucine (36).

1.2.5. L–Isoleucine
Isoleucine processes have been devised in various bacteria such as S. marcescens, C.
glutamicum ssp. flavum, and C. glutamicum. In S. marcescens, resistance to isoleucine
hydroxamate and α-aminobutyric acid led to derepressed L-threonine deaminase (TD) and
acetohydroxyacid synthase AHAS) and production of 12 g L-1 of isoleucine (37). Further work
involving transductional crosses into a threonine-overproducer yielded isoleucine at 25 g L-1
(24).
Feedback regulation in C. glutamicum was eliminated (38) by replacing the native threonine
dehydratase gene ilvA with the feedback resistant gene from E. coli. By introducing additional
copies of genes encoding branched amino acid biosynthetic enzymes, lysine- or threonine-
producing strains were converted into L-isoleucine producers with improved titers (39– 41).
Amplification of the wild-type threonine dehydratase gene ilvA in a threonine-producing strain of
Corynebacterium lactofermentum led to isoleucine production (42).
A threonine-overproducing strain of C. glutamicum was sequentially mutated to resistance
to thiaisoleucine, azaleucine, and aminobutyric acid; it produced 10 g L-1 of isoleucine (43).
Metabolic engineering studies involving overexpression of biosynthetic genes were useful in
improving isoleucine production by this species. Colon et al. (42) obtained an isoleucine-
producing strain by cloning multiple copies of hom (encoding HDI), and wild-type ilvA (encoding
TD) into a lysine-overproducer, and by increasing HK (encoded by thrB); a titer of 15 g L-1
isoleucine was obtained. Independently, Morbach et al. (44) cloned three copies of the
feedback-resistant HD gene (hom) and multicopies of the deregulated TD gene (ilvA) in a
deregulated lysine producer of C. glutamicum, yielding an isoleucine producer (13 g L-1) with no
threonine production and reduced lysine production. Application of a closed loop control fed-
batch strategy raised production to 18 g L-1 (45). Further metabolic engineering work involving
amplification of feedback inhibition-insensitive biosynthetic enzymes converted lysine

5
overproducers and threonine overproducers into C. glutamicum strains yielding 30 g L-1 of
isoleucine (46).
C. glutamicum ssp. flavum studies employed resistance to α-amino- β-hydroxyvaleric acid
and the resultant mutant produced 11 g L-1 (47). D-Ethionine resistance was used by Ikeda et
al. (48) to yield a mutant producing 33 g L-1 in a fermentation continuously fed with acetic acid.
Accumulated L-isoleucine in the cell is excreted via a two-component export system BrnFE,
which is regulated by the global regulator Lrp. Overexpression of Lrp and BrnFE in C.
glutamicim led to 27 g L-1 production in fed-batch fermentation (49). A C. glutamiiun strain
overexpressing the threonine dehydratase (ilvA1), and acetohydroxy acid synthase (ilvBN1)
feed-back resistant genes, as well as the ppnk1 gene, encoding NAD kinase (Strain
IWJ001/pDXW-8-ilvBN1-ilvA1-ppnk1), led to a 32 g L-1 isoleucine production in a 72 h fed-batch
fermentation (50). Recently in a Brevibacterium flavum strain KM011 (Met-+Ethr+α-
ABr+LysL+AECr), betaine, vitamin B12 and Vitamin B5 concentrations were optimized for
isoleucine production in shake flasks. Under these conditions, a máximum of 13.35 g L-1
isoleucine were produced. However, a production of 35.26 g L-1 was obtained with optimized
conditions using a 5-L fermenter (51).

1.2.6. L-Alanine
Lee et al. (52) 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 Bacillus sphaericus alanine dehydrogenase gene (alaD). The strain produced L-
alanine in 27 h with a yield on glucose of 0.63 g g-1 and a maximum productivity of 2 g L-1 h-1.
Further work has raised the titer to 114 g L-1. A genetically engineered E. coli W (strain, XZ132),
with a D-lactate dehydrogenase replaced by an alanine dehydrogenase from Geobacillus
stearothermophilus and deletions in the methylglyoxal synthase (mgsA) and the catabolic
alanine racemase (dadX) genes, produced 1,279 mmol alanine (53). Deletion of both enzymes
reduced low levels of lactate and conversion of L- to D-alanine. A thermo-regulated genetic
switch designed to dynamically control the expression of L-alanine dehydrogenase (alaD) from
G. stearothermophilus on the E. coli B0016-060BC chromosome, led to an L-alanine titer of
120.8 g L-1 with higher overall and oxygen-limited volumetric productivities of 3.09 and 4.18 g L-1
h-1, respectively, using glucose as the sole carbon source (54).

1.2.7. L –Proline
Proline-hyperproducing strains of bacteria, exhibiting reduced proline-mediated feedback
inhibition of -glutamyl kinase (GK) activity (a result of single-base pair substitutions in the
bacterial proB gene coding region), have been isolated based on their resistance to toxic proline
analogs (L-azetidine-2-carboxylic acid and 3,4-dehydro-DL-proline), compounds which inhibit
GK activity while not interfering with protein synthesis. Cloning of the three genes of proline
biosynthesis in E. coli on multicopy plasmids and selection of mutants of such plasmid-
containing strains to resistance to 3,4-dehydroproline led to a process producing 20 g L-1 proline
(55).
A mutant of S. marcescens resistant to 3,4-dehydroproline, thiazolidine-4-carboxylate, and
azetidine-2-carboxylate and unable to utilize proline produced 50–55 g L-1 L-proline (56).
Cloning of a gene bearing the dehydroproline-resistance locus on a plasmid yielded a
recombinant strain of S. marcescens producing 75 g L-1 (57). Further development work
increased the production to over 100 g L-1 (58).
A sulfaguanidine-resistant mutant of C. glutamicum ssp. flavum produced 35 g L-1proline
(59). When a glutamate-producing strain of C. glutamicum was grown under modified

6
conditions, it made 48 g L-1 (60). A strain of Corynebacterium acetoacidophilum produced 108 g
L-1 proline when grown in the presence of glutamate (61).

1.2.8. 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) (62). When proline was added, the hydroxyproline titer reached 41 g
L-1, with a yield of 87% based on the amount of proline added. Optimization of the gene codons
and vectors of proline-4-4-hydroxylase (P4H) in a recombinant E. coli BL21 strain and chemo-
physical combination mutagenesis, allowed isolation of strain NA45, able to grow in glycerol as
a sole carbon source. With further systemic optimization of nutritional elements, the strain
produced 25.4 g L−1 trans-4-hydroxy-L-proline at 48 h in fed batch mode in a 5 L fermentor (63).

1.3. Nucleotides
Nucleotide fermentations became commercially important due to the activity of two purine
ribonucleoside 5’-monophosphates, namely guanylic acid (GMP) and inosinic acid (IMP), as
enhancers of flavor. Titers of IMP and GMP have reached 30 g L-1 (64). The techniques used to
achieve such production are similar to those used for amino acid fermentations. In Japan, 2,500
tons of GMP and IMP are produced annually with a market of $350 million. GMP can also be
made by bioconversion of xanthylic acid (XMP). Genetic modification of Corynebacterium
ammoniagenes involving transketolase, an enzyme of the nonoxidative branch of the pentose
phosphate pathway, resulted in the accumulation of 39 g L-1 of XMP (65).

1.4. Vitamins
Riboflavin. Production of riboflavin (vitamin B2) reached over 6,000 tons per year and a titer of
20 g L-1 by overproducers such as the yeast-like molds, Eremothecium ashbyii and Ashbya
gossypii. A bacterial process using C. ammoniagenes (previously Brevibacterium
ammoniagenes) was developed by cloning and overexpressing the organisms own riboflavin
biosynthetic genes (66) and its own promoter sequences. The resulting culture produced 15 g L-
1
riboflavin in 3 days.
Genetic engineering of a Bacillus subtilis strain, already containing purine analog-resistance
mutations, led to improved production of riboflavin (67). The industrial strain of B. subtilis was
produced by (1) making purine analog-resistance mutations to increase guanosine triphosphate
(GTP; a precursor) production and (2) using a riboflavin analog (roseflavin)-resistance mutation
in ribC that deregulated the entire pathway (68). Resultant production was over 25 g L-1. A
genome-wide transcript expression analysis (69) was successfully used to discover new targets
for further improvement of the fungus A. gossypii (70). The authors identified 53 genes of known
function, some of which could clearly be related to riboflavin production. A metabolic
engineering approach overexpressing the ribA (encoding 3,4-dihydroxy-2-butanone 4-
phosphate synthase) gene in B. subtilis RB50::[pRF69]n[pRF93] strain, led to productions of 15
g L-1 riboflavin (71).
Biotin has traditionally been made 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 (72). Sequential mutation of S. marcescens to resistance to the biotin antimetabolite
acedomycin (= actithiazic acid) led to mutant strain SB412, which produced 20 mg L-1 of biotin
(73). 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-1in a fed-batch fermention along with 600 mg L-1 of biotin vitamers. Later advances led to
production by recombinant S. marcescens of 600 mg L-1 of biotin (74). A process using an E.

7
coli mutant resistant to -hydroxynorvaline (a threonine antimetabolite) yielding 970 mg L-1 has
been patented (75). Biotin production was further increased to over 1 g L-1 by the use of a B.
subtilis strain resistant to 5-(2-thenyl) pentanoic acid (a biotin analog) and overexpressing
several bio genes.
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 (L-AA). Annual production is 110,000
tons generating revenue of over 600 million dollars. 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 (76). A
better process was devised independently which converted 40 g L-1 of glucose into 20 g L-1 of 2-
KGA (77). 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 (78). Further mutation to suppress the L-idonate pathway and
to improve the promoter led to the production of 130 g L-1 of 2-KGA from 150 g L-1 of sorbitol.
Further improvement of the strain was possible by suppressing the L-idonate pathway (79). An
Erwinia herbicola transformed with the 2,5-diketo-d-gluconate reductase gene from
Corynebacterium sp., produced up to 120 g L-1 2-KGA in less than 120 h with the help of
glucose dehydrogenase, gluconate dehydrogenase, ketogluconate dehydrogenase, and 2,5-
diketo-d-gluconate reductase (80). Similar to the chemical conversion of 2-KGA to vitamin C by
lactonization, X. campestris probably lactonizes 2-KGA under oxidative stress to form L-AA.
Using this method, 20.4 g L-1 L-AA were found in the extracellular broth which corresponds to
the 14-fold amount of that detected in yeast cells directly synthesizing L-AA from D-glucose
(81).
Vitamin B12 production depends on avoidance of feedback repression by vitamin B12. The
vitamin is industrially produced by Propionibacterium shermanii or Pseudomonas denitrificans.
The early stage of the P. shermanii fermentation is conducted under anaerobic conditions in the
absence of the precursor 5,6-dimethylbenzimidazole. These conditions prevent vitamin B12
synthesis and allow for the accumulation of the intermediate, cobinamide. Then, the culture is
aerated and dimethylbenzimidazole is added, converting cobinamide to the vitamin. In the P.
denitrificans fermentation, the entire process is carried out under low levels of oxygen. A high
level of oxygen results in an oxidizing intracellular environment, which represses formation of
the early enzymes of the pathway. Production of vitamin B12 has reached levels of 150 mg L-1,
10 tons per year, and a world market of $71 million.
Other vitamins. Recombinant E. coli, transformed with genes encoding pantothenic acid
(vitamin B5) biosynthesis, and resistant to salicylic and/or other acids, produce 65 g L-1 of D–
pantothenic acid from glucose using alanine as precursor (82). A total of 7,000 tons per year are
made chemically and microbiologically. Thiamine (vitamin B1) is produced synthetically at 4,000
tons per year. Pyridoxine (vitamin B6) is made chemically at 2,500 tons per year.

1.5. Carotenoids
Carotenoid production processes have been extensively studied (83), but they have had
difficulty in economically challenging chemical methods. Of over 600 microbial carotenoids, only
-carotene and astaxanthin are produced industrially by fermentation (84). A semi-industrial -
carotene process was developed using mated cultures of Blakeslea trispora plus and minus
strains. -Carotene was produced at 1 g L-1 in the early 1960s (85). By the addition of
carotogenic chemicals and antioxidants, the titer was raised to over 3 g L-1 (86). Processes in
development include those yielding -carotene, lycopene, zeaxanthin, and astaxanthin. Some
have been improved by metabolic engineering and directed evolution (87–89). Metabolic

8
engineering of E. coli has led to strains forming 0.2 g L-1 of lycopene (90). Lutein, a xanthophyll
carotenoid with antioxidant properties, had sales as a food colorant of $150 million in the USA
(91). It is thought that this carotenoid prevents age-related macular degeneration and cataracts.
It is made from petals of marigold, but microalgae are a potential new source.
Chemical production of trans-astaxanthin has a selling price of $2,000 kg (92) and a market
of over $100 million per year. It is mainly used for pigmentation of salmonids raised in
aquaculture, a multibillion dollar industry (93). Astaxanthin can be made by the yeast Phaffia
rhodozyma (Xanthophyllomyces dendrorhous) and the microalga Haematococcus pluvialis.
Genetically improved strains of P. rhodozyma produce 10 mg g-1 cells in industrial fermentors.
Recent improvements in astaxanthin production have been published by de la Fuente et al. (94)
and Rodríguez-Sáiz et al. (95) to get maximal astaxathin titers of 420 mg L-1 when X.
dendrorhous is fermented under continuous white light. Recently astaxanthine production has
been reported with an engineered C. glutamicum strain. Volumetric productivities of up to about
0.4 mg L-1 h-1 reported in simple shaking flask cultures by the recombinant strain compare
favorably with those reported for the commercially used production hosts such as the green
microalga H. pluvialis, the red yeast X. dendrorhous and the recombinant E. coli. (96).

1.6. Organic Acids

Microbial production of organic acids is an excellent approach for obtaining building-block
chemicals from renewable carbon sources (97). 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 (98).

1.6.1. Citric Acid

Citric acid has a market of $2 billion. About 1 million tons per year are produced annually by
Aspergillus niger and yeasts. The commercial process employs A. niger in media deficient in
iron and manganese. Other factors contributing to high citric acid production are a high
intracellular concentration of fructose 2,6-biphosphate, inhibition of isocitrate dehydrogenase,
and low pH (1.7–2.0). In approximately 4–7 days, the major portion (80%) of the sugar (glucose
or sucrose) provided is converted into citric acid. A. niger titers have reached over 200 g L-1
(99).
Alternative processes have been developed with Candida yeasts, especially from
hydrocarbons. Such yeasts are able to convert n-paraffins to citric and isocitric acids in
extremely high yields (150–170% on a weight basis). Titers as high as 225 g L-1 have been
reached (100). Anastasiadis and Rehm (101) reported production levels of 250 g L-1 with
Candida oleophila ATCC 20177 under submerged continuous fermentation, with glucose as
carbon source.

1.6.2. Acetic Acid

Titers of acetic acid reached 53 g L-1 with genetically engineered E. coli (102) and 83 g L-1 with a
Clostridium thermoaceticum mutant (103). 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% (1.8–4 g L-1 h-1) and the titer by 40% (68–97 g L-
1
) (104).

1.6.3. Lactic Acid

Whole genome shuffling was used to improve the acid tolerance of a commercial lactic acid-
producing Lactobacillus sp. (105). Further approaches using this recursive protoplast fusion
technique yielded strains of Lactobacillus rhamnosus ATCC 11443 with improved glucose

9
tolerance (160–200 g L-1 glucose), 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 Bacillus amyloliquefaciens ATCC 23842 produced a fusant that could utilize liquefied
cassava bagasse starch directly to yield a titer of 40 g L-1 of lactic acid with a 96% conversion of
starch to lactic acid (106).
Although lactobacilli make more lactic acid than Rhizopus oryzae, they produce mixed
isomers. The fungus, however, produces L-(+) lactic acid exclusively. The yield is about 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 and
production was increased to 78 g L-1, whereas the undesirable coproduct ethanol was reduced
from 10.6 to 8.7 g L-1 (107). A recombinant Saccharomyces cerevisiae strain containing six
copies of bovine L-lactate dehydrogenase produced 122 g L-1 from sugar cane with an optical
purity of 99.9% or higher (108). Expression of the same bovine enzyme and a deletion of the
pyruvate decarboxylase gene in Kluyveromyces lactis produced 109 g L-1 (109).
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 (110). 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 (111).

1.6.4. Succinic Acid

Sanchez et al. (112) 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. Metabolic engineering of Mannheimia succiniciproducens led to
a strain producing 52 g L-1of succinic acid at a yield of 1.16 mol mol-1 glucose and a productivity
of 1.8 g L-1 h-1 in fed-batch culture (113). A metabolically engineered succinate-producing strain
of E. coli yielded 58 g L-1succinate in a 59 h fed-batch fermentation under aerobic conditions
(114). The average succinate yield was 0.94 mol mol-1 of glucose, the average productivity was
1.08 g L-1 h-1, and the average specific activity was 90 mg g-1 h-1. A titer of 99 g L-1with a
productivity of 1.3 g L-1 h-1 has been reached with recombinant E. coli (115).

1.6.5. Other Organic 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 (116). S. cerevisiae normally produces 2 g L-1
of malic acid from fumaric acid. However, a recombinant strain containing a cloned fumarase
gene was able to produce 125 g L-1 with a yield of almost 90% (117). Microbial fermentation
titers of some other organic acids are 135 g L-1 pyruvic acid, 107 g L-1 fumaric acid, 90 g L-1
shikimic acid, 69 g L-1 dehydroshikimic acid, 85 g L-1 itaconic acid, 504 g L-1 gluconic acid, 106 g
L-1 propionic acid, 68 g L-1 oxalic acid, and 136 g L-1 glyceric acid. 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 (118). Product concentrations
reached 200 g L-1 and problematic side reactions such as unsaturation, hydroxylation and chain-
shortening did not occur.

1.7. Alcohols
Ethanol. Fermentation of sugars by S. cerevisiae in the case of hexoses, and Kluyveromyces
fragilis or Candida species with lactose or a pentose, results in the production of ethanol. Under
optimum conditions, approximately 120 g L-1 ethanol can be obtained. Such a high
concentration slows down growth and the fermentation ceases.

10
 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 (119). After
three rounds of genome shuffling, a strain was obtained which was able to grow on plates 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.
In silico metabolic models have been used to overcome the redox imbalance in S.
cerevisiae engineered with the Xyl1 and Xyl2 genes from Pichia stipitis (120, 121).
Overexpression of both genes led to an accumulation of NADH and a shortage of NADPH.
Deletion of NADP+-dependent glutamate dehydrogenase (GGH1) and overexpression of NAD+-
dependent GDH2 led to an increase in ethanol production using xylose as fermentation
substrate. An in silico genome-scale gene insertion strategy was used to improve ethanol
production and decrease the production of byproducts glycerol and xylitol (122). Introduction of
glyceraldehyde-3-phosphate dehydrogenase in S. cerevisiae led to a 58% reduction in glycerol,
a 33% reduction in xylitol, and a 24% increase in ethanol production.
When biomass is used as a carbon source for ethanol production, its breakdown results in
liberation of acetic acid. The acid interferes with ethanol production. Tolerance of Candida
krusei GL560 to acetic acid was improved by genome shuffling (123). 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.
E. coli was converted into an ethanol producer (43 g L-1) by cloning the alcohol
dehydrogenase II and pyruvate decarboxylase genes from Zymomonas mobilis (124). 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 (125).
Maximum theoretical yield was 81–86% and a titer of 47 g L-1 of ethanol was produced from 100
g L-1 of cellulose.
Recombinant strains of E. coli, Zymomonas, and Saccharomyces can convert corn fiber
hydrolysate to 21–35 g L-1 ethanol with yields of 0.41–0.50 ethanol per gram of sugar consumed
(126). For a recombinant E. coli strain making 35 g L-1, time was 55 h and yield was 0.46 g
ethanol per gram of available sugar, which is 90% of the attainable maximum.
1,3-Propanediol. A strain of Clostridium butyricum converts glycerol to 1,3-propanediol
(PDO) at a yield of 0.55 g g-1glycerol consumed (127). A major metabolic engineering feat was
carried out in E. coli leading to a culture growing on glucose and producing PDO at 135 g L-1,
with a yield of 51% and a rate of 3.5 g L-1 h-1 (128). To do this, eight new genes were introduced
to convert dihydroxyacetone phosphate (DHAP) into PDO. Production was further improved by
modifying 18 E. coli genes, including regulatory genes. PDO is the monomer used to chemically
synthesize industrial polymers such as polyurethanes and the polyester fiber Sorono™ by
DuPont. This bioplastic is polytrimethylene terephthalate (3GT polyester) made by reacting
terephthalic acid with PDO (129). PDO is also used as a polyglycol-like lubricant and as a
solvent.
D-Mannitol is a naturally occurring polyol, widely used in the food, chemical, and
pharmaceutical industries. A whole cell bioconversion of D-fructose to D-mannitol was
developed by metabolic engineering of E. coli (130). The mdh gene encoding mannitol
dehydrogenase from Leuconostoc pseudomesenteroides and the fdh gene encoding formate
dehydrogenase from Mycobacterium vaccae were coexpressed 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-1 h-1 and a molar yield of 84–92% with no by-
products. 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 (131). Supplementation of the medium used for mannitol production by Candida
magnolia with Ca2+ and Cu2+ increased production up to 223 g L-1 (132).

11
 Sorbitol, also called D-glucitol, is 60% as sweet as sucrose and is used 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 or
syrup with a 50:50 mixture of glucose and fructose. It is also produced by extraction from
seaweed as a by-product of alginate and iodine manufacture. However, excellent microbial
processes have been developed (133). Toluenized (permeabilized) cells of Z. mobilis produce
290 g L-1 of sorbitol and 283 g L-1 of gluconic acid from a glucose and fructose mixture in 16 h
with yields near 95% for both products (134). Other leading organisms are recombinant C.
glutamicum at 285 g L-1, Lactobacillus intermedius at 227 g L-1, C. magnoliae at 223 g L-1, and
many others producing between 100 and 200 g L-1. 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 (135).
n-Butanol is a good alternative fuel additive as it has two more carbons than ethanol, which
results in an energy content about 40% higher. Also, automobile engines do not require
modification until the percentage of butanol reaches over 40% of the total automobile fuel. In
contrast, modification is required when ethanol is added to gasoline at levels exceeding 15%.
Butanol can be obtained from the acetone–butanol–ethanol fermentation of Clostridium
beijerinkii or Clostridium acetobutylicum. Butanol-resistant mutants showed increased
production of butanol and acetone (136). Biochemical engineering modifications were able to
increase total acetone, butanol, and ethanol production (ABE) to 69 g L-1 (137). A mutant in the
presence of added acetate was able to produce almost 21 g L-1 butanol and 10 g L-1 of acetone
from glucose (138).
Because butanol’s octane number is lower than that of ethanol and the octane number
increases with methyl branching and double bonds, other higher alcohols are also being
considered as biofuels, e.g., branched C 4 and C 5 alcohols. They are also desirable because
of their higher energy density, lower vapor pressure, and lower hygroscopicity as compared to
ethanol (139). They include isopropanol, 1-propanol, 1-butanol (n-butanol), isobutanol (2-
methyl-propanol), 3-methyl-1-butanol, 2-methyl-1-butanol, isopentanol (3-methyl-1 butanol), and
isopentenol (3-methyl-3-buten-1-ol). A novel screening method based on overcoming the
toxicity associated with the accumulation of prenyl diphosphate was used to screen a library of
19,000 clones harboring fragments of Bacillus genomic DNA (140). Two genes, yhfR and nudF,
coding for proteins capable of overcoming the toxicity associated with accumulating IPP and
DMAPP were isolated. Both protein products have an affinity for IPP and DMAPP, converting
them into isopentenol. Clostridium beijerinckii (Clostridium butylicum) produces 20 g L-1 of 1-
butanol and 2 g L of isopropanol as part of a mixed product. Recombinant E. coli can produce
4.9 g L-1 of isopropanol. Recently, a new strategy for the production of these alcohols has been
reported (141). This approach is based on the diversion of 2-keto acid intermediates from the
endogenous amino acid pathway to alcohol biosynthesis especially that of isobutanol. As a
result, engineered E. coli can produce 22 g L-1 of isobutanol in 110 h with a yield of 86% of the
theoretical maximum.
Other alcohols. The noncariogenic, noncaloric, and diabetic-safe sweetener erythritol has
70–80% of the sweetness of sucrose. It can be produced by Aureobasidium sp. (165 g L-1), an
acetate-negative mutant of Yarrowia lipolytica (170 g L-1), a C. magnoliae osmophilic mutant
(187 g L-1), the osmophile Trichosporon sp. (88 g L-1), Torula sp. (200 g L-1), and the yeast
Pseudozyma tsukubaensis (245 g L-1) (142).
Xylitol is a naturally occurring sweetener with anti-cariogenic properties, which is used for
some diabetes patients. It can be produced by chemical reduction of D-xylose or by
fermentation. Xylitol production at 150 g L-1 was obtained with Candida guilliermondii 2,581 at
pH 6.0 and shaking at 60 rpm (143).

12
2. Secondary Metabolites
As a group that includes antibiotics, pesticides, pigments, toxins, pheromones, enzyme
inhibitors, immunomodulating agents, receptor antagonists and agonists, pesticides, antitumor
agents, immunosuppressants, cholesterol-lowering agents, plant protectants, and animal and
plant growth factors, these metabolites have tremendous economic importance. This
remarkable group of compounds is produced by certain restricted taxonomic groups of
organisms and is usually formed as mixtures of closely related members of a chemical family.

2.1. Antibiotics
Antibiotics are the most well-known secondary metabolites and have a tremendous importance.
The most well known are the β-lactams, tetracyclines, aminoglycosides, chloramphenicol,
macrolides, and other polyketides, polyenes, glycopeptides, among others. They have been
crucial in the increase in average life expectancy in the USA from 47 years in 1900 to 74 for
males and 80 for women in 2000 (144). More than 350 agents have reached the market as
antimicrobials. The global market for finished antibiotics has reached $35 billion.

2.1.1. β–Lactams
The β-lactams are the most important class of antibiotics in terms of use. Included are the
penicillins, cephalosporins, cephamycins, clavulanic acid, and the carbapenems. Many of the
current penicillins and cephalosporins are semisynthetic. All of the above are of great
importance in chemotherapy of bacterial infections.
β-Lactamases of pathogenic bacteria are the major cause of resistance development and
there are over 450 such enzymes. However, β-lactams are still very useful due to the discovery
of β-lactamase inhibitors. Although clavulanic acid is a β-lactam compound, it has only low
antibacterial activity, but is used widely as an inhibitor of β-lactamase, in combination with β-
lactam antibiotics. Conventional strain improvement by protoplast fusion of auxotrophic strains
yielded a fusant producing 30-fold more clavulanic acid than the wild type (145). Inactivation of
two G-3-P dehydrogenases, encoded by gap1 and gap2 by targeted gene disruption, doubled
clavulanic acid production (146). Also, increased dosage of biosynthetic genes ceas and cs2
(147) or overexpression of positive regulatory genes increased production two- to threefold
(148, 149).
Yield improvements have been achieved through different strategies. Thus, protoplast
fusion between strains of Penicillium chrysogenum yielded a higher-producing strain of penicillin
G (150). Metabolic engineering was used to replace the normal promoter with the ethanol
dehydrogenase promoter (151), increasing penicillin production up to 30-fold.
Protoplast fusion was also been carried out with strains of Acremonium chrysogenum to
obtain a strain that produced 40% more cephalosporin C than the parent (152). Production was
also improved by cloning multiple copies of cyclase (153) or the pcbC and the cefEF genes
(154).
Two improved cephamycin C-producing strains from Nocardia lactamdurans were fused to
obtain cultures, which produced 10–15% more antibiotic (155). Overexpression of lat, encoding
lysine-aminotransferase also led to an overproducing strain (156). High-level expression of
ccaR, a positive regulatory gene in Streptomyces clavuligerus (157) led to a two- to threefold
increase in antibiotic production.
Chemical methods had traditionally been used to produce 7-aminocephalosporanic acid (7-
ACA) and 7-aminodeacetoxycepalosporanic acid (7-ADCA), chemical precursors of
semisynthetic cephalosporins. These processes are being replaced by safer microbiological
processes. Transformation of P. chrysogenum with bacterial cefD and cefE genes allowed the
production of deacetoxycephalosporin C (DAOC) (158), another key intermediate in the
commercial production of semisynthetic cephalosporins. Also, cloning of cefE from S.
clavuligerus or cefEF and cefG from Acremonium chrysogenum into P. chrysogenum fed with

13
adipic acid as side-chain precursor (159) resulted in the formation of several adipyl-6/7-
intermediates. Enzymatic removal of the adipoyl side chain led to the production of 7-ADCA.
Disruption and one-step replacement of the cefEF gene of an industrial 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 (160). An E. coli strain containing the D-amino acid
oxidase gene from Trigonopsis variabilis and the glutaryl-7-aminocephalosporanic acid acylase
gene from Pseudomonas sp. was able to convert cephalosporin C directly to 7-ACA (161).
Natural carbapenems, such as thienamycin, are made by Streptomyces cattleya , Erwinia
carotovora subsp. carotovora , Serratia sp., and Photorhabdus luminescens (162).
Carbapenems are resistant to attack by most -lactamases. The commercial carbapenems are
made synthetically and include imipenem, meropenem, and ertapenem. Thienamycin is one of
the most potent and broadest in spectrum of all antibiotics known today. Although a -lactam, it
is not a member of the penicillins or cephalosporins. It is active against aerobic and anaerobic
bacteria, both Gram positive and Gram negative, including Pseudomonas. This novel structure
was isolated in Spain from a new soil species, which was named S. cattleya (163). Interestingly,
this culture also produces penicillin N and cephamycin C. Also used to combat β -lactamase
containing pathogens are new carbapenems, such as the recently approved doripenem (S-
4661) which has broad spectrum activity against resistant bacteria including Pseudomonas
aeruginosa.

2.1.2. Other Antibiotics

The biosynthesis of polyketide macrolides has been subjected to genetic engineering (164).
This group of compounds includes antibiotics such as erythromycin, oleandomycin, pikromycin,
tylosin, and amphotericin B. Reverse metabolic engineering increased erythromycin production
by Aeromicrobium erythreum (165). The technique is also known as inverse metabolic
engineering and as combinatorial engineering. Tylosin production was increased up to 60% in
Streptomyces fradiae by transposing a second copy of tylF, encoding macrocin O -
methyltransferase, into a neutral site on its chromosome (166).
Genetic engineering of the nystatin biosynthetic pathway yielded polyenes with high
antifungal activity, which are less toxic than amphotericin B (167). The production of antibiotics
in heterologous hosts via combinatorial biosynthesis is becoming very popular in antibiotic
production and discovery (168). New derivatives of antibiotics have been obtained after the
biosynthetic paths were elucidated and the biosynthetic genes isolated (169). Over 200 new
polyketides have been made by combinatorial biosynthesis (170, 171). The discovery of new
antibiotics has also been achieved by genetic recombination between producers of different or
even the same antibiotics (172– 174).
Combinatorial biosynthesis been used to construct macrolides with new sugar moieties
(175, 176). 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 spp. calichensis. The gene
encodes TDP-glycero-hexulose aminotransferase. Transfer of a 12.6 kb DNA fragment from the
tetracenomycin C-producing Streptomyces glaucescens to Streptomyces lividans resulted in
tetracenomycin C production by the latter (177). 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 Streptomyces olivaceus into the
polyketide synthase (PKS)-deleted mutant of the urdamycin producer, S. fradiae and into the
mithramycin producer, Streptomyces argillaceus (178). The cosmid contains a
glycosyltransferase gene whose enzyme has broad substrate specificity and thus produces
hybrid products containing different D- and L-sugars. For more than 35 years, vancomycin and
teicoplanin were the only antibiotics active against multidrug-resistant Gram-positive bacteria.

14
Their use became severely limited by an increase in multidrug resistance. One group of narrow-
spectrum compounds are the streptogramins which are synergistic pairs of antibiotics made by
a single microbial strain. The pairs are constituted by a (Group A) polyunsaturated macrolactone
containing an unusual oxazole ring and a dienylamide fragment and a (Group B) cyclic
hexadepsipeptide possessing a 3-hydroxypicolinoyl exocyclic fragment. Such streptogramins
include virginiamycin and pristinamycin (179). Pristinamycin, made by Streptomyces
pristinaespiralis, is a mixture of a cyclodepsipeptide (pristinamycin I) and a polyunsaturated
macrolactone (pristinamycin II). Increasing resistance to pristinamycin in the pristinamycin
producer S. pristinaespiralis was combined with genome shuffling to increase pristinamycin
production ninefold (180).
An important new strategy to improve the discovery of new antibiotics is genome mining, which
has come about due to advances in microbial genomics (181). Mining of whole genome
sequences and genome scanning allows the rapid identification of more than 450 clusters of
genes in antibiotic-producing cultures encoding biosynthesis of new bioactive products and the
prediction of structure based on gene sequences (182, 183). These efforts include mining of
whole genome sequences, genome scanning, heterologous expression, and discovery of novel
chemistry. Genomics will also provide a huge group of new targets against which natural
products can be screened (184). Up to February 2017, more than 16,000 complete microbial
genome sequences were available in one or two scaffolds. Of them, 411 belong to fungi, and
699 to actinobacteria. Currently, 69 streptomycete genomes have been sequenced (185). The
smaller genome corresponds to Stretomyces xiamenensis with 5.95 Mb size and the biggest is
Streptomyces rapamyonicus with 12.7 Mb.
Consensus sequences can be obtained through alignments from the enzyme domains
and used to construct Hidden Markov Models (HMM) of the chemical structure to predict
SMILES. For this purpose, many online and stand-alone sources are freely available. Some of
them include antiSMASH 2.0 (186), which detects 24 types of secondary metabolites.
CLUSEAN, NaPDoS, NP.search, Bagel2 are used for lantipeptides, while PKS/NRPS Analysis,
SBSPKS (187) and SMURF pipeline were developed for fungal systems. AntiSMASH is one of
the most popular softwares since it is friendly and easy going.

2.2. Antitumor Agents

Microorganisms have played a crucial role in identifying compounds with therapeutic benefit
against cancer (188). Most of the important compounds used for chemotherapy of tumors are
microbially produced antibiotics mainly made by actinomycetes. Among the most well known
are actinomycin D (dactinomycin), anthracyclines (including daunorubicin, doxorubicin,
epirubicin, pirirubicin, idarubicin, valrubicin, and amrubicin), glycopeptolides (bleomycin and
phleomycin), mitomycin C, anthracenones (mithramycin, streptozotocin, and pentostatin), the
enediyne calicheamycin attached to a monoclonal antibody (Mylotarg ®) and recently, the
epothilones.
Novel anthracyclines have been produced by metabolic engineering, i.e., cloning genes
from antitumor-producing species into other producing or nonproducing strains, or by blocking
deoxysugar biosynthesis. A new anthracycline, 11-hydroxyaclacinomycin 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 (189). The hybrid molecule showed greater activity against leukemia and melanoma
than aclacinomycin A. Another hybrid molecule produced was 2″-amino-11-
hydroxyaclacinomycin Y, which was highly active against tumors (190). Additional new
anthracyclines have been made by introducing DNA from Streptomyces purpurascens into
Streptomyces galilaeus, both of which normally produce known anthracyclines (191). Novel
anthracyclines were produced by cloning DNA from the nogalomycin producer, Streptomyces
nogalater, into S. lividans and into an aclacinomycin-negative mutant of S. galilaeus (192).

15
Cloning of the actI, actIV and actVII genes from Streptomyces coelicolor into the 2-
hydroxyaklavinone producer, S. galilaeus 31,671 yielded novel hybrid metabolites,
desoxyerythrolaccin and 1- O -methyl-desoxyerythrolaccin (193). Similar studies yielded the
novel metabolite aloesaponarin II (194).
Epirubicin (4´-epidoxorubicin) is a semisynthetic anthracycline with less cardiotoxicity than
doxorubicin (195). Genetic engineering of a blocked S. peucetius strain provided a new method
to produce it (196). The gene introduced was avrE of the avermectin-producing Streptomyces
avermitilis or the eryBIV genes of the erythromycin producer, Saccharopolyspora erythrea.
These genes and the blocked gene in the recipient are involved in deoxysugar biosynthesis.
Taxol ®, a diterpene alkaloid, is approved for breast and ovarian cancer and acts by
blocking depolymerization of microtubules. In addition, taxol promotes tubulin polymerization
and inhibits rapidly dividing mammalian cancer cells (197). Taxol was originally isolated from the
bark of the Pacific yew tree (Taxus brevifolia), but it took six trees of 100 years of age to treat
one cancer patient (198). It is now produced by plant cell culture or by semisynthesis from
taxoids made by Taxus species. Early genetic engineering of S. cerevisiae yielded no taxadiene
(the taxol precursor) because too little of the intermediate, geranylgeranyl diphosphate, was
formed. When the Taxus canadensis geranylgeranyl diphosphate synthase gene was
introduced, 1 mg L-1 of taxadiene was obtained (199). More recent metabolic engineering
studies (200) yielded a S. cerevisiae strain producing over 8 mg L-1 taxadiene and 33 mg L-1
geranylgeraniol. Taxol has sales of $1.6 billion per year.
Epothilones are an important group of new anticancer agents produced by the
myxobacterium, Sorangium cellulosum (201). Rounds of classical mutation and screening
followed by recursive protoplast fusion resulted in fusants able to produce 130-fold more
epothilone B compared to the starting strain. Epothilones have a mode of action similar to Taxol
and, very importantly, are active against Taxol-resistant tumors.

2.3. Cholesterol-Lowering Agents

The largest segment of the pharmaceutical business is that of cholesterol-lowering drugs,
amounting to about 30% of global sales. The first member of the fungal statins, i.e., compactin,
was discovered in cultures of Penicillium brevicompactum (202) and Penicillium citrinum (203).
A few years later, the more active methylated form of compactin known as lovastatin (monacolin
K, mevinolin, Mevacor™), was isolated from broths of Monascus ruber and Aspergillus terreus
(204, 205). Simvastatin (Zocor™), a semisynthetic derivative of lovastatin, reached a market of
over $7 billion. Pravastatin, a product of compactin bioconversion, attained sales of $5 billion.
The synthetic statin, atorvastatin (Lipitor™), became the world’s leading drug at $12 billion per
year.
Recently, association analysis, a strategy that integrates transcriptional and metabolic
profiles, led to an improvement in lovastatin production of over 50% (206). Improvement was
achieved by increasing the dosage of lovastatin biosynthetic genes and of regulatory genes for
secondary metabolism.

2.4. Antihelmintics
Microbially produced polyethers such as monensin, lasalocid, and salinomycin dominate the
coccidiostat market.
The avermectins, a family of secondary metabolites having both antihelmintic and
insecticidal activities, produced by S. avermitilis, have a market of over 1 billion dollars per year.
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.

16
 Although the Merck Laboratories had earlier developed a commercially useful synthetic
product, thiobenzole, they had enough foresight to also examine microbial broths for
antihelmintic activity and found a nontoxic fermentation broth which killed the intestinal
nematode, Nematospiroides dubius, in mice. The S. avermitilis culture, which was isolated by
Omura and coworkers at the Kitasato Institute in Japan (207), produced a family of secondary
metabolites having both antihelmintic and insecticidal activities. These were discovered by
Merck scientists and named “avermectins.” They are disaccharide derivatives of macrocyclic
lactones with exceptional activity against parasites, i.e., at least ten times higher than any
synthetic antihelmintic agent known. They have activity against both nematode and arthropod
parasites in sheep, cattle, dogs, horses, and swine.
Incorporation of multiple copies of afsR2, a global regulatory gene, from S. lividans into
wild-type S. avermitilis increased avermectin production by 2.3-fold (208). Transposon
mutagenesis was used to eliminate production of the troublesome toxic oligomycin in S.
avermitilis (209). DNA shuffling of the ave C gene of S. avermitilis gave an improved ratio of the
undesirable CHC-B2 to the desirable CHC-B1 of 0.07:1. This was an improvement of 21-fold
over the ratio with the starting strain (210).
A semisynthetic 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 (211).
A new avermectin, called Doramectin (=cyclohexyl avermectin B1), was developed at Pfizer
by the technique of mutational biosynthesis (212). Indeed, it was the first commercially
successful example of mutational biosynthesis.

2.5. Immunosuppressive Agents

Cyclosporin A was originally discovered as a narrow spectrum antifungal peptide produced by
the mold, Tolypocladium niveum (previously Tolypocladium inflatum). Discovery of
immunosuppressive activity led to its use in heart, liver, and kidney transplants and to the
overwhelming success of the organ transplant field. Sales of cyclosporin A have reached $1.5
billion. Although cyclosporine A had been the only product on the market for many years, two
other products, produced by actinomycetes, provided new opportunities. These are rapamycin
(=sirolimus) (213) and the independently discovered FK-506 (tacrolimus) (214). They are both
narrow spectrum polyketide macrolide antifungal agents, which are 100-fold more potent that
cyclosporin as immunosuppressants and less toxic. FK-506 and rapamycin have been used
clinically for many years. FK-506 had a market of $2 billion in 2007. Mutants developed by
increasing resistance to FK-506 produce higher titers (215).
Genome shuffling using mutants of the rapamycin producer, Streptomyces hygroscopicus,
as well as interspecies fusion of protoplasts of S. hygroscopicus D7-804 and Streptomyces
erythreus ZJU325, generated improved rapamycin-producing strains (216). Two genes of the
rapamycin biosynthetic cluster in S. hygroscopicus, i.e., rap G and rap H, encode positive
regulatory proteins for rapamycin production (217). Overexpression of either gene increased
rapamycin formation, whereas their deletions eliminated rapamycin biosynthesis. They act by
affecting the promoter of the operon.

2.6. Bioinsecticides
The insecticidal bacterium, Bacillus thuringiensis (BT), owes its activity to its crystal protein
produced during sporulation. Crystals plus spores had been applied to plants for years to
protect them against lepidopteran insects. BT preparations are highly potent, some 300 times
more active on a molar basis than synthetic pyrethroids and 80,000 times more active than

17
organophosphate insecticides. In 1993, BT represented 90% of the biopesticide market and had
annual sales of $125 million.
A very important insecticide is Spinosad (Naturalyte ®) produced by Saccharopolyspora
spinosa and used for protection of crops and feedstock animals. Spinosad is a mixture of two
tetracyclic macrolides containing forosamine and tri- O -methyl rhamnose with different levels of
methylation on the polyketide moiety. The two components are spinosyns A and D, which differ
by a methyl group at position 6 of the polyketide. Spinosad is an excellent nontoxic agricultural
insecticide. Genome shuffling has been used for strain improvement (218).

3. Recombinant Proteins: Biopharmaceuticals

Biopharmaceutical proteins can be categorized into four major groups: (1) protein therapeutics
with enzymatic activity (e.g., insulin), (2) protein vaccines, (3) protein therapeutics with special
targeting activity (e.g., monoclonal antibodies), and (4) protein diagnostics (e.g., biomarkers)
(219). Biologics accounted for over $80 billion in sales in 2008. Six of these therapeutic proteins
were among the best selling drugs in the USA in that year. Monoclonal antibodies and Fc-fusion
proteins made up 43% of this market value.
By means of genetic engineering, desired proteins are massively generated to meet the
copious demands of industry (220). Protein quality, functionality, production speed, and yield
are the most important factors to consider when choosing the right expression system for
recombinant protein production.
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 yeasts) (221).
On the other hand, 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 of
the human type. Although yeasts, molds, and insect cells are generally unable to provide
mammalian glycosylation, the methylotrophic yeast, Pichia pastoris, has been genetically
engineered to produce a human type of glycosylation (222).
Directed evolution of proteins, has been reviewed by Yuan et al. (223). Strategies include
DNA shuffling, whole genome shuffling, heteroduplex, random chimeragenesis of transient
templates, assembly of designed oligonucleotides, mutagenic and unidirectional reassembly,
exon shuffling, Y-ligation-based block shuffling, nonhomologous recombination, and the
combining of rational design with directed evolution.

3.1. Bacteria
Bacterial systems are used to make somatostatin, insulin, bovine growth hormone for veterinary
applications, −1 antitrypsin, interleukin-2, tumor necrosis factor, -interferon, and -interferon.
E. coli is one of the earliest and most widely used hosts for the production of heterologous
proteins (224). As early as 1993, recombinant processes of E. coli were responsible for almost
$5 billion worth of products, i.e., insulin, human growth hormone, interferons, and G-CSF.
Advantages of E. coli include rapid growth, rapid expression, ease of culture and genome
modifications, low cost, and high product yields (225). It is used for massive production of many
commercialized proteins. This system is excellent for functional expression of non-glycosylated
proteins.
E. coli genetics are far better understood than those of any other microorganism. Recent
progress in the fundamental understanding of transcription, translation, and protein folding in E.
coli, together with the availability of improved genetic tools, are making this bacterium more
valuable than ever for the expression of complex eukaryotic proteins. Its genome can be quickly
and precisely modified with ease, promotor control is not difficult, and plasmid copy number can
be readily altered. This system also features alteration of metabolic carbon flow, avoidance of
incorporation of amino acid analogs, formation of intracellular disulfide bonds, and reproducible

18
performance with computer control. E. coli can accumulate recombinant proteins up to 80% of
its dry weight and survives a variety of environmental conditions. Recombinant protein
production in E. coli can be increased by mutations which eliminate acetate production (226).
Avecia Biologics achieved a titer of 14 g L-1 of recombinant protein using E. coli (227).
The value of transcriptome analysis in process improvement, was shown by Choi et al.
(228). They analyzed an E. coli process yielding human insulin-like growth factor 1 fusion
protein (IGF-If) in a high density culture. Of 200 or so genes whose expression was
downregulated after induction, the ones involved in biosynthesis of amino acids or nucleotides
were studied. Amplification of two of these, prsA (encoding PRPP synthetase) and glpF
(encoding the glycerol transporter), raised product formation from 1.8 to 4.3 g L-1.
Bacilli have yields as high as 3 g L-1. The organisms used are usually Bacillus megaterium,
B. subtilis, and Bacillus brevis. Staphylococcus carnosus can produce 2 g L-1 of secreted
mammalian protein.
An improved Gram-negative host for recombinant protein production has been developed
using Ralstonia eutropha (229). 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. Another
useful bacterium is Pseudomonas fluorescens MB101 developed by Dowpharma (230). This
system has produced 4 g L-1 of TNF-α.

3.2. Yeasts
Yeasts, the single-celled eukaryotic fungal organisms, are often used to produce recombinant
proteins that are not produced well in E. coli because of problems dealing with folding or the
need for glycosylation. The major advantages of yeast expression systems are listed in Table 2.
The yeast strains are genetically well characterized and are known to perform many
posttranslational modifications. They are easier and less expensive to work with than insect or
mammalian cells and are easily adapted to fermentation processes.
The two most utilized yeast strains are S. cerevisiae and the methylotrophic yeast P.
pastoris. Glucose oxidase from A. 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.
P. pastoris has the desirable qualities of dense growth and methanol-induced expression
and secretion of recombinant proteins. It is used for the commercial production of non-
glycosylated human serum albumin and glycosylated vaccines. Strains have been developed
which are capable of human type N-glycosylation and such products are already in clinical
testing. High recombinant protein yields can be obtained with P. pastoris, e.g., 10 g L-1 of tumor
necrosis factor, (231) 14.8 g L-1 of gelatin, 15 g L-1 of mouse collagen (232), and E. coli phytase
and Candida parapsilosis lipase/acetyltransferase at 6 g L-1. Bacterial proteins such as
intracellular tetanus fragment C were produced as 27% of protein with a titer of 12 g L-1 (233).
Production of serum albumin in S. cerevisiae amounted to 0.15 g L-1, whereas in P. pastoris the
titer was 10 g L-1 (234). Indeed, claims have been made that P. pastoris can make 20–30 g L-1 of
recombinant proteins (235).

Table 2
Advantages of yeast expression systems
Stable strains
High density of growth
Durability
High production titers and yields
Protein glycosylation

19
 Reasonable cost
Product processing similar to that of mammalian cells
Suitable for isotopically labeled proteins
Suitable for S–S-rich proteins
Protein folding assisted
Chemically defined media supporting rapid growth

Heterologous gene expression in another methylotroph Hansenula polymorpha yielded 13.5

g L-1 of phytase, and other proteins were made at levels over 10 g L-1. Among the advantages of
methylotrophic yeasts over S. cerevisiae as a cloning host are the following: (1) higher protein
productivity, (2) avoidance of hyperglycosylation, (3) growth in reasonably strong methanol
solutions that would kill most other microorganisms, (4) a system that is cheap to set up and
maintain, and (5) integration of multicopies of foreign DNA into chromosomal DNA yielding
stable transformants (236).

3.3. Filamentous Fungi (Molds)

Filamentous fungi are attractive hosts for recombinant DNA technology because of their ability
to secrete high levels of bioactive proteins with posttranslational processing such as
glycosylation. A. niger excretes 25 g L-1 of native glucoamylase (99, 237). Foreign genes can be
incorporated via plasmids into chromosomes of the filamentous fungi where they integrate
stably into the chromosome as tandem repeats providing superior long-term genetic stability. An
excellent comprehensive review of heterologous expression in Aspergillus, has been published
by Lubertozzi and Keasling (238). Like mammalian cells, fungi possess the cellular machinery
for translation of proteins, protein folding, and posttranslational modification. Like bacteria, they
are easy to culture. Genetic development of aspergilli has included (1) transformation systems,
(2) expression constructs, (3) targeted integration and copy number manipulation, (4)
promoters, (5) improved gene design, (6) engineering of proteases, secretion, and
glycosylation, and (7) tools for tagging, targeting, and silencing of genes.
A 1,000-fold increase in phytase production was achieved in A. niger by the use of
recombinant technology (239). Recombinant Aspergillus oryzae can produce 2 g L-1 of human
lactoferrin (240) and 3.3 g L-1 of Mucor renin (241). Production of human lactoferrin (242) by
Aspergillus awamori via rDNA technology and classical strain improvement amounted to 2 g L-1
of extracellular protein (240). A. niger glucoamylase was made by A. awamori at 4.6 g L-1.
The fungus Chrysosporium lucknowense has been genetically converted into a non-
filamentous, less viscous, low protease producing strain that is capable of producing very high
yields of heterologous proteins (243). Dyadic International, Inc., the company responsible for the
development of the C. lucknowense system, claims protein production levels of up to 100 g L-1
of native extracellular protein.

3.4. 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 NS0
murine myeloma cells (244), (2) baby hamster kidney (BHK) cells for production of cattle foot-
and-mouth disease vaccine, (3) green monkey kidney cells for polio vaccine (245), and (4)
human cell lines such as human embryonic kidney (HEK) cells. NSO is a nonsecreting subclone
of the NS-1 mouse melanoma cell line. By 2006, production of therapeutic proteins by
mammalian systems reached $20 billion (246).
Animal-free, protein-free, and even chemically defined media with good support of
production have been developed (247). Protein production by mammalian cells (CHO) went

20
from 5–50 mg L-1 in 1985 to 50–500 mg L-1 1in 1995 and to 5 g L-1 in 2005 (248). A number of
mammalian processes are producing 3–5 g L-1 of recombinant protein (249) and in some cases,
protein titers have reached 10 g L-1 in industry (250), including antibodies (251). 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 (252) and then later, 27 g L-1
of a monoclonal antibody (253). Protein production of over 20 g L-1 has been achieved in serum-
free medium, but the production of 2–3 g L-1 in such media is more usual.

3.5. Insect Cells

Insect cells are able to carry out more complex posttranslational 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 proteins of mammalian origin (254).
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.
Baculovirus-assisted insect cell expression offers many advantages as follows: (1)
Eukaryotic posttranslational modifications without complication, including phosphorylation, N-
and O-glycosylation, correct signal peptide cleavage, proper proteolytic processing, acylation,
palmitylation, myristylation, amidation, carboxymethylation, and prenylation (255, 256). (2)
Proper protein folding and S–S bond formation, unlike the reducing environment of E. coli
cytoplasm. (3) High expression levels. The virus contains a gene encoding the protein
polyhedron, which is made at very high levels normally and is not necessary for virus
replication. The gene to be cloned is placed under the strong control of the viral polyhedrin
promoter, allowing expression of heterologous protein of up to 30% of cell protein. (4) Easy
scale up with high density suspension culture. (5) Safety, expression vectors are prepared from
the baculovirus which can attack invertebrates but not vertebrates or plants. (6) Lack of limit on
protein size. (7) Efficient cleavage of signal peptides (8). Simultaneous expression of multiple
genes (257).
Production of recombinant proteins with the baculovirus expression vector system in insect
cells reached 600 mg L-1 in 1988 (258). Later information indicated that the baculovirus insect
cell system can produce 11 g L-1 of recombinant protein (259). Recombinant insect cell cultures
have yielded over 200 proteins encoded by genes from viruses, bacteria, fungi, plants, and
animals (260).

4. Enzymes
Over the years, high titers of enzymes were obtained using “brute force” mutagenesis and
random screening of microorganisms. Recombinant DNA technology acted as a boon for the
enzyme industry in the following ways (261): (1) plant and animal enzymes could be made by
microbial fermentations, e.g., chymosin; (2) enzymes from organisms difficult to grow or handle
genetically were now produced by industrial organisms such as species of Aspergillus and
Trichoderma, and K. lactis, S. cerevisiae, Y. lipolytica, and Bacillus licheniformis (e.g.,
thermophilic lipase was produced by A. oryzae and Thermoanaerobacter cyclodextrin glycosyl
transferase by Bacillus); (3) enzyme productivity was increased by the use of multiple gene
copies, strong promoters, and efficient signal sequences; (4) production of a useful enzyme
from a pathogenic or toxin-producing species could now be done in a safe host; and (5) protein
engineering was employed to improve the stability, activity, and/or specificity of an enzyme.
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 different applications including detergent, food and starch processing industry are

21
recombinant proteins (262). Recombinant molds are one of the main sources of enzymes for
industrial applications. Yields as high as 4.6 g L-1 have been reached for several hosts including
A. niger, A. oryzae, A. awamori, C. lucknowense, and A. chrysogenum.
Plant phytase (263), produced in recombinant A. niger was used as a feed for 50% of all
pigs in Holland. A 1,000-fold increase in phytase production was achieved in A. niger by the use
of recombinant technology (239). Mammalian chymosin was cloned and produced by A. niger or
E. coli and recombinant chymosin was approved in the USA; its price was half that of natural
calf chymosin.
Three fungal recombinant lipases are currently used in the food industry. They are from
Rhizomucor miehi, Thermomyces lanuginosus, and Fusarium oxysporum and are produced in
A. oryzae. They are used for laundry cleaning, inter-esterification of lipids and esterification of
glucosides producing glycolipids which have applications as biodegradable nonionic surfactants
for detergents, skin care products, contact lenses and as food emulsifiers. Washing powders
have been improved in activity and low temperature operation has been achieved by the
application of recombinant DNA technology and site-directed mutagenesis of genes encoding
proteases and lipases (264, 265). The first commercial recombinant lipase used in a detergent
was from Humicola lanuginose. The gene was cloned into the A. oryzae genome.
A multicopy plasmid of B. subtilis was used to increase by 2,500- fold the production of an
α-amylase from B. amyloliquefaciens (266). An exoglucanase from Cellulomonas fimi was
overproduced in E. coli to a level of over 20% of cell protein (267). The same host has also
been used to clone the endo--glucanase components from Thermomonospora and Clostridium
thermocellum as well as the cellobiohydrolase I gene of Trichoderma reesei (268). P. pastoris
was engineered to produce and excrete S. cerevisiae invertase into the medium at 100 mg L-1
(269). Self-cloning of the xylanase gene in S. lividans resulted in a sixfold overproduction of the
enzyme (270).
The properties of many enzymes have been modified by random mutagenesis and
screening of microorganisms over the years leading to changes in substrate specificity,
feedback inhibition, kinetic parameters (Vmax, Km or Ki), pH optimum, thermostability, and carbon
source inhibition. Based on this information, more rational techniques such as site-directed
mutagenesis were used to introduce single changes in amino acid sequences yielding similar
types of changes in a large variety of enzymes. Modification of eight amino acids increased heat
tolerance and temperature stability at 100°C of a protease from Bacillus stearothermophilus
(271). Interestingly, all mutations were far from the active site of the enzyme.
Molecular breeding techniques (e.g., DNA shuffling and DNA family shuffling) are being
currently used to generate enzymes with improved properties such as activity and stability at
different pH values and temperatures (272), increased or modified enantioselectivity (273),
altered substrate specificity (274), stability in organic solvents (275), novel substrate specificity
and activity (276), increased biological activity of protein pharmaceuticals and biological
molecules (277) as well as novel vaccines (278, 279). Two proteins from directed evolution work
were already on the market by year 2,000 (280). These were green fluorescent protein of
Clontech (281) and Novo Nordisk’s LipoPrime ® lipase.

5. Closing Remarks
The fermentation industry developed slowly from the beginning of the twentieth century to the
early 1970s using brute force mutagenesis followed by screening or selection. However, the
birth of the era of recombinant DNA (rDNA) in 1971–1973 catalyzed a major change in the way
useful processes could be developed. Production of primary metabolites was markedly
improved by modern genetic techniques. Environmentally friendly fermentations replaced
chemical synthesis to a great extent. Of great interest has been the application of rDNA
technology to the production of secondary metabolites and to the elucidation of their
biosynthetic pathways. Tools include transposition mutagenesis, targeted deletions, genetic

22
recombination via combinatorial biosynthesis, transcriptome analysis, proteomics,
metabolomics, metabolic engineering, etc. Genes encoding many enzymes have been cloned
and the enzymes have been expressed at levels hundreds of times higher than those naturally
produced. Random redesign techniques have generated enzymes with improved properties
including activity, stability, increased or modified enantioselectivity, altered substrate specificity,
etc. An entirely new field of industrial microbiology has arisen out of rDNA, i.e., the
biopharmaceutical industry which is the most rapidly expanding segment of the biological
industry, especially that of monoclonal antibodies. The best is yet to come from the fantastic
combination of industrial microbiology and rDNA technology. Many of the new techniques are
carried out by small companies and academic groups who could play a major role in rescuing us
from the antibiotic crisis that we are now experiencing. Furthermore, we look forward to its role
in eventually replacing the environmentally dangerous energy sources that we live with today,
i.e., petroleum, coal, etc., with future biofuels made from agricultural and forest biomass.

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