DNA shuffling

DNA shuffling is a way to rapidly propagate beneficial mutations in a directed evolution experiment. It is

used to rapidly increase DNA library size.

Procedure
DNAse I is first used to fragment a set of parent genes into pieces of 50-100 bp in length. This is then
followed by a PCR without primers- the pieces that have overlapping areas anneal to each other and are
then extended by DNA polymerase.

Several rounds of this PCR extension are allowed to occur, after some of the DNA molecules reach the
size of the parental genes. These genes can then be amplified with another PCR, this time with the
addition of primers that are designed to complement the ends of the strands. The primers may have
additional sequences added to their 5' ends, such as sequences for restriction enzyme recognition sites
needed for ligation into a cloning vector.

It is possible to recombine portion of these genes to generate hybrids or chimeric forms with unique

properties, this is called DNA shuffling.

Shuffling methods
Restriction enzymes

1. Restriction enzymes that cut in similar places are used to digest members of the gene family
2. DNA fragments are joined together with DNA ligase
3. Large numbers of hybrids are produced which can be tested for unique properties

DNase 1

1. Different members of the gene family are fragmented using DNase 1 followed by PCR
2. During PCR different members of the family are cross-primed, DNA fragments with
high homology will anneal to each other
3. The hybrids generated are then used generate a library of mutants which are tested for unique
properties
Anticipatory evolution and DNA shuffling

DNA shuffling has proven to be a powerful technique for the directed evolution of proteins. A mix of theoretical and applied research
has now provided insights into how recombination can be guided to more efficiently generate proteins and even organisms with altered
functions.

Proteins are machines created by evolution, but it is unclear just how finely evolution has guided their sequence, structure, and function.
It is undoubtedly true that individual mutations in a protein affect both its structure and its function and that such mutations can be fixed
during evolutionary history, but it is also true that there are other elements of protein sequence that have been acted upon by evolution.
For example, the genetic code appears to be laid out so that mutations and errors in translation are minimally damaging to protein
structure and function [1]. Could the probability that a beneficial mutation is found and fixed in the population also have been
manipulated during the course of evolution, so that the proteins we see today are more capable of change than the proteins that may have
been cobbled together following the 'invention' of translation? Have proteins, in fact, evolved to evolve? There is already some evidence
that bacteria are equipped to evolve phenotypes that are more capable of further adaptation (reviewed in [2,3,4]). For example, mutator
[5] and hyper-recombinogenic [6] strains arise as a result of selection experiments. The development of DNA shuffling (reviewed in
[7,8]) and the appearance of several recent papers using this technique [9,10,11] provide us with a surprising new opportunity to ask and
answer these fundamental questions at the level of individual genes, and perhaps even genomes.
DNA shuffling, a method for in vitro recombination, was developed as a technique to generate mutant genes that would encode proteins
with improved or unique functionality [12,13]. It consists of a three-step process that begins with the enzymatic digestion of genes,
yielding smaller fragments of DNA. The small fragments are then allowed to randomly hybridize and are filled in to create longer
fragments. Ultimately, any full-length, recombined genes that are recreated are amplified via the polymerase chain reaction. If a series of
alleles or mutated genes is used as a starting point for DNA shuffling, the result is a library of recombined genes that can be translated
into novel proteins, which can in turn be screened for novel functions. Genes with beneficial mutations can be shuffled further, both to
bring together these independent, beneficial mutations in a single gene and to eliminate any deleterious mutations. Although multiple,
beneficial mutations could potentially be generated just as well by serial mutagenesis and screening, DNA shuffling is much quicker: for
example, the starting population of a library generated by mutagenic PCR typically contains 70-99% nonfunctional variants [14],
whereas most variants formed by DNA shuffling are functional. Thus, DNA shuffling should allow a streamlined exploration of
sequence space and acquisition of novel protein phenotypes easily, as has indeed proven to be the case for a number of protein targets
[15,16].
Beyond biotechnology applications, DNA shuffling can potentially be used to recapitulate natural recombination and to ask whether
recombination generally leads to better or novel proteins. In this regard, DNA shuffling can be carried out not only with genes that are
closely related alleles, but also with a group of phylogenetically related genes that may differ by up to 40%, a process known as family
shuffling [15]. As mentioned above, it was strongly suspected that by starting with a population of genes already known to be functional,
family shuffling could move the most beneficial mutations into the same gene and thus quickly optimize or alter protein function. In fact,
however, this intuition should hold true only if mutant alleles can generally either act in an additive or synergistic fashion. If mutant
alleles are neutral or interfere with each other, then there will be no generic benefit to recombination.
In order to address this hypothesis, Joern et al. [9] have developed a novel technique for mapping recombination events by probe-
hybridization analysis. Shuffled libraries were generated by crossing genes for several dioxygenase: toluene dioxygenase, todC1C2;
tetrachlorobenzene dioxygenases, tecA1A2; and biphenyl dioxygenase, bphA1A2.Shuffled variants from the three-parent library were
screened for toluene dioxygenase activity, and randomly selected variants were sequenced to determine the actual number of crossovers
that had occurred to give rise to functional and nonfunctional variants. Unsurprisingly, it was found that crossovers commonly occurred
in regions of high homology: although regions that contained ten or more common, identical residues made up less than 10% of the
lengths of the genes, over 60% of the crossovers occurred in these regions. Interestingly, it was found that the number of crossover
events did not correlate with protein function, suggesting that individual segments of a protein might act independently during evolution
[9]. It is also possible that the proteins were so closely related to one another that multiple crossovers did not reduce or alter
functionality.
Building on these results, Voigt et al. [10] hypothesized that functional genes derived by DNA shuffling (and perhaps by natural
recombination) should preserve clustered sets of structural interactions (the so-called 'schemas') of the original protein (Figure 1a). In
order to validate this hypothesis, the authors developed an algorithm that attempted to predict the effect of crossover events at specific
sites in a gene. In particular, the algorithm assessed which amino acids were close to one another in both the primary and the tertiary
protein structure and predicted which interaction subsets could be manipulated in a way that minimally disrupted protein structure and
function. This analysis results in a 'schema profile' for the proteins, which indicates the amount of disruption to the schemas that
recombination at each point along the sequence will cause (Figure1b). Several proteins that had previously been evolved in vitro by
family shuffling were evaluated, and the schema profiles of these proteins correlated well with the experimentally determined crossover
points [14].

This algorithm was then used to generate schema profiles between two β-lactamases, TEM-1 and PSE-4, which confer ampicillin
resistance and share only 40% amino-acid sequence identity. Hybrid enzymes that had varying degrees of recombination between
schemas were then constructed, and the recombined variants were transformed into bacteria, which were assayed for ampicillin
resistance. The most resistant hybrids contained recombined genes with crossovers that had been predicted in advance to occur between
schemas [10].
What is particularly surprising is not that DNA shuffling occurs between domains; even a brief observation of the three-dimensional
structures of proteins immediately suggests that recombinational breakpoints will probably have the smallest effect on protein function if
they occur outside of major structural units found by Voigt et al. [16] (although certain breakpoints between structural subunits, such as
in the middle of α helices, would probably not have been predicted without schema profiling). Rather, the amazing thing is that proteins
have evolved so that they are by and large composed of structural domains that can undergo recombination. As Voigt et al. [10] point
out, Gô [17,18] found a correlation between intron locations and structural domains. This was expanded on by Gilbert and his co-
workers [19], who advanced the notion that proteins could be modularly constructed from structural domains as an attempt to explain the
origin of introns. Although the 'introns early' hypothesis has long since been shown to be implausible [20,21,22], the original notion that
introns could act as buffers for recombination is still intellectually compelling, and it may be consistent with the results of Voigt et
al. [10].
Interestingly, to the extent that proteins have evolved as modular machines that are capable of taking advantage of recombination during
their evolutionary history, the very mathematical models propagated by Joern et al. [9] and Voigt et al. [10] may be unnecessary. 'Blind'
DNA shuffling between closely related proteins may already be more than good enough to generate proteins with novel phenotypes. For
example, we have evolved a β-glucuronidase in vitro to switch its substrate specificity from β-glucuronides to β-galactosides and have
achieved an over 500-fold increase in activity towards the new substrate [23]. This catalytic conversion was achieved in three rounds of
shuffling and screening, but further rounds of selection failed to achieve greater cleavage of β-galactosides. The initial library of this
selection was constructed using mutagenic PCR, and a large fraction of the population was inactive, yet the catalytic specificity of the
selection produced a switch of over 52-million-fold in substrate preference.
Similarly, new experiments from Zhang et al. [11] provide additional evidence that blind shuffling is fully capable of functional
improvement, not just at the protein level, but even at the organismal level. These researchers coupled classical strain improvement
(mutation and selection) with genetic recombination. Protoplast fusion results in very efficient recombination between the genomes
of Streptomyces species, and iterative protoplast fusion results in the reassortment of multiple markers between species. To show the
power of this new method, aStreptomyces strain producing the complex polyketide antibiotic tyiosin was selected for improved function
and was then forced to undergo the equivalent of sexual reproduction. The genomes of several surviving mutants were shuffled after
every round of selection to generate a combinatorial library of organisms that could again be screened for improved function. A strain
generated by only two rounds of shuffling could produce tylosin at a rate comparable to strains that had undergone 20 rounds of classical
selection. These results demonstrate that genome shuffling will probably lead to changes and improvements in organismal function as
radical as those that have previously been observed for proteins.
Overall, these results further support the idea that evolution can act reflexively - that is, to enhance its own ability to act. From the results
of Arnold and co-workers [9,10], it is possible that regions that fall between predicted schema might be conserved in sequence in order to
facilitate recombination; this hypothesis could be checked directly by database analysis. The application of the techniques described by
Arnold and co-workers [9,10], del Cardayré and co-workers [11], and others may allow researchers to more effectively design libraries
for screening. A large fraction of the products generated in traditional screening or even shuffling reactions are nonfunctional. Schema
profiling and pathway shuffling may eventually make it possible to design directed evolution experiments in which structural and
metabolic subunits are preserved, thereby limiting the exploration of sequence space largely to functional molecules. Ultimately, these
advances should expand our understanding of natural genetic processes and thereby allow biologists to generate novel proteins and
pathways in a fraction of the time that nature or conventional breeding would take.
A graphical representation of the relationship between protein structure and schemas. (a) Theβ-
lactamase protein is shown divided into different colored substructures (schemas), which are derived
from the schema profile of the protein. (b) An example of a schema profile for a (simpler) hypothetical
protein. Peaks correlate with positions in the protein where recombination will be maximally
disruptive; valleys correlate with positions that are predicted to minimally disrupt the structure and
function of the protein. (c) Intron structure may correlate with schema structure. To the extent it is
now possible to calculate schema profiles, it can be hypothesized that introns (white) may generally
fall at minima while exons (black) may generally contain larger disruption values.
Tailoring vectors through DNA shuffling

Rational vector design is pivotal for successful human gene therapy and the application of
high-throughput methods may provide a means for the efficient realization of vectors with
desired properties. A method involving DNA shuffling represents a powerful new paradigm
in this regard.
DNA shuffling of Cry proteins
Introduction:
Bacillus thuringiensis (Bt), is a gram-positive soil bacterium, with a genome size of 2.4 to 5.7 million basepairs.
The prevalence of this strain is not restricted and has been isolated worldwide from many habitats, including soil,
stored-product dusts, insects, deciduous and coniferous leaves. Bacillus thuringiensis forms parasporal crystals
during the stationary phase of its growth cycle. These crystals are specifically toxic to certain orders and species of
insects, like Lepidoptera, Diptera, and Coleoptera. Many different strains of Bt have been shown to produce these
inclusions of insecticidal crystal protein (ICP). Bt also produces antibiotic compounds that have antifungal
activity. (1)

During sporulation, it synthesizes a cytoplasmic inclusion containing one or more proteins that are toxic to insect
larvae. Upon completion of sporulation the parent bacterium lyses to release the spore and the inclusion. In these
inclusions, the toxins exist as inactive protoxins. When the inclusions are ingested by insect larvae, the alkaline pH
solubilizes the crystal. The protoxin is then converted in to an active toxin after processing by the host proteases
present in the midgut. (1)

It has been indicated that the activated toxin binds to insect-specific receptors exposed on the surface of the plasma
membrane of midgut epithelial cells and then inserts into the membrane to create transmembrane pores that cause
cell swelling and lysis and eventually death of the insect. (1)

Due to their high specificity for these unique receptors on the membrane of the gut epithelial cells, these toxins
(delta-endotoxins) are harmless to non-target insects and the end-user and are compatible with integrated pest
management programs. The fact that they are proteins ensures that they are readily biodegraded.

The Cry Gene Family:

These toxins can be categorized under the -endotoxins, which is highly specific to only certain insects. The family
of genes coding for this toxin is the Cry gene family. A common characteristic of the cry genes is their expression
during the stationary phase. Their products generally accumulate in the mother cell compartment to form a crystal
inclusion that can account for 20-30% of the dry weight of the sporulated cells. The high level of synthesis and
coordination with the stationary phase are controlled by a variety of mechanisms occurring at the transcriptional,
posttranscriptional and posttranslational levels. (1)

 At the transcriptional level, the development of sporulation is controlled by the successive activation of
sigma factors.

 At the posttranscriptional level, the stability of the mRNA is enhanced by the formation of a stemloop
structure during termination of transcription. This protects the mRNA from the activity of exonucleases
present in the cell from degrading the mRNA. The 5’ end of the mRNA is protected due to the presence of
the perfect Shine-Dalgarno sequence designated as STAB-SD. The interaction with the 16s rRNA of the
30s subunit of the ribosome confers stability to the mRNA.

 At the posttranslational level, these proteins form crystalline inclusions in the mother cell compartment.
Depending on the protoxin composition, the crystals have various forms. This ability to crystallize helps in
protecting the protein itself from premature proteolytic degradation.
In recent years, various Cry gene products have been discovered, and a phylogenetic tree was generated. The figure
below gives an idea about the relatedness of the Cry genes (2). (revised nomenclature for the Cry genes was used.)

Fig 1. Relatedness Dendrogram of Bt crystal proteins on the basis of alignments of the full-length protein sequences. More closely related sequences
or groups are connected by branch points closer to the right. Each protein is described by a name and the corresponding GenBank accession number of its
DNA sequence.
Bacillus thuringiensis toxins: regulation, activities and structural diversity. H. Ernest Schnepf. Current Opinion in Biotechnology, 1995, 6:305-
312.

It was clearly observed that there was a high degree of sequence similarity among the proteins in the N-terminal
regions which, confers toxicity to the protein. As an illustration, the aminoacid sequences of Cry proteins from
different subspecies of the B.t strain was compared and a sequence alignment was done using the software
CLUSTALW. It was observed that not only the N-terminal region was conserved, but there were blocks in the C-
terminal region, which were also highly conserved.

Fig 2. Highly conserved regions of the aminoacid sequence for the cry proteins among different subspecies. (The genes are Cry1B, Cry1A, Cry1D
and Cry4B in order, all proteins weigh approximately 130 kd and are composed of approximately 1170 Aminoacids).

For these conserved blocks, the consensus sequence denotes the position at which atleast 75% of the aligned proteins in the group have an identical or
conserved aminoacid (indicated by shading). An uppercase letter within the consensus sequence indicates that atleast 75% of the residue at that position are
identical, while a lower case letter indicates that atleast 75% of the residue are conserved . Conserved aminoacids are those that fall into the following
groups:

a: A, G, S, T or P :: d: D, E, N or Q :: f: F, W or Y :: i: I, L, M or V and k: K or R.

Structure-Function Interpretation of the Cry Proteins:

The Cry toxin has three domains which are, from N to C terminus, a seven helix bundle, (Domain I), a triple anti-
parallel beta sheet domain (Domain II) and a beta-sheet sandwich (Domain III). (1)

The core of the molecule is built from five sequence blocks, which are a highly conserved feature of all the Bt toxins
indicating that all the proteins in this Cry family will adopt the same general fold.

The long, hydrophobic and amphipathic alpha helices of Domain I is equipped for transmembrane pore formation.
The seven alpha helix domain I structure resembles the pore forming domain of Colicin A and is important for the
membrane insertion step.

Pore formation is initiated by insertion of a helical hairpin (alpha4/alpha5) from domain I with subsequent
association of alpha4/alpha5 hairpins from several molecules to form an oligomeric helical bundle pore with a radius
of 5-10 Angstroms.
Before one or more of these Cry helices can insert into the membrane to initiate oligomerization and pore formation,
a major conformational change must occur, since in the water soluble pre-insertion form all the hydrophobic faces of
the Cry Domain I helical bundle face inwards.

Membrane penetration occurs in two steps: binding to a specific receptor exposed on the membrane surface,
followed by insertion of the delta-endotoxin protein into the membrane leading to pore formation.

The three beta sheet structure (beta prism) of domain II is involved in receptor binding and specificity
determination. This is further supported by reports that domain II shared the same structural fold with three
carbohydrate binding proteins: the vitelline membrane outer layer protein I from hen's eggs, the plant lectin jacalin
and the Maclura pomifera agglutinin.

Domain III of the Bt toxin (see below) may also be a determinant of insect specificity/receptor binding.

Fig 3. Schematic ribbon diagram structure of the CryA toxin. The three domains and their suggested functions are indicated. Shaded segments
correspond to the five conserved sequence blocks (1–5). The position of the -sheets of domain II are indicated near the protruding loops of each sheet

The striking similarity between the structure of domain II of the Bt toxins and the three dimensional structures of
two known lectins suggests that insecticidal specificity might be determined by the carbohydrate affinity of the
domain II lectin fold. A recent discovery that domain III is also a lectin-like domain suggests that the insecticidal
specificity of these toxins could be determined by two lectin-like domains acting in concert or independently.

To fully realize the potential of Bacillus thuringiensis -endotoxins as biopesticides, progress is required in several
areas. First, we must increase the yield or efficiency of toxin protein production. Second, we must gain a sufficient
understanding of the mechanism of toxicity to allow engineering of the toxins for maximum activity. Third, we must
continue to isolate new strains with novel toxin structures and activities either on known B. thuringiensis targets or
on pests thought to be insensitive to B. thuringiensis. And fourth, we must gain a better understanding of the
mechanism, and management, of insect resistance to B. thuringiensis toxins. All the requirement can be easily met
by the process of DNA shuffling of the Cry genes.

 
DNA shuffling:
DNA shuffling is a powerful process for directed evolution, which generates diversity by recombination. DNA
family shuffling mimics and extends classical breeding methods by recombining more than two parental genes, or
genes from different species, in a single DNA shuffling reaction. In this method, the gene is subjected to random
mutations and is then screened for improved ones.

The previous methods of DNA shuffling (3) incorporated, the Single Sequence Shuffling, where a pool of
homologous genes with different point mutations (induced by error prone PCR, or by Oligo nucleotide directed
mutagenesis) is cut in to fragments by DNase I Digestion, followed by reassembly of the fragments in to full genes
by using PCR with and without primers (discussed below).

It was found that the sequence space for searching for this method was very less when compared to the sequence
space available in DNA family shuffling. (4)

Fig 4: Searching sequence space by family shuffling versus by single sequence shuffling. Single sequence shuffling yields clones with a few point
mutations and the library members are 97-99% identical. Family shuffling causes sequence block exchange, which yields genes that have greater diversity.
At equal library size, the sampling space is larger for DNA family shuffling than the single sequence shuffling, thereby allowing promising areas to be
found.

DNA shuffling of a family of genes from diverse species accelerates directed evolution. Willem P.C. Stemmer etal. Nature 1998,391:288-291.

The process of DNA family shuffling (4,5) is illustrated in the diagram (see next page). A detailed protocol of how this is carried out is discussed in the
experimental protocol section.
 
Fig 5. DNA shuffling methodology. The first step of this method is to randomly fragment a population of related genes using DNAse I. This produces
fragments of various lengths that, after denaturation, hybridize to form an equal mixture of 5' and 3' overhangs. Using PCR techniques, the 5' overhang
fragments can be extended by Taq DNA polymerase—leaving the 3' overhang fragments unaffected. As a consequence of this extension, the average
fragment length increases during each cycle. Recombination occurs, when a fragment derived from one template primes a template with a different
sequence. Green dots represent beneficial mutations and red dots represent deleterious mutations. The colored bars indicate recombinations of portions of
three parents into recombinant progeny.

Applications of DNA shuffling to pharmaceuticals and vaccines. Phillip A Patten, Russell J Howard, Willem PC Stemmer. Current Opinion in
Biotechnology 1997, 8:724-733.

Objective:
The following characteristics of the Bt toxin are to be achieved, using DNA family shuffling.

 To attack wide range of insects by making the protein recognize an increased range of receptors in the host
mid gut cells.
 To increase it’s toxicity by using less amount of protein.

Experimental Protocol:
The following is the detailed experimental protocol adopted for DNA Shuffling of the Cry genes.

1. Get DNA sequences of Cry proteins of interest and design degenerate primers : Download the
sequences of the Cry protein of interest from the sequence database (from EMBL and SWISS-PROT) and do a
sequence alignment of them using CLUSTALW, to find out conserved regions in the sequences and design the
primer.

2. Use these primers to amplify the genes in the chosen Bt subspecies : Use the designed primer and
perform a PCR to amplify the genes present in the Bt subspecies. Run an agarose gel electrophoresis and elute the
amplified genes in the gel. Purification of the fragments is done, by transferring on to DE81 ion-exchange paper
(Whatman), elution with NaCl, followed by ethanol precipitation.

3. Clone it in E.coli and express it : Clone the genes in E.coli, in a suitable cloning site and express the
protein to check its activity. Screen out the ones, which do not produce functional protein and retained the properly
cloned colonies. (Screening is done, based on the protein’s ability to bring about toxicity).

4. Amplify the genes in these colonies using the previously designed primer in a PCR.

5. Substrate for the DNA shuffling experiment : The substrate for the experiment will be the PCR
amplified genes in which the primers are removed, to get only the full genes.

6. DNase I digestion of the substrate and purification of bases, 10-50 bases in length : Using DNase
I, digest the substrate in to small fragments. Purify the fragments by elution (whose, length are 10-50 bases in
length) after running an agarose gel electrophoresis. The purification is done, by transferring on to DE81 ion-
exchange paper (Whatman), elution with NaCl, followed by ethanol precipitation.

7. PCR without primers : Resuspend the purified fragments in a PCR mixture without primers, and perform the
reaction. The templates are the sequences purified in step 4. Each cycle causes a template switch and provides a
ground for recombination of fully functional blocks. This results in the sequences to be almost unique.

8. PCR with primers : In order to selectively amplify the full genes, which have been obtained, perform a PCR
with the originally designed primers.
9. Purification of full genes :Run an agarose gel electrophoresis, to get a sharp band, corresponding to the full
gene. Purify it by transferring onto DE81 ion-exchange paper (Whatman), followed by elution with NaCl, and
precipitation using ethanol.

10. Cloning : After digestion of the PCR products with suitable restriction enzymes and gel purification, clone
them in to E.coli cells.

11. Design a high throughput screen to select functional proteins (qualitatively).

12. Select best mutants by quantitative analysis.

Increasing genetic diversity by shuffling fragments of a family of

homologous genes or a pool of randomly mutated sequences arising
from a single gene:
(A) The "classical" DNA-shuffling strategy2 begins by fragmenting a pool of double-stranded
parent genes with DNase I. Selection of small fragments by size fractionation maximizes the
probability of multiple recombination events occurring, as the fragments cross-prime each
other in a round of PCR amplification. The full-length, diversified products are then obtained
by PCR amplification with terminal primers. (B) RACHITT begins with DNase I fragmentation
and size fractionation of single-stranded DNA, and hybridization—in the absence of
polymerase—to a complementary single-stranded scaffold. Any overlapping fragments leave
single-stranded overhangs that are trimmed down. The gaps between fragments are filled
in; the fragments are then ligated, yielding a pool of full-length, diversified single strands
hybridized to the scaffold. This scaffold, which is synthesized so as to include uracil (U), can
be efficiently fragmented so as to preclude its amplification. With PCR, it is replaced by a
new strand that is complementary to the diversified strand, and the whole is amplified.

Semi-Synthetic DNA Shuffling and Doramectin

Background
Doramectin is a drug used to treat gastrointestinal roundworms, lungworms, eyeworms, grubs, and
sucking lice in cattle. The drug is one of several avermectins, compounds used for the treatment of
parasites in animals and river blindness in humans. Over 1 billion dollars is spent each year on
avermectin derivatives in the United States.

Avermectins are produced by the soil-borne bacteria Streptomyces avermitilis. The

subspecies Streptomyces is the largest genus of antibiotic-producing bacteria; erythromycin, neomycin,
streptomycin, and tetracycline were all originally derived from species of Streptomyces.

The Experiment

The Goal
Researchers Stutzman-Engwall et al. began their experiment with a strain of S. avermitilis capable of
producing doramectin from supplemented cyclohexancaroxylic acid. This strain produced doramectin in
two forms: CHC-B1, the most useful form of doramectin, and CHC-B2, a related compound less effective
as an antiparasital than CHC-B1. The ratio of ineffective CHC-B2 to effective CHC-B1 produced by this
strain was 1:1.

The researchers sought to engineer a strain of S. avermitilis that would produce higher yields of the B1
form of doramectin. The team had already identified that the gene aveC was responsible for the B2:B1
ratio. This knowledge led the team to conduct directed evolution upon the aveC gene.

In the Lab
The researchers conducted three rounds of directed evolution on the aveC gene. To generate a mutant
library of the gene, the researchers used the mutagenic agent, Mutazyme, and semi-synthetic DNA
shuffling (see following section). The mutant aveC genes were inserted into gene replacement vectors,
which were then transformed into S. avermitilis cells. These cells were diluted into 96 well plates for high
throughput culturing under conditions suitable for doramectin production. Selection of the best mutants
was conducted by testing the doramectin output and B2:B1 ratios of each well (quantifiable with MS/MS).
DNA isolated was isolated from the best mutants and the aveC gene was amplified using PCR. The
amplified DNA from these mutants was recombined using semi-synthetic shuffling, subjected to random
mutagenesis, and resubmitted to selection.

After three rounds of directed evolution, the resulting strains of S. avermitilis produced doramectin with a
B2 to B1 ratio of 0.07:1 (Fig. 1); This ratio was significantly lower than the ratio in wild-type strain of S.
avermitilis (1:1).

(Stutzman-Engwall et al., 2005 - Permission Pending)

Figure 1 - Doramectin production profiles of thirteen strains of S. avermitilis transformed with the most
evolved form of the aveC gene after three rounds of directed evolution. Numbers above each bar
represent ratios of CHC-B2 to doramectin produced by the strain. Fermentation analysis for each strain
was conducted in a 30 mL shake flask under humidity for 12-14 days. Each strain in which wild-
type aveC had been replaced by the evolved aveC displayed a significant reduction in the B2:B1
(B2:Doramectin) ratio.

Conclusion
Semi-synthetic shuffling proved successful in quickly and efficiently improving the function of
the aveC gene. The method also appears adept for recombining beneficial mutations when using directed
evolution to improve genes (Fig. 4) and allows for some degree of rational input into directed evolution
through the use of oligonucleotides in shuffling.
DNA shuffling as a tool for protein crystallization

The success of structural studies performed on an individual target in small scale or on many targets in
the systemwide scale of structural genomics depends critically on three parameters: (i) obtaining an
expression system capable of producing large quantities of the macromolecule(s) of interest, (ii) purifying
this material in soluble form, and (iii) obtaining diffraction-quality crystals suitable for x-ray analysis. The
attrition rate caused by these constraints is often quite high. Here, we present a strategy that addresses
each of these three parameters simultaneously. Using DNA shuffling to introduce functional sequence
variability into a protein of interest, we screened crude lysate supernatants for soluble variants that retain
enzymatic activity. Crystallization trials performed on three WT and eight shuffled enzymes revealed two
variants that crystallized readily. One of these was used to determine the high-resolution structure of the
enzyme by x-ray analysis. The sequence diversity introduced through shuffling efficiently samples crystal
packing space by modifying the surface properties of the enzyme. The approach demonstrated here does
not require guidance as to the type of mutation necessary for improvements in expression, solubility, or
crystallization. The method is scaleable and can be applied in situations where a single protein is being
studied or in high-throughput structural genomics programs. Furthermore, it should be readily applied to
structural studies of soluble proteins, membrane proteins, and macromolecular complexes.
Random DNA Shuffing Using Recombination
DNA shuffling methods have been used to allow recombination between existing genes for related enzymes
(sometimes even from different species). This creates new genes through, what has been described
as molecular sex. 
The technique is being used to develop drugs, dyes, fragrances, flavours and cosmetics. Using these
recombined genes, enzymes can be produced in fastgrowing organisms suitable for large scale production.
An important example is the development of metabolic pathways for synthesis of carotenoid synthesis.

DNA shuffing as a method

1. Ancestral gene 2. Natural evolution 3. Species 4
4. Species 3 5. Species 2 6. Species 1
7. DNA shuffling 8. Hybrid genes