Download - Bioremediation and synthetic biology
Bioremediation is a technology that utilizes
the metabolic potential of microorganisms
to clean up contaminated environments.
It constitutes an attractive alternative to
physicochemical methods of remediation,
may be less expensive, less harmful to
the environment, etc.
Here are some examples:
Marine petroleum hydrocarbon degradation:
Spilled-oil bioremediation experiments show that the bacteria
involved in degradation are some gropus of α-Proteobacteria, Pseudomonas and Cycloclasticus groups (γ-Proteobacteria)
and the genus Alcanivorax.
Metal bioremediation
Studies show that the major groups of bacteria capable of removing
metals from the environment are α-Proteobacteria ,Actinobacteria and some some species of the genus Ralstonia,
like Ralstonia metallidurans o Ralstonia eutropha.
However, although a large number of microorganisms have been
isolated in recent years that are able to degrade compounds
previously considered to be non-degradable, there are a number of
factors, some non-biological, that contribute to the persistence of
some pollutants in the environment, one of which is the fact that
current pathways for the metabolism of xenobiotics are not optimal.
This is particularly true for highly toxic pollutants such as dioxins,
dibenzofurans and PCBs, for which effective pathways have not yet
been described. Moreover, most microbial activities that can serve
as the basis of biotechnological applications do not function
optimally under process conditions and can almost always be
improved.
Thus, biotechnology allows the design of improved biocatalysts
involves different aspects of optimization.
In some cases, a simple two- or three member
consortium is obtained, one member of which may
carry out the initial catabolic reaction , and another of
wich may complete the sequence.
Such consortia have been developed for the
mineralization of bicyclic aromatics such as
chlorinated biphenyls (PCBs), chlorinated dibenzo-
furans and aminonaphthalenesulfonates
But the metabolic “division of labour” in co-
cultures of aerobic microorganims may not
constitute the most effective situation for the
bioremediation in natural environment .
A variety of strategies for designing new or imprived catalysts for
bioremediation have been developed over recent years:
The simplest strategy is improving the biodegradative performance of
a consortium (a mixed bacterial culture) through the addition of a
“specialist” organism; in this case, a consortium is designed Consortia that
exhibit novel catabolic activitie.
The most effective strategy is the transfer of catabolic
genes from its original host to an appropriate
recipient , that result in the combination of a central
pathway with a pathway sequence that enable a new
substrate to be channelled into the central pathway.
Gene cloning generally circumvents barrier to natural gene
transfer and, of course, involves precisely
predertermined genes and expression signals.
Plasmid cloning
vector may,
however, suffer from
the same instability
as natural plasmids
and, moreover, have
antibiotic-resistance
selection makers,
which are
undesirable from
enviromental
applications.
For these reasons , minitransposon cloning vector have been developed to insert heterologous genes stably into the
chromosomes of host bacteria without the use of antibiotic-resistance makers or, more recently, with makers that can be
selectively eliminated after gene transfer.
As the transposase gene is not cotransferred, the transposon vector do not
cause sequence instability or rearrangements at the site of transposition, not
do they mediate inmunyty to transposición. They can therefore be used for
multiple, sequential cloning evets in the same host organism.
The University of Minnesota Biocatalysis/Biodegradation Database
should soon offer a systematic display of theoretical routes from one substrate
to a specific intermediate or central metabolite. http://umbbd.msi.umn.edu/index.html
Some bioremediation processes require an increase in the rate of pollutant removal.
Achieving this goal involves identifying the enzymatic or regulatory step fo the pathway that is rate limiting, followed by experimental
elevation of the activity of the rate-limiting protein through an increase in the transcription or translation of its gene, or in its
stability or kinetic properties.
Transcription of the genetic determinants of metabolic pathways, which are usually organized in operons, is generally controled by positively-acting regulatory proteins
that are activated by pathway substrates or metabolites.
Mutants of regulatory proteins have been produced that either mediate higher levels of transcription than the wild-tipe regulator or respod to new effectors.
The use of artificial regulatory systems allows the expression of catabolic genes to be uncoupled from the signals that ordinarily control their expression and offers
considerable flexibility for process control.
Thus, the use of starvation-induced promoters can uncouple gene expression from growth and augment catabolic activity in nutrient-limited environments or when target
pollutants fall below certain thresholds.
Protein engineering can be exploited to improve an enzyme´s stability specifity and kinetic properties.
However, the number of degradative enzimes whose structure has been elucidated still small and this constitutes a major limitation for
rational protein design.
An alternative to improve enzyme activity is combining the best attributes of related enzymes is to extange subunits or subunit
sequences.
A more recently developed and powerful alternative method for obtaining proteins with new activities involves shuffling their gene
sequences.
It is sometimes assumed that a major problem in the use of designed inoculants is their poor competitiveness in natural environments.
Nevertheless, improving inoculant survival is an important goal in the further development of bacterial inocula for biotechnological
applications in the environment, where the microorganisms are exposed to a variety of stresses such as toxic metals, solvents and
extremes of temperature and pH.
The combination of resistance to environmental stresses and catabolic phenotypes in appropriate bacterial strains is expected to yield
microbial catalysts with significantly improved survival characteristics in hostile habitats. For example, solvent-resistant bacteria able to mineralize hydrophobic pollutants have recently
been engineered.
However, the most striking case is the use of Deinococcus radiodurans
for the metal remediation in radioactive mixed waste environments.
The high cost of remediating radioactive waste sites from nuclear
weapons production has stimulated the development of
bioremediation strategies using this bacteria, the most radiation
resistant organism known.
Using genetic engineering, deinococos have been used in
bioremediation to consume and digest solvents and heavy metals,
even in highly radioactive areas.
The mercuric reducing bacterial gene of Escherichia coli (merA) was
cloned in the deinococo to detoxify ionic mercury commonly found in
radioactive wastes from the manufacture of nuclear weapons. The
same engineers developed a kind of deinococo able to detoxify
mercury and toluene in mixed radioactive wastes.
Synthetic biology is the engineering of biology: the synthesis of
complex, biologically based (or inspired) systems which display
functions that do not exist in nature.
This engineering perspective may be applied at all levels of the
hierarchy of biological structures – from individual molecules to
whole cells, tissues and organisms. In essence, synthetic
biology will enable the design of ‘biological systems’ in a
rational and systematic way.
In 2002 researchers at SUNY Stony Brook succeeded in synthesizing the 7741 base poliovirus genome from its published sequence, producing the first synthetic organism. This took about two years of painstaking
work.
In 2003 the 5386 bp genome of the bacteriophage Phi X 174 was assembled in about two weeks.
In 2007 it was reported that several companies were offering the synthesis
of genetic sequences up to 2000 bp long, for a price of about $1 per base pair and a turnaround time of less than two weeks.
As of the present date, September 2009, the price has dropped to less than $0.50 per base pair with some improvement in turn around time. Not
only is the price judged lower than the cost of conventional cDNA cloning, the economics make it practical for researchers to design and purchase multiple variants of the same sequence to identify genes or
proteins with optimized performance.
Recently, a group of scientists headed by Craig Venter have created a cell
controlled entirely by man-made genetic instructions -- the latest step
toward creating life from scratch. The achievement is a landmark in the
emerging field of "synthetic biology," which aims to control the behavior of
organisms by manipulating their genes.