Project: Production of Omega 3 Fatty Acids using E. coli and Feedstock

This project delves into the production of Omega 3 fatty acids using Escherichia coli (E. coli) as the organism and lignocellulosic hydrolysate as the feedstock. The assignment begins with an introduction to Omega 3 fatty acids, their chemical characteristics, and the properties of lignocellulosic hydrolysate, highlighting the importance of these components in various industrial applications, including biofuels. The project then focuses on metabolic engineering strategies to optimize the production of Omega 3 fatty acids within E. coli, including the modification of metabolic pathways at the DNA level, and the optimization of central carbon metabolism. Furthermore, the project examines enzyme engineering, detailing how specific enzymes in the pathway can be engineered to improve product yield, providing examples from existing literature. The discussion covers both transcriptional and post-transcriptional regulation, highlighting the role of enzyme engineering in understanding and fine-tuning the significant components in the metabolic pathways. The analysis includes an overview of the functional reversal of the β-oxidation cycle and the dynamic sensor-regulator system (DSRS) to enhance fatty acid synthesis. The project concludes by demonstrating how these engineering techniques can be translated into industrial applications, such as the production of lubricants and diesel from lignocellulosic biomass, thus emphasizing the practical implications of the research.

PRODUCTION OF OMEGA 3 FATTY ACIDS 1
PRODUCTION OF OMEGA 3 FATTY ACIDS USING E.COLI AS YOUR
ORGANISM AND lignocellulosichydrolysate AS YOUR FEEDSTOCK
by [Name]
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PRODUCTION OF OMEGA 3 FATTY ACIDS 2
Introduction
Question 1: Introduction: What is the background of the product and
feedstock? What are their chemical characteristics?
Background of the product and feedstock
Omega 3 fatty acids are a polyunsaturated type of fat, which typically means
that the chemical structure of their carbon atoms has more double bonds. Most of
the natural Omega 3 bulk is found in fish, but some does exist in plants such as
walnuts, hemp, and flaxseed (Demirbas, 2009, p.89). Although the latter is quite
different, when it gets into the body, it gets converted into the type found within
fish. Scientists have no clear image of how the Omega 3 affects the human body,
but it has been proven to assist with cell growth. On the other hand, lignocellulose
hydrolysate facilitates an abundant renewable resource during generation of
polymers, chemicals, and biofuels. The feedstock includes residues from pulp
mills, biorefineries, forestry, and agriculture. Due to the need for a consistent
source of energy, there has been a significant focus of researches on the
biosynthesis of microbial fatty acids with the aim of producing Omega 3 fatty acids
and other substitute derivatives of diesel.
Chemical characteristics

PRODUCTION OF OMEGA 3 FATTY ACIDS 3
As an organism that is industrially significant and one that has the best result
of fatty acid biosynthesis, Escherichia coli has been regularly chosen as a producer
for these studies and several conclusions have been made in the field of E.coli bio-
fuels or omega three fatty acid biosynthesis.Omega-3 fatty acids are one of the
most researched nutrients. Linolenic acid (ALA- C18:3, omega-3) is not produced
in human body synthesis and thus has to be consumed orally in the diet. ALA is the
precursor of two essential bio-active long-chain polyunsaturated fatty acids (LC-
PUFA), which are the docosahexaenoic acid (DHA- C22:6, omega-3) and
eicosapentaenoic acid (EPA- C20:5, omega-3). ALA is converted to DHA and
EPA through the occurrence of desaturation reactions and enzymatic elongation,
where Δ-5 desaturase, Δ-6 desaturase,andelongase enzymes are involved. Omega 3
fatty acids are regarded as conditionally essential because usually, their
synthesized quantity is insufficient to meet human needs. When it comes to the
lignocellulosichydrolysate feedstock, it is ideally a non-edible plant material that is
primarily composed of hemicellulose and polysaccharides cellulose(Huffer, 2012,
p.30). Accurate compositional analysis of this feedstock and a good measurement
of its biomass are of prime significance as it is directly proportional to the omega-3
fatty acids yield.
2.Metabolic engineering:How will you modify the metabolic pathways of
your chosen organism to perform the reaction stated with optimal efficiency?

PRODUCTION OF OMEGA 3 FATTY ACIDS 4
Describe how this will be done at the DNA level, and what effect this will have
at the proteome and metabolomic levels. Note that heterologous genes will
need to be added to perform the reaction, but you must also address how
central carbon metabolism will be optimised.?
Modification of the Metabolic Pathways of the Organism
The metabolic engineering of the E.coli organism involves an iterative
process of analysis and synthesis, where progressively refined fatty acids strains
are constructed and designed with regards to past knowledge. Based on intuitive
guesses and existing literature evidence from Lee et al (2011), several strategies
are utilized to modify and improve the production of omega-3 fatty acids. The
strategies include regulation of the available precursors like malonyl-CoA and
malonyl-ACP (Lee et al. 2011b) as well reversal of -oxidation pathway genes
fade and fadD to avoid any degradation of the FAs. With reference to Yu et al.
(2011), it was apparent that overrepresentation of chain-elongation genes fabG,
fabZ and fabA encoded for the FAB pathway will also be performed. Moreover,
expression of native E.colithioesterasestesA and tesBwith the inclusion of
heterologous plant thioesterases from U.californica and C.camphorum has been
identified to produce omega-3 acids with unique carbon chain length (Yu et al.
2011, p.30). Research by Lee and other scientists showed that optimal expression

PRODUCTION OF OMEGA 3 FATTY ACIDS 5
of E.coli plant thioesteraseis guided by assumptions of ribosomal binding sites as
well as new thioesterases identifications such as the recently discovered
E.colithioesterase gene, fadM, which is involved in the β-oxidation pathway;
reveals improvement of both long-chain and medium-chain omega-3 fatty acids
production(Lee et al. 2011, p.22). However, the removal and regulation of the
competitive pathway towards acetate did not improve the flux of the omega-3 FAs,
thus proving the reversal of the beta oxidation cycle is the best method of
metabolic engineering. The coexistence and association of the positive
interventions mentioned before are usually used to boost the production of FA
from the E.coli organism monumentally. For instance, Lennen et al. (2010)
observed that 1,2g/L of omega-3 FAs (14% of theoretical yield) was produced with
deletion of fadE and fadD-oxidation gene of cytosolic tesAthioesterase
overexpression.
Modification at DNA level
In an elegant depiction of a metabolic engineering approach that is system-
wide, the biological system in existence was redesigned by reversal engineering of
the -oxidation gene pathways in E.coli; therefore leading to a noticeable increase
of the production yield of omega-3 fatty acids. With regards to Lee et al. (2011),
the cellular system was then reprogrammed by manipulating global regulators.

PRODUCTION OF OMEGA 3 FATTY ACIDS 6
Thus, mutations in AtoCregulon and FadR were brought forth to present the -
oxidation pathway enzymes when FAs are absent. The crp gene that is native was
replaced with a cAMP-independent mutant to alleviate the cataboliterepression in
the case of glucose present. To relieve the ArcA-mediated repression caused by
oxygen availability, the ArcA gene had to be deleted(Lee et al. 2011, p.22). In
association with deletion of the native fermentation and the degradation pathway of
the FA and the expression of selected gene pathway, extracellular C20-C22
omega-3 FAs were yielded at the titer of 7g/L in the bioreactor, with the medium
produce of mineral salts being 0.28g/g glucose (80% theoretical yield). Therefore,
redesigning and modifying the native FA biosynthesis in E.coli by utilizing a CoA-
based functional reversal of-oxidation which provides an efficient basement for
the production of the FAs.
Adopting the Dellomonaco (2011) conventional “push and pull” approach
enhanced the acetyl-CoA availability, minimizing the drains of acetyl-CoA, while
at the same time eliminating the competitive and overexpressed product pathways.
Ultimately, this led to an elongated-chain with nearly 100% maximum yield
theoretically for the production of the omega-3 fatty acids. Expression of fabZ in
the encoding of β-hydroxyacyl-ACP dehydratase improves the FA titer and
produce by drawing the carbon flux towards FA elongation cycle. Naturally
existing FA-sensing factors of transcription regulate and coordinate the

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degradation or synthesis of the omega-3 FAs at the transcription level
(Dellomonaco, Clomburg & Miller 2011, p.65). Whereby FabR antagonizes the
production of FA by suppressing fabA and fabB FAB genes and DNA, vice versa
for the FadR factor of transcription is true. By deletion of fabR and expression of
fadR, there was an indirect up-regulation of elongation reactions which eventually
showed an improvement in the FA yield and titer. In addition to the FAB terminal
pathway, some focus has been put on the central carbon metabolism to manipulate
and augment omega-3 FA production,but there has been little success. San et al.
(2012) proved that redirection of the TCA cycle flux (removal of fumAC,
gltA,andsucC) towards the production of fatty acids had a significant improvement
on the FA yield.
Furthermore, interruption of the gene and DNA in the glycolytic pathway
was also revealed to be a strategic manipulation of the genetic system (San et al.,
2012, p.27). In general, the combination of the fabZexpression and the removal of
sucC in a knockout strain of fadD boosted the production of omega-3 fatty acids to
5.7g/L C20-C22 (DHA – EPA) with a glucose yield of 0.38g/g (100% theoretical
yield). The technology used in the metabolic engineering of E.coli has been
translated through industrial collaborations toproduce synthesized lubricants and
diesel from lignocellulosic biomass.

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3. Enzyme engineering: State how an enzyme in the pathway could be
engineered to improve the product yield. Give examples from the literature of
this approach being used and describe the structure-function relationship for
the starting enzyme and the optimised, engineered enzyme.
Enzyme engineering in the Pathways, with examples of Literature use
Both transcriptional and post-transcriptional handling in E. colistrictly
regulates the omega-3 fatty acids biosynthesis. Engineering tools often fail to
provide any gene regulation inference; however, enzyme engineering plays an
essential role in understanding, modeling and fine-tuning the significant
components in the metabolic pathways. According to Clomburg et al. (2012) such
core pieces of the pathways enable the completion of specified performance
criteria, for instance acquiring the desired phenotypes, once integrated into broader
biological systems. The synthetic engineering approach straightens the enzymatic
pathways of the E.coli organisms and lignocellulosic hydrolysate feedstock,
allowing the optimized transfer from one system to another. As illustrated by
(Clomburg et al. 2012, p.25), one can use of a bottom-up strategy to restructure a
β-oxidation cycle functional reversal for producing omega-3 fatty acids through the
assembly of self-contained and well-defined enzymes such as acetyl-CoA
carboxylase and FA synthase (FAS), when composing the pathway. Functional

PRODUCTION OF OMEGA 3 FATTY ACIDS 9
reversal of the cycle is made up of thiolase (AtoB, FadA), enoyl-CoA hydratase
(FabB), 3-hydroxyacyl-CoA dehydrogenase (FabB), and acyl-CoA dehydrogenase
(YdiO, FadE, egTER). Jing et al. (2011) proved that each intermediate CoA in the
oxidation cycle can be converted to fatty acids with the thioesterase termination
pathways. In the aftermath of the in-vitro kinetic characterization, egTER, AtoB,
and FabB were assembled in the E.coli together with the existing thioesterase
termination pathway, yielding 3.43g/L butyrate and 0.35g/g (a 74% theoretical
yield); which is a great improvement. The in-vitro kinetic analysis of the enzymes
revealed the ability of FadAthiolase on the longer FA chains like acyl-CoA(Jing et
al 2011, p.7). For the enzyme synthesis of longer chain carboxylic acids, a
functional reversal could be done on multiple β-oxidation cycles through
integrating AtoB, FadBA,andegTER into a host carbon strain. The success in
similar self-contained enzyme units in the β-oxidationcycle functional reversal
provides a platform for the efficient improvement and production of omega-3 FAs
using enzyme engineering techniques.
Despite the genetic engineering advent, Zhang et al. (2012) insisted that
metabolic imbalance with decreased expression pathway genes may become a clog
or bottleneck in biosynthetic enzyme pathways. Enormously increased levels of
gene expression overwork cellular resources due to unnecessary cell maintenance,
instead of focusing the resources to yield the desired chemicals and fatty acids.

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Researchers have developed the dynamic sensor-regulator system (DSRS) to allow
active control of FAs synthesis and the biodiesels derived from E.coli (Zhang et al.
2012a, p.35). An acyl-CoA/FA sensor was engineered on the basis of the FadR
protein and related regulator in the pathway; which was meant to improve the limit
of the dynamic ranges of the native promoters that are FadR-regulated. Enzyme
engineered biosensors responded initially to acyl-CoA and served the purpose of
an indirect FA sensor (Jing et al 2011, p.11). With the introduction of this
biosensor, the omega-3 FA-yielding E.coli strain with a fadE reversal and a tesA
expression produced 3.8g/L FA (~56% theoretical yield). FadR overexpression
maximally tuned the expression levels of the fatty acids pathway enzymes for the
improved production of the FAs.
Enzyme engineering enables the systematic investigation and analysis of
pathway limitations and removes any bottlenecks that may tightly regulate the
production of the omega-3 FAs. Xu et al. (2013) believed that the specified
expressions of enzymatic reactions may enhance the carbon flux towards the
corresponding and precursor products. The depletion or accumulation of any
intermediates should be avoided to ensure no loss of pathway productivity or cell
viability. Recently, the bio-engineering workers applied modular enzymatic and
synthetic strategies to improve the fatty acids pathway transcription, which
consisted of intermediary acetyl-CoA activation, upstream acetyl-CoA, and fatty

PRODUCTION OF OMEGA 3 FATTY ACIDS 11
acid synthase modules (Xu et al. 2013, p 10). Moreover, the efficiency of
translation could be increased by customization of the ribosomal binding sites in
the modular fatty acid pathways; thus improving the production of the omega-3
fatty acids. The combination of these tools in enzyme engineering yielded 8.6g/L
fatty acids (~22% theoretical yield) in the fermentation fed-batch.
4. Immobilization: How you will immobilise the cells? What are the
engineering constraints governing the immobilisation?
Immobilization of the E.coli Cells and the Engineering Constraints
The method utilized to immobilize the Escherichia cells is by using porous
biomass support particles (BSPs), which is pretty convenient and simple to
perform. This is because it relies on the adhesive cells inherent ability, due to their
growth, to form films all around the support material. In recent researches by Kim
et al. (2011), the immobilization of E.coli cells by using BSPs was determined to
be a shake-flask culture. The density of the immobilized cells was evaluated by
measuring the activity of their intracellular lactate dehydrogenase (LDH) (Kim et

PRODUCTION OF OMEGA 3 FATTY ACIDS 12
al. 2011, p.9). As the E.coli K12 cells were unsuccessful and constraint when being
retained within the reticulated PVF (polyvinyl formal) resin BSPs with relatively
small pore matrices, coating the BSP surface with a variety of polymers was
determined as a way of improving cell attachment. The major constraint witnessed
with this method is the limitation during the diffusion of products and substrates
through the E.coli cell wall, followed by poor holding capacity of the porous
biomass. Regardless of the engineering constraints of retaining, the results
indicated that E.coli cells are effectively immobilized with resin BSPs by the
electrostatic association between positively charged polymers on the BSP surface
and the negatively charged ions in the E.coli cells.
5. In particular, what size will the particles containing the immobilised cells be,
and why? You will need to use maths to justify this.
Math Justification of the Size of the Particles in the Immobilized Cells
After BSP immobilization, the average size of the E.coli cells particles has a
2.5 ratio of length-to-width increase from original size. The average population of
particles in all immobilization stages is a ratio of 3.9 from original population after
electrostatic association. Therefore, it is clear that the E.coli cells fall perfectly in
the size realm that provides minimum resistance to movement. Actually, of all the
immobilized cells, 21% are spherical, 10% of the particles are coccoid, while the
rest are rod-like. This favors the researchers’ notion that motility of particles favors