Report: Enzyme Engineering in E. coli for Omega 3 Fatty Acid Synthesis

This report delves into the enzyme engineering of omega 3 fatty acids in E. coli, focusing on the enzymes involved in the biosynthesis pathway. It examines the roles of key enzymes like Acetyl-CoA carboxylase, Malonyl-CoA: acyl carrier proteins transacylase, and various synthases and dehydrases, detailing their impact on fatty acid production. The report then highlights the potential of engineering mutant acyl-CoA thioesterase I (TesA) to enhance omega 3 fatty acid yields, supported by literature examples. It discusses the structure-function relationships of the enzyme and the engineered mutants, demonstrating how modifications can improve catalytic activity and overall production rates. The study emphasizes the importance of optimizing the ratio of enzymes and engineering their catalytic activity to achieve maximum omega 3 fatty acid synthesis. The report concludes by illustrating how metabolic engineering of E. coli can produce high yields of omega 3 fatty acids from lignocellulosic hydrolysate, presenting this as a promising approach for biofuel and industrial product development. The report is a valuable resource for students and researchers interested in biotechnology and metabolic engineering, offering a detailed overview of the subject with supporting references and findings from the literature.

ENZYME ENGINEERING IN ECOLI TO PRODUCE OMEGA 3 FATTY ACIDS FROM
LIGNOCELLULOSIC HRDROLYSATE
Student name
Chemistry
Lecturer
Institution
25th January, 2019
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Introduction
Fatty acids are very suitable ingredients in production of biofuels, industrial products and
consumer products leading to a lot of studies towards production of microbial oleochemicals.
Microbial production is beneficial in that it takes little time to produce, cost- effective
production process and is a direct process. These advantages of microbial production of fatty
acids have increased investigations that purpose to increase their production through better
engineered processes (Kwang et al. 2016).
Impact of each enzyme on the manufacturing of omega 3 fatty acids
Acetyl-CoA carboxylase
This is the first enzyme and is the one responsible for increasing the length of the chain and
facilitates transfer of acetyl-Coa to de-novo fatty acid where biosynthesis occur. Two
subcomplexes are produced by purifying four subunits which are in unstable complex. They
are the biotin carboxylase-biotin carboxyl carrier protein comprising of AccC and AccB, the
other one is the carboxyl transferase which consists AccA and AccD. This reaction can occur
in two different reactions with the carboxylation of biotin under activation of ATP in
presence of Mg2+-ions, secondly there is movement of the carboxyl group to acetyl-CoA
amounting to malonyl-CoA (Selwood & Jaffe 2012).
The subunits are supposed to be synthesized in equimolar quantities therefore there should be
strict regulation of transcription of accABCD.Carboxylation of acetyl-Coa requires energy
because it is driven by ATP cleavage. AccB and AccC which constitute mRNA have their
transcription being more so that it can be auto regulated by the N-terminal domain of
AccB.Studies show that acyl-ACP with spans of C6 to C20 inhibit the enzyme activity of the
E. coli acetyl-CoA carboxylase (Desbois & Smith 2010).
Malonyl-CoA: acyl carrier proteins (ACPs) transacylase
It catalyses the exchange of malonyl-moiety to acyl carrier proteins which guides it to
approach unsaturated fat neogenesis and unsaturated fat chain extension. Research
demonstrates that cancellation of gene fabD (gene of ACP transacylase) is dangerous and its
overexpression in E coli encourages the alteration of structure of the unsaturated fats. The
shifted extents of palmitoleic acid and cis-vaccenic acid is brought about by malonyl-ACP
pool adding to movement of 3-ketoacyl-acyl carrier proteins synthase II (FabF), an enzyme
in charge of tie stretching of C16:1 to C18:1. Measure of free unsaturated fat is expanded by
about 11% when there is overexpression of the Escherichia coli gene fabD, concurrently with
acyl-CoA thioesterase I contrasted with when there is overexpression of thioesterase quality
alone (Amiri-Jami & Griffiths 2011) .The fab D quality interpreted in E. coli prompts a
somewhat adjusted unsaturated fat piece. The extent of palmithe toleic acid reductions though
the extent of cis-vaccenic acid increments.
FabB, FabF and FabH: 3-ketoacyl-ACP synthase I, II and III
This enzyme through buildup of fatty acyl-acyl carrier proteins with malonyl-acyl carrier
proteins catalyzes the development of 3-ketoacyl-ACP. FabH start the main cycle of chain
prolongation amid unsaturated fat biosynthesis while FabF and FabB play out the ensuing
lengthening steps (Nikaido & Takatsuka 2009).
FabA and FabZ: 3-hydroxyacyl-ACP dehydrase
This enzyme perform dehydration of 3-hyroxyacyl-ACP (Kosa & Ragauskas 2011). Trans-2-
decenoyl-ACP is isomerized by FabA into cis-3-decenoyl-ACP making the primary response
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towards amalgamation of unsaturated fats. FabA speeds up the deciccation of saturated 3-
hydroxyacyl-ACPs with various chain spans and its overexpression builds the measure of
immersed unsaturated fats. It can likewise get dehydrated 3-hydroxydecanoyl-CoA with a
movement of 11 percent in contrast with 3-hydroxydecanoyl-ACP. FabZ performs drying out
of 3-hydroxymyristoyl-ACP (Walther & Farese 2012).
FabI: enoyl-ACP reductase
This enzyme encoded by FabI speeds up the decrease of 2-enoyl-ACP to fatty acyl-ACP to
the detriment of NADPH + H+ or NADH + H+. Palmitoyl-ACP hinder the enoyl-ACP
reductace at focus which is around 50 times high. Too much of the fabI gene does not lead to
any development imperfection and does not build the cell lipid, palmitic acid or stearic acid
substance (Wymann & Schneiter, 2008).
ACP, ACP synthase and ACP phosphodiesterase
A phosphopantethein class is appended to a serine of the deciphered apo-ACP by the activity
of the acyl carrier proteins synthase (AcpS) in order to get the physiologically dynamic
frame. The physiological work of ACP is to separate unsaturated fat biosynthesis where all
transitionals are bound to acyl carrier proteins from unsaturated fat catabolism. In escherichia
coli, acyl carrier proteins speaks to 0.25% of every single soluble protein. Overexpression of
acpS prompts end of cell development which is because of solid hindrance of the glycerol-3-
phosphate acyltransferase. Heterologous articulation of acpP provides some capabilities, as it
has been demonstrated that the outflow of acpP from Azospirillum brasilense modifies the
Escherichia coli unsaturated fat profile and the substance of C18:1 is expanded to 2-overlay
at 30°C (Walther & Farese, 2012).
Impact of engineered enzyme on the quantity of omega 3 fatty acids
The enzyme that I will design is mutant acyl-CoA thioesterase I (TesA) in light because it has
high action in E coli (Wymann & Schneiter 2008). This was concentrated by developing a
detecting framework with a combination protein of antibiotic medication obstruction and red
fluorescent protein under the control of FadR responsive promoter which selects suitable
mutants. TesA mutant that produces double the quantity of omega 3 fatty acids was separated
from an error-prone PCR mutant collection of E.coli TesA. The kinetic analysis showed that
replacement of Arg64 with Cys64 in the enzyme causes roughly double increase in catalytic
activity.
In order to increase the rate of production of omega 3 fatty acids it is more preferable to
increase the ratio of every component including fatty acid synthase and TesA which enhances
optimal production. Engineering the catalytic activity of TesA is also necessary than just
increasing the enzyme expression in order to enhance omega 3 fatty acids (Kwang et al.
2016).
When acyl-Coa thioesterase was combined with acetyl-Coa carboxylase and acyl-Coa
synthase removed, an engineered E coli strain was established to easily synthesize omega 3
fatty acids. From induced culture, under shake flask conditions 244.8mg/l of omega 3 fatty
acids were acquired. Mass production of hydroxy fatty acids was obtained when fatty acid
hydroxylase was brought into the strain. Ultimately engineered strain in the culture broth
gathered up to 58.7 mg/L of hydroxy fatty acids. Almost 24 % of the Free Fatty Acids that
was produced by the thioesterase ezyme were transformed to Hydoxy Fatty Acids.
Investigation on Fatty acid constitution revealed that the Hyroxy Fatty Acids mainly
composed of 9-hydroxydecanoic acid, 11-hydroxydodecanoic acid, 10-hydroxyhexadecanoic
acid and 12-hydroxyoctadecanoic acid. The strain was finally fermented leading increase of
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amount of fatty acids from 244.8mg/l to 548 mg/L thus making this a better enzyme
engineering route (Yuhin et al. 2016).
Conclusion
Omega 3 fatty acids produced in metabolically engineered Escherichia coli cells have been
beneficial since it is being converted into various molecules such as consumable products,
industrial products and fuels.
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References
Amiri-Jami, M. & Griffiths, M., 2011. Amiri-Jami M, Griffiths MW. Recombinant production of omega-
3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1.. [Online]
Available at: 10.1111/j.1365-2672.2010.04817.x
[Accessed 24 01 2019].
De Bhowmick, G., Sarmah, A. & Sen, R., 2018. Lignocellulosic biorefinery as a model for sustainable
development of biofuels and value added products. Bioresource Technology, pp. 1144-1154.
Desbois, A. & Smith, V., 2010. Antibacterial free fatty acids: activities, mechanisms of action and
biotechnological potential.. Appl Microbiol Biotechnol, Volume 85, pp. 1629-1642.
Kosa, M. & Ragauskas, A., 2011. Lipids from heterotrophic microbes ,advances in metabolism
research. Trends Biotechnol., s.l.: PubMed.
Kwang, S., Sangwoo, K. & K.L, S., 2016. Biotechnology for Biofuels. [Online]
Available at: https://doi.org/10.1186/s13068-016-0622-y
[Accessed 25 01 2019].
Nikaido, H. & Takatsuka, Y., 2009. Mechanisms of RND multidrug efflux pumps. Biochim Biophys, pp.
769-781.
Selwood, T. & Jaffe, E., 2012. Dynamic dissociating homo-oligomers and the control of protein
fuction, 519(2), pp. 131-43.
Walther, T. & Farese, R., 2012. Lipid droplets and cellular lipid metabolism. pp. 686-714.
Wymann, M. & Schneiter, R., 2008. Lipid signalling in disease, s.l.: PubMed.
Yuhin, C. et al., 2016. metabolic engineering of Escherichia coli for the production of hyroxy fatty
acids from glucose. BMC Biotechnology, pp. 1-9.
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