WHATS THE PROBLEM?

Our project deals with an everyday problem described by the following scenario: You want to enjoy a refreshing shower in the morning, but your hairy roommate clogged the drain again? You would like to have a relaxed bubble bath after a long day, but there are bad odors coming out of the pipe system?

The general practice now:
1.You try the ‘hot water method’ to flush the drain… nothing happens.
2.You remember what your mother taught you, so you put a fancy mix of vinegar, baking soda and some magic into the drain… a mysterious creature arises, but nothing else happens.
3.In the end there is just one thing left – you put the nasty chemical mixture of the stinky cleaning agent down the drain. The corrosive cloud disappears and you can finally take your shower… still trying not to breath in the acrid fumes.

Pipe clogging and closure occurs in every household as well as in industry. Various components contribute to the pipe closure, such as fat and hair. Commercial chemical pipe cleaners contain aggressive substances, which are harmful to the environment and health and also can lead to pipe breaks. Biological alternatives (purified enzymes) have been researched for years, without reaching the efficiency of chemical cleaners. In addition, the purification of the enzymes is very complex and cost intensive.

OUR SOLUTION

We pursue a system-biological solution which is based on an intact microbial system. Inspired by different interesting projects we decided to develope a keranolytic Escherichia coli. The projects of past iGEM-teams were part of our investigation too and we are already co-working with iGEM-team OLS_Canmore 2015/2016 to exchange information. We would like to use the knowledge of these groundworks to develop a new type of enzymatic cleaning agent by complementing and combining ideas out of different scientific sources. To differ from other projects we are aiming to engineer Escherichia coli to express enzymes like esterases, lipases and keratinases into the medium. Due to the extracellular expression expensive and time-consuming purification steps could be avoided and the enzymes could be secreted directly into the drain.As a special feature we want to use the metabolites of the enzymatic degradation to produce a lovely rose-scent to refresh your whole room. In addition this reaction could also be used to indicate that the enzymes are working efficiently on your drainage problem.

PART I - ESTERASES and LIPASES

Hair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. For the first degradation step we choose a combination of esterases and lipases. We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. Additionally we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. For the extracellular secretion of the enzymes we attached different signal sequences (pelB, OmpA and phoA) that were provided on the iGEM plates.

EstCS2

EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing these construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay.

LipB

LipB showed an enzyme activity of 2,8 U/mL in the supernatant. First, we repeated the enzyme activity assay from the iGEM TU Darmstadt 2012 to determine the esterase with the highest enzyme activity.

TliA

The lipase that is used for this project is the TliA lipase from Pseudomonas fluorescens. TliA is secreted by the ABC (ATP binding cassette) export system (prtDEF gene cluster) from Dickeya dadantii (formerly known as Erwinia chrysanthemi). The LARD secretion tag for the ABC export system is added directly to the TliA gene, which hopefully leads to a good export yield and a high extracellular activity. In order to control gene expression, the TliA gene is expressed by the pBAD promoter, which can be induced by arabinose. The prtDEF gene cluster is expressed by a constitutive promoter of mediocre strength. So, the ABC export system is always expressed at a certain level and can export the LARD-tagged TliA lipase, if expressed.Eom et al. have already shown extracellular activity of TliA when secreted with the ABC export system. So, if the TliA lipase is expressed, it is exported out of the cell and degrades the greasy layer surrounding the hair, which makes the keratin accessible for the keratinases.

PART II - KERATINASES

Keratinases are promising enzymes that find their applicability in agro-industrial, pharmaceutical and biomedicals fields. We want to profit from them by applying them on clugged pipes full of hair that is a common issue in lots of households. Keratinases are enzymes that are capable of degrading hair. Hair mostly consists of alpha-Keratin. Many different keratinases produced by different Bacilli, Actinobacteria and fungi have been reported. All of them vary by having their specific biochemical and biophysical properties e.g. temperature and pH activity range. Using their proteolytic capability that destroys hair we want to use them to avoid chemical compounds that are recently mainly applied to cleanse tubes. By hydrolization of disulfide bonds keratin degrades. This is due to confirmation changes which leads to an exposure of more sites for keratinase action (Satyanarayana et al. 2013, Vignardes et al. 1999). Previous projects from iGEM Teams such as Sheffield 2014, Team Canmore 2015 and Team Canmore 2016 helped us to tackle this problem as they were regarding similar problems. Still there are a lots of things that have to be improved as these Teams were facing different issues. The Team of Sheffield stumbled over the problem that the keratinase colonies were either not producing or exporting the protein in a functional form. While Canmore accomplished to succeed to show a certain Keratinase activity, due to time limitation they were not able to show quanititative but qualititative observations of keratine degredation. Based on these already promising results we want to focus on improving the keratinases using different promotors that regulate a successful protein secretations without being toxic to the host organism. For efficient hair degradation we chose different keratinases (KerUS, KerA, KerBL and KerP). Moreover we combined this keratinases with different anderson-promotors (BBa_K206000, BBa_J23114 and BBaJ23102) and signal sequences (PelB, OmpA and PhoA) for extracellular transport of the keratinases.

Inclusion bodies: extracellular transport as limiting step

Besides the fact that Escherichia coli is the most commonly used expression system for recombinant protein production, especially extracellular transport of expressed proteins appears to be one of the most significant barriers to generate necessary levels (Ni and Chen 2009). Often reported, recombinant protein production in Escherichia coli is following by formation of intracellular inclusion bodies, as in the case of recombinant keratinase expression in Escherichia coli as well(Wang, Swaisgood, and Shih 2003).Although the mechanism of extracellular protein secretion in Escherichia coli is still not understood completely, in some cases researchers could achieve high concentrations using different signal peptids like e.g OmpA (Choi and Lee 2004).

keratinase kerP

Keratinase kerP is a 33 kDa monomeric protein. Ker P is a serine protease. Serine proteases are protelytic enzymes, charaterizised by a reactive serine side chain (Kraut 1977). The family contains many diffrent enzymes with wide spread functions. Most of keratinases are serine proteases capable to degrade recalcitrant protein like nails, hair, feathers (Sharma and Gupta 2010b). KS-1 has his optimal activity at pH = 9 and 60 C.

PART III - A LOVELY SCENT OF ...

The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (1). In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (1,2,3). For our project we discussed different approaches and choose two different scents: rose and limonene.

... ROSE FRAGRANCE

As first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (98%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor (1). Therefor this odor can act as an indicator for keratin degradation. In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli (1). This pathway was used for our project and comprised four steps (Fig.1):

Aminotransferase (ARO 8) for transamination of L-phenylalanine to phenylpyruvate

2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde

Aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-Phenylethanol

Alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc.

.. LIMONENE FRAGRANCE

Limonene is a well-known cyclic monoterpene which can occur in two optical forms (2). (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy (2). The (L)-Limonene form has a more harsh fir-like odor with a lemon-note (2). For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli (Fig.2).