Engineering substrate specificity into a promiscuous ancestral diterpene synthase

Terpene synthases are a class of enzymes that catalyse the cyclisation of linear unsaturated hydrocarbons into a plethora of cyclic structures, of which many are industrially relevant as fragrances, flavours or due to their medical properties. Despite the structural and functional diversity of their products, members of this enzyme family show a high degree of structural and functional similarity, which is why the rules defining product and substrate specificity are not fully understood. Recently, we have used ancestral sequence reconstruction to design a hypothetical molecular ancestor of spiroviolene synthase - a diterpene synthase from Streptomyces violens that converts the linear C-20 precursor geranylgeranyl pyrophosphate (GGPP) to spiroviolene1. This ancestral enzyme shares 77 % sequence identity with the modern wild-type enzyme from S. violens. Compared to the modern enzyme, the ancestral enzyme showed increased thermostability and an additional new reactivity with the shorter C-15 substrate farnesyl pyrophosphate (FPP) that the modern enzyme did not display. To the best of our knowledge, this is the first time ancestral sequence reconstruction was used on a diterpene cyclase. Based on the ancestral enzyme, a library of enzyme-variants was designed with the aim to influence the substrate specificity of the promiscuous ancestral enzyme. We identified several variants that showed substantial preference for the native substrate GGPP (modern enzyme-like), which demonstrates that the subtle GGPP-preference of the ancestral enzyme could be “evolved” to the GGPP-specific modern enzyme phenotype. Most interestingly, we were also able to identify a few variants that showed reversed substrate preference for FPP over GGPP and thus demonstrated “evolvability” of the ancestor towards the unpreferred shorter substrate. Taken together these findings suggest that the hypothesis of promiscuous evolvable ancestral enzymes might be appropriate for this member of the diterpene synthase family. Moreover, it opens up the exciting prospect of using ancestral sequence reconstruction as a tool to engineer enzyme specificity – either by introducing new desired functionalities next to an already existing one or by reprogramming existing promiscuity towards a desired substrate specificity. Please click Additional Files below to see the full abstract

Systematic screening for the biocatalytic hydration of fatty acids from different oily substrates by Elizabethkingia meningoseptica oleate hydratase through a Design-of-experiments approach

The edible plant oils production is associated with the release of different types of by-products. The latter represent cheap and available substrates to produce valuable compounds, such as flavours and fragrances, biologically active compounds and bio-based polymers. Elizabethkingia meningoseptica Oleate hydratases (Em_OhyA) can selectively catalyze the conversion of unsaturated fatty acids, specifically oleic acid, into hydroxy fatty acids, which find different industrial applications. In this study, Design-of-experiment (DoE) strategy was used to screen and identify conditions for reaching high yields in the reaction carried out by Escherichia coli whole-cell carrying the recombinant enzyme Em_OhyA using Waste Cooking Oils (WCO)-derived free fatty acids (FFA) as substrate. The identified reaction conditions for high oleic acid conversion were also tested on untreated triglycerides-containing substrates, such as pomace oil, sunflower oil, olive oil and oil mill wastewater (OMW), combining the triglyceride hydrolysis by the lipase from Candida rugosa and the E. coli whole-cell containing Em_OhyA for the production of hydroxy fatty acids. When WCO, sunflower oil and OMW were used as substrate, the one-pot bioconversion led to an increase of oleic acid conversion compared to the standard reaction. This work highlights the efficiency of the DoE approach to screen and identify conditions for an enzymatic reaction for the production of industrially-relevant products

Exploitation of Microalgae Biomass Under an Integrated Biorefinery Approach

As known, microalgae are an appealing source of chemicals and high-value compounds which find application in nutraceuticals, cosmetics and pharmaceutics. Fatty acids (FA), in particular, have drawn attention to the possibility of employing them as a source of biodiesel alternatively to fossil fuels. In addition, several lipid derivatives have been found in microalgae and may be employed in several biotechnological applications. Hydroxy fatty acids can be substrates for several industrial applications thanks to their functionalization, which increases their reactivity and, for this reason, can be used as functional building blocks to produce a multitude of bio-based materials. Recently, a promising method for the chemical modification of unsaturated-FAs (U-FA) has appeared. In fact, U-FA may be modified by members of the hydratase enzyme family to produce saturated and unsaturated hydroxy fatty acids with high stereo- and regio-selectivity. These enzymes are able to introduce a water molecule to the double bond present in the free fatty acids (FFA) Oleic Acid (OA), Linoleic Acid (LA), producing 10-hydroxy fatty acids (10-hydroxy-FAs). Furthermore, the carbohydrate component of the microalgal biomass may be converted into furfuryl compounds and, in particular in 5-hydroxyl methyl furfural (5-HMF). This is one of the chemical bio-compound different from petroleum-derived ones with the highest added value and may be obtained through lignocellulosic biomasses or hexoses sugars through acid catalysis. It is defined platform molecule because it is the precursor of several compounds for the chemical industry. In this work, we aimed to optimize a circular bioprocess by performing, starting from the same biomass, two different processes: the biotransformation of microalgal FFAs through the employment of a genetically modified E. coli on one side, and the conversion of the remaining biomass in furfuryl products. The first process allowed the production of very interesting lipid derivatives with biotechnological applications, including 10 hydroxy-stearic acid and 10-hydroxy-octadecenoic acid. The second process was obtained through heterogeneous catalysis based on niobium phosphate. This procedure represents a high-innovative application of microalgal biomass and allows the simultaneous exploitation of FAs and carbohydrates. This may result in an increase in the commercial value of microalgal biomass

Polyester derivatives of unsaturated fatty acids and process for their production

The present invention relates to a polyester consisting of hydroxyacid monomer units wherein the hydroxyacid monomer unit is 10-hydroxy stearic acid (10-HSA) having a hydroxyl group in position C-10 involved in the ester bond, wherein said hydroxyacid monomer units are present in a number ranging from 10 to 100 and/or the polyester has a number average molecular weight ranging from 2,800 to 28,000 Da, said number average molecular weight being determined by means of the SEC method. The invention also comprises the process for the preparation and use of said polyester

SELECTIVE BIOCATALYTIC HYDRATION OF FATTY ACIDS FROM WASTE COOKING OILS FOR HYDROXY FATTY ACIDS AND POLYESTER SYNTHESIS

The development of biorefinery approaches is of great relevance for the sustainable production of valuable compounds. In accordance with circular economy principles, waste cooking oils (WCOs) are renewable resources and biorefineries feedstocks contributing to a reduced impact on the environment. Commonly, this waste is wrongly disposed of into municipal sewage systems creating problems for the environment and increasing treatment costs in wastewater treatment plants. In this study, regenerated WCOs intended for the production of biofuel were transformed through a chemo-enzymatic approach to produce hydroxy fatty acids, which were further used in polycondensation reaction for polyester production. Escherichia coli whole cell biocatalyst containing the recombinantly produced Elizabethkingia meningoseptica Oleate hydratase (Em_OhyA) was used for the biocatalytic hydration of crude WCOs-derived unsaturated free fatty acids for the production of hydroxy fatty acids. Further hydrogenation reaction and methylation of the crude mixture allowed the production of (R)-10-hydroxystearic acid methyl ester that was further purified through column chromatography with the production of a high purity (> 90 %) product. Different intermediates were analyzed by GC-MS, HPLC and NMR to identify the specificity of the biocatalytic reaction and the stability of the compounds during further chemical transformation. The purified (R)-10-hydroxystearic acid methyl ester was polymerized through a polycondensation reaction for the production of the corresponding polyester which was analyzed by SEC and DSC to characterize both molecular weight and physical properties. This work highlights the potential of enzymes for selective transformation of waste products and for the production of bio-based polyesters through a biorefinery approach

SELECTIVE BIOCATALYTIC HYDRATION OF FATTY ACIDS FROM WASTE COOKING OILS FOR HYDROXY FATTY ACIDS AND POLYESTER SYNTHESIS

The development of biorefinery approaches is of great relevance for the sustainable production of valuable compounds.1 In accordance with circular economy principles, waste cooking oils (WCOs) are renewable resources and biorefineries feedstocks contributing to a reduced impact on the environment.2 Commonly, this waste is wrongly disposed of into municipal sewage systems creating problems for the environment and increasing treatment costs in wastewater treatment plants. In this study, regenerated WCOs intended for the production of biofuels3 were transformed through a chemo-enzymatic approach to produce hydroxy fatty acids, which were further used in polycondensation reaction for polyester production. Escherichia coli whole cell biocatalyst expressing the recombinant Elizabethkingia meningoseptica Oleate hydratase (Em_OhyA)4 was used for the biocatalytic hydration of crude WCOs-derived unsaturated free fatty acids for the production of hydroxy fatty acids. Further hydrogenation reaction and methylation of the crude mixture allowed the production of (R)-10-hydroxystearic acid methyl ester that was further purified through column chromatography with the production of a high purity (> 90 %) product. Different intermediates were analyzed by GC-MS, HPLC and NMR to identify the specificity of the biocatalytic reaction and the stability of the compounds during further chemical transformation. The purified (R)-10-hydroxystearic acid methyl ester was polymerized through a polycondensation reaction for the production of the corresponding polyester which was analyzed by SEC and DSC to characterize both molecular weight and physical properties. This work highlights the potential of enzymes for the selective transformation of waste products and for the production of bio-based polyesters through a biorefinery approach

Characterization of a new alpha/beta hydrolase from Pelosinus fermentans for polyesters hydrolysis

A new alpha/beta hydrolase gene from Pelosinus fermentans (Pfe_Lip) was optimized against Escherichia coli codon usage and cloned into the pET26b(+) vector, containing a 6xHis-Tag downstream the polylinker, and transformed into the bacterial cells. The Pfe_Lip is a member of the serine hydrolases containing a modification in the common –GxSxG– motif in the first glycine to have the –AxSxG– motif. This enzyme can be grouped in the I.5 subfamily (Nardini M, 1999). To improve the expression of this enzyme in the soluble fraction, a lactose-containing medium was used for the expression of the enzyme. Western blotting was carried out for revelation of the enzyme with an antibody against the His-Tag and further purification was carried out by means of Immobilized Ion Metal Affinity Chromatography (IMAC) with Nickel-coated columns for the binding of the His-Tagged enzyme. The Pfe_Lip purified was tested in hydrolase activity assay with p-nitrophenylbutyrate (p-NPB) (Herrero Acero E, 2013). Pfe_Lip had the pH optimal at 8 (0.1 M sodium phosphate buffer). Analysis of homology shows high identity with enzymes containing a zinc ion in the structure and a lid covering the active site, containing serine, aspartic acid and histidine. The enzyme was able to hydrolyse polyesters like Ecoflex based on HPLC quantification of the solubilized hydrolysis products

Truncation of an esterase enhances the hydrolysis on polyesters: Less is more

Polymers, and especially polyesters, frequently used for one-way applications like packaging are preferably biodegradable, albeit non-biodegradable polyesters are mostly used, namely polyethylene terephthalate (PET). Biodegradability of the aliphatic/aromatic copolyester poly(butylene adipate-co- terephthalate) (PBAT) has been investigated, showing biological decomposability under composting conditions. However, little is known about its anaerobic hydrolysis, while large amounts of food packaging end up in biogas plants. Two different enzymes belonging to the carboxylesterase superfamily from Clostridium botulinum (Cbotu_EstA) and from Pelosinus fermentans, were reported to actively hydrolyze PBAT, while they failed to act on PET. Yet, enzymes would allow mild decomposition of widely used PET enabling recycling of the monomeric building blocks. The enhancement of the hydrolase activity with regard to polyester hydrolysis can be achieved by fusion of hydrophobic domains, improving the biocatalyst adsorption on the hydrophobic polymer surface, or by substitution of specific residues, enlarging the active site of the enzyme. Interestingly, analysis of the 3D structure of Cbotu_EstA revealed the presence of an extra domain at the N-terminus of the enzyme which covered the lid structure and a hydrophobic patch. The deletion of the Cbotu_EstA N-terminal domain can satisfactory combine the enlarging of the active site and the presence of hydrophobic domain on the surface of the enzyme for improved sorption properties. Surface engineering successfully produced a highly active Cbotu_EstA variant (del71Cbotu_EstA) which was able to hydrolyze PET. Truncation of the N-terminal domain of Cbotu_EstA improved the adsorption of the enzyme on hydrophobic polyester surfaces and enhanced their hydrolysis eight times more compared to the wild-type enzyme, based on released monomers quantification