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      Towards biotechnological production of bio-based low molecular weight esters: a patent review

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      a , b , a , a ,
      RSC Advances
      The Royal Society of Chemistry

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          Abstract

          Low molecular weight (LMW) esters, like ethyl acetate, methyl formate or butyl acetate, are widespread bulk chemicals in many industries. Each of them is currently produced in huge amounts (millions of tons per year scale) starting from fossil-based feedstock and they are used mainly because of their low toxicity and complete biodegradability. Energy transition is just half of the story on the path of fighting climate change: 45% of the global greenhouse gas emissions are caused by the production and use of all the products, materials and food necessary for modern human life. If the world is to reach its climate goals, there is the need to leave underground a significant proportion of the fossil feedstock and minimize environmental impacts of chemical manufacturing. This is the reason why a lot of efforts have been made to find novel routes for LMW esters production starting from renewable raw materials ( e.g. biomasses or off-gases) and exploiting low-impact manufacturing, such as microbial fermentation or enzymatic reactions. This review reports the most significant patents, in the field of white biotechnology, that will hopefully lead to the commercialization of bio-based LMW esters as well as novel strategies, current problems to be solved, newer technologies, and some patent applications aiming at possible future developments.

          Abstract

          Low molecular weight (LMW) esters are widespread bulk chemicals in many industries. This review presents the most significant patents and patent applications related to the biotechnological production of LMW esters.

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          Most cited references23

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          The Saccharomyces cerevisiae EHT1 and EEB1 genes encode novel enzymes with medium-chain fatty acid ethyl ester synthesis and hydrolysis capacity.

          Fatty acid ethyl esters are secondary metabolites produced by Saccharomyces cerevisiae and many other fungi. Their natural physiological role is not known but in fermentations of alcoholic beverages and other food products they play a key role as flavor compounds. Information about the metabolic pathways and enzymology of fatty acid ethyl ester biosynthesis, however, is very limited. In this work, we have investigated the role of a three-member S. cerevisiae gene family with moderately divergent sequences (YBR177c/EHT1, YPL095c/EEB1, and YMR210w). We demonstrate that two family members encode an acyl-coenzymeA:ethanol O-acyltransferase, an enzyme required for the synthesis of medium-chain fatty acid ethyl esters. Deletion of either one or both of these genes resulted in severely reduced medium-chain fatty acid ethyl ester production. Purified glutathione S-transferase-tagged Eht1 and Eeb1 proteins both exhibited acyl-coenzymeA:ethanol O-acyltransferase activity in vitro, as well as esterase activity. Overexpression of Eht1 and Eeb1 did not enhance medium-chain fatty acid ethyl ester content, which is probably due to the bifunctional synthesis and hydrolysis activity. Molecular modeling of Eht1 and Eeb1 revealed the presence of a alpha/beta-hydrolase fold, which is generally present in the substrate-binding site of esterase enzymes. Hence, our results identify Eht1 and Eeb1 as novel acyl-coenzymeA:ethanol O-acyltransferases/esterases, whereas the third family member, Ymr210w, does not seem to play an important role in medium-chain fatty acid ethyl ester formation.
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            Scale-up of industrial biodiesel production to 40 m(3) using a liquid lipase formulation.

            In this work, we demonstrate the scale-up from an 80 L fed-batch scale to 40 m(3) along with the design of a 4 m(3) continuous process for enzymatic biodiesel production catalyzed by NS-40116 (a liquid formulation of a modified Thermomyces lanuginosus lipase). Based on the analysis of actual pilot plant data for the transesterification of used cooking oil and brown grease, we propose a method applying first order integral analysis to fed-batch data based on either the bound glycerol or free fatty acid content in the oil. This method greatly simplifies the modeling process and gives an indication of the effect of mixing at the various scales (80 L to 40 m(3) ) along with the prediction of the residence time needed to reach a desired conversion in a CSTR. Suitable process metrics reflecting commercial performance such as the reaction time, enzyme efficiency, and reactor productivity were evaluated for both the fed-batch and CSTR cases. Given similar operating conditions, the CSTR operation on average, has a reaction time which is 1.3 times greater than the fed-batch operation. We also showed how the process metrics can be used to quickly estimate the selling price of the enzyme. Assuming a biodiesel selling price of 0.6 USD/kg and a one-time use of the enzyme (0.1% (w/woil ) enzyme dosage); the enzyme can then be sold for 30 USD/kg which ensures that that the enzyme cost is not more than 5% of the biodiesel revenue. Biotechnol. Bioeng. 2016;113: 1719-1728. © 2016 Wiley Periodicals, Inc.
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              Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

              Background Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/T7 and XylS/Pm promoters allowed optimisation of their expression levels. Conclusion Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced 42.8 mM (3.8 g/L) ethyl acetate from glucose with an unprecedented efficiency, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.
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                Author and article information

                Journal
                RSC Adv
                RSC Adv
                RA
                RSCACL
                RSC Advances
                The Royal Society of Chemistry
                2046-2069
                18 September 2024
                12 September 2024
                18 September 2024
                : 14
                : 40
                : 29472-29489
                Affiliations
                [a ] Department of Biotechnology and Biosciences, University of Milano-Bicocca Piazza della Scienza 2 Milano 20126 Italy immacolata.serra@ 123456unimib.it m.zago@ 123456campus.unimib.it zago@ 123456astrobiosolvent.com paola.branduardi@ 123456unimib.it +390264484140
                [b ] Soft Chemicals S.r.l., ASTROBIO™ Division Via Sandro Pertini 14, Arsago Seprio Varese 21010 Italy
                Author information
                https://orcid.org/0000-0003-1873-4873
                https://orcid.org/0000-0003-4115-7015
                https://orcid.org/0000-0002-4036-033X
                Article
                d4ra04131c
                10.1039/d4ra04131c
                11409443
                39297040
                0baa2996-1543-47e0-bfe7-9892c0e3961d
                This journal is © The Royal Society of Chemistry

                This article is licensed under a Creative Commons Attribution-Non Commercial 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

                History
                : 5 June 2024
                : 6 September 2024
                Page count
                Pages: 18
                Funding
                Funded by: Università degli Studi di Milano-Bicocca, doi 10.13039/501100002954;
                Award ID: Unassigned
                Categories
                Chemistry
                Custom metadata
                Paginated Article

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