Enhancement of castor oil biotransformation into aroma by Yarrowia lipolytica mutants

The food industry has a great interest in biotechnological production of γ- decalactone by Yarrowia lipolytica, due to its increasing consumers acceptability in comparison with similar products obtained by chemical synthesis. This yeast is able to produce γ-decalactone by transformation of a hydroxylated C18 fatty acid. However, lower yields of γ-decalactone were obtained (up to 4–5 gL-1), mainly due the degradation of newly synthesized lactone and the partial use of ricinoleic acid or intermediate at the C10 level, which is simultaneously the precursor for other γ-lactones. Thus, the purpose of this work is to enhance the biotransformation of castor oil, source of ricinoleic acid, into γ-decalactone exploring different operation mode strategies in bioreactor (batch and fed-batch) and compare the yields obtained with wild type strain with those achieved by mutant strains. Different experiments were conducted in a 3.7-L bioreactor using an aeration rate of 5.1 L min-1, agitation 650 rpm and pH 6.0 (previously optimized conditions [1]). The influence of castor oil concentration and cell density on γ-decalactone production was investigated. Two different cell and castor oil concentrations (30 g L-1 and 60 gL-1) were used for the biotransformation. In the expectation of achieving higher γ-decalactone concentrations, a step-wise fed-batch strategy was also attempted. In a first approach, this study was conducted with Yarrowia lipolytica W29 (ATCC20460) and the highest γ-decalactone productivity of 215.4 mg L-1 h-1 was obtained in a batch mode of operation with 60 g L-1 of cells and 60 g L-1 of castor oil. After that, γ-decalactone production with two Yarrowia lipolytica mutants was studied. Experiments performed with Y. Lipolytica MTLY40-2P, with a deletion of all the POX 3–5 genes and a multicopy insertion of POX2 [2], resulted in an increased accumulation and an inhibition of γ-decalactone degradation. Since this yeast is also known to be a lipase producer and these enzymes catalyze the hydrolysis of triacylglycerides into glycerol and free fatty acids, a Y. lipolytica JMY3010 mutant, that overexpress extracellular lipase by the LIP2 gene (encoded the main extracellular lipase activity) cloned under the control of the TEF promoter [3], as also used. With these different approaches is possible to increase aroma productivity and a greater enhance in γ-decalactone production was achieved (up to 7-9 gL-1) through conjugation of a bioprocess optimization and genetic engineering approach

Immobilization of Yarrowia lipolytica for aroma production from castor oil

The main aim of this study was to compare different materials for Y. lipolytica immobilization that could be used in the production of γ-decalactone (a peach-like aroma) in order to prevent the toxic effect both of the substrate and the aroma upon the cells. Therefore, cells adsorption onto pieces of methyl polymethacrylate and of DupUM® was studied and further used in the biotransformation of castor oil into γ-decalactone. The highest aroma concentration was obtained with immobilized cells in DupUM®, where reconsumption of the aroma by the cells was prevented, contrarily to what happens with free cells. This is a very promising result for γ-decalactone production, with potential to be used at an industrial level since the use of immobilized cells system will facilitate the conversion of a batch process into a continuous mode keeping high cell density and allowing easier recovery of metabolic products.The authors acknowledge Fundacao para a Ciencia e Tecnologia (FCT) for the financial support provided (SFRH/BD/63701/2009)

Bioprocess optimization for the production of aromatic compounds with metabolically engineered hosts: recent developments and future challenges

The most common route to produce aromatic chemicals organic compounds containing at least one benzene ring in their structure is chemical synthesis. These processes, usually starting from an extracted fossil oil molecule such as benzene, toluene, or xylene, are highly environmentally unfriendly due to the use of non-renewable raw materials, high energy consumption and the usual production of toxic by-products. An alternative way to produce aromatic compounds is extraction from plants. These extractions typically have a low yield and a high purification cost. This motivates the search for alternative platforms to produce aromatic compounds through low-cost and environmentally friendly processes. Microorganisms are able to synthesize aromatic amino acids through the shikimate pathway. The construction of microbial cell factories able to produce the desired molecule from renewable feedstock becomes a promising alternative. This review article focuses on the recent advances in microbial production of aromatic products, with a special emphasis on metabolic engineering strategies, as well as bioprocess optimization. The recent combination of these two techniques has resulted in the development of several alternative processes to produce phenylpropanoids, aromatic alcohols, phenolic aldehydes, and others. Chemical species that were unavailable for human consumption due to the high cost and/or high environmental impact of their production, have now become accessible.info:eu-repo/semantics/publishedVersio

Yarrowia lipolytica as a microbial host for flavors and fragrances production

This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and of the project TUBITAK/0009/2014 and by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Lipase-mediated hydrolysis of castor oil on its biotransformation into γ-decalactone by Yarrowia lipolytica

γ-Decalactone is a peach-like flavour compound that can be obtained biotechnologically by the biotransformation of ricinoleic acid. Castor oil is the substrate most usually used in the biotechnological production of γ-decalactone and it needs to be hydrolyzed in order to release ricinoleic acid. That biotransformation can be carried out by various microorganisms, such as the non-conventional yeast Yarrowia lipolytica, considered as non-pathogenic and as GRAS by the FDA. In order to increase the availability of the substrate to the cells for the production of γ-decalactone, castor oil previously hydrolyzed can be used. This hydrolysis may be promoted by enzymatic action, more specifically by lipases. The purpose of this work is to study the influence of the lipases-mediated castor oil hydrolysis, in aroma production, using different commercial lipases and a lipase produced by the yeast. Firstly, the enzymatic hydrolysis of castor oil by different commercial lipases (CALB L, Lipozyme TL IM and Lipolase 100T) was studied, under different operating conditions (pH and temperature) and Lipozyme TL IM was the most adequate enzyme to hydrolyze castor oil, at the optimal operating conditions of pH 8 and 27 ˚C (95.4%). Furthermore, different strategies for γ-decalactone production in flask experiments were also investigated, namely the addition of previously hydrolyzed castor oil to the culture medium, the addition of an immobilized lipase to the biotransformation medium and finally, the pre-addition of an inducer of lipase production (olive oil) to the biotransformation medium. As result, the process was faster when lipase was involved in any form, since the maximum of aroma concentration was attained at 140 h and 185 h of batch process with lipase and without lipase addition, respectively. However, no significant improvements in the γ-decalactone global yields and productivities were obtained (productivities varied from 8 ± 1 mg/L h to 9 ± 1 mg /L h in all conditions tested)

Strategies for increasing aroma production from castor oil by Yarrowia lipolytica

Tese de doutoramento em Engenharia Química e BiológicaThe aromatic compounds produced by biotechnological processes are increasingly accepted by consumers because they are considered as “natural” compounds. In addition, they are of great interest due to the high yields obtained comparatively to chemical processes. γ-Decalactone is an aromatic compound of industrial interest, resulting from the peroxisomal β-oxidation of ricinoleic acid. This fatty acid, the major constituent of castor oil, is the precursor most commonly used in the biotechnological production of this aroma. Although there are many works described in the literature about aroma production, several factors remain to be fully understood and optimized in order to improve γ- decalactone production. Thus, this work initially aimed to study the influence of lipase in castor oil hydrolysis and the consequent impact in the aroma production. Lipozyme TL IM® revealed to be an efficient lipase to hydrolyze castor oil (95.4 % of hydrolysis in 48 h). In spite of the higher aroma concentration obtained without lipase, the process was faster when Lipozyme TL IM® was involved, resulting in similar productivities. One of the limitations in the development of an industrial process for γ-decalactone production is the toxicity of the substrate and the lactone. The immobilization by adsorption of Y. lipolytica W29 in methyl polymetacrilate and DupUm® was studied in flasks batch cultures. After selecting the best conditions for cell immobilization, free and immobilized cells were used in the biotransformation of ricinoleic acid into γ-decalactone. The results demonstrated an improvement in γ-decalactone concentration with adsorbed Y. lipolytica cells on DupUM® since a greater amount of γ-decalactone was accumulated in the medium compared to free cells. In this case the use of the extracellular lipase Lipozyme TL IM® in the biotransformation medium was crucial to allow castor oil hydrolysis, without it the substrate access to the cells would be impossible. Furthermore, immobilized cells hold a stable γ- decalactone production after being stored for 30 days at 4 ºC. Also after reuse in three consecutive biotransformations, γ-decalactone concentration was ca. 80 % of that in the first cycle, indicating that immobilized cells could be reused for at least three cycles. The production of γ-decalactone in batch cultures of Y. lipolytica W29 free cells was studied at bioreactor level and the preformance in stirred tank (STR) and airlift bioreactors was compared. The oxygen mass transfer was characterized and the positive influence of kLa on γ-decalactone productivity was demonstrated for both bioreactors. A 2-fold increase in γ-decalactone concentration was achieved in the airlift when compared to STR; but, in this last bioreactor the production rate was higher. Morphological characterization of the yeast cells by image analysis showed that pneumatic agitation causes less impact in the cells morphology than mechanical agitation. A predominance of loose cells and quite irregular structures was observed in the STR. So, the airlift bioreactor presents potential interest for larger scale production, with important cost savings, due to the reduction of power input consumption. Finally, the performance of Y. lipolytica strains with modifications in the lipid metabolism at the β-oxidation pathway (acyl-CoA oxidases) and the triglyceride hydrolysis (LIP2 overexpression) using castor oil as substrate was monitored in the STR bioreactor. Depending on genotype, degradation of γ- decalactone was prevented. Also, a faster initial rate of aroma production was obtained with strain overexpressing LIP2 due to the fast hydrolysis of castor oil and release of ricinoleic acid. Step-wise fedbatch cultures improved γ-decalactone concentration only for MTLY40-2P strain, for which a 1.7-fold increase in γ-decalactone final concentration was achieved. The present work brings new insights on the biotechnological production of γ-decalactone contributing with some different strategies for increasing aroma production leading to a greater γ- decalactone concentration.Os compostos aromáticos produzidos por via biotecnológica são cada vez mais aceites pelos consumidores, uma vez que são considerados “naturais”, sendo esta uma mais valia perante as atuais preferencias do mercado. Além disso, os rendimentos obtidos por esta via são superiores aos obtidos na produção por síntese química. A γ−decalactona é um composto aromático de elevado interesse industrial que resulta da β-oxidação peroxissomal do ácido ricinoleico. Este ácido gordo, principal constituinte do óleo de rícino, é o precursor mais utilizado para a produção biotecnológica deste aroma. Embora existam muitos trabalhos descritos na literatura neste tópico, são vários os fatores cujo efeito ainda permanace por compreender e, consequentemente optimizar podendo permitir melhorar a produção da γ-decalactona. Assim, este trabalho teve como objetivo inicial estudar a influência da lípase na hidrólise do óleo de rícino e consequente impacto na produção do aroma. Foi utilizada a enzima Lipozyme TL IM® que foi eficiente na hidrólise do óleo de rícino (95.4 % de hidrólise em 48 h). Apesar dos resultados obtidos demonstrarem que a produção de aroma mais elevada é obtida na ausência de lípase, o processo foi mais rápido quando a Lipozyme TL IM® esteve envolvida, resultando em produtividades idênticas. Uma das limitações para o desenvolvimento de um processo industrial para a produção de γ- decalactona é a toxicidade do substrato e da própria lactona. A imobilização de Y. lipolytica W29 por adsorção em polimetilmetacrilato e DupUm® foi estudada, em modo batch em matraz. Depois de selecionadas as melhores condições de imobilização, foi comparada a produção de γ-decalactona com células livres e imobilizadas. Os resultados obtidos demonstraram que a melhor abordagem para aumentar a concentração de γ-decalactona é a adsorção de células em DupUm® uma vez que, nestas condições, a acumulação de aroma obtida foi semelhante às células livres. Neste caso, a utilização da lípase extracelular Lipozyme TL IM® no meio de biotransformação foi crucial para a hidrólise do óleo de rícino, sem a qual o acesso do substrato às células seria impossível. Além disso, as células imobilizadas podem ser armazenadas até 30 dias a 4 ºC mantendo a capacidade de produção de γ- decalactona. Após reutilização das células em três biotransformações consecutivas, a concentração de γ-decalactona foi de aproximadamente 80 % do valor obtido no primeiro ciclo, indicando que as células imobilizadas podem ser reutilizadas, pelo menos, durante três ciclos. A produção de aroma com células livres de Y. lipolytica W29 em modo batch foi testada em dois biorreatores, tanque agitado (STR) e airlift. A transferencia de oxigénio foi caracterizada em ambos os reatores assim como a influência positiva do kLa na produção da γ-decalactona. A concentração de γ-decalactona duplicou no bioreactor airlift em comparação com o STR, no entanto produtividades mais elevadas são obtidas para o STR. A caracterização morfológica das células por análise da imagem mostrou que a agitação pneumática causa menos impacto na morfologia das células do que a agitação mecânica, sendo que no STR foram observadas, predominantemente, estruturas bastante irregulares. Assim, o biorreator airlift apresenta elevado interesse para a produção de aroma em grande escala uma vez que permite reduzir os custos de operação devido à redução do consumo de energia. Por fim, foi monitorizado o desempenho de estirpes mutantes de Y. lipolytica que possuem deleções génicas no metabolismo dos lípidos na via de β-oxidação (acil-CoA oxidases) e na hidrólise dos triglicéridos (subexpressão do gene Lip2), utilizando óleo de rícino como substrato. Dependendo do génótipo, a degradação da γ-decalactona foi impedida. Além disso, uma maior velocidade de produção do aroma foi observada para a estirpe que subexpressa o gene Lip2, uma vez que houve uma hidrólise mais rápida do óleo de rícino e consequente libertação do ácido ricinoleico. O modo de operação semi-contínuo com alimentação intermitente permitiu aumentar a produção de γ-decalactona apenas para a estirpe MTLY40-2P, para a qual se observou um aumento de 1.7 vezes na concentração final de γ-decalactona. Assim, o presente trabalho apresenta novas prespetivas sobre a produção biotecnológica da γ- decalactona, contribuindo com diferentes estratégias que conduziram a um aumento na produção de aroma e permitiram obter uma elevada concentração de γ-decalactona (7 g L-1).Fundação para a Ciência e Tecnologia (FCT) bolsa de doutoramento (SFRH/BD/63701/2009). Ao Projeto “BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes", REF. NORTE-07-0124-FEDER-000028, cofinanciado pelo Programa Operacional Regional do Norte (ON.2 – O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER)

Biotransformação do óleo de rícino em aromas por Yarrowia lipolytica

Dissertação de mestrado integrado em Engenharia BiológicaOs compostos aromáticos produzidos por processos biotecnológicos são cada vez mais aceites pelo mercado consumidor, por serem considerados naturais. Além disso, são processos de grande interesse devido aos elevados rendimentos que apresentam relativamente aos processos de extracção a partir de fontes naturais. A γ-decalactona é um composto aromático de interesse industrial, que resulta da β-oxidação peroxisomal do ácido ricinoleico. Este ácido gordo, maior constituinte do óleo de rícino, é o precursor mais vulgarmente utilizado na produção biotecnológica deste aroma. Para que o substrato (óleo de rícino) esteja mais disponível para as células o utilizarem na produção de γ-decalactona, pode utilizar-se óleo de rícino hidrolisado. Esta hidrólise pode ser promovida por acção enzimática, mais especificamente por lipases. O objectivo geral do presente trabalho consiste na avaliação do papel das lipases, comerciais e produzidas pela levedura, na hidrólise do óleo de rícino e consequente impacto na produção de γ-decalactona. Inicialmente foi feita a validação do método experimental utilizado para determinar a actividade lipolítica, tendo-se concluído que o método que utiliza como substrato o p-nitrofenil laurato não era preciso. Assim, nos ensaios posteriores de determinação da actividade lipolítica foi empregue o substrato p-nitrofenil butirato, que forneceu resultados mais precisos. Realizaram-se ensaios de caracterização da actividade de várias enzimas comerciais solúveis e imobilizadas. Os ensaios tiveram como finalidade avaliar a actividade lipolítica das diferentes enzimas e permitiram concluir que a lipase de C. rugosa (5.873 U*mg-1 enzima) apresenta uma actividade bastante superior à das outras enzimas analisadas, Lipozyme TL IM, Lipolase 100 T e Lipase CALB L. Com o intuito de estudar quais as condições que conduzem a um maior grau de hidrólise do óleo de rícino realizaram-se ensaios com as diferentes lipases comerciais estudadas, tendo-se variado as condições de temperatura, pH e concentração de óleo de rícino. A maior percentagem de hidrólise (95.37 %) foi obtida quando se utilizou a enzima Lipozyme TL IM, a pH 8 e 27 ºC, ao fim de 45 horas. Nos ensaios de produção de lipase por diferentes estirpes de Y. lipolytica observou-se que a produção de enzima é favorecida quando o meio de produção de lipase é adicionado ao meio de crescimento celular, sem ocorrer centrifugação das células na transferência entre meios. Foram comparados vários substratos na indução de lipase e verificou-se que, para a estirpe Yarrowia lipolytica W29, foi o ricinoleato de metilo que se revelou o melhor indutor para a produção de lipase, nas condições analisadas. Nos ensaios de biotransformação do óleo de rícino em γ-decalactona verificou-se que a etapa inicial de hidrólise do óleo não se revelou crucial para o aumento da produtividade. A maior produção de γ-decalactona (1600 mg*mL-1) foi obtida usando óleo de rícino não hidrolisado e sem adição de lipase extracelular, sendo que a produtividade foi melhorada pelo aumento da velocidade de agitação.The aromatic compounds produced by biotechnological processes are increasingly accepted by consumers, because they are considered as natural compounds. In addition, they are of great interest due to the high yields obtained comparatively to chemical processes. γ-Decalactone is an aromatic compound of industrial interest, resulting from the peroxisomal β-oxidation of ricinoleic acid. This fatty acid, the major constituent of castor oil, is the precursor most commonly used in the biotechnological production of this aroma. In order to increase the availability of the substrate (castor oil) to the cells for the production of γ-decalactone, hydrolyzed castor oil can be used. This hydrolysis can be promoted by enzymatic action, more specifically by lipases. The main goal of the present work is to assess the role of lipases, both commercial and produced by the yeast, in the hydrolysis of castor oil and the consequent impact on the production of γ-decalactone. Initially, a validation of the experimental method used to determine the lipolytic activity was developed, and it was concluded that the method using the substrate p-nitrophenyl laurate was not accurate. Thus, in the subsequent experiments to determine the lipolytic activity, the substrate p- nitrophenyl butyrate was used, providing more accurate results. Experiments were performed in order to determine the activity of several commercial enzymes, soluble and immobilized. The aim of those essays was to evaluate the lipolytic activity of the different enzymes. The results allowed to conclude that, from all enzymes analyzed, the lipase of C. rugosa (5.873 U*mg-1 enzyme) presents the highest activity. It was also aim of this work to study the conditions that lead to a greater hydrolysis rate of castor oil. Thus, assays were performed with different commercial lipases previously studied, under different operating conditions, such as temperature, pH and castor oil concentration. The largest percentage of hydrolysis (95.37 %) was achieved when using the enzyme Lipozyme TL IM, at pH 8 and 27 ºC, after 45 hours. In lipase production essays, carried out by different strains of Y. lipolytica, lipase production was improved when the components of the production medium were added to the inoculum culture without harvesting and centrifuging cells in the transfer between both media.Different substrates were tested as lipase inducers and it was observed that, for the strain Yarrowia lipolytica W29, methyl ricinoleate revealed to be the best lipase inducer, for the conditions tested. The biotransformation of castor oil into γ-decalactone was not improved by the initial step of oil hydrolysis. The highest production of γ-decalactone (1600 mg*mL-1) was obtained using non-hydrolyzed castor oil and without adding extracellular lipase.Moreover, the productivity was improved by increased agitation rates

Immobilization of whole cells of Yarrowia lipolytica for citric acid production

Yarrowia lipolytica is one of the most extensively studied ‘‘non-conventional’’ yeast species, highlighted by their participation in bioremediation processes and production of lipases, γ-decalactone and citric acid. The use of immobilized cells presents several advantages over free cells in bioprocesses. In this work, Y. lipolytica entrapment in calcium alginate was applied for citric acid production from glycerol. The influence of sodium alginate and calcium chloride concentrations on the immobilization efficiency was investigated, being selected 4 % of sodium alginate and 0.5 M of calcium chloride, the best conditions. Afterwards, citric acid production was assessed at several spheres diameter and cells concentration, but no significant differences of final citric acid concentration were observed

Application of the yeast Yarrowia lipolytica for food additives production

[Excerpt] Introduction: Yarrowia lipolytica is a nonconventional, aerobic and dimorphic yeast with many biotechnological applications due to the wide range of substrates that can use as carbon source and the ability to produce a large variety of metabolites with industrial interest. It can usually be found in environments containing hydrophobic substrates, such as alkanes and fats. It can also be isolated from cheeses, yoghurts, kefir, soy sauce, meat and shrimp salads. Y. lipolytica has been proved to be a robust cell for the biotechnological production of compounds that can be used as additives in food industry, such as organic acids (citric acid), enzymes (such as proteases and lipases), biosurfactants, sweeteners (such as erythritol and mannitol), and aroma and fragrances compounds. [...] Introduction: Yarrowia lipolytica is a nonconventional, aerobic and dimorphic yeast with many biotechnological applications due to the wide range of substrates that can use as carbon source and the ability to produce a large variety of metabolites with industrial interest. It can usually be found in environments containing hydrophobic substrates, such as alkanes and fats. It can also be isolated from cheeses, yoghurts, kefir, soy sauce, meat and shrimp salads. Y. lipolytica has been proved to be a robust cell for the biotechnological production of compounds that can be used as additives in food industry, such as organic acids (citric acid), enzymes (such as proteases and lipases), biosurfactants, sweeteners (such as erythritol and mannitol), and aroma and fragrances compounds. [...We would like to thank the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and through the bilateral cooperation project FCT/NKFIH 2017/2018, COMPETE 2020 (POCI-01-0145-FEDER-006684) and BiotecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio