Final Report Summary - LIPOYEASTS (Mobilising the enzymatic potential of hydrocarbonoclastic bacteria and the oleaginous yeast Yarrowia lipolytica to create a powerful cellular production platform for
Executive summary:
The LIPOYEASTS project aimed at developing a versatile fermentation platform for the conversion of lipid feedstocks into wax esters (WE), isoprenoid-derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs), and free hydroxyl fatty acids (HFAs). Different lipid stocks (petroleum, alkane, vegetable oil, fatty acid) and combinations thereof were assessed with the aim of modulating the composition of the produced added-value products. The fermentation platform, Y. lipolytica, selected for this project is well-studied and genetically amenable yeast known for its ability to grow on a variety of lipid substrates. Furthermore, Yarrowia is considered a GRAS organism and already used for biotechnological applications like e.g. citric acid production and heterologous protein expression (Forster et al., 2007; Madzak et al., 2004). Hence, processes developed in this research can be readily translated into commercial processes. Yarrowia's extensive lipid metabolism allows it in response to metabolic conditions to either degrade, store or modify exogenous lipid substrates via distinct compartmentalised metabolic routes. Recent research efforts directed at elucidating its lipid metabolism (reviewed in Fickers et al., 2005) together with available genomic data make it now possible to intercept its branched lipid metabolism for biotechnological purposes.
Project context and objectives:
In previous studies, we have deleted several key metabolic steps in Yarrowia lipolytica resulting in the accumulation of lipid intermediates as potential precursors for conversion into added-value derivatives. Additional engineering efforts were proposed by the LIPOYEASTS project to further optimise and tailor Yarrowia's lipid metabolism to create new branch points for interception and increase metabolic flux along those engineered routes.
We have set the following goals and objectives:
1) Accumulate of acyl-CoA and 3 -hydroxyacyl-CoA intermediates with desired acyl chain lengths.
2) Block the biosynthesis of competing TAGs from lipid feed stocks.
3) Block competing oxidation of fatty acids into undesired dicarboxylic acids.
4) Convert lipid intermediates into PHA by introducing bacterial phaC synthase genes into Yarrowia.
5) Produce HFAs by introducing hydroxyl -acyl-CoA thioesterases into Yarrowia.
6) Produce WE by heterologous expression of bacterial WE synthases along with a cognate acyl-CoA synthetase in Yarrowia.
7) Convert lipids into carotenoids by introduction of crt genes into Yarrowia.
8) Produce polyenic carotenoid ester by introduction of isoprenoid -specific WS CoA-synthetase (Acs) and the carotenoid genes crt in Yarrowia.
9) Discover and characterise novel aliphatic enzymes by metagenomic screening and express them in Yarrowia production strains.
10) Design a pilot-scale production system for production of targeted compounds by Yarrowia strains from lipids.
11) Test the targeted compounds for their applicational value (antibacterial and prebiotic effects assessed).
Project results:
Our team has isolated and characterised several highly active aliphatic hydrocarbon modifying enzymes from hydrocarbonoclastic and other producing bacteria that have been moved into Yarrowia for lipid transformation. Metagenomic library from oil-polluted environments has been constructed and additional lipid modifying enzymes with novel activities and specificities have been expressed in Yarrowia's modified genetic backgrounds. Enzyme activities will be later optimised, if necessary, to specific catalytic needs using rational and / or evolutionary protein design approaches. During the first reporting period, we have constructed the delta pox1-6 delta lro1 delta dga1 strain as a storage lipid deficient strain. However, the lipid analysis of the double acyltransferase knock out mutant in the Pold or in the delta pox1-6 delta lro1 delta dga1 genetic context, as presented below, clearly demonstrated that there is still significant production of storage lipids. Further work on TAG synthesis pathway revealed that in contrast to what could be expected; the ARE1 and ARE2 genes are also involved in the TAG synthesis pathway. Therefore, during the second year we have introduced additional deletions into Yarrowia into its ARE1 and ARE2 genes in the pox knockout mutant to completely abolish neutral lipid synthesis and thus to improve the accumulation of acyl-CoA precursors (delta pox1-6, delta dga1, delta lro1, delta are1, delta are2). Based on this and other genetic backgrounds of Yarrowia, we have constructed Yarowia lipolytica strains engineered for the production of all target compounds (PHA, HFA, wax esters, carotenoids and carotenoid wax esters). We found that POX phenotypes significantly affect the PHA production yields in Yarrowia, but not PHA composition (Haddouche et al., 2010). The quantitative contribution of the distinct Aox enzymes to PHA production depends on the fatty acid chain length used as substrate. Some genetic modifications resulted in high PHA yields (circa 12% of PHA from CDW), one of the highest yields achieved for recombinant yeasts so far (Haddouche et al., 2011).
As functional screening of novel PHA synthases in metagenomic libraries did not result in isolation of novel enzymes, we have suggested and carried out experiments with codon-optimised PHA synthase.
Here significant improve of PHA yields has been achieved by expressing a bacterial PHA synthase codon-optimised for the expression in Yarrowia (circa 20-25% of PHA from CDW). Further improve of PHA yields under optimal growth conditions is anticipated. Thus, protein expression in Yarrowia is another very crucial factor controlling PHA yields. It still needs to be determined whether codon optimisation of other bacterial PHA synthases and their subsequent expression in Yarrowia can result in further increase of the biopolymer yields. We have also identified a scope of lipids and fatty acids for production of PHA by using Yarrowia. Four of these carbon sources namely tetradecanoic acid, oleic acid, rapseed oil and olive oil, have resulted in good quantities of PHA, as compared with all recombinant PHA-producing yeasts engineered so far. These fatty acids and lipids are also abundant feedstocks widely used in industrial processes. Moreover, we have established a protocol to recover fungal PHA, as well as have studied the biopolyester's material properties. These results help us to assign industrial niche for these yeast-derived polymers.
Engineering of recombinant wax ester production has been continued beyond the project duration by developing more sensitive and reproducible analytical methods for extracting and quantification of wax esters from Yarrowia. We are currently conducting final experiments to validate wax ester production levels in the various yeast strains that were engineered during the project period and anticipate manuscript submission later this year. In addition, we have licensed our wax ester / biodiesel production technology to a biotechnology company (LS9). We have also demonstrated carotenoid production in Yarrowia, however production levels are not significantly higher than what has been reported in other microorganisms. Thus the production level of carotenoid lycopene (10-15 mg/g CDW) obtained in Yarrowia is comparable to the yields obtained in metabolically engineered E. coli. We therefore decided to focus our efforts on wax ester production which has a larger industrial impact as demonstrated by the recent licensing of this technology.
Brief description of the main results:
1) Recombinant Yarrowia strains with modified pool of acyl-CoA and 3-hydroxyacyl-CoA intermediates with desired acyl chain lengths constructed.
2) Biosynthesis of competing TAG's from lipid feed stocks blocked.
3) Conversion of lipid intermediates into PHA in Yarrowia achieved.
5) PHA yields of circa 20-25% CDW obtained, which are highest ever achieved so far for recombinant yeasts.
5) Recombinant Yarrowia strains for production of HFAs constructed.
6) Recombinant Yarrowia strains with the genes for WE biosynthesis constructed.
7) Conversion lipids into carotenoids and carotenoid esters in Yarrowia achieved.
8) Recovery protocol and material properties of biopolymers established.
Potential impact:
Recombinant Yarrowia strains with modified pool of acyl-CoA and 3-hydroxyacyl-CoA intermediates with desired acyl chain lengths have been constructed and biosynthesis of competing TAG's from lipid feed stocks have been blocked. These strains have been used as a basis to construct all other Yarrowia's strains producing target compounds. The fatty acids metabolism in these strains has thus been significantly streamlined for the production of wax esters, biopolyesters and other compounds, which can be now exploited on industrial level. Moreover, these strains can be used for the construction of any other interesting lipid-derived monomers and polymers produced from cheap lipid feedstocks.
Conversion of lipid intermediates into PHA in Yarrowia has been achieved. PHA yields of circa 20-25% CDW obtained. Genetic engineering of yeasts for the production of PHAs has been tried out in a number of yeast species, including Saccharomyces cerevisiae, Pichia pastoris etc. However, PHA yields achieved in those experiments were significantly low, not exceeding 1% of PHA from total dry weight. Thus, due to the use of highly efficient strains and highly efficient enzymes constructed under the project, we were able to significantly increase the biopolymer yields achieving highest polymer yields ever produced by yeasts. This rational engineering of beta-oxidation, TAG biosynthesis and other lipid modifying metabolic routes results prove the importance of intracellular lipid fluxes in PHA biosynthesis and can be used as a very efficient strategy to design further genetic manipulations towards higher PHA yields.
Recombinant Yarrowia strains for production of HFAs have been constructed. Although this line of research did not result in significant yields of the product, it still has shown the importance and competition of the beta-oxidation cycle in the generation of 3-hydroxy fatty acids, and further genetic manipulations affecting beta-oxidation cycle could be done to increase the yields of these interesting compounds.
Recombinant Yarrowia strains with the genes for WE biosynthesis have been constructed. We have managed to achieve good yields of wax esters, but also found out that the WE yields are highly dependent on the type of wax ester synthase, type of promoter and feedstocks used. By simple manipulation of growth media, we established optimal growth condition for the production of wax esters. Moreover, in order to eliminate the need to add ethanol as a co-substrate to the Yarrowia cultures, we have engineered a recombinant ethanol production pathway into Yarrowia.
Conversion of lipids into carotenoids and carotenoid esters in Yarrowia has been achieved. The genes encoding the lycopene biosynthetic pathway were successfully cloned into Yarrowia. Although we are still quantifying and optimising lycopene production levels, the preliminary results show that the carotenoid yields achieved in Yarrowia are comparable with the ones produced by recombinant E.coli strains. Moreover, we have engineered a biosynthetic pathway for the production of purple colored C30 carotenoid diapolycopene-dial and diacid in Yarrowia.
Recovery protocol and material properties of biopolymers have been established. The Yarrowia's strain producing highest yields of PHA was used to develop a strategy to recover the biopolymer. Several methods have been applied with little success due to the significant impermeability of fungal cells to solvents. Still, extended exposure to solvents finally resulted in extraction of good amounts of the biopolymer, which has been analysed to obtain its physical properties and exact chemical structure. Future efforts should be applied to optimise the extraction procedure to avoid the extensive use of solvents.
To summarise, the LIPOYEASTS project has resulted in a number of avenues potentially leading to industrial exploitation. The most promising results were developed concerning the use of Yarrowia lipolytica for the production of PHA biopolymers and wax esters, the latter technology has already been licensed to a biotechnology company. The knowledge generated by the project was also widely disseminated through scientific publications, scientific conferences, and through the project website.
List of websites: http://www.lipoyeasts.ugent.be/
The LIPOYEASTS project aimed at developing a versatile fermentation platform for the conversion of lipid feedstocks into wax esters (WE), isoprenoid-derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs), and free hydroxyl fatty acids (HFAs). Different lipid stocks (petroleum, alkane, vegetable oil, fatty acid) and combinations thereof were assessed with the aim of modulating the composition of the produced added-value products. The fermentation platform, Y. lipolytica, selected for this project is well-studied and genetically amenable yeast known for its ability to grow on a variety of lipid substrates. Furthermore, Yarrowia is considered a GRAS organism and already used for biotechnological applications like e.g. citric acid production and heterologous protein expression (Forster et al., 2007; Madzak et al., 2004). Hence, processes developed in this research can be readily translated into commercial processes. Yarrowia's extensive lipid metabolism allows it in response to metabolic conditions to either degrade, store or modify exogenous lipid substrates via distinct compartmentalised metabolic routes. Recent research efforts directed at elucidating its lipid metabolism (reviewed in Fickers et al., 2005) together with available genomic data make it now possible to intercept its branched lipid metabolism for biotechnological purposes.
Project context and objectives:
In previous studies, we have deleted several key metabolic steps in Yarrowia lipolytica resulting in the accumulation of lipid intermediates as potential precursors for conversion into added-value derivatives. Additional engineering efforts were proposed by the LIPOYEASTS project to further optimise and tailor Yarrowia's lipid metabolism to create new branch points for interception and increase metabolic flux along those engineered routes.
We have set the following goals and objectives:
1) Accumulate of acyl-CoA and 3 -hydroxyacyl-CoA intermediates with desired acyl chain lengths.
2) Block the biosynthesis of competing TAGs from lipid feed stocks.
3) Block competing oxidation of fatty acids into undesired dicarboxylic acids.
4) Convert lipid intermediates into PHA by introducing bacterial phaC synthase genes into Yarrowia.
5) Produce HFAs by introducing hydroxyl -acyl-CoA thioesterases into Yarrowia.
6) Produce WE by heterologous expression of bacterial WE synthases along with a cognate acyl-CoA synthetase in Yarrowia.
7) Convert lipids into carotenoids by introduction of crt genes into Yarrowia.
8) Produce polyenic carotenoid ester by introduction of isoprenoid -specific WS CoA-synthetase (Acs) and the carotenoid genes crt in Yarrowia.
9) Discover and characterise novel aliphatic enzymes by metagenomic screening and express them in Yarrowia production strains.
10) Design a pilot-scale production system for production of targeted compounds by Yarrowia strains from lipids.
11) Test the targeted compounds for their applicational value (antibacterial and prebiotic effects assessed).
Project results:
Our team has isolated and characterised several highly active aliphatic hydrocarbon modifying enzymes from hydrocarbonoclastic and other producing bacteria that have been moved into Yarrowia for lipid transformation. Metagenomic library from oil-polluted environments has been constructed and additional lipid modifying enzymes with novel activities and specificities have been expressed in Yarrowia's modified genetic backgrounds. Enzyme activities will be later optimised, if necessary, to specific catalytic needs using rational and / or evolutionary protein design approaches. During the first reporting period, we have constructed the delta pox1-6 delta lro1 delta dga1 strain as a storage lipid deficient strain. However, the lipid analysis of the double acyltransferase knock out mutant in the Pold or in the delta pox1-6 delta lro1 delta dga1 genetic context, as presented below, clearly demonstrated that there is still significant production of storage lipids. Further work on TAG synthesis pathway revealed that in contrast to what could be expected; the ARE1 and ARE2 genes are also involved in the TAG synthesis pathway. Therefore, during the second year we have introduced additional deletions into Yarrowia into its ARE1 and ARE2 genes in the pox knockout mutant to completely abolish neutral lipid synthesis and thus to improve the accumulation of acyl-CoA precursors (delta pox1-6, delta dga1, delta lro1, delta are1, delta are2). Based on this and other genetic backgrounds of Yarrowia, we have constructed Yarowia lipolytica strains engineered for the production of all target compounds (PHA, HFA, wax esters, carotenoids and carotenoid wax esters). We found that POX phenotypes significantly affect the PHA production yields in Yarrowia, but not PHA composition (Haddouche et al., 2010). The quantitative contribution of the distinct Aox enzymes to PHA production depends on the fatty acid chain length used as substrate. Some genetic modifications resulted in high PHA yields (circa 12% of PHA from CDW), one of the highest yields achieved for recombinant yeasts so far (Haddouche et al., 2011).
As functional screening of novel PHA synthases in metagenomic libraries did not result in isolation of novel enzymes, we have suggested and carried out experiments with codon-optimised PHA synthase.
Here significant improve of PHA yields has been achieved by expressing a bacterial PHA synthase codon-optimised for the expression in Yarrowia (circa 20-25% of PHA from CDW). Further improve of PHA yields under optimal growth conditions is anticipated. Thus, protein expression in Yarrowia is another very crucial factor controlling PHA yields. It still needs to be determined whether codon optimisation of other bacterial PHA synthases and their subsequent expression in Yarrowia can result in further increase of the biopolymer yields. We have also identified a scope of lipids and fatty acids for production of PHA by using Yarrowia. Four of these carbon sources namely tetradecanoic acid, oleic acid, rapseed oil and olive oil, have resulted in good quantities of PHA, as compared with all recombinant PHA-producing yeasts engineered so far. These fatty acids and lipids are also abundant feedstocks widely used in industrial processes. Moreover, we have established a protocol to recover fungal PHA, as well as have studied the biopolyester's material properties. These results help us to assign industrial niche for these yeast-derived polymers.
Engineering of recombinant wax ester production has been continued beyond the project duration by developing more sensitive and reproducible analytical methods for extracting and quantification of wax esters from Yarrowia. We are currently conducting final experiments to validate wax ester production levels in the various yeast strains that were engineered during the project period and anticipate manuscript submission later this year. In addition, we have licensed our wax ester / biodiesel production technology to a biotechnology company (LS9). We have also demonstrated carotenoid production in Yarrowia, however production levels are not significantly higher than what has been reported in other microorganisms. Thus the production level of carotenoid lycopene (10-15 mg/g CDW) obtained in Yarrowia is comparable to the yields obtained in metabolically engineered E. coli. We therefore decided to focus our efforts on wax ester production which has a larger industrial impact as demonstrated by the recent licensing of this technology.
Brief description of the main results:
1) Recombinant Yarrowia strains with modified pool of acyl-CoA and 3-hydroxyacyl-CoA intermediates with desired acyl chain lengths constructed.
2) Biosynthesis of competing TAG's from lipid feed stocks blocked.
3) Conversion of lipid intermediates into PHA in Yarrowia achieved.
5) PHA yields of circa 20-25% CDW obtained, which are highest ever achieved so far for recombinant yeasts.
5) Recombinant Yarrowia strains for production of HFAs constructed.
6) Recombinant Yarrowia strains with the genes for WE biosynthesis constructed.
7) Conversion lipids into carotenoids and carotenoid esters in Yarrowia achieved.
8) Recovery protocol and material properties of biopolymers established.
Potential impact:
Recombinant Yarrowia strains with modified pool of acyl-CoA and 3-hydroxyacyl-CoA intermediates with desired acyl chain lengths have been constructed and biosynthesis of competing TAG's from lipid feed stocks have been blocked. These strains have been used as a basis to construct all other Yarrowia's strains producing target compounds. The fatty acids metabolism in these strains has thus been significantly streamlined for the production of wax esters, biopolyesters and other compounds, which can be now exploited on industrial level. Moreover, these strains can be used for the construction of any other interesting lipid-derived monomers and polymers produced from cheap lipid feedstocks.
Conversion of lipid intermediates into PHA in Yarrowia has been achieved. PHA yields of circa 20-25% CDW obtained. Genetic engineering of yeasts for the production of PHAs has been tried out in a number of yeast species, including Saccharomyces cerevisiae, Pichia pastoris etc. However, PHA yields achieved in those experiments were significantly low, not exceeding 1% of PHA from total dry weight. Thus, due to the use of highly efficient strains and highly efficient enzymes constructed under the project, we were able to significantly increase the biopolymer yields achieving highest polymer yields ever produced by yeasts. This rational engineering of beta-oxidation, TAG biosynthesis and other lipid modifying metabolic routes results prove the importance of intracellular lipid fluxes in PHA biosynthesis and can be used as a very efficient strategy to design further genetic manipulations towards higher PHA yields.
Recombinant Yarrowia strains for production of HFAs have been constructed. Although this line of research did not result in significant yields of the product, it still has shown the importance and competition of the beta-oxidation cycle in the generation of 3-hydroxy fatty acids, and further genetic manipulations affecting beta-oxidation cycle could be done to increase the yields of these interesting compounds.
Recombinant Yarrowia strains with the genes for WE biosynthesis have been constructed. We have managed to achieve good yields of wax esters, but also found out that the WE yields are highly dependent on the type of wax ester synthase, type of promoter and feedstocks used. By simple manipulation of growth media, we established optimal growth condition for the production of wax esters. Moreover, in order to eliminate the need to add ethanol as a co-substrate to the Yarrowia cultures, we have engineered a recombinant ethanol production pathway into Yarrowia.
Conversion of lipids into carotenoids and carotenoid esters in Yarrowia has been achieved. The genes encoding the lycopene biosynthetic pathway were successfully cloned into Yarrowia. Although we are still quantifying and optimising lycopene production levels, the preliminary results show that the carotenoid yields achieved in Yarrowia are comparable with the ones produced by recombinant E.coli strains. Moreover, we have engineered a biosynthetic pathway for the production of purple colored C30 carotenoid diapolycopene-dial and diacid in Yarrowia.
Recovery protocol and material properties of biopolymers have been established. The Yarrowia's strain producing highest yields of PHA was used to develop a strategy to recover the biopolymer. Several methods have been applied with little success due to the significant impermeability of fungal cells to solvents. Still, extended exposure to solvents finally resulted in extraction of good amounts of the biopolymer, which has been analysed to obtain its physical properties and exact chemical structure. Future efforts should be applied to optimise the extraction procedure to avoid the extensive use of solvents.
To summarise, the LIPOYEASTS project has resulted in a number of avenues potentially leading to industrial exploitation. The most promising results were developed concerning the use of Yarrowia lipolytica for the production of PHA biopolymers and wax esters, the latter technology has already been licensed to a biotechnology company. The knowledge generated by the project was also widely disseminated through scientific publications, scientific conferences, and through the project website.
List of websites: http://www.lipoyeasts.ugent.be/
