Synthesis of jojoba-like wax esters in metabolically engineered strains of Saccharomyces cerevisiae

Yeast has a long-standing tradition in human history as a production organism of choice. Besides being used for the production of fermented products like bread or beer, it has also been extensively explored for the production of proteins and chemicals. In the past, yeast research in this respect was often focused on the production of biofuels as an alternative to fossil fuels to enable the independence of crude oil. However, crude oil also functions as a source for a wide range of chemicals.Some of these chemicals can be substituted by alternatives derived from plant oils, like jojoba oil. Jojoba seeds contains approximately 50% (w/w) oil, which consists mostly (up to 97%) of wax esters (WEs). Minor parts include phytosterols, triacylglycerols (TAGs) and fatty alcohols (FOHs). This makes jojoba exceptional, since plants usually accumulate TAGs as storage compounds. Jojoba oil can among other applications be used in cosmetic and personal care products as well as lubricants. Currently, around 4,000 tons/year of jojoba oil is produced, with an estimated demand of up to 200,000 tons/year. Because of this, oil produced from the jojoba plant will not be enough to meet the demand in the future, even if huge land areas in various parts of the world are planted. Therefore, jojoba oil production in modified microorganisms represents a very promising approach. In this thesis, the yeast Saccharomyces cerevisiae was explored as a production organism for jojoba-like WEs. Jojoba-like WEs are naturally derived from the fatty acid (FA) metabolism of the plant, more specifically from very long-chain monounsaturated fatty acids (VLCMUFAs) with a carbon chain length of 20/22 (C20/C22). These VLCMUFAs can be converted to FOHs by the action of fatty acyl reductases (FARs). WEs are synthesized by wax synthases (WSs), which esterify an activated FA (fatty acyl-CoA) with a FOH molecule.In this thesis, the synthesis of jojoba-like WEs in S. cerevisiae was established by making use of various enzymes derived from bacterial and plant sources as well as tuning S. cerevisiae FA metabolism towards the increased synthesis of VLCMUFAs. In this way a S. cerevisiae strain was created that produces 14.38 +/- 1.76 mg WEs/g CDW. Of these WEs, 39.2 mol% are jojoba-like diunsaturated C38:2-WEs to C42:2-WEs, with the most abundant ones being C42:2-WEs (18.3 mol% of all WE species). These are also the most abundant WEs in natural jojoba oil (46.8 mol% of all WE species)

Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals

Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae. VLCFAs can be selectively modified towards the fatty alcohol docosanol (C22H46O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l(-1) in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner

Increasing jojoba-like wax ester production in Saccharomyces cerevisiae by enhancing very long-chain, monounsaturated fatty acid synthesis

Abstract Background Fatty acids (FAs) with a chain length of more than 18 carbon atoms (> C18) are interesting for the production of specialty compounds derived from these FAs. These compounds include free FAs, like erucic acid (C22:1-Δ13), primary fatty alcohols (FOHs), like docosanol (C22:0-FOH), as well as jojoba-like wax esters (WEs) (C38-WE to C44-WE), which are esters of (very) long-chain FAs and (very) long-chain FOHs. In particular, FAs, FOHs and WEs are used in the production of chemicals, pharmaceuticals and cosmetic products. Jojoba seed oil is highly enriched in diunsaturated WEs with over 70 mol% being composed of C18:1–C24:1 monounsaturated FOH and monounsaturated FA moieties. In this study, we aim for the production of jojoba-like WEs in the yeast Saccharomyces cerevisiae by increasing the amount of very long-chain, monounsaturated FAs and simultaneously expressing enzymes required for WE synthesis. Results We show that the combined expression of a plant-derived fatty acid elongase (FAE/KCS) from Crambe abyssinica (CaKCS) together with the yeast intrinsic fatty acid desaturase (FAD) Ole1p leads to an increase in C20:1 and C22:1 FAs in S. cerevisiae. We also demonstrate that the best enzyme candidate for C24:1 FA production in S. cerevisiae is a FAE derived from Lunaria annua (LaKCS). The combined overexpression of CaKCS and Ole1p together with a fatty acyl reductase (FAR/FAldhR) from Marinobacter aquaeolei VT8 (MaFAldhR) and a wax synthase (WS) from Simmondsia chinensis (SciWS) in a S. cerevisiae strain, overexpressing a range of other enzymes involved in FA synthesis and elongation, leads to a yeast strain capable of producing high amounts of monounsaturated FOHs (up to C22:1-FOH) as well as diunsaturated WEs (up to C46:2-WE). Conclusions Changing the FA profile of the yeast S. cerevisiae towards very long-chain monounsaturated FAs is possible by combined overexpression of endogenous and heterologous enzymes derived from various sources (e.g. a marine copepod or plants). This strategy was used to produce jojoba-like WEs in S. cerevisiae and can potentially be extended towards other commercially interesting products derived from very long-chain FAs

Biosynthesis of very long-chain fatty alcohols and wax esters in metabolically engineered strains of Saccharomyces cerevisiae

The objective of our research is the biosynthesis of very long-chain fatty alcohols (VLCFAlcs) and wax esters (WEs) in S. cerevisiae. VLCFAlcs and WEs have a broad application range and can be used for many commercial purposes. The applications of WEs include personal care products, lubricants, varnishes, inks, detergents, resins & plastics. WEs can also be used for coatings (for fruits & pills) and as an oil phase in formulas containing active compounds to enhance the efficiency of topical drugs.VLCFAlcs, like docosanol, are used as an emollient, emulsifier and thickener in cosmetics as well as a nutritional supplement. Unfortunately, at present most of the possible applications are limited to cosmetic and medical products due to the high price for WE isolation from their natural host, the plant Simmondsia chinensis. Because of this fact, a renewable approach for low-cost production of VLCFAlcs and WEs in a well-studied organism like S. cerevisiae is desirable. The in vitro and in vivo synthesis of different WEs up to C36 has already been shown in S. cerevisiae, but these WEs do not show the mentioned desired properties for commercial use. The in vivo synthesis of WE up to C44 in S. cerevisiae has so far only been achieved after substrate feeding. In our approach we demonstrate that the heterologous expression of specific fatty acyl-CoA reductases (FARs), enzymes required for alcohol synthesis, and wax synthases (WSs), enzymes responsible for WE synthesis, allow the in vivo synthesis of VLCFAlcs up to C22 and VLCWEs up to C42

Investigation of putative regulatory acetylation sites in Fas2p of Saccharomyces cerevisiae

Yeast metabolism is highly regulated, in part via coordinated reprogramming of metabolism on a transcriptional level, for example in response to environmental changes. Furthermore, regulation occurs on the protein level via posttranslational modifications directly affecting enzymatic activity – a mode of regulation that has the benefit of being very fast in response to environmental changes. One group of posttranslational modification that has been suggested to have a high impact on regulation of metabolism are acetylations. Around 4000 distinct protein acetylation sites have been found in Saccharomyces cerevisiae, many of which are located in central metabolic enzymes. However, reports on the verification of regulatory roles of specific acetylation sites on these metabolic enzymes have yet to emerge. This study investigates putative regulatory acetylation sites on Fas2p, which in concert with Fas1p is responsible for cytosolic fatty acid (FA) biosynthesis in S. cerevisiae. Fas2p stands out as one of the most highly acetylated proteins in yeast and is located at a branchpoint of acetyl-CoA metabolism. The amino acids (AAs) glutamine (Q) and arginine (R) were introduced to mimic a constitutively acetylated or non-acetylatable state at three separate lysine sites (K) (K83, K173 and K1551) confirmed to be acetylated in two independent studies, either separately or simultaneously. The results suggest that the residue replacement system in the specific case interferes with the enzymatic function of the fatty acid synthase (FAS), as QQQ and RRR triple mutants both reduce the amount of secreted free fatty acids (FFAs) in a faa1∆ faa4∆ yeast deletion mutant. The K173Q substitution significantly decreased C16 FA species at the expense of C18 FAs, while no such change could be observed for the corresponding K173R modification

Establishing very long-chain fatty alcohol and wax ester biosynthesis in Saccharomyces cerevisiae

Wax esters (WEs) are neutral lipids and can be used for a broad range of commercial applications, including personal care products, lubricants, or coatings. They are synthesized by enzymatic reactions catalyzed by a fatty acyl reductase (FAR) and a wax ester synthase (WS). At present, commercially used WEs are mainly isolated from Simmondsia chinensis (jojoba), but the high extraction costs and limited harvest areas constrain their use. The use of FARs in combination with different WSs to achieve a synthesis of jojoba-like WEs in bacteria and yeast has been reported previously, but the products were restricted to C28-C36 WEs. These rather short WEs make up only a very small percentage of the total WEs in natural jojoba oil. The synthesis of longer chain WEs (up to C44) in Saccharomyces cerevisiae has so far only been achieved after substrate feeding. Here we identified new routes for producing very long-chain fatty alcohols (VLCFOHs) up to a chain length of C22 by heterologous expression of a FAR derived from Apis mellifera (AmFAR1) or Marinobacter aquaeolei VT8 (Maqu_2220) in S. cerevisiae and achieved maximum yields of 3.22 \ub1 0.36 mg/g cell dry weight (CDW) and 7.84 \ub1 3.09 mg/g CDW, respectively, after 48 h. Moreover, we enabled the synthesis of jojoba-like WEs up to a chain length of C42, catalyzed by a combination of Maqu_2220 together with the WS from S. chinensis (SciWS) and the S. cerevisiae elongase Elo2p, with a maximum yield of 12.24 \ub1 3.35 mg/g CDW after 48 h

Establishing very long-chain fatty alcohol and wax ester biosynthesis in Saccharomyces cerevisiae

Wax esters (WEs) are neutral lipids and can be used for a broad range of commercial applications, including personal care products, lubricants, or coatings. They are synthesized by enzymatic reactions catalyzed by a fatty acyl reductase (FAR) and a wax ester synthase (WS). At present, commercially used WEs are mainly isolated from Simmondsia chinensis (jojoba), but the high extraction costs and limited harvest areas constrain their use. The use of FARs in combination with different WSs to achieve a synthesis of jojoba-like WEs in bacteria and yeast has been reported previously, but the products were restricted to C28-C36 WEs. These rather short WEs make up only a very small percentage of the total WEs in natural jojoba oil. The synthesis of longer chain WEs (up to C44) in Saccharomyces cerevisiae has so far only been achieved after substrate feeding. Here we identified new routes for producing very long-chain fatty alcohols (VLCFOHs) up to a chain length of C22 by heterologous expression of a FAR derived from Apis mellifera (AmFAR1) or Marinobacter aquaeolei VT8 (Maqu_2220) in S. cerevisiae and achieved maximum yields of 3.22 \ub1 0.36 mg/g cell dry weight (CDW) and 7.84 \ub1 3.09 mg/g CDW, respectively, after 48 h. Moreover, we enabled the synthesis of jojoba-like WEs up to a chain length of C42, catalyzed by a combination of Maqu_2220 together with the WS from S. chinensis (SciWS) and the S. cerevisiae elongase Elo2p, with a maximum yield of 12.24 \ub1 3.35 mg/g CDW after 48 h

Biosynthesis of insect sex pheromone precursors via engineered β-oxidation in yeast

Mating disruption with insect sex pheromones is an attractive and environmentally friendly technique for pest management. Several Lepidoptera sex pheromones have been produced in yeast, where biosynthesis could be accomplished by the expression of fatty acyl-CoA desaturases and fatty acyl-CoA reductases. In this study, we aimed to develop yeast Yarrowia lipolytica cell factories for producing Lepidoptera pheromones which biosynthesis additionally requires β-oxidation, such as (Z)-7-dodecenol (Z7-12:OH), (Z)-9-dodecenol (Z9-12:OH), and (Z)-7-tetradecenol (Z7-14:OH). We expressed fatty acyl-CoA desaturases from Drosophila melanogaster (Dmd9) or Lobesia botrana (Lbo_PPTQ) and fatty acyl-CoA reductase from Helicoverpa armigera (HarFAR) in combinations with 11 peroxisomal oxidases of different origins. Yeast cultivations were performed with supplementation of methyl myristate (14:Me). The oxidase Lbo_31670 from L. botrana provided the highest titers of (Z)-7-dodecenoate, (Z)-9-dodecenoate, and (Z)-7-tetradecenoate. However, no chain-shortened fatty alcohols were produced. The mutation of fatty acid synthase (Fas2p(I1220F)) to increase myristate production did not lead to targeted fatty alcohol production. The problem was solved by directing the reductase into peroxisomes, where the strain with Dmd9 produced 0.10 ± 0.02 mg/l of Z7-12:OH and 0.48 ± 0.03 mg/l of Z7-14:OH, while the strain with Lbo_PPTQ produced 0.21 ± 0.03 mg/l of Z9-12:OH and 0.40 ± 0.07 mg/l of Z7-14:OH. In summary, the engineering of β-oxidation in Y. lipolytica allowed expanding the portfolio of microbially produced insect sex pheromones

Pre-operative knee extensor and flexor torque after secondary ACL rupture: a comparative retrospective analysis

Abstract Background Secondary anterior cruciate ligament (ACL) ruptures are a relevant clinical concern after surgical treatment of a primary ACL rupture. However, there is a lack of scientific evidence related to the role of muscle strength prior to revision surgery in a second ACL rupture. The aim of this study was to assess differences in knee extensor and flexor strength in patients before primary and secondary ACL reconstruction compared to healthy controls. Methods In total, n = 69 age, weight and sex matched individuals were included in the study: n = 23 patients with isolated primary ACL rupture, n = 23 with secondary ACL rupture, and n = 23 matched healthy controls. Maximal isokinetic knee extension and flexion torque normalized to body mass was assessed for both legs. Results For patients with secondary ACL ruptures, torques were reduced in the non-injured (extension: 1.94 Nm/kg vs. 2.46 Nm/kg, p < 0.05, flexion: 1.25 Nm/kg vs. 1.59 Nm/kg, p < 0.05) and the injured leg (extension: 1.70 Nm/kg vs. 2.46 Nm/kg, p < 0.05, flexion: 1.14 Nm/kg vs. 1.59 Nm/kg, p < 0.05) compared to healthy controls. For patients with a primary ACL rupture torques were reduced in the non-injured (extension: 1.92 Nm/kg vs. 2.46 Nm/kg, p < 0.05, flexion: 1.24 Nm/kg vs. 1.59 Nm/kg, p < 0.05) and the injured leg (extension: 1.38 Nm/kg vs. 2.46 Nm/kg, p < 0.05, flexion: 1.01 Nm/kg vs. 1.59 Nm/kg, p < 0.05) compared to healthy controls. There were no differences between patients with primary and secondary ruptures, except of the knee extension on the injured leg showing higher values after a secondary ACL rupture (1.38 Nm/kg vs. 1.70 Nm/kg, p < 0.05). Conclusions The findings indicate that maximal knee torques were significantly reduced in patients with primary and secondary ACL ruptures before surgical reconstruction for the non-injured and injured leg as compared to healthy controls. Further investigations are needed to assess strength abilities before and after a second revision within a prospective design