An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo production of 1-octanol in Saccharomyces cerevisiae

An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo... Background: The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase- mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway. R1834K Results: The previously engineered short-chain acyl-CoA producing yeast Fas1 /Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involve- ment of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl- −1 CoA was converted to 1-octanol up to a titer of 26.0 mg L in a 72-h fermentation. The additional accumulation of −1 90 mg L octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was sup- plied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escheri- −1 chia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L . −1 However, in growth tests concentrations even lower than 50.0 mg L turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake. Conclusions: By providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells. Keywords: Fatty alcohol, 1-octanol, Carboxylic acid reductase, Biofuel, Octanoic acid, Caprylic acid, Fatty acid synthase, Short-chain fatty acids, Yeast, Saccharomyces cerevisiae *Correspondence: e.boles@bio.uni-frankfurt.de Faculty of Biological Sciences, Institute of Molecular Bioscience, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 2 of 12 [5–9], 1-octanol has acquired special attention as substi- Background tute for diesel and jet fuels [10–13]. Previous studies [10, Dwindling fossil resources and a growing global energy 14] compared various characteristics of fossil-derived demand, especially in the sector of human mobility as well as bio-derived diesel fuels with saturated short- and transportation, are leading to economic and envi- and medium-chain alcohols, and showed that 1-octanol ronmental burdens. This development poses a serious exhibits best matching overall properties compared to threat for the environment with respect to emissions of ethanol or other long-chain alcohols. greenhouse gases and particulate matter from traditional Various approaches are under investigation for the fuels like gasoline and diesel [1, 2]. An alternative is the microbial synthesis of higher unbranched alcohols with development of sustainable and regenerative fuels from respect to the origin of the saturated carbon chain and renewable feedstocks. However, those substitutes are the formation of the terminal hydroxyl group. They are not always compatible with the existing infrastructure based to some extent on entirely different metabolic for distribution or with traditional vehicle engines [3], pathways in bacterial as well as yeast systems (Fig.  1). but may require technical modifications of engines due Most approaches aim at harvesting acyl chain for the syn- to different physicochemical properties and combustion thesis of the higher alcohol. Two major biochemical strat- behaviors. Therefore, current research focuses on the egies can be distinguished for the acyl chain synthesis: (1) application of so-called drop-in biofuels. They are consid - The alpha-ketoacid route [15] exerts the recursive elon - ered as complete replacements of fossil fuels or as addi- gation of alpha-ketoacids by one carbon atom with an tives for blending due to similar characteristics regarding adapted leucine biosynthesis pathway and gives access to critical parameters [4, 5]. Among a comprehensive port- saturated carbon chains from C to C [16]. (2) Fatty acid folio of approved molecules from microbial production 3 9 Fig. 1 Common pathways for microbial higher alcohol production. Three different recursive metabolic pathways, fatty acid synthesis (a), reverse β-oxidation (b), or an artificial pathway engineered from l -leucine biosynthesis with enzymes LeuA to LeuD (c) can serve to provide different precursors with carbon chain lengths as indicated. Reaction steps for which shifted chain length control has successfully been reported are highlighted in red. For description and abbreviations, see text Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 3 of 12 synthesis or an artificially induced reverse β-oxidation of the desired 1-octanol from product mixtures later on. [17, 18] exerts two-carbon elongation of beta-ketoacids Akhtar et al. [14] were first in publishing the introduction for saturated acyl chain creation. of a thioesterase, Tes3 from Anaerococcus tetradius, in a In all these cases, the desired alcohol must be formed CAR/Ahr-expressing E. coli, which is highly specific for in a final reaction step via reduction of a correspond - C - and C -acyl thioesters. In this way, they harvested 6 8 ing precursor, either CoA-thioester, ACP-tethered thi- immature fatty acyl-ACPs from the bacterial fatty acid −1 oester, or free carboxylate. Fatty acyl-CoA reductases biosynthesis and obtained up to 62  mg  L 1-octanol −1 (FAR) and acyl-ACP reductases (AAR) proved to be suit- besides 29 mg L 1-hexanol with laboratory E. coli BL21 able for the reduction of both thioester species, which cells [14]. they directly reduce to the corresponding alcohol [17, Yeast as the applied organism offers certain advantages 19–26] and often do not discriminate between ACP over the hitherto exclusively reported E. coli systems for and CoA as carrier [27]. A free carboxylate is compara- microbial 1-octanol production. S. cerevisiae is highly bly unreactive, but can be efficiently converted to a C robust, shows a high tolerance to stress in fermentative n-1 aldehyde by α-dioxygenases (αDOX) under decarboxy- processes, and ferments sugars at low pH values [42, 43]. lative elimination of the terminal carboxyl group [28] or Also, many genetic manipulation tools are established to a C aldehyde by carboxylic acid reductases (CAR) which make yeast a very attractive organism for meta- [29–31]. The CAR-enzyme family requires a phospho - bolic engineering [44]. Several studies have been dealing pantetheinylation by a phosphopantetheinyl transferase with the overproduction of long-chain fatty acids and to be active [32]. The aldehydes can be further reduced derivatives in S. cerevisiae [24–26, 30, 45]. to alcohols by various aldehyde reductases (ALR) or Due to the spatial encapsulation of fatty acid synthe- alcohol dehydrogenases (ADH) [6, 33]. A broad appli- sis in fungi in a barrel-shaped multienzyme complex cability of all these reducing enzymes is facilitated by a [46–50], the strategy of hydrolyzing short/medium-chain promiscuous substrate acceptance of FARs/AARs [19, acyl-ACP with specific thioesterases, as e.g., Tes3, is dif - 20, 23, 34, 35] as well as CAR [31, 36, 37] and ALR/ADH ficult to apply for this system. In previous studies, this [30]. In aiming for microbial short/medium-chain alco- was addressed by introducing a structurally different hol production, the bottleneck does therefore rather lie metazoan fatty acid synthase with fused thioesterase [51] in the supply of the respective short/medium-chain acyl or the incorporation of a thioesterase in the fungal fatty chain than the downstream reducing enzymes. This is acid synthase’s reaction chamber [52]. We have recently reflected in the variety of publications about fatty alco - reported the rational engineering of the type I fatty acid hol production derived from the naturally defined pool synthase (FAS) from S. cerevisiae for the selective pro- of fatty acids in the C /C -range [23, 25, 38], while duction of short/medium-chain fatty acids (C or C ) 16 18 6 8 work on short/medium-chain alcohol production is rare. [53] based on a preceding in vitro/in silico approach [54]. Marcheschi et  al. [39] reported the production of mixed Mutations in the selected enzymatic domains, controlling −1 C –C -alcohols with the α–ketoacid route using mutated fatty acid chain length, led to a total yield of 245 mg L 3 8 LeuA variants in E. coli, but with a maximal 1-octanol (C or C fatty acid) and a specificity for C fatty acids of 6 8 8 −1 yield of 15  mg  L , which accounts for only 0.1% in this 90% (of secreted fatty acids), resembling to date the most mixture. Higher titers were reported using a reverse efficient and most specific short/medium-chain fatty β-oxidation route and an aldehyde/alcohol dehydroge- acids producing yeast strains [51, 52, 55, 56]. We con- nase AdhE2 from Clostridium acetobutylicum for reduc- sider this strain as ideal platform to produce 1-octanol. −1 ing the obtained CoA-ester to give 65  mg  L 1-octanol Here, we show that combining an octanoyl-CoA pro- as one component of a C –C alcohol mixture in E. coli ducing engineered FAS from yeast with heterologously 2 10 −1 [40] or even 100  mg  L as minor product after further expressed carboxylic acid reductases and aldehyde reduc- metabolic optimizations [18]. For the direct usage of tases, together with endogenous thioesterases and alco- fatty acyl-CoAs as precursors, Sheng et al. [41] reported hol dehydrogenases, enables efficient de novo production an elaborate strategy to escape from the normal product of 1-octanol from glucose with S. cerevisiae. spectrum of mainly C /C . They directed a fatty acyl- 16 18 CoA reductase to the peroxisome to capture medium- Results and discussion chain substrates from β-oxidation, which resulted in the Biosynthesis of 1‑octanol from glucose −1 production of 1  g  L C10, C and C fatty alcohols in In this study, we engineered a synthetic pathway to pro- 12 16 S. cerevisiae [41]. In spite of the success of producing duce 1-octanol from glucose in S. cerevisiae in which short/medium-chain alcohols, these examples illustrate the chain length of the fatty alcohol is determined by the difficulty of chain length control that causes dissipa - the product release of a mutated yeast FAS, namely R1834K tion of synthetic capacity and finally requires separation Fas1 /Fas2 [53]. Host strain RPY21 which exhibits Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 4 of 12 −1 −1 −1 deletions of both FAS genes, FAS1 and FAS2, as well as to 26.0 ± 3.6  mg  L (2.8 ± 0.3  mg  L OD ) after 72  h the gene FAA2 encoding a short/medium-chain fatty (Fig.  3a), which was also indicated by a strong specific acyl-CoA synthetase, was transformed with centromeric smell of the cell cultures. The production of 1-octanol R1834K vectors expressing FAS1 and FAS2 under control confirmed that S. cerevisiae contains suitable endogenous of their native promoters. We had previously reported ADHs/ALRs for the reduction of 1-octanal (Fig.  2) as R1834K that the FAS mutant version of FAS in concert shown earlier [29]. Besides 1-octanol, also small amounts −1 with the thioesterase activities of three short-chain acyl- of 1-hexanol (5.5 ± 0.6 mg L after 72 h; data not shown) R1834K CoA:ethanol acyltransferases, Eht1, Eeb1, and Mgl2, could be determined. This was expected since FAS produces high amounts of octanoic acid [53]. To avoid also produces small amounts of hexanoic acid which subsequent degradation of octanoic acid, we additionally is then also reduced by CAR to its corresponding alde- deleted FAA2 which is required for the re-activation of hyde [14, 31, 53]. In contrast, although decanoic acid was R1834K octanoic acid to octanoyl-CoA initiating its degradation another side product of FAS [53], decanol was not by β-oxidation [57]. Interestingly, it has been reported detected. before that the deletion of FAA2 already in a wild-type It is worthy to mention that even the control strain FAS background leads to the production of low amounts RPY21, expressing wild-type FAS and overexpress- R1834K of octanoic acid [58]. In the F AS mutant back- ing CAR and Sfp, secreted 1-octanol in small amounts −1 ground, the deletion of FAA2 resulted in an increase of (3.9 ± 0.1 mg L ) (Fig. 3a), which has also been reported −1 up to 25% in octanoic acid production (301 mg L ; data before in a comparable setup [29]. Possibly, this is the not shown). To produce 1-octanol from free octanoic result of the small amounts of octanoic acid produced by acid, we overexpressed a heterologous carboxylic acid wild-type FAS or the mitochondrial type II FAS system in reductase (CAR) from Mycobacterium marinum under S. cerevisiae [53, 54, 59], especially together with deletion −1-−392 the control of the strong and constitutive HXT7 of FAA2 as in strain RPY21 [58]. promotor fragment together with the phosphopanteth- To determine the limiting factors in 1-octanol produc- R1834K einyl transferase Sfp from Bacillus subtilis under the tion in strain RPY21/FAS overexpressing CAR and control of the strong PGK1 promoter on a multicopy vec- Sfp, we furthermore analyzed the accumulation of free R1834K tor in RPY21 expressing Fas1 /Fas2 [14]. CAR uses octanoic acid in the culture medium, and detected a con- −1 NADPH and ATP in order to reduce free fatty acids to siderable titer of 90.3 ± 6.8  mg  L after 72  h (Fig.  3c). the corresponding aldehydes [31] (Fig.  2) and must be Compared to the same strain without CAR which accu- −1 activated by a phosphopantetheine transferase: in this mulated 118.9 ± 7.3  mg  L octanoic acid, this reveals study, Sfp, which attaches the prosthetic group 4′-phos- that 76% of precursor substrate remained unused for phopantetheine to the enzyme [31, 32]. The aldehydes octanol production, suggesting a bottleneck either in the can be further reduced to primary alcohols by endog- CAR or the ADH/ALR reactions, or losses due to secre- enous alcohol dehydrogenases (ADH) or aldehyde reduc- tion of octanoic acid out of the cells. tases (ALR) in yeast [29, 33]. The decreased maximal optical density after 72  h of R1834K Cultivation of the yeast cells in potassium phosphate the yeast culture RPY21/FAS overexpressing CAR buffered medium at pH 6.5 with 2% glucose under and Sfp indicated that 1-octanol or the other pathway aerobic conditions resulted in the accumulation of intermediates octanoic acid and octanal might have a 1-octanol in the extracellular medium with titers of up toxic effect on the yeast cells and inhibit their growth Fig. 2 Metabolic pathway for 1-octanol production in S. cerevisiae from glucose via fatty acid biosynthesis. A mutant version of S. cerevisiae FAS R1834K (Fas1 /Fas2) produces octanoyl-CoA which is hydrolyzed by endogenous thioesterases ( TE) to the free octanoic acid [53]. A heterologous carboxylic acid reductase (CAR) from M. marinum then converts the free fatty acid to octanal which is further reduced to 1-octanol by endogenous alcohol dehydrogenases (ADH) and aldehyde reductases (ALR) [29]. CAR must be activated by the phosphopantetheinyl tranferase Sfp from Bacillus RK R1834K subtilis [31]. Heterologous or mutated enzymes are marked in blue. F AS means FAS Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 5 of 12 a bc 30 20 15 80 0 0 0 0 20 40 60 80 0 20 40 60 80 time [h] time [h] RK RK FAS FAS FAS CAR CAR vector R1834K Fig. 3 Production of 1-octanol with S. cerevisiae. Strain RPY21 expressing mutated F AS as well as CAR and Sfp from a high-copy plasmid (black), was grown for 72 h in buffered YPD medium. For reference, analogous combinations of wild-type FAS, CAR, and Sfp (red) as well as R1834K mutated FAS with empty vector instead of CAR and Sfp are shown (blue). Error bars reflect the standard deviations from three biological replicates. OD (a) and extracellular 1-octanol concentrations (b) were analyzed at different time points. c Final octanoic acid concentration in the RK R1834K extracellular culture medium after 72 h. FAS means FAS (Fig.  3b). To test the inhibitory effects, different con - Re‑uptake of secreted octanoic acid from the medium centrations of 1-octanol, octanal, and octanoic acid To rule out that the secretion of octanoic acid into the were added to yeast cultures of the wild-type strain culture medium is a limiting factor for 1-octanol produc- BY4741 and growth curves were determined (Fig.  4). tion, we analyzed the transport of octanoic acid back into −1 Indeed, at the lowest tested concentration of 50 mg L the cell and its further conversion to 1-octanol by over- of 1-octanol or octanal in the culture medium, slightly expressing CAR and Sfp in the wild-type strain BY4741 −1 inhibited growth of the yeast cells was observed, and in buffered media supplemented with 90 mg L octanoic −1 growth was completely prevented at a concentration of acid. After 24 h, 11.0 ± 0.8 mg L 1-octanol was detected −1 150 mg L (Fig. 4a, b). In contrast, octanoic acid is less (Fig.  5a), showing that octanoic acid can be transported toxic, and moderate cell growth was even observed at a back into the cell and subsequently be reduced by CAR −1 concentration of 400 mg L (Fig. 4c). together with ADHs/ALRs to 1-octanol. Nevertheless, the concentration of octanoic acid in the medium also ab c 15 15 15 10 10 10 5 5 5 0 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 time [h] time [h] time [h] -1 -1 -1 -1 0 mg L 100 mg L 0 mg L 200 mg L -1 -1 -1 -1 50 150 100 400 mg L mg L mg L mg L Fig. 4 Inhibitory effects of 1-octanol, octanal, and octanoic acid added to the culture medium of strain BY4741. The yeast strain BY4741 was cultivated for 24 h in buffered YPD medium supplemented with different concentrations of a 1-octanol b octanal, or c octanoic acid. Error bars reflect the standard deviations from two biological replicates. OD was analyzed at different time points RK FAS CAR FAS CAR RK FAS vector -1 OD 1-octanol [mg L ] OD OD 600 600 OD -1 octanoic acid [mg L ] Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 6 of 12 a b 15 120 medium + C acid 10 80 vector + C acid CAR + C acid 5 40 CAR 0 0 0 20 40 60 80 0 20 40 60 80 time [h] time [h] Fig. 5 Uptake and conversion analysis of octanoic acid. a 1-Octanol production and b octanoic acid consumption of S. cerevisiae BY4741 expressing CAR and Sfp from a multicopy plasmid. Cultivation was performed for 72 h in buffered YPD medium without and with supplementation −1 of 90 mg L octanoic acid. For reference, noninoculated cultivation medium was treated analogously. Error bars reflect the standard deviations from three biological replicates decreased in the absence of a CAR (Fig.  5b), indicating in 1-octanol production is also reflected in a decreased alternative reaction pathways for octanoic acid conver- maximal optical density of the cell culture after 72  h sion in S. cerevisiae. Probably, this is due to degradation compared to the control strain without Ahr (Fig.  6b), of octanoic acid via β-oxidation in strain BY4741 [57, 60]. probably due to the negative effect of 1-octanol on cell Moreover, the amount of 1-octanol in the extracellular growth (Fig.  4a). When the 1-octanol titers are nor- medium also decreased after 24 h (Fig. 5a). This might be malized to the final OD of the cultures, overexpres- due to its reconversion to octanal by endogenous alco- sion of Ahr resulted in a threefold increase of 1-octanol −1 −1 −1 hol dehydrogenases which then might be oxidized, e.g., (7.9 ± 0.4  mg  L OD compared to 2.8 ± 0.3  mg  L −1 by the dehydrogenase Hfd1, which has been reported to OD after 72  h). Furthermore, the presence of Ahr convert at least long-chain aliphatic aldehydes to carbox- nearly completely prevented the accumulation of octa- −1 ylic acids [33]. noic acid (0.6 ± 0.0  mg  L after 72  h) compared to the −1 strain without Ahr (113.2 ± 2.6  mg  L ) (Fig.  6c). This Overexpression of an aldehyde reductase Ahr from E. coli result revealed that the reduction of octanal to 1-octanol increases 1‑octanol production by endogenous ADHs and ALRs was a limiting step in The previous experiment showed that octanoic acid can the original pathway. freely diffuse or is transported in both directions across the yeast plasma membrane, and therefore should not In situ extraction of 1‑octanol be limiting 1-octanol production. Therefore, we tested Since already minor concentrations of 1-octanol inhib- octanal reduction as a limiting factor in 1-octanol pro- ited growth of the yeast cells (Fig.  4a), which makes duction. For this reaction, S. cerevisiae contains various further improvements toward higher 1-octanol titers endogenous ADHs/ALRs, successfully employed for the difficult, one possibility to circumvent this problem aldehyde–alcohol conversion in the C –C range already could be an in  situ extraction of released 1-octanol in 8 18 earlier [29]. However, little is known about the efficien - a two-phase fermentation using an organic solvent as cies of these enzymes to short/medium-chain fatty alde- the secondary phase. An appropriate solvent is dode- hydes, especially to octanal. It has been shown by Akhtar cane which was shown to have no negative effects on et  al. [31] that the aldehyde reductase Ahr from E. coli yeast growth (Fig.  7) [61, 62] and was already used for accepts a broad range of aliphatic aldehydes (C to C ). production of long-chain fatty alcohols [22, 24]. To test 4 16 We overexpressed Ahr from E. coli under the control of if dodecane as secondary phase can improve growth in −1-−392 the HXT7 promotor fragment from a high-copy the presence of 1-octanol by trapping it out of the yeast plasmid together with the plasmid encoding for CAR culture, different concentrations of 1-octanol were R1834K and Sfp in strain RPY21/FAS under aerobic condi- added to yeast cultures of the wild-type strain BY4741 tions. Overexpression of Ahr led to a twofold increase overlaid with 20% dodecane, and growth curves were −1 in the absolute 1-octanol titer (49.5 ± 0.8  mg  L after determined (Fig.  7). Indeed, this time, the addition of −1 72 h) in the extracellular medium (Fig. 6a). This increase even 150  mg  L 1-octanol to the yeast culture with -1 1-octanol [mg L ] -1 octanoic acid [mg L ] Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 7 of 12 ab c 60 20 30 10 0 0 0 20 40 60 80 0 20 40 60 80 time [h] time [h] RK RK FAS FAS FAS CAR CAR CAR Ahr Ahr vector R1834K Fig. 6 Increase in 1-octanol production in a yeast strain by additional overexpression of heterologous Ahr. Strain RPY21/FAS expressing CAR and Sfp together with Ahr from E. coli from a high-copy plasmid (black), was grown for 72 h in buffered YPD medium. For comparison, analogous R1834K combinations of wild-type FAS with CAR, Sfp, and Ahr (red) as well as mutated F AS with empty vector instead of Ahr are shown (blue). Error bars indicate the standard deviation from three biological replicates. a OD and 1-octanol b were analyzed at different time points. c Final RK R1834K octanoic acid concentration after 72 h. FAS means FAS the dodecane phase were analyzed. We found that in the two-phase fermentation, no 1-octanol was detect- -1 able in the aqueous phase revealing the efficient extrac - 0 mg L -1 tion of 1-octanol. However, analysis of 1-octanol in the 0 mg L + Dd dodecane layer showed that 1-octanol production was -1 50 mg L + Dd reduced by about 25% compared to the strain cultivated -1 100 mg L + Dd without dodecane (Fig.  8a) (it should be noted that the 5 -1 150 mg L + Dd concentration of 1-octanol determined in the dodecane phase was calculated on the volume of the aqueous phase of the culture). This is likely due to the sequestra - 0 5 10 15 20 25 tion of octanoic acid into the dodecane phase (Fig. 8a). time [h] R1834K Cultivation of the strain RPY21/FAS without the Fig. 7 Growth behavior of strain BY4741 in the presence of 20% dodecane and different 1-octanol concentrations. BY4741 was 1-octanol pathway (which yields higher titers of octa- cultivated up to 24 h in buffered YPD medium overlaid with noic acid) in buffered medium overlaid with 20% dode - 20% dodecane (Dd) and supplemented with different 1-octanol cane confirmed the partial accumulation of octanoic concentrations. For comparison, also the growth curve of a culture acid in the organic phase (Fig.  8b). Although addition without dodecane was determined (gray). Error bars reflect the of dodecane could circumvent the inhibitory effect of standard deviations from two biological replicates. OD was analyzed at different time points added 1-octanol on growth (Fig.  7), surprisingly, this was not the case when 1-octanol was produced by the cells themselves (Fig.  8a). Here, the growth of the 20% dodecane did not affect growth. Based on these 1-octanol producing yeast cultures with dodecane was results, a two-phase fermentation with the 1-octanol only slightly improved. Taken together, in  situ extrac- producing strain was performed using dodecane as tion could not improve 1-octanol production and the a secondary phase for in  situ extraction. To achieve speedup of the downstream pathway for octanoic acid this, we overexpressed Ahr, CAR, and Sfp from high- production is necessary to compensate the loss of octa- R1834K copy plasmids in strain RPY21/FAS under aero- noic acid into the organic layer. The results also suggest bic conditions in buffered media overlaid with and that beside the toxic effect of 1-octanol, its production without 20% of dodecane. After 72  h, the growth was somehow also inhibits growth of the cells which might determined by OD measurements, and the amounts 600 partially be connected to a limited supply of ATP and of fatty acids and fatty alcohols in the aqueous and in the cofactor NADPH required for octanoic acid pro- duction as well as for the CAR and Ahr reactions. RK FAS CAR Ahr FAS CAR Ahr RK FAS CAR vector OD -1 1-octanol [mg L ] OD -1 octanoic acid [mg L ] OD OD Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 8 of 12 RK RK a b FAS CAR Ahr FAS 15 150 25 aqueous 30 phase 10 100 dodecane phase 5 50 0 0 0 0 0 dodecane - + - + - + 20% R1834K Fig. 8 In situ extraction of 1-octanol and octanoic acid in a two-phase fermentation of yeast. Strain RPY21/FAS overexpressing CAR and Sfp R1834K together with Ahr (a) and control strain RPY21/FAS (b) were grown for 72 h, in buffered YPD medium overlaid with (+) and without (−) 20% of dodecane. Error bars reflect the standard deviations from two biological replicates. After 72 h, the final OD (·) and the final concentrations RK R1834K of 1-octanol and octanoic acid were analyzed. FAS means FAS . The gray areas indicate concentrations of 1-octanol or octanoic acid, respectively, calculated on the basis of the volume of the aqueous phase of the culture, but based on the amounts measured in the dodecane phase. The white-colored areas indicate concentrations measured directly in the aqueous phase homologous recombination were transformed accord- Conclusion ing to protocols by Gietz and Schiestl [65]. This is the first study to report on dedicated produc - tion of 1-octanol in S. cerevisiae. We achieved the nearly selective production of 1-octanol in S. cer- Growth experiments evisiae by combining a proprietary C -acid producing The strain BY4741 was pregrown in YPD medium buff - R1834K FAS -mutant with a two-step reduction pathway ered with 100 mM potassium phosphate and adjusted to composed of CAR, Sfp, and Ahr [14, 53]. In the pro- a pH of 6.5 as described in Gajewski et  al. [53] without cess of the current study, the chain length specificity supplementation of free FAs. After washing steps, the of the carbon chain-providing FAS is the decisive step main culture (50 mL YPD in 300 mL flasks; two biological to ensure specific 1-octanol production. Nevertheless, replicates) was inoculated to OD = 0.1, supplemented toxicity of 1-octanol and a negative effect of its produc - with different concentrations of 1-octanol (0, 50, 100, −1 −1 tion on growth of the cells pose fresh challenges for and 150 mg L ), octanal (0, 50, 100, and 150 mg L ), or −1 further optimizations. octanoic acid (0, 100, 200, and 400  mg  L ) and aerobi- cally shaken for 24  h at 180 r.p.m. at 30  °C. OD was analyzed at different time points. Methods Yeast strain, media, and transformation S. cerevisiae fermentations The haploid S. cerevisiae strain RPY21 used in this Saccharomyces cerevisiae strains were cultured in YPD study for fatty alcohol production has a BY background medium as described in Gajewski et al. [53] without sup- and is based on BY.PK1238_1A_KO with knocked out plementation of free FAs. The medium was additionally FAS1 and FAS2 from previous studies [53]. The dele - buffered with 100 mM potassium phosphate and adjusted tion of the fatty acyl-CoA synthetase Δfaa2 was gener- to a pH of 6.5. To test the 1-octanol production of the ated using the CRISPR–Cas9 system [63]. The relevant different strains, a preculture was inoculated in buff - genotype of RPY21 is Matα; ura3Δ0; his3Δ0; leu2Δ0; ered YPD medium and grown aerobically to exponen- TRP1; lys2Δ0; MET15; fas1::uptag-kanMX4-downtag; tial phase. After washing steps, the main culture (50 mL fas2::uptag-kanMX4-downtag; Δfaa2. RPY21 was trans- YPD with respective antibiotics; two to three biological formed as described in Gajewski et al. [53]. Selection of replicates) was inoculated to OD = 0.1 and aerobically yeast transformants was done on the defined synthetic shaken for 72 h at 180 r.p.m. and 30 °C. complete media (SCD) as described in Bruder et al. [64] For the in situ extraction, the main culture (25 mL YPD without leucine and histidine containing the respec- with respective antibiotics; two biological replicates) was −1 −1 tive antibiotics (200 µg mL hygromycin; 100 µg mL inoculated to OD = 0.1 and overlaid with 20% dode- nourseothricin sulfate). The strain BY4741 and the cane (5  mL). Fatty alcohols, fatty acids, and OD were strain CEN.PK2-1C for plasmid construction by analyzed at different time points. The final 1-octanol -1 1-octanol [mg L ] -1 octanoic acid [mg L ] -1 octanoic acid [mg L ] Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 9 of 12 concentration after 72  h was normalized to the final four PCR fragments with 30 bp overlaps. Yeast was trans- −1 OD value (mg L  OD ). formed as described above with a mixture of these frag- 600 600 ments generated by PCR using primers shown in Table 2. Plasmid and strain construction The assembled plasmids were recovered by yeast DNA The plasmids used in this study are listed in Table  1. FAS- preparations and transformed into E. coli for amplifica - related plasmids are described in Gawjeski et  al. [53]. tion by standard procedures. The construction of the Genes encoding carboxylic acid reductase CAR from M. plasmid pRS62N-Ahr was performed by Gibson assem- marinum (UniProt: B2HN69) and the phosphopanteth- bly as described in Gibson et al. [67]. The bacterial gene einyl transferase Sfp from B. subtilis (UniProt: P39135) Ahr [previously known as yigB (UniProt: P27250)] was were codon-optimized according to the yeast glycolytic amplified from chromosomal DNA of E. coli DH5α using codon usage [66]. The plasmid pRS62H–CAR–Sfp was primers shown in Table 2. assembled by homologous recombination in yeast with Fatty alcohol extraction For the extraction of fatty alcohols from the culture medium, the cells were separated from the medium (8000 Table 1 List of plasmids used in this study rcf, 3  min). 500  µL supernatant was mixed with 1  mL −1 ethyl acetate containing 50  mg  L heptanol as internal Plasmid name Description References standard and thoroughly shaken. After centrifugation Sc pRS315-FAS1-Wt CEN6ARS4, AmpR, LEU2, p - FAS1-Wt- [53] FAS1 (5000 rcf, 2 min), 500 µL of the organic phase was trans- FAS1 ferred to a gas chromatography (GC) vial. For the deter- Sc R1834K pRS315-FAS1-RK CEN6ARS4, AmpR, LEU2, p - FAS1 - [53] FAS1 mination of the amount of fatty alcohols in the dodecane FAS1 Sc layer, 100  µL of the dodecane overlay was mixed with pRS313-FAS2-Wt CEN6ARS4, AmpR, HIS3, p - FAS2-Wt- [53] FAS2 −1 t 900  µL ethyl acetate containing 50  mg  L heptanol as FAS2 pRS62H 2µ, hphNT1, AmpR, truncated HXT7 pro- [68] internal standard in a GC vial. −1-−392 motor (p ) and FBA1 terminator HXT7 pRS62 N 2µ, natNT2, AmpR, shortened HXT7 pro- [68] Fatty acid extraction and derivatization −1-−392 motor (p ) and CYC1 terminator HXT7 For the extraction of free fatty acids present in the cul- −1-−392 Mm Bs pRS62H-CAR-Sfp pRS62H; p - CAR -t ; p - Sfp- This study HXT7 FBA PGK1 ture medium, the cells were separated from the medium PGM2 −1-−392 Ec (3500  rcf, 10  min). An internal standard (0.2  mg hepta- pRS62 N-Ahr pRS62N; p - Ahr-t This study HXT7 CYC1 noic acid) was added to 10  mL supernatant and mixed Genes from Saccharomyces cerevisiae (Sc), Mycobacterium marinum (Mm), E. coli with 1  mL 1  M HCl and 2.5  mL methanol–chloroform (Ec), and Bacillus subtilis (Bs) are indicated by prefixes in superscript. hphNT1 hygromycin resistance, Amp ampicillin resistance, natNT2 nourseothricin sulfate solution (1:1). The solution was vigorously shaken (5 min) resistance Table 2 Relevant primers for plasmid construction Primer name Sequence 5′–3′ Amplicon SHP35_pPGK1_forAAC CCT GGC GTT ACC CAA CTT AAT CGC CTT GCAG PGK1 promotor CAT GTT TGC AAA AAG AAC AAA ACT G CBP160_pPGK1_revTGT TTT ATA TTT GTT GTA AAA AGT AGA TAA TTAC PGK1 promotor CBP35_tPGM2_forAAC GAA TGA TTT ACT AAT GGC PGM2 terminator SHP36_tPGM2_revGAA ATC GGC AAA ATC CCT TAT AAA TCA AAA GAAT PGM2 terminator AGA CAA AAA ACT CGG GGT AGG TAA T SHP54_Ahr_forCAA AAA CAA AAA GTT TTT TTA ATT TTA ATC AAAAA Ahr ORF ATG TCG ATG ATA AAA AGC TAT GCC SHP53_Ahr_revATG TAA GCG TGA CAT AAC TAA TTA CAT GAC TCGA Ahr ORF GTC AAA AAT CGG CTT TCA ACAC DR_Faa2GGA AGA ATG CAG GTT ACA AAA AAC GGA TAA GAA Donor-DNA fragment for FAA2 CAA ACT TGT TTC GAA ATG TAC TTA TGA CGA TTTGG deletion AAC ACA TTC AAA CTA GAA AAA ACT TTG ATG TA CC_FAA2-revCGT AAG GTT TCA AAA TCT TCG ATC ATT TAT CTTTC Amplification of a CRISPR-Cas9 ACT GCG GAG plasmid pRCCN for deletion of FAA2, reverse CC_FAA2-fwGAA GAT TTT GAA ACC TTA CGG TTT TAG AGC TAGAA Amplification of a CRISPR-Cas9 ATA GCA AGT TAA AAT AAG G plasmid pRCCN for deletion of FAA2, forward The abbreviations within the primer names were used as follows: forward primer (fw) and reverse primer (rev) Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 10 of 12 and then centrifuged for 10 min at 3.000 rcf. The chloro - extraction experiment with dodecane as second phase, form layer was recovered and evaporated overnight. The the column Elite 5MS capillary column (30 m × 0.25 mm, methylation of the fatty acids was performed as described film thickness 1.00 µm, Perkin Elmer, Germany) was used in Ichihara and Fukubayashi [69]. The samples were dis - for analysis of FAMEs and fatty alcohols. The tempera - solved in 200 µL of toluene, mixed with 1.5 mL of meth- tures of the injector and detector were 250 and 300  °C, anol and 300  µL of 8.0% (w/v) HCl solution [conc. HCl respectively. The following temperature program was (35%  w/w; 9.7  mL) was diluted with 41.5  mL of metha- applied for FAMEs: 50  °C for 5  min, increase at the rate −1 nol], vortexed vigorously, and incubated at 100 °C for 3 h of 10 °C min to 120 °C and hold for 5 min; increase at −1 to form fatty acid methyl ester (FAME). After cooling the rate of 15  °C  min to 220  °C, and hold for 10  min, −1 under ice for 10 min, 1  mL H O and 1  mL hexane were increase at the rate of 20 °C min to 300 °C, and hold for added to the sample. The mixture was shaken thoroughly, 5 min. For fatty alcohols, the following temperature pro- and the organic phase was transferred to a GC vial. gram was applied: 50 °C for 5 min, increase at the rate of −1 For the derivatization of the fatty acids from the 20 °C min to 220 °C and hold for 2 min; increase at the −1 in  situ extraction experiment, the derivative reagent rate of 20 °C min to 300 °C, and hold for 5 min. Bis(trimethylsilyl)-trifluoroacetamide (BSTFA) was used. After separation of the cells from the medium (3500 rcf, Abbreviations 10 min), 50 µL 1 M HCl and 0.01 mg heptanoic acid as an OD : optical density at 600 nm; PGK1: 3-Phosphoglycerate kinase; HXT7: internal standard were added to 500 µL supernatant and high-affinity glucose transporter; PGM2: phosphoglucomutase; CYC1: cytochrome c; FBA1: fructose-1,6-bisphosphate aldolase; FAS: fatty acid mixed with 1 mL ethyl acetate. The ethyl acetate layer was synthase; FAA2: medium-chain fatty acyl-CoA synthetase; Ahr: aldehyde evaporated overnight, and the samples were dissolved reductase; CAR : carboxylic acid reductase; ALR: aldehyde reductase; ADH: in 100  µL ethyl acetate and mixed with 100  µL BSTFA. alcohol dehydrogenase; Sfp: phosphopantetheinyl transferase. For the determination of the amount of fatty acids in the Authors’ contributions dodecane layer, 20  µL of the dodecane layer was mixed SH, MF, MG, MO, and EB conceived the study. SH conducted the experiments. with 80 µL ethyl acetate, 0.01 mg of heptanoic acid as an SH, MO, and EB analyzed the data. SH, MF, and EB wrote the paper. All authors read and approved the manuscript. internal standard, and 100 µL BSTFA. The derivatization was done for 45  min at 80  °C. After cooling at 4  °C for Author details 15 min, the samples were analyzed by GC. Faculty of Biological Sciences, Institute of Molecular Bioscience, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecu- lar Life Sciences, Cluster of Excellence “Macromolecular Complexes”, Goethe GC‑FID analysis of FAMEs and fatty alcohols University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt, Germany. The gas chromatographic measurements were carried out on a Perkin Elmer Clarus 400 system (Perkin Elmer, Acknowledgements We thank Renata Pavlovic for providing the yeast strain RPY21. Germany) equipped with an Elite FFAP capillary column (30  m × 0.25  mm, film thickness: 0.25  µm; PerkinElmer, Competing interests Germany) and a flame ionization detector (Perkin Elmer, E.B. and M.G. are inventors of EP patent application No. 15 162 192.7 filed on April 1, 2015, and of EP patent application No. 15 174 342.4 filed on June Germany). 1  μL of the sample was analyzed after split 26, 2015, by Goethe-University Frankfurt, concerning short-chain acyl-CoA injection (1:10); helium was used as carrier gas (90 kPa). producing FAS variants. There are no other competing interests. For fatty acid methyl ester (FAME) quantification, the Availability of data and materials temperatures of the injector and detector were 200 and The datasets used and/or analyzed during the current study are available from 250  °C, respectively. The following temperature pro - the corresponding author upon reasonable request. gram was applied: heating to 50  °C for 5  min; increase −1 Consent for publication of 10 °C min to 120 °C and hold for 5 min; increase at −1 Not applicable. the rate of 15  °C  min to 180  °C and hold for 10  min; −1 increase at the rate of 20 °C min to 220 °C, and hold for Ethics approval and consent to participate Not applicable. 7 min. FAME were identified and quantified by compari - son with authentic standard substances. For fatty alcohol Funding quantification, an initial temperature of 50 °C was main - The project underlying this report is financially supported by the German Federal Ministry of Food and Agriculture following a decision of the German tained for 5 min, followed by an increase at the rate of −1 Bundestag under the Grant Number 22026315; the responsibility for the 20 °C min to 210 °C and kept constant for 5 min. After content of this publication lies with the authors. −1 a further increase at the rate of 20  °C  min to 230  °C, the temperature was kept constant for 6  min. The tem - Publisher’s Note peratures of both the injector and detector were 250  °C. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Fatty alcohols were identified and quantified by compari - son with authentic standard substances. For the in  situ Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 11 of 12 Received: 12 December 2017 Accepted: 23 May 2018 20. Willis RM, Wahlen BD, Seefeldt LC, Barney BM. Characterization of a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8: a bacterial enzyme catalyzing the reduction of fatty acyl-CoA to fatty alcohol. Biochemistry. 2011. https ://doi.org/10.1021/bi200 8646. 21. Rowland O, Domergue F. 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An engineered fatty acid synthase combined with a carboxylic acid reductase enables de novo production of 1-octanol in Saccharomyces cerevisiae

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Chemistry; Biotechnology; Plant Breeding/Biotechnology; Environmental Engineering/Biotechnology; Renewable and Green Energy; Renewable and Green Energy; Microbiology
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Abstract

Background: The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase- mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway. R1834K Results: The previously engineered short-chain acyl-CoA producing yeast Fas1 /Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involve- ment of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl- −1 CoA was converted to 1-octanol up to a titer of 26.0 mg L in a 72-h fermentation. The additional accumulation of −1 90 mg L octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was sup- plied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escheri- −1 chia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L . −1 However, in growth tests concentrations even lower than 50.0 mg L turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake. Conclusions: By providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells. Keywords: Fatty alcohol, 1-octanol, Carboxylic acid reductase, Biofuel, Octanoic acid, Caprylic acid, Fatty acid synthase, Short-chain fatty acids, Yeast, Saccharomyces cerevisiae *Correspondence: e.boles@bio.uni-frankfurt.de Faculty of Biological Sciences, Institute of Molecular Bioscience, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 2 of 12 [5–9], 1-octanol has acquired special attention as substi- Background tute for diesel and jet fuels [10–13]. Previous studies [10, Dwindling fossil resources and a growing global energy 14] compared various characteristics of fossil-derived demand, especially in the sector of human mobility as well as bio-derived diesel fuels with saturated short- and transportation, are leading to economic and envi- and medium-chain alcohols, and showed that 1-octanol ronmental burdens. This development poses a serious exhibits best matching overall properties compared to threat for the environment with respect to emissions of ethanol or other long-chain alcohols. greenhouse gases and particulate matter from traditional Various approaches are under investigation for the fuels like gasoline and diesel [1, 2]. An alternative is the microbial synthesis of higher unbranched alcohols with development of sustainable and regenerative fuels from respect to the origin of the saturated carbon chain and renewable feedstocks. However, those substitutes are the formation of the terminal hydroxyl group. They are not always compatible with the existing infrastructure based to some extent on entirely different metabolic for distribution or with traditional vehicle engines [3], pathways in bacterial as well as yeast systems (Fig.  1). but may require technical modifications of engines due Most approaches aim at harvesting acyl chain for the syn- to different physicochemical properties and combustion thesis of the higher alcohol. Two major biochemical strat- behaviors. Therefore, current research focuses on the egies can be distinguished for the acyl chain synthesis: (1) application of so-called drop-in biofuels. They are consid - The alpha-ketoacid route [15] exerts the recursive elon - ered as complete replacements of fossil fuels or as addi- gation of alpha-ketoacids by one carbon atom with an tives for blending due to similar characteristics regarding adapted leucine biosynthesis pathway and gives access to critical parameters [4, 5]. Among a comprehensive port- saturated carbon chains from C to C [16]. (2) Fatty acid folio of approved molecules from microbial production 3 9 Fig. 1 Common pathways for microbial higher alcohol production. Three different recursive metabolic pathways, fatty acid synthesis (a), reverse β-oxidation (b), or an artificial pathway engineered from l -leucine biosynthesis with enzymes LeuA to LeuD (c) can serve to provide different precursors with carbon chain lengths as indicated. Reaction steps for which shifted chain length control has successfully been reported are highlighted in red. For description and abbreviations, see text Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 3 of 12 synthesis or an artificially induced reverse β-oxidation of the desired 1-octanol from product mixtures later on. [17, 18] exerts two-carbon elongation of beta-ketoacids Akhtar et al. [14] were first in publishing the introduction for saturated acyl chain creation. of a thioesterase, Tes3 from Anaerococcus tetradius, in a In all these cases, the desired alcohol must be formed CAR/Ahr-expressing E. coli, which is highly specific for in a final reaction step via reduction of a correspond - C - and C -acyl thioesters. In this way, they harvested 6 8 ing precursor, either CoA-thioester, ACP-tethered thi- immature fatty acyl-ACPs from the bacterial fatty acid −1 oester, or free carboxylate. Fatty acyl-CoA reductases biosynthesis and obtained up to 62  mg  L 1-octanol −1 (FAR) and acyl-ACP reductases (AAR) proved to be suit- besides 29 mg L 1-hexanol with laboratory E. coli BL21 able for the reduction of both thioester species, which cells [14]. they directly reduce to the corresponding alcohol [17, Yeast as the applied organism offers certain advantages 19–26] and often do not discriminate between ACP over the hitherto exclusively reported E. coli systems for and CoA as carrier [27]. A free carboxylate is compara- microbial 1-octanol production. S. cerevisiae is highly bly unreactive, but can be efficiently converted to a C robust, shows a high tolerance to stress in fermentative n-1 aldehyde by α-dioxygenases (αDOX) under decarboxy- processes, and ferments sugars at low pH values [42, 43]. lative elimination of the terminal carboxyl group [28] or Also, many genetic manipulation tools are established to a C aldehyde by carboxylic acid reductases (CAR) which make yeast a very attractive organism for meta- [29–31]. The CAR-enzyme family requires a phospho - bolic engineering [44]. Several studies have been dealing pantetheinylation by a phosphopantetheinyl transferase with the overproduction of long-chain fatty acids and to be active [32]. The aldehydes can be further reduced derivatives in S. cerevisiae [24–26, 30, 45]. to alcohols by various aldehyde reductases (ALR) or Due to the spatial encapsulation of fatty acid synthe- alcohol dehydrogenases (ADH) [6, 33]. A broad appli- sis in fungi in a barrel-shaped multienzyme complex cability of all these reducing enzymes is facilitated by a [46–50], the strategy of hydrolyzing short/medium-chain promiscuous substrate acceptance of FARs/AARs [19, acyl-ACP with specific thioesterases, as e.g., Tes3, is dif - 20, 23, 34, 35] as well as CAR [31, 36, 37] and ALR/ADH ficult to apply for this system. In previous studies, this [30]. In aiming for microbial short/medium-chain alco- was addressed by introducing a structurally different hol production, the bottleneck does therefore rather lie metazoan fatty acid synthase with fused thioesterase [51] in the supply of the respective short/medium-chain acyl or the incorporation of a thioesterase in the fungal fatty chain than the downstream reducing enzymes. This is acid synthase’s reaction chamber [52]. We have recently reflected in the variety of publications about fatty alco - reported the rational engineering of the type I fatty acid hol production derived from the naturally defined pool synthase (FAS) from S. cerevisiae for the selective pro- of fatty acids in the C /C -range [23, 25, 38], while duction of short/medium-chain fatty acids (C or C ) 16 18 6 8 work on short/medium-chain alcohol production is rare. [53] based on a preceding in vitro/in silico approach [54]. Marcheschi et  al. [39] reported the production of mixed Mutations in the selected enzymatic domains, controlling −1 C –C -alcohols with the α–ketoacid route using mutated fatty acid chain length, led to a total yield of 245 mg L 3 8 LeuA variants in E. coli, but with a maximal 1-octanol (C or C fatty acid) and a specificity for C fatty acids of 6 8 8 −1 yield of 15  mg  L , which accounts for only 0.1% in this 90% (of secreted fatty acids), resembling to date the most mixture. Higher titers were reported using a reverse efficient and most specific short/medium-chain fatty β-oxidation route and an aldehyde/alcohol dehydroge- acids producing yeast strains [51, 52, 55, 56]. We con- nase AdhE2 from Clostridium acetobutylicum for reduc- sider this strain as ideal platform to produce 1-octanol. −1 ing the obtained CoA-ester to give 65  mg  L 1-octanol Here, we show that combining an octanoyl-CoA pro- as one component of a C –C alcohol mixture in E. coli ducing engineered FAS from yeast with heterologously 2 10 −1 [40] or even 100  mg  L as minor product after further expressed carboxylic acid reductases and aldehyde reduc- metabolic optimizations [18]. For the direct usage of tases, together with endogenous thioesterases and alco- fatty acyl-CoAs as precursors, Sheng et al. [41] reported hol dehydrogenases, enables efficient de novo production an elaborate strategy to escape from the normal product of 1-octanol from glucose with S. cerevisiae. spectrum of mainly C /C . They directed a fatty acyl- 16 18 CoA reductase to the peroxisome to capture medium- Results and discussion chain substrates from β-oxidation, which resulted in the Biosynthesis of 1‑octanol from glucose −1 production of 1  g  L C10, C and C fatty alcohols in In this study, we engineered a synthetic pathway to pro- 12 16 S. cerevisiae [41]. In spite of the success of producing duce 1-octanol from glucose in S. cerevisiae in which short/medium-chain alcohols, these examples illustrate the chain length of the fatty alcohol is determined by the difficulty of chain length control that causes dissipa - the product release of a mutated yeast FAS, namely R1834K tion of synthetic capacity and finally requires separation Fas1 /Fas2 [53]. Host strain RPY21 which exhibits Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 4 of 12 −1 −1 −1 deletions of both FAS genes, FAS1 and FAS2, as well as to 26.0 ± 3.6  mg  L (2.8 ± 0.3  mg  L OD ) after 72  h the gene FAA2 encoding a short/medium-chain fatty (Fig.  3a), which was also indicated by a strong specific acyl-CoA synthetase, was transformed with centromeric smell of the cell cultures. The production of 1-octanol R1834K vectors expressing FAS1 and FAS2 under control confirmed that S. cerevisiae contains suitable endogenous of their native promoters. We had previously reported ADHs/ALRs for the reduction of 1-octanal (Fig.  2) as R1834K that the FAS mutant version of FAS in concert shown earlier [29]. Besides 1-octanol, also small amounts −1 with the thioesterase activities of three short-chain acyl- of 1-hexanol (5.5 ± 0.6 mg L after 72 h; data not shown) R1834K CoA:ethanol acyltransferases, Eht1, Eeb1, and Mgl2, could be determined. This was expected since FAS produces high amounts of octanoic acid [53]. To avoid also produces small amounts of hexanoic acid which subsequent degradation of octanoic acid, we additionally is then also reduced by CAR to its corresponding alde- deleted FAA2 which is required for the re-activation of hyde [14, 31, 53]. In contrast, although decanoic acid was R1834K octanoic acid to octanoyl-CoA initiating its degradation another side product of FAS [53], decanol was not by β-oxidation [57]. Interestingly, it has been reported detected. before that the deletion of FAA2 already in a wild-type It is worthy to mention that even the control strain FAS background leads to the production of low amounts RPY21, expressing wild-type FAS and overexpress- R1834K of octanoic acid [58]. In the F AS mutant back- ing CAR and Sfp, secreted 1-octanol in small amounts −1 ground, the deletion of FAA2 resulted in an increase of (3.9 ± 0.1 mg L ) (Fig. 3a), which has also been reported −1 up to 25% in octanoic acid production (301 mg L ; data before in a comparable setup [29]. Possibly, this is the not shown). To produce 1-octanol from free octanoic result of the small amounts of octanoic acid produced by acid, we overexpressed a heterologous carboxylic acid wild-type FAS or the mitochondrial type II FAS system in reductase (CAR) from Mycobacterium marinum under S. cerevisiae [53, 54, 59], especially together with deletion −1-−392 the control of the strong and constitutive HXT7 of FAA2 as in strain RPY21 [58]. promotor fragment together with the phosphopanteth- To determine the limiting factors in 1-octanol produc- R1834K einyl transferase Sfp from Bacillus subtilis under the tion in strain RPY21/FAS overexpressing CAR and control of the strong PGK1 promoter on a multicopy vec- Sfp, we furthermore analyzed the accumulation of free R1834K tor in RPY21 expressing Fas1 /Fas2 [14]. CAR uses octanoic acid in the culture medium, and detected a con- −1 NADPH and ATP in order to reduce free fatty acids to siderable titer of 90.3 ± 6.8  mg  L after 72  h (Fig.  3c). the corresponding aldehydes [31] (Fig.  2) and must be Compared to the same strain without CAR which accu- −1 activated by a phosphopantetheine transferase: in this mulated 118.9 ± 7.3  mg  L octanoic acid, this reveals study, Sfp, which attaches the prosthetic group 4′-phos- that 76% of precursor substrate remained unused for phopantetheine to the enzyme [31, 32]. The aldehydes octanol production, suggesting a bottleneck either in the can be further reduced to primary alcohols by endog- CAR or the ADH/ALR reactions, or losses due to secre- enous alcohol dehydrogenases (ADH) or aldehyde reduc- tion of octanoic acid out of the cells. tases (ALR) in yeast [29, 33]. The decreased maximal optical density after 72  h of R1834K Cultivation of the yeast cells in potassium phosphate the yeast culture RPY21/FAS overexpressing CAR buffered medium at pH 6.5 with 2% glucose under and Sfp indicated that 1-octanol or the other pathway aerobic conditions resulted in the accumulation of intermediates octanoic acid and octanal might have a 1-octanol in the extracellular medium with titers of up toxic effect on the yeast cells and inhibit their growth Fig. 2 Metabolic pathway for 1-octanol production in S. cerevisiae from glucose via fatty acid biosynthesis. A mutant version of S. cerevisiae FAS R1834K (Fas1 /Fas2) produces octanoyl-CoA which is hydrolyzed by endogenous thioesterases ( TE) to the free octanoic acid [53]. A heterologous carboxylic acid reductase (CAR) from M. marinum then converts the free fatty acid to octanal which is further reduced to 1-octanol by endogenous alcohol dehydrogenases (ADH) and aldehyde reductases (ALR) [29]. CAR must be activated by the phosphopantetheinyl tranferase Sfp from Bacillus RK R1834K subtilis [31]. Heterologous or mutated enzymes are marked in blue. F AS means FAS Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 5 of 12 a bc 30 20 15 80 0 0 0 0 20 40 60 80 0 20 40 60 80 time [h] time [h] RK RK FAS FAS FAS CAR CAR vector R1834K Fig. 3 Production of 1-octanol with S. cerevisiae. Strain RPY21 expressing mutated F AS as well as CAR and Sfp from a high-copy plasmid (black), was grown for 72 h in buffered YPD medium. For reference, analogous combinations of wild-type FAS, CAR, and Sfp (red) as well as R1834K mutated FAS with empty vector instead of CAR and Sfp are shown (blue). Error bars reflect the standard deviations from three biological replicates. OD (a) and extracellular 1-octanol concentrations (b) were analyzed at different time points. c Final octanoic acid concentration in the RK R1834K extracellular culture medium after 72 h. FAS means FAS (Fig.  3b). To test the inhibitory effects, different con - Re‑uptake of secreted octanoic acid from the medium centrations of 1-octanol, octanal, and octanoic acid To rule out that the secretion of octanoic acid into the were added to yeast cultures of the wild-type strain culture medium is a limiting factor for 1-octanol produc- BY4741 and growth curves were determined (Fig.  4). tion, we analyzed the transport of octanoic acid back into −1 Indeed, at the lowest tested concentration of 50 mg L the cell and its further conversion to 1-octanol by over- of 1-octanol or octanal in the culture medium, slightly expressing CAR and Sfp in the wild-type strain BY4741 −1 inhibited growth of the yeast cells was observed, and in buffered media supplemented with 90 mg L octanoic −1 growth was completely prevented at a concentration of acid. After 24 h, 11.0 ± 0.8 mg L 1-octanol was detected −1 150 mg L (Fig. 4a, b). In contrast, octanoic acid is less (Fig.  5a), showing that octanoic acid can be transported toxic, and moderate cell growth was even observed at a back into the cell and subsequently be reduced by CAR −1 concentration of 400 mg L (Fig. 4c). together with ADHs/ALRs to 1-octanol. Nevertheless, the concentration of octanoic acid in the medium also ab c 15 15 15 10 10 10 5 5 5 0 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 time [h] time [h] time [h] -1 -1 -1 -1 0 mg L 100 mg L 0 mg L 200 mg L -1 -1 -1 -1 50 150 100 400 mg L mg L mg L mg L Fig. 4 Inhibitory effects of 1-octanol, octanal, and octanoic acid added to the culture medium of strain BY4741. The yeast strain BY4741 was cultivated for 24 h in buffered YPD medium supplemented with different concentrations of a 1-octanol b octanal, or c octanoic acid. Error bars reflect the standard deviations from two biological replicates. OD was analyzed at different time points RK FAS CAR FAS CAR RK FAS vector -1 OD 1-octanol [mg L ] OD OD 600 600 OD -1 octanoic acid [mg L ] Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 6 of 12 a b 15 120 medium + C acid 10 80 vector + C acid CAR + C acid 5 40 CAR 0 0 0 20 40 60 80 0 20 40 60 80 time [h] time [h] Fig. 5 Uptake and conversion analysis of octanoic acid. a 1-Octanol production and b octanoic acid consumption of S. cerevisiae BY4741 expressing CAR and Sfp from a multicopy plasmid. Cultivation was performed for 72 h in buffered YPD medium without and with supplementation −1 of 90 mg L octanoic acid. For reference, noninoculated cultivation medium was treated analogously. Error bars reflect the standard deviations from three biological replicates decreased in the absence of a CAR (Fig.  5b), indicating in 1-octanol production is also reflected in a decreased alternative reaction pathways for octanoic acid conver- maximal optical density of the cell culture after 72  h sion in S. cerevisiae. Probably, this is due to degradation compared to the control strain without Ahr (Fig.  6b), of octanoic acid via β-oxidation in strain BY4741 [57, 60]. probably due to the negative effect of 1-octanol on cell Moreover, the amount of 1-octanol in the extracellular growth (Fig.  4a). When the 1-octanol titers are nor- medium also decreased after 24 h (Fig. 5a). This might be malized to the final OD of the cultures, overexpres- due to its reconversion to octanal by endogenous alco- sion of Ahr resulted in a threefold increase of 1-octanol −1 −1 −1 hol dehydrogenases which then might be oxidized, e.g., (7.9 ± 0.4  mg  L OD compared to 2.8 ± 0.3  mg  L −1 by the dehydrogenase Hfd1, which has been reported to OD after 72  h). Furthermore, the presence of Ahr convert at least long-chain aliphatic aldehydes to carbox- nearly completely prevented the accumulation of octa- −1 ylic acids [33]. noic acid (0.6 ± 0.0  mg  L after 72  h) compared to the −1 strain without Ahr (113.2 ± 2.6  mg  L ) (Fig.  6c). This Overexpression of an aldehyde reductase Ahr from E. coli result revealed that the reduction of octanal to 1-octanol increases 1‑octanol production by endogenous ADHs and ALRs was a limiting step in The previous experiment showed that octanoic acid can the original pathway. freely diffuse or is transported in both directions across the yeast plasma membrane, and therefore should not In situ extraction of 1‑octanol be limiting 1-octanol production. Therefore, we tested Since already minor concentrations of 1-octanol inhib- octanal reduction as a limiting factor in 1-octanol pro- ited growth of the yeast cells (Fig.  4a), which makes duction. For this reaction, S. cerevisiae contains various further improvements toward higher 1-octanol titers endogenous ADHs/ALRs, successfully employed for the difficult, one possibility to circumvent this problem aldehyde–alcohol conversion in the C –C range already could be an in  situ extraction of released 1-octanol in 8 18 earlier [29]. However, little is known about the efficien - a two-phase fermentation using an organic solvent as cies of these enzymes to short/medium-chain fatty alde- the secondary phase. An appropriate solvent is dode- hydes, especially to octanal. It has been shown by Akhtar cane which was shown to have no negative effects on et  al. [31] that the aldehyde reductase Ahr from E. coli yeast growth (Fig.  7) [61, 62] and was already used for accepts a broad range of aliphatic aldehydes (C to C ). production of long-chain fatty alcohols [22, 24]. To test 4 16 We overexpressed Ahr from E. coli under the control of if dodecane as secondary phase can improve growth in −1-−392 the HXT7 promotor fragment from a high-copy the presence of 1-octanol by trapping it out of the yeast plasmid together with the plasmid encoding for CAR culture, different concentrations of 1-octanol were R1834K and Sfp in strain RPY21/FAS under aerobic condi- added to yeast cultures of the wild-type strain BY4741 tions. Overexpression of Ahr led to a twofold increase overlaid with 20% dodecane, and growth curves were −1 in the absolute 1-octanol titer (49.5 ± 0.8  mg  L after determined (Fig.  7). Indeed, this time, the addition of −1 72 h) in the extracellular medium (Fig. 6a). This increase even 150  mg  L 1-octanol to the yeast culture with -1 1-octanol [mg L ] -1 octanoic acid [mg L ] Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 7 of 12 ab c 60 20 30 10 0 0 0 20 40 60 80 0 20 40 60 80 time [h] time [h] RK RK FAS FAS FAS CAR CAR CAR Ahr Ahr vector R1834K Fig. 6 Increase in 1-octanol production in a yeast strain by additional overexpression of heterologous Ahr. Strain RPY21/FAS expressing CAR and Sfp together with Ahr from E. coli from a high-copy plasmid (black), was grown for 72 h in buffered YPD medium. For comparison, analogous R1834K combinations of wild-type FAS with CAR, Sfp, and Ahr (red) as well as mutated F AS with empty vector instead of Ahr are shown (blue). Error bars indicate the standard deviation from three biological replicates. a OD and 1-octanol b were analyzed at different time points. c Final RK R1834K octanoic acid concentration after 72 h. FAS means FAS the dodecane phase were analyzed. We found that in the two-phase fermentation, no 1-octanol was detect- -1 able in the aqueous phase revealing the efficient extrac - 0 mg L -1 tion of 1-octanol. However, analysis of 1-octanol in the 0 mg L + Dd dodecane layer showed that 1-octanol production was -1 50 mg L + Dd reduced by about 25% compared to the strain cultivated -1 100 mg L + Dd without dodecane (Fig.  8a) (it should be noted that the 5 -1 150 mg L + Dd concentration of 1-octanol determined in the dodecane phase was calculated on the volume of the aqueous phase of the culture). This is likely due to the sequestra - 0 5 10 15 20 25 tion of octanoic acid into the dodecane phase (Fig. 8a). time [h] R1834K Cultivation of the strain RPY21/FAS without the Fig. 7 Growth behavior of strain BY4741 in the presence of 20% dodecane and different 1-octanol concentrations. BY4741 was 1-octanol pathway (which yields higher titers of octa- cultivated up to 24 h in buffered YPD medium overlaid with noic acid) in buffered medium overlaid with 20% dode - 20% dodecane (Dd) and supplemented with different 1-octanol cane confirmed the partial accumulation of octanoic concentrations. For comparison, also the growth curve of a culture acid in the organic phase (Fig.  8b). Although addition without dodecane was determined (gray). Error bars reflect the of dodecane could circumvent the inhibitory effect of standard deviations from two biological replicates. OD was analyzed at different time points added 1-octanol on growth (Fig.  7), surprisingly, this was not the case when 1-octanol was produced by the cells themselves (Fig.  8a). Here, the growth of the 20% dodecane did not affect growth. Based on these 1-octanol producing yeast cultures with dodecane was results, a two-phase fermentation with the 1-octanol only slightly improved. Taken together, in  situ extrac- producing strain was performed using dodecane as tion could not improve 1-octanol production and the a secondary phase for in  situ extraction. To achieve speedup of the downstream pathway for octanoic acid this, we overexpressed Ahr, CAR, and Sfp from high- production is necessary to compensate the loss of octa- R1834K copy plasmids in strain RPY21/FAS under aero- noic acid into the organic layer. The results also suggest bic conditions in buffered media overlaid with and that beside the toxic effect of 1-octanol, its production without 20% of dodecane. After 72  h, the growth was somehow also inhibits growth of the cells which might determined by OD measurements, and the amounts 600 partially be connected to a limited supply of ATP and of fatty acids and fatty alcohols in the aqueous and in the cofactor NADPH required for octanoic acid pro- duction as well as for the CAR and Ahr reactions. RK FAS CAR Ahr FAS CAR Ahr RK FAS CAR vector OD -1 1-octanol [mg L ] OD -1 octanoic acid [mg L ] OD OD Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 8 of 12 RK RK a b FAS CAR Ahr FAS 15 150 25 aqueous 30 phase 10 100 dodecane phase 5 50 0 0 0 0 0 dodecane - + - + - + 20% R1834K Fig. 8 In situ extraction of 1-octanol and octanoic acid in a two-phase fermentation of yeast. Strain RPY21/FAS overexpressing CAR and Sfp R1834K together with Ahr (a) and control strain RPY21/FAS (b) were grown for 72 h, in buffered YPD medium overlaid with (+) and without (−) 20% of dodecane. Error bars reflect the standard deviations from two biological replicates. After 72 h, the final OD (·) and the final concentrations RK R1834K of 1-octanol and octanoic acid were analyzed. FAS means FAS . The gray areas indicate concentrations of 1-octanol or octanoic acid, respectively, calculated on the basis of the volume of the aqueous phase of the culture, but based on the amounts measured in the dodecane phase. The white-colored areas indicate concentrations measured directly in the aqueous phase homologous recombination were transformed accord- Conclusion ing to protocols by Gietz and Schiestl [65]. This is the first study to report on dedicated produc - tion of 1-octanol in S. cerevisiae. We achieved the nearly selective production of 1-octanol in S. cer- Growth experiments evisiae by combining a proprietary C -acid producing The strain BY4741 was pregrown in YPD medium buff - R1834K FAS -mutant with a two-step reduction pathway ered with 100 mM potassium phosphate and adjusted to composed of CAR, Sfp, and Ahr [14, 53]. In the pro- a pH of 6.5 as described in Gajewski et  al. [53] without cess of the current study, the chain length specificity supplementation of free FAs. After washing steps, the of the carbon chain-providing FAS is the decisive step main culture (50 mL YPD in 300 mL flasks; two biological to ensure specific 1-octanol production. Nevertheless, replicates) was inoculated to OD = 0.1, supplemented toxicity of 1-octanol and a negative effect of its produc - with different concentrations of 1-octanol (0, 50, 100, −1 −1 tion on growth of the cells pose fresh challenges for and 150 mg L ), octanal (0, 50, 100, and 150 mg L ), or −1 further optimizations. octanoic acid (0, 100, 200, and 400  mg  L ) and aerobi- cally shaken for 24  h at 180 r.p.m. at 30  °C. OD was analyzed at different time points. Methods Yeast strain, media, and transformation S. cerevisiae fermentations The haploid S. cerevisiae strain RPY21 used in this Saccharomyces cerevisiae strains were cultured in YPD study for fatty alcohol production has a BY background medium as described in Gajewski et al. [53] without sup- and is based on BY.PK1238_1A_KO with knocked out plementation of free FAs. The medium was additionally FAS1 and FAS2 from previous studies [53]. The dele - buffered with 100 mM potassium phosphate and adjusted tion of the fatty acyl-CoA synthetase Δfaa2 was gener- to a pH of 6.5. To test the 1-octanol production of the ated using the CRISPR–Cas9 system [63]. The relevant different strains, a preculture was inoculated in buff - genotype of RPY21 is Matα; ura3Δ0; his3Δ0; leu2Δ0; ered YPD medium and grown aerobically to exponen- TRP1; lys2Δ0; MET15; fas1::uptag-kanMX4-downtag; tial phase. After washing steps, the main culture (50 mL fas2::uptag-kanMX4-downtag; Δfaa2. RPY21 was trans- YPD with respective antibiotics; two to three biological formed as described in Gajewski et al. [53]. Selection of replicates) was inoculated to OD = 0.1 and aerobically yeast transformants was done on the defined synthetic shaken for 72 h at 180 r.p.m. and 30 °C. complete media (SCD) as described in Bruder et al. [64] For the in situ extraction, the main culture (25 mL YPD without leucine and histidine containing the respec- with respective antibiotics; two biological replicates) was −1 −1 tive antibiotics (200 µg mL hygromycin; 100 µg mL inoculated to OD = 0.1 and overlaid with 20% dode- nourseothricin sulfate). The strain BY4741 and the cane (5  mL). Fatty alcohols, fatty acids, and OD were strain CEN.PK2-1C for plasmid construction by analyzed at different time points. The final 1-octanol -1 1-octanol [mg L ] -1 octanoic acid [mg L ] -1 octanoic acid [mg L ] Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 9 of 12 concentration after 72  h was normalized to the final four PCR fragments with 30 bp overlaps. Yeast was trans- −1 OD value (mg L  OD ). formed as described above with a mixture of these frag- 600 600 ments generated by PCR using primers shown in Table 2. Plasmid and strain construction The assembled plasmids were recovered by yeast DNA The plasmids used in this study are listed in Table  1. FAS- preparations and transformed into E. coli for amplifica - related plasmids are described in Gawjeski et  al. [53]. tion by standard procedures. The construction of the Genes encoding carboxylic acid reductase CAR from M. plasmid pRS62N-Ahr was performed by Gibson assem- marinum (UniProt: B2HN69) and the phosphopanteth- bly as described in Gibson et al. [67]. The bacterial gene einyl transferase Sfp from B. subtilis (UniProt: P39135) Ahr [previously known as yigB (UniProt: P27250)] was were codon-optimized according to the yeast glycolytic amplified from chromosomal DNA of E. coli DH5α using codon usage [66]. The plasmid pRS62H–CAR–Sfp was primers shown in Table 2. assembled by homologous recombination in yeast with Fatty alcohol extraction For the extraction of fatty alcohols from the culture medium, the cells were separated from the medium (8000 Table 1 List of plasmids used in this study rcf, 3  min). 500  µL supernatant was mixed with 1  mL −1 ethyl acetate containing 50  mg  L heptanol as internal Plasmid name Description References standard and thoroughly shaken. After centrifugation Sc pRS315-FAS1-Wt CEN6ARS4, AmpR, LEU2, p - FAS1-Wt- [53] FAS1 (5000 rcf, 2 min), 500 µL of the organic phase was trans- FAS1 ferred to a gas chromatography (GC) vial. For the deter- Sc R1834K pRS315-FAS1-RK CEN6ARS4, AmpR, LEU2, p - FAS1 - [53] FAS1 mination of the amount of fatty alcohols in the dodecane FAS1 Sc layer, 100  µL of the dodecane overlay was mixed with pRS313-FAS2-Wt CEN6ARS4, AmpR, HIS3, p - FAS2-Wt- [53] FAS2 −1 t 900  µL ethyl acetate containing 50  mg  L heptanol as FAS2 pRS62H 2µ, hphNT1, AmpR, truncated HXT7 pro- [68] internal standard in a GC vial. −1-−392 motor (p ) and FBA1 terminator HXT7 pRS62 N 2µ, natNT2, AmpR, shortened HXT7 pro- [68] Fatty acid extraction and derivatization −1-−392 motor (p ) and CYC1 terminator HXT7 For the extraction of free fatty acids present in the cul- −1-−392 Mm Bs pRS62H-CAR-Sfp pRS62H; p - CAR -t ; p - Sfp- This study HXT7 FBA PGK1 ture medium, the cells were separated from the medium PGM2 −1-−392 Ec (3500  rcf, 10  min). An internal standard (0.2  mg hepta- pRS62 N-Ahr pRS62N; p - Ahr-t This study HXT7 CYC1 noic acid) was added to 10  mL supernatant and mixed Genes from Saccharomyces cerevisiae (Sc), Mycobacterium marinum (Mm), E. coli with 1  mL 1  M HCl and 2.5  mL methanol–chloroform (Ec), and Bacillus subtilis (Bs) are indicated by prefixes in superscript. hphNT1 hygromycin resistance, Amp ampicillin resistance, natNT2 nourseothricin sulfate solution (1:1). The solution was vigorously shaken (5 min) resistance Table 2 Relevant primers for plasmid construction Primer name Sequence 5′–3′ Amplicon SHP35_pPGK1_forAAC CCT GGC GTT ACC CAA CTT AAT CGC CTT GCAG PGK1 promotor CAT GTT TGC AAA AAG AAC AAA ACT G CBP160_pPGK1_revTGT TTT ATA TTT GTT GTA AAA AGT AGA TAA TTAC PGK1 promotor CBP35_tPGM2_forAAC GAA TGA TTT ACT AAT GGC PGM2 terminator SHP36_tPGM2_revGAA ATC GGC AAA ATC CCT TAT AAA TCA AAA GAAT PGM2 terminator AGA CAA AAA ACT CGG GGT AGG TAA T SHP54_Ahr_forCAA AAA CAA AAA GTT TTT TTA ATT TTA ATC AAAAA Ahr ORF ATG TCG ATG ATA AAA AGC TAT GCC SHP53_Ahr_revATG TAA GCG TGA CAT AAC TAA TTA CAT GAC TCGA Ahr ORF GTC AAA AAT CGG CTT TCA ACAC DR_Faa2GGA AGA ATG CAG GTT ACA AAA AAC GGA TAA GAA Donor-DNA fragment for FAA2 CAA ACT TGT TTC GAA ATG TAC TTA TGA CGA TTTGG deletion AAC ACA TTC AAA CTA GAA AAA ACT TTG ATG TA CC_FAA2-revCGT AAG GTT TCA AAA TCT TCG ATC ATT TAT CTTTC Amplification of a CRISPR-Cas9 ACT GCG GAG plasmid pRCCN for deletion of FAA2, reverse CC_FAA2-fwGAA GAT TTT GAA ACC TTA CGG TTT TAG AGC TAGAA Amplification of a CRISPR-Cas9 ATA GCA AGT TAA AAT AAG G plasmid pRCCN for deletion of FAA2, forward The abbreviations within the primer names were used as follows: forward primer (fw) and reverse primer (rev) Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 10 of 12 and then centrifuged for 10 min at 3.000 rcf. The chloro - extraction experiment with dodecane as second phase, form layer was recovered and evaporated overnight. The the column Elite 5MS capillary column (30 m × 0.25 mm, methylation of the fatty acids was performed as described film thickness 1.00 µm, Perkin Elmer, Germany) was used in Ichihara and Fukubayashi [69]. The samples were dis - for analysis of FAMEs and fatty alcohols. The tempera - solved in 200 µL of toluene, mixed with 1.5 mL of meth- tures of the injector and detector were 250 and 300  °C, anol and 300  µL of 8.0% (w/v) HCl solution [conc. HCl respectively. The following temperature program was (35%  w/w; 9.7  mL) was diluted with 41.5  mL of metha- applied for FAMEs: 50  °C for 5  min, increase at the rate −1 nol], vortexed vigorously, and incubated at 100 °C for 3 h of 10 °C min to 120 °C and hold for 5 min; increase at −1 to form fatty acid methyl ester (FAME). After cooling the rate of 15  °C  min to 220  °C, and hold for 10  min, −1 under ice for 10 min, 1  mL H O and 1  mL hexane were increase at the rate of 20 °C min to 300 °C, and hold for added to the sample. The mixture was shaken thoroughly, 5 min. For fatty alcohols, the following temperature pro- and the organic phase was transferred to a GC vial. gram was applied: 50 °C for 5 min, increase at the rate of −1 For the derivatization of the fatty acids from the 20 °C min to 220 °C and hold for 2 min; increase at the −1 in  situ extraction experiment, the derivative reagent rate of 20 °C min to 300 °C, and hold for 5 min. Bis(trimethylsilyl)-trifluoroacetamide (BSTFA) was used. After separation of the cells from the medium (3500 rcf, Abbreviations 10 min), 50 µL 1 M HCl and 0.01 mg heptanoic acid as an OD : optical density at 600 nm; PGK1: 3-Phosphoglycerate kinase; HXT7: internal standard were added to 500 µL supernatant and high-affinity glucose transporter; PGM2: phosphoglucomutase; CYC1: cytochrome c; FBA1: fructose-1,6-bisphosphate aldolase; FAS: fatty acid mixed with 1 mL ethyl acetate. The ethyl acetate layer was synthase; FAA2: medium-chain fatty acyl-CoA synthetase; Ahr: aldehyde evaporated overnight, and the samples were dissolved reductase; CAR : carboxylic acid reductase; ALR: aldehyde reductase; ADH: in 100  µL ethyl acetate and mixed with 100  µL BSTFA. alcohol dehydrogenase; Sfp: phosphopantetheinyl transferase. For the determination of the amount of fatty acids in the Authors’ contributions dodecane layer, 20  µL of the dodecane layer was mixed SH, MF, MG, MO, and EB conceived the study. SH conducted the experiments. with 80 µL ethyl acetate, 0.01 mg of heptanoic acid as an SH, MO, and EB analyzed the data. SH, MF, and EB wrote the paper. All authors read and approved the manuscript. internal standard, and 100 µL BSTFA. The derivatization was done for 45  min at 80  °C. After cooling at 4  °C for Author details 15 min, the samples were analyzed by GC. Faculty of Biological Sciences, Institute of Molecular Bioscience, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecu- lar Life Sciences, Cluster of Excellence “Macromolecular Complexes”, Goethe GC‑FID analysis of FAMEs and fatty alcohols University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt, Germany. The gas chromatographic measurements were carried out on a Perkin Elmer Clarus 400 system (Perkin Elmer, Acknowledgements We thank Renata Pavlovic for providing the yeast strain RPY21. Germany) equipped with an Elite FFAP capillary column (30  m × 0.25  mm, film thickness: 0.25  µm; PerkinElmer, Competing interests Germany) and a flame ionization detector (Perkin Elmer, E.B. and M.G. are inventors of EP patent application No. 15 162 192.7 filed on April 1, 2015, and of EP patent application No. 15 174 342.4 filed on June Germany). 1  μL of the sample was analyzed after split 26, 2015, by Goethe-University Frankfurt, concerning short-chain acyl-CoA injection (1:10); helium was used as carrier gas (90 kPa). producing FAS variants. There are no other competing interests. For fatty acid methyl ester (FAME) quantification, the Availability of data and materials temperatures of the injector and detector were 200 and The datasets used and/or analyzed during the current study are available from 250  °C, respectively. The following temperature pro - the corresponding author upon reasonable request. gram was applied: heating to 50  °C for 5  min; increase −1 Consent for publication of 10 °C min to 120 °C and hold for 5 min; increase at −1 Not applicable. the rate of 15  °C  min to 180  °C and hold for 10  min; −1 increase at the rate of 20 °C min to 220 °C, and hold for Ethics approval and consent to participate Not applicable. 7 min. FAME were identified and quantified by compari - son with authentic standard substances. For fatty alcohol Funding quantification, an initial temperature of 50 °C was main - The project underlying this report is financially supported by the German Federal Ministry of Food and Agriculture following a decision of the German tained for 5 min, followed by an increase at the rate of −1 Bundestag under the Grant Number 22026315; the responsibility for the 20 °C min to 210 °C and kept constant for 5 min. After content of this publication lies with the authors. −1 a further increase at the rate of 20  °C  min to 230  °C, the temperature was kept constant for 6  min. The tem - Publisher’s Note peratures of both the injector and detector were 250  °C. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Fatty alcohols were identified and quantified by compari - son with authentic standard substances. For the in  situ Henritzi et al. Biotechnol Biofuels (2018) 11:150 Page 11 of 12 Received: 12 December 2017 Accepted: 23 May 2018 20. Willis RM, Wahlen BD, Seefeldt LC, Barney BM. Characterization of a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8: a bacterial enzyme catalyzing the reduction of fatty acyl-CoA to fatty alcohol. Biochemistry. 2011. https ://doi.org/10.1021/bi200 8646. 21. Rowland O, Domergue F. 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Biotechnology for BiofuelsSpringer Journals

Published: Jun 1, 2018

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