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Improved fatty aldehyde and wax ester production by overexpression of fatty acyl-CoA reductases.

ABSTRACT: BACKGROUND:Fatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different properties are needed for the construction of tailored synthetic pathways for the production of various compounds. RESULTS:Fatty aldehyde production in Acinetobacter baylyi ADP1 was increased by the overexpression of three different FARs: a native A. baylyi FAR Acr1, a cyanobacterial Aar, and a putative, previously uncharacterized dehydrogenase (Ramo) from Nevskia ramosa. The fatty aldehyde production was followed in real-time inside the cells with a luminescence-based tool, and the highest aldehyde production was achieved with Aar. The fate of the overproduced fatty aldehydes was studied by measuring the production of wax esters by a native downstream pathway of A. baylyi, for which fatty aldehyde is a specific intermediate. The wax ester production was improved with the overexpression of Acr1 or Ramo compared to the wild type A. baylyi by more than two-fold, whereas the expression of Aar led to only subtle wax ester production. The overexpression of FARs did not affect the length of the acyl chains of the wax esters. CONCLUSIONS:The fatty aldehyde production, as well as the wax ester production of A. baylyi, was improved with the overexpression of a key enzyme in the pathway. The wax ester titer (0.45 g/l) achieved with the overexpression of Acr1 is the highest reported without hydrocarbon supplementation to the culture. The contrasting behavior of the different reductases highlight the significance of in vivo characterization of enzymes and emphasizes the possibilities provided by the diversity of FARs for pathway and product modulation.

SUBMITTER: Lehtinen T

PROVIDER: S-EPMC5806253 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Improved fatty aldehyde and wax ester production by overexpression of fatty acyl-CoA reductases.

Lehtinen Tapio T Efimova Elena E Santala Suvi S Santala Ville V

Microbial cell factories 20180208 1

<h4>Background</h4>Fatty aldehydes are industrially relevant compounds, which also represent a common metabolic intermediate in the microbial synthesis of various oleochemicals, including alkanes, fatty alcohols and wax esters. The key enzymes in biological fatty aldehyde production are the fatty acyl-CoA/ACP reductases (FARs) which reduce the activated acyl molecules to fatty aldehydes. Due to the disparity of FARs, identification and in vivo characterization of reductases with different proper ...[more]

PMID: 29422050

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Project description:BACKGROUND:Triacylglycerols (TAGs) and wax esters (WEs) are important neutral lipids which serve as energy reservoir in some plants and microorganisms. In recent years, these biologically produced neutral lipids have been regarded as potential alternative energy sources for biofuel production because of the increased interest on developing renewable and environmentally benign alternatives for fossil fuels. In bacteria, the final step in TAG and WE biosynthetic pathway is catalyzed by wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT). This bifunctional WS/DGAT enzyme is also a key enzyme in biotechnological production of liquid WE via engineering of plants and microorganisms. To date, knowledge about this class of biologically and biotechnologically important enzymes is mainly from biochemical characterization of WS/DGATs from Arabidopsis, jojoba and some bacteria that can synthesize both TAGs and WEs intracellularly, whereas little is known about WS/DGATs from eukaryotic microorganisms. RESULTS:Here, we report the identification and characterization of two bifunctional WS/DGAT enzymes (designated TrWSD4 and TrWSD5) from the marine protist Thraustochytrium roseum. Both TrWSD4 and TrWSD5 comprise a WS-like acyl-CoA acyltransferase domain and the recombinant proteins purified from Escherichia coli Rosetta (DE3) have substantial WS and lower DGAT activity. They exhibit WS activity towards various-chain-length saturated and polyunsaturated acyl-CoAs and fatty alcohols ranging from C10 to C18. TrWSD4 displays WS activity with the lowest Km value of 0.14 ?M and the highest kcat/Km value of 1.46 × 105 M-1 s-1 for lauroyl-CoA (C12:0) in the presence of 100 ?M hexadecanol, while TrWSD5 exhibits WS activity with the lowest Km value of 0.96 ?M and the highest kcat/Km value of 9.83 × 104 M-1 s-1 for decanoyl-CoA (C10:0) under the same reaction condition. Both WS/DGAT enzymes have the highest WS activity at 37 and 47 °C, and WS activity was greatly decreased when temperature exceeds 47 °C. TrWSD4 and TrWSD5 are insensitive to ionic strength and reduced WS activity was observed when salt concentration exceeded 800 mM. The potential of T. roseum WS/DGATs to establish novel process for biotechnological production of WEs was demonstrated by heterologous expression in recombinant yeast. Expression of either TrWSD4 or TrWSD5 in Saccharomyces cerevisiae quadruple mutant H1246, which is devoid of storage lipids, resulted in the accumulation of WEs, but not any detectable TAGs, indicating a predominant WS activity in yeast. CONCLUSIONS:This study demonstrates both in vitro WS and DGAT activity of two T. roseum WS/DGATs, which were characterized as unspecific acyltransferases accepting a broad range of acyl-CoAs and fatty alcohols as substrates for WS activity but displaying substrate preference for medium-chain acyl-CoAs. In vivo characterization shows that these two WS/DGATs predominantly function as wax synthase and presents the feasibility for production of WEs by heterologous hosts.

Project description:Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 ± 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 ± 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon- or nitrogen-limited medium. However, the ?fcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ?13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C17 versus C16). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of high-value neutral lipids.IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.

Dataset Information

Improved fatty aldehyde and wax ester production by overexpression of fatty acyl-CoA reductases.

Publications

Improved fatty aldehyde and wax ester production by overexpression of fatty acyl-CoA reductases.

Similar Datasets

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