Engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters

Background Production of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production. Results We first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing. Conclusion We achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.

Engineering Oleaginous Yeast Rhodosporidium Toruloides for Overproduction of Fatty Acid Ethyl Esters

BackgroundProduction of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70 % biomass as lipids, Rhodosporidium toruloides represents an attractive host for FAEEs production. ResultsWe first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase /acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs in shake-flask fermentation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, by fed-batch fermentation in a 1-L fermenter, the titer of FAEEs reached 9.2 g/L. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing.ConclusionWe achieved the highest FAEEs production in yeast with a final titer of 9.2 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid derived molecules.

Directed evolution of a bacterial WS/DGAT acyltransferase: improving tDGAT from Thermomonospora curvata

Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.

Biosynthesis of Long Chain Alkyl Diols and Long Chain Alkenols in Nannochloropsis spp. (Eustigmatophyceae)

We investigated potential biosynthetic pathways of long chain alkenols (LCAs), long chain alkyl diols (LCDs), and long chain hydroxy fatty acids (LCHFAs) in Nannochloropsis oceanica and Nannochloropsis gaditana, by combining culturing experiments with genomic and transcriptomic analyses. Incubation of Nannochloropsis spp. in the dark for 1 week led to significant increases in the cellular concentrations of LCAs and LCDs in both species. Consistently, 13C-labelled substrate experiments confirmed that both LCA and LCD were actively produced in the dark from C14–18 fatty acids by either condensation or elongation/hydroxylation, although no enzymatic evidence was found for the former pathway. Nannochloropsis spp. did, however, contain (i) multiple polyketide synthases (PKSs) including one type (PKS-Clade II) that might catalyze incomplete fatty acid elongations leading to the formation of 3-OH-fatty acids, (ii) 3-hydroxyacyl dehydratases (HADs), which can possibly form Δ2/Δ3 monounsaturated fatty acids, and (iii) fatty acid elongases (FAEs) that could elongate 3-OH-fatty acids and Δ2/Δ3 monounsaturated fatty acids to longer products. The enzymes responsible for reduction of the long chain fatty acids to LCDs and LCAs are, however, unclear. A putative wax ester synthase/acyl coenzyme A (acyl-CoA): diacylglycerol acyltransferase is likely to be involved in the esterification of LCAs and LCDs in the cell wall. Our data thus provide useful insights in predicting the biosynthetic pathways of LCAs and LCDs in phytoplankton suggesting a key role of FAE and PKS enzymes.

Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

ABSTRACTRecently, we isolated a novelStreptomycesstrain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed inEscherichia coli. The purified enzyme AtfG25showed acyltransferase activity with C12- or C16-acyl-CoA, C12to C18alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA fromAcinetobacter baylyibut has high sequence similarities to WS/DGATs from otherStreptomycesspecies. To investigate the impact of AtfG25on lipid accumulation, the respective gene,atfG25, was inactivated inStreptomycessp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that AtfG25has an important, but not exclusive, role in TAG biosynthesis in the novelStreptomycesisolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study.IMPORTANCEA novelStreptomycesstrain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected acyltransferase were studied. As discussed in this paper, and in contrast to many other bacteria, streptomycetes seem to possess a complex metabolic network to synthesize lipids, whereof crucial steps are still largely unknown. This paper therefore provides insights into a range of topics, including extremophile bacteria, the physiology of lipid accumulation, and the biotechnological production of bacterial lipids.