Microbial Oil Research Articles

An Evolved Strain of the Oleaginous Yeast Rhodotorula toruloides, Multi-Tolerant to the Major Inhibitors Present in Lignocellulosic Hydrolysates, Exhibits an Altered Cell Envelope.

The presence of toxic compounds in lignocellulosic hydrolysates (LCH) is among the main barriers affecting the efficiency of lignocellulose-based fermentation processes, in particular, to produce biofuels, hindering the production of intracellular lipids by oleaginous yeasts. These microbial oils are promising sustainable alternatives to vegetable oils for biodiesel production. In this study, we explored adaptive laboratory evolution (ALE), under methanol- and high glycerol concentration-induced selective pressures, to improve the robustness of a Rhodotorula toruloides strain, previously selected to produce lipids from sugar beet hydrolysates by completely using the major C (carbon) sources present. An evolved strain, multi-tolerant not only to methanol but to four major inhibitors present in LCH (acetic acid, formic acid, hydroxymethylfurfural, and furfural) was isolated and the mechanisms underlying such multi-tolerance were examined, at the cellular envelope level. Results indicate that the evolved multi-tolerant strain has a cell wall that is less susceptible to zymolyase and a decreased permeability, based on the propidium iodide fluorescent probe, in the absence or presence of those inhibitors. The improved performance of this multi-tolerant strain for lipid production from a synthetic lignocellulosic hydrolysate medium, supplemented with those inhibitors, was confirmed.

Production of Single-Cell Oil from a Local Isolate Bacillus subtilis Using Palm Fronds.

This study, conducted at the Department of Biology, University of Anbar, Iraq, focuses on addressing the escalating issue of contamination and aims to acquire microbial oils to alleviate the global shortage in plant and animal oil production, utilizing environmental waste as a carbon source to reduce global pollution and select efficient local bacterial isolates of Bacillus subtilis for the production of single-cell oil (SCO) using local soil and environmental waste as a carbon source. Four isolates were selected as the best in producing single-cell oil, with the isolate with code C4 standing out as it recorded the highest production. It is worth noting that all these isolates belong to the bacteria type Bacillus subtilis. Palm fronds were found to be the most suitable environmental residue for SCO production compared to other waste materials (wheat straw and wheat bran). Submerged cultures were used to improve SCO production, with optimal conditions determined as pH 7, a temperature of 30°C, carbon source concentration of 3 g/100 ml, inoculum volume of 3 ml/100 ml, inoculum density of 20 × 107 cells, and an incubation period of 72 hours. The Soxhlet extraction method was used to obtain the oil, which was found to contain high percentages of unsaturated fatty acids, particularly linoleic acid (46.030%) and palmitoleic acid (16.579%). The oil was highly soluble in chloroform and ethanol but insoluble in water. The saponification test indicated the potential for soap production from the oil. This comprehensive research addresses the need for locally sourced and sustainable SCO production, offering insights into the selection of efficient bacterial isolates, the optimization of cultivation conditions, and the valuable properties of the resulting SCO. The significance of this study lies in the production of single-cell oil from soil-isolated Bacillus subtilis bacteria for use in food applications.

Topraktan İzole Edilen Bacillus sp. Suşlarının Lipid Üretim Kapasitelerinin Taranması ve Farklı Boyama Teknikleri ile Lipid Boyama

Microbial lipids have been attracting more and more attention in recent years as promising raw 
 materials for the production of biodiesel and value-added compounds. In the current economic and 
 environmental situation, finding new renewable sources of lipids will be crucial. Recent research has 
 necessitated the search for new efficient microbial producers with lipid production efficiency. For this 
 purpose,50 Bacillus sp. strains previously isolated from the soil were screened for their lipid production 
 capacity. As a result of the production using a single-cell oil production medium, only two Bacillus spp. 
 strains showed growth. These bacteria were named as Bacillus sp. E40 and Bacillus sp. E226. Bacteria 
 were then characterized in terms of their biomass, lipid yields, and lipid contents. The biomass of 
 Bacillus sp. E40 and E226 bacterial isolates, were 0,28 and 0,22 g.L-1, respectively. The highest lipid 
 content was reached by E226 with 54.5%. Bacillus sp. E40 lipid content was determined as 46.4%. In 
 addition, both intracellular lipid staining and colony staining demonstrated the presence of lipids. With 
 this study, it was reported for the first time that local Bacillus sp. strains isolated from soil have lipid 
 producing capacity.

State-of-the-art in the accumulation of lipids and other bioproducts from sustainable sources by Yarrowia lipolytica

AbstractEnergy and environmental issues related to conventional fossil-derived products and fuels have led researchers to focus on alternative, more environmentally-friendly processes, such as the production of microbial oils from renewable feedstocks or even pollutants as sustainable sources of biofuels, allowing to progressively move away from the use of fossil fuels. Among the oleaginous yeasts, Yarrowia lipolytica is a highly promising cell factory and microbial oil producer because of its high capacity to accumulate lipids for subsequent biofuel production. Y. lipolytica also stands out for its ability to assimilate various carbon sources, even at low cost, reaching lipid concentrations of at least 30% by weight with non-genetically modified strains, and even much higher values with engineered organisms. Among others, fatty acids have attracted recent interest as substrates for their lower cost and possible production from pollutants compared to sugars. This review pays special attention to some of those emerging carbon sources, i.e., carboxylic acids and even greenhouse gases. Besides, another focus is to provide detailed up to date information on the main characteristics and factors that most influence the fermentation process of this yeast, with the ultimate aim of optimising the bioconversion process and the synthesis of useful metabolites. Besides, the reader will find comprehensive information on the industrial applicability of the synthesised lipids, in addition to the production of biofuels. Apart from lipids, other metabolites of interest that can be synthesised by Y. lipolytica are also discussed.

Lipid Production from Native Oleaginous Yeasts Isolated from Southern Chilean Soil Cultivated in Industrial Vinasse Residues.

In this research, six strains of oleaginous yeasts native to southern Chile were analyzed for their biotechnological potential in lipid accumulation. For this purpose, the six strains, named PP1, PP4, PR4, PR10, PR27 and PR29, were cultivated in a nitrogen-deficient synthetic mineral medium (SMM). Then, two strains were selected and cultivated in an industrial residual "vinasse", under different conditions of temperature (°C), pH and carbon/nitrogen (C/N) ratio. Finally, under optimized conditions, the growth kinetics and determination of the lipid profile were evaluated. The results of growth in the SMM indicate that yeasts PP1 and PR27 presented biomass concentrations and lipid accumulation percentages of 2.73 and 4.3 g/L of biomass and 36.6% and 45.3% lipids, respectively. Subsequently, for both strains, when cultured in the residual vinasse under optimized environmental conditions, biomass concentrations of 14.8 ± 1.51 g/L (C/N 80) and 15.83 ± 0.57 g/L (C/N 50) and lipid accumulations of 28% and 30% were obtained for PP1 and PR27, respectively. The composition of the triglycerides (TGs), obtained in the culture of the yeasts in a 2 L reactor, presented 64.25% of saturated fatty acids for strain PR27 and 47.18% for strain PP1. The saturated fatty acid compositions in both strains are mainly constituted of fatty acids, myristic C 14:0, heptadecanoic C 17:0, palmitic C 16:0 and stearic C 18:0, and the monounsaturated fatty acids constituted of oleic acid C 18:1 (cis 9) (28-46%), and in smaller amounts, palmitoleic acid and heptadecenoic acid. This work demonstrates that the native yeast strains PP1 and PR27 are promising strains for the production of microbial oils similar to conventional vegetable oils. The potential applications in the energy or food industries, such as aquaculture, are conceivable.

Two-stage syngas fermentation into microbial oils and β-carotene with Clostridium carboxidivorans and engineered Yarrowia lipolytica

An engineered Yarrowia lipolytica strain was able to simultaneously accumulate microbial oils and β-carotene from both acetic and butyric acids produced from syngas fermentation by Clostridium carboxidivorans, through the Wood-Ljungdahl pathway, with CO and CO2 as carbon sources. It was first determined that Y. lipolytica showed no inhibition at concentrations < 16 g/L of a mixture of VFAs (Volatile Fatty Acids) containing acetic, butyric, and even hexanoic acids. The yeast was then grown in a bioreactor with a culture medium derived from acetogenic syngas (CO, CO2, H2) fermentation containing all three acids. Y. lipolytica consumed a total of 91 g/L VFAs, progressively supplied through eight successive additions of these acids to the reactor. A maximum lipid content of 36.18% g/g cell was achieved at 61 g/L VFAs consumed and with an airflow rate of 2.0 vvm. Besides, a total of 88.4 mg/g cell and 759 mg/L β-carotene content and concentration were achieved at about 80 g/L VFAs consumed and with a 2.0 vvm airflow rate. It was concluded that; a non-limiting air flow favors the metabolic pathway for β-carotene formation against lipid accumulation.

Bioimprinted lipase-catalyzed synthesis of medium- and long-chain structured lipids rich in docosahexaenoic acid for infant formula.

Medium- and long-chain structured lipids (MLSLs) rich in docosahexaenoic acid (DHA) were obtained in shorter reaction time by acidolysis of single-cell oil (DHASCO) from Schizochytrium sp. with caprylic acid (CA) using a lipase bioimprinted with fatty acids as a catalyst. The conditions for preparation of the bioimprinted lipase for the acidolysis reaction were firstly optimized and the activity of the obtained lipase was 2.17 times higher than that of the non-bioimprinted. The bioimprinted lipase was then used as a catalyst and the reaction conditions were optimized. Under the optimal conditions, the equilibrium could be achieved in 4h, and the total and sn-1,3 CA contents in the product were 29.18% and 42.34%, respectively, and the total and sn-2 DHA contents were 46.26% and 70.12%, respectively. Such MLSLs rich in sn-1,3 CA and sn-2 DHA are beneficial for DHA absorption, and thus have potential for use in infant formula.

Yeast-driven valorization of agro-industrial wastewater: an overview.

The intensive industrial and agricultural activities currently on-going worldwide to feed the growing human population have led to significant increase in the amount of wastewater produced. These effluents are high in phosphorus (P), nitrogen (N), chemical oxygen demand (COD), biochemical oxygen demand (BOD), and heavy metals. These compounds can provoke imbalance in the ecosystem with grievous consequences to both the environment and humans. Adequate treatment of these wastewaters is therefore of utmost importance to humanity. This can be achieved through valorization of these waste streams, which is based on biorefinery idea and concept of reduce, reuse, and recycle for sustainable circular economy. This concept uses innovative processes to produce value-added products from waste such as wastewater. Yeast-based wastewater treatment is currently on the rise given to the many characteristics of yeast cells. Yeasts are generally fast growing, and they are robust in terms of tolerance to stress and inhibitory compounds, in addition to their ability to metabolize a diverse range of substrates and create a diverse range of metabolites. Therefore, yeast cells possess the capacity to recover and transform agro-industrial wastewater nutrients into highly valuable metabolites. In addition to remediating the wastewater, numerous value-added products such as single cell oil (SCO), single cell proteins (SCPs), biofuels, organic acid, and aromatic compounds amongst others can be produced through fermentation of wastewater by yeast cells. This work thus brings to limelight the potential roles of yeast cells in reducing, reusing, and recycling of agro-industrial wastewaters while proffering solutions to some of the factors that limit yeast-mediated wastewater valorization.

From straw to salmon: a technical design and energy balance for production of yeast oil for fish feed from wheat straw

BackgroundAquaculture is a major user of plant-derived feed ingredients, such as vegetable oil. Production of vegetable oil and protein is generally more energy-intensive than production of the marine ingredients they replace, so increasing inclusion of vegetable ingredients increases the energy demand of the feed. Microbial oils, such as yeast oil made by fermentation of lignocellulosic hydrolysate, have been proposed as a complement to plant oils, but energy assessments of microbial oil production are needed. This study presents a mass and energy balance for a biorefinery producing yeast oil through conversion of wheat straw hydrolysate, with co-production of biomethane and power.ResultsThe results showed that 1 tonne of yeast oil (37 GJ) would require 9.2 tonnes of straw, 14.7 GJ in fossil primary energy demand, 14.6 GJ of process electricity and 13.3 GJ of process heat, while 21.5 GJ of biomethane (430 kg) and 6 GJ of excess power would be generated simultaneously. By applying economic allocation, the fossil primary energy demand was estimated to 11.9 GJ per tonne oil.ConclusionsFossil primary energy demand for yeast oil in the four scenarios studied was estimated to be 10–38% lower than for the commonly used rapeseed oil and process energy demand could be met by parallel combustion of lignin residues. Therefore, feed oil can be produced from existing non-food biomass without causing agricultural expansion.

Expanding the genetic toolbox for Cutaneotrichosporon oleaginosus employing newly identified promoters and a novel antibiotic resistance marker

BackgroundCutaneotrichosporon oleaginosus is an oleaginous yeast that can produce up to 80% lipid per dry weight. Its high capacity for the biosynthesis of single cell oil makes it highly interesting for the production of engineered lipids or oleochemicals for industrial applications. However, the genetic toolbox for metabolic engineering of this non-conventional yeast has not yet been systematically expanded. Only three long endogenous promoter sequences have been used for heterologous gene expression, further three dominant and one auxotrophic marker have been established.ResultsIn this study, the structure of putative endogenous promoter sequences was analyzed based on more than 280 highly expressed genes. The identified motifs of regulatory elements and translational initiation sites were used to annotate the four endogenous putative promoter sequences D9FADp, UBIp, PPIp, and 60Sp. The promoter sequences were tested in a construct regulating the known dominant marker hygromycin B phosphotransferase. The four newly described promoters and the previously established GAPDHp successfully initiated expression of the resistance gene and PPIp was selected for further marker development. The geneticin G418 resistance (aminoglycoside 3’-phosphotransferase, APH) and the nourseothricin resistance gene N-acetyl transferase (NAT) were tested for applicability in C. oleaginosus. Both markers showed high transformation efficiency, positive rate, and were compatible for combined use in a successive and simultaneous manner.ConclusionsThe implementation of four endogenous promoters and one novel dominant resistance markers for C. oleaginosus opens up new opportunities for genetic engineering and strain development. In combination with recently developed methods for targeted genomic integration, the established toolbox allows a wide spectrum of new strategies for genetic and metabolic engineering of the industrially highly relevant yeast.

Alternative stable microbiome state triggered by the introduction of functional microbes in oil reservoirs drives sustainable microbial enhanced oil recovery

Microbial enhanced oil recovery (MEOR) is a promising substitute for other enhanced oil recovery methods in terms of sustainable development. However, the response mechanism of the microbial community to the introduction of functional microbes remains unclear, which limits MEOR strategic planning to achieve a long-term effect. In this study, we reveal the existence of alternative stable states in an oil reservoir for the first time and examine the correlation between the microbiota status and the oil production increment. There existed a basal state A and alternative state B in the reservoir microbial community, and this resulted in a differential microbial oil displacement effect. The oil production increased by up to 50% in basal state A, because Halomonas dominated most niches and restrained other functional microbes. The introduction of biosurfactant-producing bacteria triggered the microbiota status to shift to the alternative state B associated with higher oil recovery. In state B, the high microbial diversity and positive interaction among Dietzia, Bacillus and other species significantly activated the effectiveness of the microbial oil displacing function. Due to the hysteresis of the microbial community, it required frequent and intensive introduction of functional microbes to improve the microbiota in the reservoir and sustainably promote oil recovery. Overall, this study provides new insights into the response mechanism of the microbial community to the introduction of functional microbes in an oil reservoir. This study also provides guidance for effective management of a MEOR strategy to achieve sustainably enhanced oil production.

Microbial oil, alone or paired with β-glucans, can control hypercholesterolemia in a zebrafish model

Dyslipidemia is often associated with unhealthy dietary habits, and many mammalian studies have explored the mode of action of certain bioactive compounds such as β-glucans and n-3 PUFAs to understand their potential to normalize the lipid metabolism. There are only a few investigations that adopted omic approaches to unveil their combined effect on hypercholesterolemia. Zebrafish (Danio rerio) was used as a model organism to reveal the efficacy of Schizochytrium oil and β-glucans (from Euglena gracilis and Phaeodactylum tricornutum) against cholesterol-rich diet induced dyslipidemia. One of the folowing four diets was fed to a particular group of fish: a control high-cholesterol diet, a Schizochytrium oil diet or one of the two diets containing the oil and β-glucan. The plasma HDL, expression of hepatic genes linked to, among others, ferric ion binding and plasma phosphatidylcholines were higher and plasma cholesterol esters and triacylglycerols were lower in the microbial oil-fed fish compared to the fish fed high cholesterol diet. While the fish fed a mix of microbial oil and Euglena β-glucan had lower plasma triacylglycerols and expression of hepatic genes linked to PPAR signaling pathway and enriched biosynthesis of plasma unsaturated fatty acids, the fish fed microbial oil-Phaeodactylum β-glucan combination had lower abundance of triacylglycerols rich in saturated and mono-unsaturated fatty acids and cholesterol esters in the plasma.

Single-Cell Oil Production by Engineered Ashbya gossypii from Non-Detoxified Lignocellulosic Biomass Hydrolysate

In this work, microbial lipid production from non-detoxified Eucalyptus bark hydrolysate (EBH) with oleaginous xylose-utilizing Ashbya gossypii strains was explored. The best producing strain from a set of engineered strains was identified in synthetic media mimicking the composition of the non-detoxified EBH (SM), the lipid profile was characterized, and yeast extract and corn steep liquor (CSL) were pinpointed as supplements enabling a good balance between lipid accumulation, biomass production, and autolysis by A. gossypii. The potential of the engineered A. gossypii A877 strain to produce lipids was further validated and optimized with minimally processed inhibitor-containing hydrolysate and high sugar concentration, and scaled up in a 2 L bioreactor. Lipid production from non-detoxified EBH supplemented with CSL reached a lipid titer of 1.42 g/L, paving the way for sustainable single-cell oil production within the concept of circular economy and placing lipids as an alternative by-product within microbial biorefineries.

Two Cascade Reactions with Oleate Hydratases for the Sustainable Biosynthesis of Fatty Acid-Derived Fine Chemicals

Oleate hydratases (OHs) are of significant industrial interest for the sustainable generation of valuable fine chemicals. When combined with other enzymes in multi-step cascades, the direct formation of fatty acid congeners can be accomplished with minimal processing steps. In this study, two cascade reactions are presented, which can be applied in one-pot approaches. The first cascade was placed “upstream” of an OH derived from Rhodococcus erythropolis (OhyRe), where a lipase from Candida rugosa was applied to hydrolyze triglycerides into free fatty acids, a crucial step for OH conversion. Further, we tested the lipase–OhyRe cascade with various types of renewable triglycerides of plant and microbial origin. In this context, the most efficient conversion was observed for microbial oil from Cutaneotrichosporon oleaginosus leading the way toward its industrial application. In contrast, the second cascade was placed “downstream” of OhyRe, where a novel secondary alcohol dehydrogenase (secADH) was applied to oxidize the hydroxylated fatty acid into a fatty acid ketone. Optimal reaction parameters for the cascade with the secADH were established, which allows this to be applied to high-throughput screens. Moreover, we describe a light-dependent route, thereby extending the catalytic efficiency of the OH enzyme system.

The bioconversion of lignin derivative aldehydes into high-value aromatic alcohols and lipids via Lipomyces starkeyi

Lignin carries great potential as an attractive alternative to petroleum-based products. In particular, biological lignin conversion to various value-added products has recently gained attention owing to its promising benefits in supporting the future bioeconomy. The oleaginous yeast Lipomyces starkeyi is a promising organism due to its impressive native abilities to tolerate and metabolize various lignin monomer derivatives and its ability to synthesize single-cell oil (lipids) inside the cells. Herein, we investigated the coupling abilities of lignin monomer metabolism and lipid biosynthesis via L. starkeyi. Three representatives of lignin derivative aldehydes (4-hydroxybenzaldehyde (H-lignin), vanillin (G-lignin), and syringaldehyde (S-lignin)) were converted into their alcohol forms (4-hydroxybenzyl alcohol, vanillyl alcohol, and syringyl alcohol, respectively) without the formation of undesirable byproducts, and each was maintained in its alcohol form for the remaining cultivation. In addition, a double concentration of syringaldehyde in individual lignin fermentation promoted cell growth (17.01 g L−1) and lipid accumulation (30.72 % (w w−1)). However, mixed fermentation of all lignin aldehydes resulted in heavy inhibition of the cells. Hence, combination fermentation strategies of high initial inoculum size using a medium with a high C:N ratio were beneficial to improving the fermentation time and lipid titer increment up to 2.6-fold. Overall, this study describes the first prospective of lignin alcohol and lipid production from lignin derivative aldehydes via L. starkeyi.

Single cell protein and oil production from solid cocoa fatty acid distillates co-fed ethanol

The use of solid lipid sidestreams have been overlooked as a feedstock for the production of microbial biomass for food and feed applications and little to no recent work has examined the utilization of solid fatty acid distillates (FADs), which are a significant residue from vegetable oil processing. Yarrowia lipolytica and Rhodosporidium toruloides cultivated on cocoa fatty acid distillates (CFAD) generated final cell dry weight values > 40 g/L, with strong productivity (3.3 g/L·h) and rich protein (>45%) and lipid content (>25%). Interestingly, microbial oils were > 65% unsaturated fatty acids, compared < 20% unsaturated content in FAD. Importantly, to overcome mass-transfer limitations associated with bioconversion of solid lipid residues, ethanol was applied as a co-substrate to solubilize FAD residues. Here, FAD residues from cocoa deodorization have been demonstrated to be high energy feedstocks that represent an attractive substrate for the production of both single cell protein and oil (SCPO).

Growth Response of Non-Conventional Yeasts on Sugar-Rich Media: Part 1: High Production of Lipid by Lipomyces starkeyi and Citric Acid by Yarrowia lipolytica.

Sugar-rich waste streams, generated in very high quantities worldwide, constitute an important source of environmental pollution. Their eco-friendly conversions into a plethora of added-value compounds through the use of microbial fermentations is currently a very "hot" scientific topic. The aim of this study, was to assess the potential of single cell oil (SCO), microbial mass and citric acid (CA) production by non-conventional yeast strains growing on expired ("waste") glucose. Six yeast strains (viz. Rhodosporidium toruloides DSM 4444, Rhodotorula glutinis NRRL YB-252, R. toruloides NRRL Y-27012, Yarrowia lipolytica LFMB Y-20, Y. lipolytica ACA-DC 50109 and Lipomyces starkeyi DSM 70296) were initially grown in shake flasks with expired glucose used as substrate under nitrogen limitation, in order to "boost" the cellular metabolism towards the synthesis of SCO and CA, and their growth response was quantitatively evaluated. Initial glucose concentration (Glc0) was adjusted at c. 50 g/L. Besides Y. lipolytica, all other yeast strains produced noticeable SCO quantities [lipid in dry cell weight (DCW) ranging from 25.3% w/w to 55.1% w/w]. Lipids of all yeasts contained significant quantities of oleic acid, being perfect candidates for the synthesis of 2nd generation biodiesel. The highest DCW production (=13.6 g/L) was obtained by L. starkeyi DSM 70296, while both Y. lipolytica strains did not accumulate noticeable lipid quantities, but produced non-negligible CA amounts. The most promising CA-producing strain, namely Y. lipolytica ACA-DC 50109 was further studied in stirred-tank bioreactor systems, while the very promising DCW- and SCO-producing L. starkeyi DSM 70296 was further studied in shake flasks. Both strains were grown on media presenting higher Glc0 concentrations and the same initial nitrogen quantity as previously. Indeed, L. starkeyi grown at Glc0 = 85 g/L, produced DCWmax = 34.0 g/L, that contained lipid =34.1% w/w (thus SCO was =11.6 g/L). The strain ACA-DC 50109 in stirred tank bioreactor with Glc0 ≈ 105 g/L produced CA up to 46 g/L (yield of CA produced on glucose consumed; YCA/Glc ≈ 0.45 g/g). Finally, in fed-batch bioreactor experiment, the significant CA quantity of 82.0 g/L (YCA/Glc = 0.50 g/g) was recorded. Concluding, "waste" glucose proved to be a suitable substrate for a number of non-conventional yeast strains. Y. lipolytica ACA-DC 50109 produced significant quantities of CA while L. starkeyi DSM 70296 was a very interesting DCW- and SCO-producing candidate. These strains can be used as potential cell factories amenable to convert glucose-based residues into the mentioned metabolic compounds, that present high importance for food, chemical and biofuel facilities.

Rhamnolipid Biosurfactants Enhance Microbial Oil Biodegradation in Surface Seawater from the North Sea

Rhamnolipid Biosurfactants Enhance Microbial Oil Biodegradation in Surface Seawater from the North Sea

Microbial Oil Production from Alkali Pre-Treated Giant Reed (Arundo donax L.) by Selected Fungi

This study aimed to evaluate the microbial oil production by three selected strains, Mortierella isabellina, Cunninghamella echinulata, and Thamnidium elegans, after fermentation of an enzymatic hydrolysate from alkali pre-treated giant reed biomass, in comparison to a semi-synthetic medium, at three increasing nitrogen supplementation levels (0.14, 0.25, and 0.47 g/L). M. isabellina showed the fastest sugar consumption, the highest final cell and oil concentrations (10.9 and 5.6 g/L, respectively), as well as the highest cellular oil content, oil yield/g sugar consumed, and oil productivity (63.4%, 0.19 g/g, and 1 g/L/day, respectively) in the giant reed hydrolysate. The oil yield tended to decrease with an increasing nitrogen content in the cultures. Oleic acid was the most copious fatty acid in the oil for all the fungi. On the contrary, T. elegans exhibited the poorest performances. In particular, M. isabellina showed, respectively, the highest and lowest values of oleic and γ-linolenic acid (52.2 and 3.1%, on average). In comparison, C. echinulata and T. elegans showed much higher γ-linolenic acid content (15.3 and 21.6%, on average). Notably, the C. echinulata cultures showed by far the highest γ-linolenic acid concentration in both substrates (345 and 595 g/L in the giant reed hydrolysate and in the synthetic medium, respectively). Finally, the estimated biodiesel properties of all the oils fell within the limits of the U.S. standards, while the oil of M. isabellina only respected the tighter limits fixed by the E.U. regulations.

Genetic analysis of acyl-CoA carboxylases involved in lipid accumulation in Rhodococcus jostii RHA1.

In actinomycetes, the acyl-CoA carboxylases, including the so-called acetyl-CoA carboxylases (ACCs), are biotin-dependent enzymes that exhibit broad substrate specificity and diverse domain and subunit arrangements. Bioinformatic analyses of the Rhodococcus jostii RHA1 genome found that this microorganism contains a vast arrange of putative acyl-CoA carboxylases domains and subunits. From the thirteen putative carboxyltransferase domains, only the carboxyltransferase subunit RO01202 and the carboxyltransferase domain present in the multidomain protein RO04222 are highly similar to well-known essential ACC subunits from other actinobacteria. Mutant strains in each of these genes showed that none of these enzymes is essential for R. jostii growth in rich or in minimal media with high nitrogen concentration, presumably because of their partial overlapping activities. A mutant strain in the ro04222 gene showed a decrease in triacylglycerol and mycolic acids accumulation in rich and minimal medium, highlighting the relevance of this multidomain ACC in the biosynthesis of these lipids. On the other hand, RO01202, a carboxyltransferase domain of a putative ACC complex, whose biotin carboxylase and biotin carboxyl carrier protein domain were not yet identified, was found to be essential for R. jostii growth only in minimal medium with low nitrogen concentration. The results of this study have identified a new component of the TAG-accumulating machinery in the oleaginous R. jostii RHA1. While non-essential for growth and TAG biosynthesis in RHA1, the activity of RO04222 significantly contributes to lipogenesis during single-cell oil production. Furthermore, this study highlights the high functional diversity of ACCs in actinobacteria, particularly regarding their essentiality under different environmental conditions. KEY POINTS: • R. jostii possess a remarkable heterogeneity in their acyl-carboxylase complexes. • RO04222 is a multidomain acetyl-CoA carboxylase involved in lipid accumulation. • RO01202 is an essential carboxyltransferase only at low nitrogen conditions.