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# Engineering Ashbya gossypii strains for de novo lipid production using industrial by-products

Patricia Lozano-Martinez, Ruben M. Buey, Rodrigo Ledesma Amaro, Alberto Jimenez, Jose Luis Revuelta

► To cite this version:

Patricia Lozano-Martinez, Ruben M. Buey, Rodrigo Ledesma Amaro, Alberto Jimenez, Jose Luis Revuelta. Engineering Ashbya gossypii strains for de novo lipid production using industrial by-products. Microbial Biotechnology, 2017, 10 (2), pp.425-433. 10.1111/1751-7915.12487. hal-01603868

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# Engineering *Ashbya gossypii* strains for *de novo* lipid production using industrial by-products

Patricia Lozano-Martínez, Rubén M. Buey, Rodrigo Ledesma-Amaro,† Alberto Jiménez and José Luis Revuelta*

*Metabolic Engineering Group, Departamento de Microbiología y Genética, Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain.*

## Summary

Ashbya gossypii is a filamentous fungus that naturally overproduces riboflavin, and it is currently exploited for the industrial production of this vitamin. The utilization of A. gossypii for biotechnological applications presents important advantages such as the utilization of low-cost culture media, inexpensive downstream processing and a wide range of molecular tools for genetic manipulation, thus making A. gossypii a valuable biotechnological chassis for metabolic engineering. A. gossypii has been shown to accumulate high levels of lipids in oil-based culture media; however, the lipid biosynthesis capacity is rather limited when grown in sugar-based culture media. In this study, by altering the fatty acyl-CoA pool and manipulating the regulation of the main Δ9 desaturase gene, we have obtained A. gossypii strains with significantly increased (up to fourfold) *de novo* lipid biosynthesis using glucose as the only carbon source in the fermentation broth. Moreover, these strains were efficient biocatalysts for the conversion of carbohydrates from sugarcane molasses to biolipids, able to accumulate lipids up to 25% of its cell dry weight. Our results represent a proof of principle showing the promising potential of A. gossypii as a competitive microorganism for industrial biolipid production using cost-effective feed stocks.

Received 16 September, 2016; revised 14 November, 2016; accepted 16 November, 2016. *For correspondence. E-mail revuelta@usal.es; Tel. +34 923 29 46 71; Fax +34 923 294 876.

†Present address: Micalis Institute, INRA UMR1319, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France.
*Microbial Biotechnology* (2017) **10**(2), 425–433
doi:10.1111/1751-7915.12487

## Funding Information

This work was supported in part by BASF and by grant BIO2014-56930-P to José Luis Revuelta. Rubén M Buey is supported by a 'Ramón y Cajal' contract from the Spanish Ministerio de Economía y Competitividad.

## Introduction

During the last years, studies unravelling the lipid metabolic pathways in microorganisms have boosted the application of systems metabolic engineering to produce biolipids that could be used to produce biofuels and oleochemicals (Beopoulos *et al.*, 2011). Biolipid-derived fuels from renewable or even waste feedstocks and advanced biofuels avoid the severe inconveniences of first- and second-generation biofuels such as competition with food industry, dependence on climate season and longer processing cycle (Stephanopoulos, 2007; Beopoulos *et al.*, 2011; Peralta-Yahya *et al.*, 2012). Biolipids provide a sustainable alternative for fossil fuels that might help to reduce the carbon footprint. Additionally, bio-based oleochemicals could substitute petroleum-based chemically synthesized compounds with interest in pharma, food and polymer industries (Ledesma-Amaro *et al.*, 2014b). Thus, the combination of an engineered microorganism host and a cost-effective feedstock is nowadays a major challenge to produce biofuels and oleochemicals in an environmentally and economically feasible manner.

The model microorganism *Saccharomyces cerevisiae* and the oleaginous yeast *Yarrowia lipolytica* have been extensively manipulated by means of rational metabolic engineering approaches to optimize lipid production (Vorapreeda *et al.*, 2012; Blazeck *et al.*, 2014; Runguphan and Keasling, 2014; Kavšček *et al.*, 2015). Other oleaginous microorganisms such as *Rhodosporidium toruloides* have also been studied for fatty acid-derived products because of its natural ability to accumulate triacylglycerol (Fillet *et al.*, 2015).

Ashbya gossypii is a filamentous fungus first identified for its natural capacity to overproduce riboflavin (vitamin B₂) and, currently, more than half of the worldwide riboflavin industrial production relies on A. gossypii fermentation (Stahmann *et al.*, 2000; Schwechheimer *et al.*, 2016). A. gossypii is a very convenient fungus for industrial use because it can be readily grown in industrial waste-based culture media. These media include low-cost oils (Schwechheimer *et al.*, 2016), glycerol (Ribeiro *et al.*, 2012) or sucrose (Pridham and Raper, 1950), the main carbon source of sugarcane molasses (Hashizume *et al.*, 1966). This and other advantages have stimulated the use of A. gossypii not only for industrial scale riboflavin production, but also for nucleoside production
(Ledesma-Amaro et al., 2015a; Ledesma-Amaro et al. 2015b) and recombinant protein production (Magalhes et al., 2014).

We have previously reported *A. gossypii* strains with compromised lipid β-oxidation which are able to accumulate lipids up to 70% of the cell dry weight when grown in culture media supplemented with 2% oleic acid. Nevertheless, when grown in glucose-based media without lipid supplementation, these strains only accumulated up to 10% of their cell dry weight (Ledesma-Amaro et al., 2014a). The development of efficient biocatalyst for the production of biolipids requires not only the conversion of low-cost oily feedstocks into high-value oils, but also a high-yield conversion of carbohydrates to biolipids. In this regard, major bottlenecks exist in the biosynthesis of lipids due to feedback inhibition of lipidogenic enzymes (Fig. 1): acyl-CoA esters regulate the activity of the fatty acid synthase (FAS), the acetyl-CoA carboxylase (ACC1) and the Δ9 desaturase (OLE1; Chen et al., 2014; Neess et al., 2015). Furthermore, saturated fatty acids also exert a negative effect over the ACC1 enzyme, thus regulating their own synthesis (Qiao et al., 2015).

Here, we aimed at developing of *A. gossypii* strains that significantly increased lipid production using sugar-based culture media through the manipulation of two major bottlenecks in lipid metabolism: (i) altering the fatty acyl-CoA pool and the subsequent feedback inhibition of

lipidogenic genes and (ii) manipulating the regulation of the main Δ9 desaturase encoded by the *OLE1* gene (Fig. 1). We show that rewiring the regulation of lipogenesis can increase significantly the conversion of carbohydrates to lipids in *A. gossypii*. In addition, we demonstrate that engineered strains of *A. gossypii* are able to produce biolipids when grown on a very simple culture medium consisting of sugarcane molasses and tap water. Our results represent a proof of principle showing the promising potential of *A. gossypii* as a competitive microorganism for industrial biolipid production using cost-effective feedstocks.

## Results

Aiming at generating *A. gossypii* strains with improved lipid *de novo* biosynthesis, using glucose as the only carbon source, we have manipulated two known bottlenecks for lipid metabolism (Fig. 1): (i) insertion of the first double bond in palmitic and stearic acid by Δ9 desaturases and (ii) alteration of the intracellular concentration of the fatty acyl-CoA pool.

*OLE1 endogenous regulation is a bottleneck for the de novo lipid biosynthesis in A. gossypii*

*Ashbya gossypii* has two identified and characterized Δ9 desaturases codified by the genes *AgOLE1* and

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Schematic – simplified – representation of the lipid metabolism in *A. gossypii*. FFA stands for free fatty acids; TAG for triacylglycerol; FAS for fatty acid synthase.</figcaption></figure>
AgOLE2 that are responsible for the insertion of the first desaturation in stearic and palmitic acid (Fig. 1; Lozano-Martínez et al., 2016). The simultaneous overexpression of these two genes in A. gossypii only slightly increased total fatty acid accumulation in glucose-based medium (up to 1.2-fold with respect to wild type; Lozano-Martínez et al., 2016). Thereby, contrary to what has been reported for Y. lipolytica (Qiao et al., 2015), the overexpression of AgOLE1/OLE2 is not enough to significantly improve the lipid de novo biosynthesis in A. gossypii. We then decided to study AgOLE1 regulation.

In S. cerevisiae, MGA2 – and its homologous SPT23 – are the main regulators of OLE1: Mga2p has been shown not only to activate OLE1 transcription (Zhang et al., 1999; Jiang et al., 2001, 2002; Auld et al., 2006) but also to stabilize OLE1 mRNA transcript when the cells are grown in fatty acid free medium and destabilize it when the cells are exposed to unsaturated fatty acids (Kandasamy et al., 2004). MGA2 codifies for an endoplasmic reticulum membrane protein (Mga2p), but the C-terminal proteolytic cleavage converts Mga2p into a cytoplasmic protein that can be transported to the nucleus (Martin et al., 2007; Liu et al., 2015). The deletion of MGA2 in S. cerevisiae has modest effect on cell fitness, but the double knockout of MGA2 and its homologous SPT23 results in an inviable mutant in the absence of unsaturated fatty acids in the culture medium (Zhang et al., 1999). The overexpression of the cleaved version of MGA2 in S. cerevisiae led to a 1.2-fold increase in triacylglycerides with respect to wild type (Kaliszewski et al., 2008), most probably due to OLE1 overexpression (Chellappa et al., 2001).

Prompted by these results, we decided to investigate the effect on lipid accumulation of the manipulation of the gene ACR165W (AgMGA2), the Ashbya's homologue of S. cerevisiae MGA2/SPT23 (Dietrich et al., 2004). In glucose-based media, AgMGA2 disruption (mga2Δ) caused a slight increase in total lipid accumulation, in contrast to its overexpression (P<sub>GPD</sub>-MGA2) that showed a significant increase with respect to the wild-type strain (Table 1). These results agree with previous reports in S. cerevisiae, where the MGA2 orthologues stabilize OLE1 mRNA transcript when the cells are grown in fatty acid free medium (Kandasamy et al., 2004). On the other hand, MGA2 might destabilize OLE1 mRNA transcript when the cells are exposed to unsaturated fatty acids, as reported for S. cerevisiae (Kandasamy et al., 2004), which would result in decreased lipid accumulation (Table 1).

Interestingly, AgMGA2 disruption does not confer auxotrophy for unsaturated fatty acids (not shown), in contrast to what has been described for S. cerevisiae (Kandasamy et al., 2004). This might indicate that MGA2 might be required for maximal transcriptional activation

<table><caption>**Table 1.** Total fatty acids (TFA) in the engineered A. gossypii strains expressed as the percentage of lipids with respect to dry cell weight. Cultures were grown in MA2 media supplemented with either 8% (w/v) Glucose (MA2-8G) or 1% (w/v) Glucose + 2% (w/v) Oleic Acid (MA2-1G-2O) at 28°C for 7 days in an orbital shaker (150 r.p.m.). Numbers are the mean ± SD of two independent experiments with two replicates each. Total biomass showed no large differences among the different strains tested in this study (8.9 ± 2 mg ml⁻¹ in MA2-8G media).</caption><thead><tr><th>Strain</th><th>TFA (%), MA2-8G</th><th>TFA (%), MA2-1G-2O</th></tr></thead><tbody><tr><td>Wild type</td><td>5.30 ± 0.4</td><td>23.53 ± 0.4</td></tr><tr><td>P<sub>GPD</sub>-MGA2</td><td>8.62 ± 0.3</td><td>15.61 ± 2.1</td></tr><tr><td>ΔMGA2</td><td>6.63 ± 0.1</td><td>24.08 ± 1.5</td></tr><tr><td>MGA2-ΔC-term</td><td>10.47 ± 0.1</td><td>24.63 ± 0.9</td></tr><tr><td>P<sub>GPD</sub>-FAA1</td><td>5.90 ± 0.9</td><td>11.55 ± 1.9</td></tr><tr><td>ΔFAA1</td><td>2.17 ± 0.4</td><td>12.88 ± 2.1</td></tr><tr><td>P<sub>GPD</sub>-TES1<sup>cyt</sup></td><td>7.89 ± 0.4</td><td>n.d.</td></tr><tr><td>P<sub>GPD</sub>-ACOT5<sup>cyt</sup></td><td>9.85 ± 0.1</td><td>n.d.</td></tr><tr><td>MGA2-ΔC-term/P<sub>GPD</sub>-ACOT5<sup>cyt</sup></td><td>20.01 ± 0.2</td><td>n.d.</td></tr></tbody></table>

of OLE1/2, but it is possible that OLE1/2 might be expressed at basal levels in the absence of MGA1. In this context, MGA2 deletion would not confer auxotrophy for unsaturated fatty acids. At this point, we do not know if there are additional genes implied in OLE1/2 transcriptional activation, but no additional paralogues of MGA2 exist in the genome of A. gossypii.

The increase in lipid accumulation was significantly higher when a C-terminal truncated version of AgMGA2 was expressed (mga2-ΔC-term), reaching more than 10% of the cell dry weight (Table 1), similarly to what has been reported for S. cerevisiae (Kaliszewski et al., 2008). Remarkably, in media supplemented with oleic acid, P<sub>GPD</sub>-MGA2 strains showed a strong decrease in total lipid accumulation with respect to the wild type, in contrast to both the mga2Δ and mga2-ΔC-term strains that showed no significant differences with the wild type (Table 1).

*The fatty acyl-CoA pool is a bottleneck for the de novo lipid biosynthesis in A. gossypii*

We next decided to study how the alteration of the fatty acyl-CoA pool could influence the de novo lipid biosynthesis in A. gossypii. The intracellular acyl-CoA pool is extensively regulated by the counteraction of acyl-CoA synthetases and acyl-CoA thioesterases (Black and DiRusso, 2007; Chen et al., 2014). Acyl-CoA-thioesterases catalyse the conversion of activated fatty acids into free fatty acids, which is the reverse reaction catalysed by fatty acyl-CoA synthetases (Fig. 1).

We first disrupted the main fatty acyl-CoA synthetase in A. gossypii: AgFAA1, which is the homolog of FAA1 and FAA4 in S. cerevisiae. Our results showed that AgFAA1 is essential when the only carbon source in the
culture medium is oleic acid (data not shown) and its dis-
ruption (faa1Δ strain) significantly decreased lipid accu-
mulation as compared to the wild type when grown on
glucose-containing media (Table 1). This finding further
supports that AgFAA1 is the main acyl-CoA synthetase
in A. gossypii, in agreement with the phenotype of the
S. cerevisiae faa1Δ/faa4Δ strain, which is also unable to
grow in fatty acid-based media (Black and DiRusso,
2007). However, the marked decrease in lipid accumula-
tion in the strain faa1Δ in glucose-based media differs
from previous results reported for S. cerevisiae, where
lipid accumulation was not significantly changed with
respect to wild type (Færgeman et al., 2001; Black and
DiRusso, 2007; Chen et al., 2014). On the other hand,
our results agree with the sharp decrease observed
when the strain faa1Δ of Y. lipolytica is grown on oleic
acid-containing media (Dulermo et al., 2015). This might
indicate that AgFAA1 has additional functions, apart from
the one shown in Fig. 1, in the lipid metabolism of
A. gossypii that remain unknown. Interestingly, AgFAA1
overexpression (P<sub>GPD</sub>-FAA1) also decreased lipid accu-
mulation in A. gossypii when the strain is grown in media
containing 2% of oleic acid, despite it has no significant
effects in glucose-based media (Table 1). This might
happen because in media-containing oleic acid FAA1
overexpression might greatly increase the cytoplasmic
levels of fatty acyl-CoA that would result in the inhibition
of lipidogenic genes and, therefore, the observed
decrease in lipid accumulation. Moreover, this result
agrees with previous reports describing that the alter-
ation of the intracellular acyl-CoA pool has an inhibitory
effect on lipidogenic genes (Færgeman and Knudsen,
1997) and also induced the expression of the genes
involved in lipid degradation (Færgeman et al., 2001).

We then intended to alter the fatty acyl-CoA pool by
the overexpression of acyl-CoA-thioesterase genes.
Acyl-CoA esters are known to repress fatty acid synthe-
sis (Fig. 1) by inhibiting several lipidogenic enzymes,
such as FAS, ACC1 and OLE1 (Bortz and Lynen, 1963;
Sumper and Träuble, 1973; Choi et al., 1996; Færgeman
and Knudsen, 1997). Thereby, we hypothesized that the
decrease in the fatty acyl-CoA intracellular pool (by con-
version to free fatty acids) could upregulate lipid accu-
mulation. To test this hypothesis, we constructed two
A. gossypii strains that ectopically overexpress in their
cytoplasm two peroxisomal acyl-CoA thioesterase
enzymes: (i) P<sub>GPD</sub>-TES1<sup>cyt</sup> that overexpress A. gossypii
TES1 (the homologue of TES1 in S. cerevisiae) and (ii)
P<sub>GPD</sub>-ACOT5<sup>cyt</sup> that overexpress Mus musculus ACOT5,
which has been previously shown to increase the accu-
mulation of free fatty acids in S. cerevisiae (Chen et al.,
2014). Both genes encode fatty acyl-CoA thioesterases
involved in fatty acid degradation in the peroxisome
(Jones and Gould, 2000; Westin et al., 2004; Maeda

et al., 2006; Chen et al., 2014) and contain the ‘SKL’
prototypical C-terminal peroxisomal targeting signal
(PTS) that it is both necessary and sufficient for directing
cytosolic proteins to peroxisomes (Gould et al., 1987).
Interestingly, total fatty acid quantification of both strains
grown in glucose-based media showed a significant
increase (up to twofold) with respect to the wild type
(Table 1), suggesting that an excess of cytoplasmic fatty
acyl-CoA thioesterase activity results in a decreased
acyl-CoA pool that relieves the feedback inhibition of lipi-
dogenic genes (Fig. 1). The excess of free fatty acids in
the cytoplasm can be readily excreted to the culture
media and, accordingly, the two engineered strains
excreted approximately fivefold more fatty acids
(118.25 ± 0.4 and 122.38 ± 0.4 mg l⁻¹ for P<sub>GPD</sub>-TES1-
cyt and P<sub>GPD</sub>-ACOT5<sup>cyt</sup> respectively) than the wild type
(22.00 ± 0.6 mg l⁻¹).

We next combined the two most favourable modifica-
tions (Table 1) into a single strain (MGA2-ΔC-term/P<sub>GPD</sub>-
ACOT5<sup>cyt</sup>) and observed an additive effect of both modi-
fications (Table 1). Remarkably, this *A. gossypii* strain
accumulates more than 20% of its dry cell weight.

Sugarcane molasses are a very convenient carbon  
source for biolipid production in *A. gossypii*

The results obtained with the strain MGA2-ΔC-term/
P<sub>GPD</sub>-ACOT5<sup>cyt</sup> together with the advantages for indus-
trial use convert *A. gossypii* into a very promising
microorganism for biolipid production using sugar-based
culture media formulations. We therefore studied the use
of convenient culture media for industrial use. To this
end, we tested sugarcane molasses as the unique car-
bon source for the culture media of *A. gossypii*.

Molasses, from sugarcane or beet, mainly contains
fructose, sucrose and glucose. This sugar is an industrial
by-product from sugar manufacturing, and it is consid-
ered an ideal raw material for cheap medium culture for-
mulations (Chao *et al.*, 2013). Indeed, sugarcane
molasses have been previously proved to be acceptable
carbon sources for lipid production in *Y. lipolytica* (Gad-
jos *et al.*, 2015), as well as for ethanol and butanol pro-
duction in *S. cerevisiae* (Ni *et al.*, 2012; Arshad *et al.*,
2014).

Remarkably, in contrast to the wild-type strain which
slightly decreased lipid accumulation in molasses with
respect to glucose-based medium, the MGA2-ΔC-term/
P<sub>GPD</sub>-ACOT5<sup>cyt</sup> A. gossypii engineered strain increased
lipid accumulation up to 25% of its dry cell weight
(Fig. 2). We then quantitatively characterized the lipid
profile of the engineered strain when grown in glucose
and molasses-based culture media. As can be observed
in Fig. 3, there are no significant changes between both
media. The strain MGA2-ΔC-term/P<sub>GPD</sub>-ACOT5<sup>cyt</sup>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Comparison of total fatty acid (TFA) per cell dry weight (CDW) in the wild-type and the *MGA2-ΔC-term/P<sub>GPD</sub>-ACOT5<sup>cyt</sup>* strains. MGA2-8G and MGA2-8M stand for MA2 medium supplemented with 8% (w/v) of either glucose or sugarcane molasses respectively. The data shown represent the mean of three independent experiments with standard errors.</figcaption></figure>

showed a slight increase in saturated C16:1 and C18:1, in detriment of C16:0 and C18:0 fatty acids (Fig. 3), as an expected consequence of the upregulation of the Δ9 desaturase *AgOLE1*.

Altogether, by manipulating two genes, we have obtained an *A. gossypii* strain able to accumulate up to 25% of its dry cell weight in a very convenient culture media composed of sugarcane molasses and tap water. Furthermore, we envisage that this number can be readily increased through a systematic optimization of the culture medium composition as well as the fermentation conditions. Eventually, further genetic manipulations of this strain by means of random and/or rational modifications could also increase this number.

Discussion

Ashbya gossypii has a large capacity to accumulate
lipids when grown in media-containing oleic acid
(Ledesma-Amaro et al., 2014a). Encouraged by this

result, we aimed at further manipulating *A. gossypii* to
optimize the *de novo* lipid biosynthesis and have
obtained strains with increased *de novo* lipid biosynthe-
sis, rather than lipid accumulation. These manipulations
enable the utilization of *A. gossypii* as an efficient biocat-
alyst for the production of biolipids from sugar-based by-
products such as molasses. The most productive strain
contained only two modifications that resulted in additive
effects on lipid accumulation: a truncated version of
*MGA2*, a main regulator of *OLE1* (the main Δ9 desat-
urase in *A. gossypii*) and a heterologously expressed
murine thioesterase gene, *MmACOT5*. These modifica-
tions are expected to increase the expression of *OLE1*
and promote the lipid biosynthetic process. In addition,
the expression of a heterologous thioesterase is
expected to decrease the cytoplasmic pool of fatty acyl-
CoA, thus alleviating the feedback inhibition mechanisms
of that this metabolite exerts on lipogenesis. Up to our
knowledge, this is the first report on the combination of
the manipulation of these two genes in microorganisms
to enhance *de novo* lipid biosynthesis. We envisage that
further manipulations will readily increase the percentage
of lipid accumulation and, indeed, significant efforts are
being directed at present in our laboratory towards this
aim.

Although the achieved amount of lipid accumulation is
not as high as that reported for *S. cerevisiae* and/or
*Y. lipolytica* (Kamisaka *et al.*, 2013; Blazeck *et al.*,
2014), it must be stressed here that *A. gossypii* shows
important advantages for industrial production of lipids
compared to these yeasts that make of it a promising
competitive candidate to be taken into account. First, the
biomass from a filamentous fungus can be easily sepa-
rated from culture media by convenient filtration or sedi-
mentation techniques, easy to implement at industrial
level (Zheng *et al.*, 2012). Second, large-scale fermenta-
tions are nowadays used for riboflavin production,
demonstrating the suitability of this fungus for industrial
scale-up. Third, *A. gossypii* hyphae suffer autolysis in
the late stationary phase, and triglycerides could be

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Lipid profile in the wild-type and the *MGA2-ΔC-term/P<sub>GPD</sub>-ACOT5<sup>cyt</sup>* *A. gossypii* strains in MGA2-8G (A; MA2 medium with 8% (w/v) glucose) and MGA2-8M (B; MA2 medium with 8% (w/v) sugarcane molasses). The data shown represent the mean of three independent experiments with standard errors.</figcaption></figure>
easily recovered by centrifugation, avoiding costly cell-
disruption processes. Therefore, our results represent a
proof of principle showing that *A. gossypii* is a promising
and convenient microorganism that deserves the further
investigation of its potential use as a convenient
industrial biolipid producer.

On the other hand, one of the disadvantages of using
microbial hosts for lipid production is the global cost of
the process, which can be notably diminished with the
use of alternative feed stocks such as industrial by-pro-
ducts. Thereby, the efficient utilization of alternative
sources of carbon will have important economic advan-
tages for the scale-up of lipid production with *A. gossypii*
using a cheap and convenient culture media. Sugarcane
molasses represent a cheap industrial by-product con-
sisting on sucrose (up to 50%), nitrogen source, pro-
teins, vitamins and amino acids among others. The use
of molasses presents important advantages with respect
to other waste products such as lignocellulosic biomass,
which needs a costly pre-treatment for its consumption
by microorganisms (Stephanopoulos, 2007; Taherzadeh
and Karimi, 2008). Interestingly, *A. gossypii* can grow on
molasses without any further modification, contrary to
what happens in *Y. lipolytica* that needs the ectopic
overexpression of invertases to degrade sucrose (Gad-
jos *et al.*, 2015). Thereby, the engineered MGA2-ΔC-
term/*P*<sub>GPD</sub>-ACOT5<sup>cyt</sup> strain is a promising candidate that
deserves future attention.

The lipid profile of the engineered oleaginous strain
has a composition in monounsaturated, polyunsaturated
and saturated methyl esters that correlate with good bio-
diesel properties, that is neither high levels of polyunsat-
urated nor long-chain saturated FAs (Ramos
et al.,2009). Thereby, this strain accumulates significant
amounts of lipids suitable for biodiesel production. Fur-
thermore, we have recently reported that the modification
of the lipid profile by manipulating the elongation and
desaturation systems enhances biodiesel properties in
A. gossypii (Ledesma-Amaro et al., 2014b). Thus, future
experiments in our laboratory will be focused on the
modification of the lipid profile in our engineered oleagi-
nous strain.

Altogether, our results demonstrate that *A. gossypii* is
a very promising industrial microorganism that uses a
cost-effective feedstock to *de novo* synthetize significant
amounts of biolipids that can be used for producing bio-
fuels in an environmentally and economically feasible
manner.

**Experimental procedures**

A. gossypii strains, media and growth conditions

The *A. gossypii* strain ATCC10895 was used and con-
sidered wild-type strain. The strains were cultured at

28°C using MA2 rich medium during 7 days (Förster et al., 1999). MA2 is composed of yeast extract, bacteriophage, agar, water and glucose. In this study, for lipid accumulation, the C/N ratio was increased using 8% of glucose (MA2-8G) as carbon source instead of 2%, which is the standard formulation. Alternatively, 1% glucose and 2% oleic acid, previously emulsified by sonication in the presence of 0.02% Tween-40 (MA2-1G-2O), were used. For experiments with molasses, media was prepared with 8% sugarcane molasses (kindly provided by AB Azucarera Iberia S.L.), 0.1% of yeast extract and tap water (MA2-8M). *A. gossypii* transformation, sporulation conditions and spore isolation have been described elsewhere (Santos et al., 2005). Briefly, DNA was introduced into *A. gossypii* by electroporation, and primary transformants were isolated in selective medium. Homokaryon transformant clones were obtained by sporulation of the primary heterokaryon transformants and isolated on antibiotic-containing plates with 250 mg/l⁻¹ of gentamicin (G418). Liquid cultures were initiated from spores and were incubated on an orbital shaker at 200 r.p.m at 28°C.

Gene manipulation of A. gossypii

Gene deletion and overexpression were carried out by
the construction of recombinant integrative cassettes
(Ledesma-Amaro et al., 2014a,b). For gene deletion, a
replacement cassette with selection marker (*loxP-
KanMX-loxP* module for *G418* resistance) was used.
This selection marker is flanked by the repeated inverted
sequences *loxP*, which enable the elimination of the
selection marker by the expression of a Cre recombi-
nase (Ledesma-Amaro et al., 2014a). The deletion of the
C-terminal part of *AgMGA2* was performed by substitut-
ing this region by a *G418* antibiotic resistance marker.
For gene overexpression, a module based on the
*A. gossypii* glycerol 3-phosphate dehydrogenase pro-
moter (*P*<sub>GPD</sub>) and phosphoglycerate kinase (*T*<sub>PGK1</sub>) ter-
minator sequences, recombinogenic flanks and the
antibiotic selectable marker *loxP-KanMX-loxP*, was inte-
grated at the *STE12* locus. DNA constructs were
obtained using Golden Gate methodology (Enger et al.,
2008). Genome integration of the deletion and overex-
pression modules was confirmed by analytical PCR and
DNA sequencing.

To ectopically express *TES1* and *ACOT5* in the cyto-
sol of *A. gossypii*, we removed the C-terminal prototypi-
cal peroxisomal targeting signal (PTS) that it is both
necessary and sufficient for directing cytosolic proteins
to peroxisomes (Gould *et al*., 1987). The signal ‘SKL’
that both *TES1* and *ACOT5* contain is a prototypical
PTS and, thereby, its removal avoids peroxisome local-
ization.
## Lipid extraction and quantification

Triacylglycerols were extracted and trans-methylated from lyophilized biomass using a modification of the method described by Bligh and Dyer (Bligh and Dyer, 1959). Approximately 200 µg of dried mycelia was mixed with 1 ml of 97.5% methanol/2.5% sulfuric acid and incubated at 80°C for 90 min. The transesterification reaction was stopped by the addition of 1 ml of distilled water. The extraction was performed by mixing the samples with 0.5 ml of hexane and recovery of the upper phase after centrifugation. The hexane-soluble extracted fatty acid methyl esters dissolved were analysed by gas chromatography coupled to mass spectrometry (GC-MS) in an Agilent 7890A gas chromatograph coupled an Agilent MS200 (Agilent, Santa Clara, California, USA) mass spectrometer. A VF50 column (30 m long, 0.25 mm internal diameter and 25 µm film) was used using helium as carrier at 1 ml min⁻¹, with a split ratio of 1:20. The oven programme was as follows: an initial temperature of 90°C for 5 min, a ramp of 12°C min⁻¹ up to 190°C and a ramp of 4°C min⁻¹ up to 290°C. MS detection was from 50 to 400 Da. Fatty acids were identified by comparison with commercial fatty acid methyl ester standards (FAME32; Supelco), and total quantification of fatty acids, expressed as total fatty acids (TFA), was performed using an internal standard: 50 µg of heptadecanoic acid C17:0 (Sigma-Aldrich, Sigma-Aldrich Quimica SL, Madrid, Spain).

## Acknowledgements

This work was supported in part by BASF and by grant BIO2014-56930-P to José Luis Revuelta. Rubén M Buey is supported by a 'Ramón y Cajal' contract from the Spanish Ministerio de Economía y Competitividad. Patricia Lozano-Martínez is supported by a postgraduate fellowship (FPI Program) from the Spanish Ministerio de Economía y Competitividad. We thank María Dolores Sánchez and Silvia Domínguez for excellent technical help.

## Conflict of interest

The authors declare that they have no competing interests.

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