Abstract
The plant-sourced polyketide triacetic acid lactone (TAL) has been
recognized as a promising platform chemical for the biorefinery
industry. However, its practical application was rather limited due to
low natural abundance and inefficient cell factories for biosynthesis.
Here we report the metabolic engineering of oleaginous yeastRhodotorula toruloides for TAL overproduction. We first
introduced a 2-pyrone synthase gene from Gerbera hybrida(GhPS ) into R. toruloides and investigated the effects of
different carbon sources on TAL production. We then systematically
employed a variety of metabolic engineering strategies to increase the
flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting
its competing pathways. We found that overexpression of citrate lyase
(ACL1) improved TAL production by 45% compared to the GhPSoverexpressing strain, and additional overexpression of acetyl-CoA
carboxylase (ACC1) further increased TAL production by 29%. Finally, we
characterized the resulting strain I12-ACL1-ACC1 using fed-batch
bioreactor fermentation in glucose or oilcane juice medium with acetate
supplementation and achieved a titer of 28 g/L or 23 g/L TAL,
respectively. This study demonstrates that R. toruloides is a
promising host for production of TAL and other acetyl-CoA-derived
polyketides from low-cost carbon sources.
Keywords: Rhodotorula toruloides ; triacetic acid
lactone; 2-pyrone synthase, metabolic targets; oilcane juice
Introduction
The demand for renewable biobased products has attracted great interest
in using microbial cell factories to replace traditional petroleum-based
chemical manufacturing processes (Cardenas & Da Silva, 2014; Du et al.,
2011; H. Liu et al., 2019; Markham et al., 2018). Among the chemicals
that can be produced by microbial cell factories, polyketides represent
a large family of naturally occurring metabolites with diversified
industrial and biomedical applications. Due to the low yield by native
producers and the challenge of introducing chiral centers by chemical
catalysis, microbial bioconversion is considered a feasible approach for
large-scale production of polyketides from renewable feedstocks
(Keatinge-Clay, 2016; H. Liu et al., 2019; Robinson, 1991).
Triacetic acid lactone (TAL), also known as 4-hydroxy-6-methyl-2-pyrone,
is a simple, yet interesting, polyketide. It has been demonstrated as a
potential platform chemical for the production of commercially valuable
bifunctional chemical intermediates and end products, including
phloroglucinol (Zha et al., 2004), acetylacetone (Saunders et al.,
2015), and sorbic acid (Chia et al., 2012). TAL is currently produced by
chemical catalysis starting with the pyrolysis of acetic acid (Saunders
et al., 2015). However, its industrial application was hampered by the
detrimental catalysts and toxic byproducts (H. Liu et al., 2019).
Therefore, it is highly desirable to develop an environment-friendly
biological route for converting inexpensive substrates to TAL.
The biosynthesis of TAL is catalyzed by a type III polyketide synthase,
2-pyrone synthase (2-PS) via two iterative decarboxylation/condensation
reactions using a starter acetyl-CoA and two extender malonyl-CoA
molecules. 2-PS encoded by g2ps1 gene was first isolated fromGerbera hybrida (Abe et al., 2005; Eckermann et al., 1998), and
has been genetically introduced to conventional organismsEscherichia coli (Li et al., 2018; Tang et al., 2013; Xie et al.,
2006) and Saccharomyces cerevisiae (Cardenas & Da Silva, 2014,
2016; Saunders et al., 2015; Sun et al., 2021) for TAL production.
However, low titers were obtained in these conventional organisms due to
cellular toxicity, limited intracellular acetyl-CoA pool, or unbalanced
energy/cofactor supply (H. Liu et al., 2019).
Recent studies showed that oleaginous, nonconventional yeasts such asYarrowia lipolytica and Rhodotorula toruloides (also known
as Rhodosporidium toruloides ) can be used for efficient TAL
production due to their potential high flux through the key polyketide
precursors, acetyl-CoA, and malonyl-CoA (Abdel-Mawgoud et al., 2018;
Park et al., 2018). As a well-known lipid producer, Y .lipolytica was chosen for TAL biosynthesis via heterologous
expression of 2-pyrone synthase (Yu et al., 2018), and the best
engineered Y. lipolytica strain achieved a titer of 35.9 g/L TAL
in 280 hours and a yield of up to 43% of the theoretical yield from
glucose (Markham et al., 2018). R. toruloides is an oleaginous
basidiomycete yeast, which can grow on various sugars and produce a
broad range of lipid and nonlipid chemicals (Jagtap & Rao, 2018; Zhang
et al., 2016; Zhang et al., 2021). Compared with Y. lipolytica ,R. toruloides has a greater substrate range and natively produces
TAG at much higher titers (Jagtap & Rao, 2018; Zhang et al., 2016;
Zhang et al., 2021), but was less explored as a result of the
unannotated genome sequence (Coradetti et al., 2018; Zhu et al., 2012)
and lack of sophisticated genetic tools (Park et al., 2018).
Nevertheless, the recent progress on characterization of constitutive
promoters (Nora et al., 2019; Wang et al., 2016), development of CRISPR
based genome editing tools (Jiao et al., 2019; Otoupal et al., 2019;
Schultz et al., 2019), RNA interference tool (X. Liu et al., 2019),
genome-scale model (Dinh et al., 2019), and functional genomics
(Coradetti et al., 2018) enable us to perform metabolic engineering ofR. toruloides for production of value-added compounds (Wen et
al., 2020), specifically TAL.
In this study, we first expressed codon-optimized 2-PS genes from
various organisms in R. toruloides and investigated the
production of TAL under different culture conditions. We then created
and characterized a broad set of TAL-producing overexpression and
knockout gene targets in R. toruloides IFO0880. After
combinatorial optimization of various targets, our final strain of
I12-ACL1 -ACC1 achieved a maximum titer of 28 g/L within
120 hours in fed-batch fermentation from glucose with acetate addition.
Then, we demonstrated the feasibility of olicane juice, an inexpensive
carbon source as the substrate for TAL production, which produced 23 g/L
TAL in fed-batch fermentation. This work not only establishes R.
toruloides as a novel host organism for TAL biosynthesis but also
demonstrates its potential as a biotechnological chassis for production
of high-value chemicals from low-cost substrates.
Materials and methods
Strains, media, and chemicals
All strains used in this study are listed in Table 1. E. coliDH5α (New England Biolabs, Ipswich, MA) was used to maintain and amplify
plasmids, and cells were grown in Luria Broth (LB) medium at 37 °C, 250
rpm with 100 µg/mL ampicillin or 50 µg/mL kanamycin. R.
toruloides IFO0880 and its mutants were grown at 30 °C, 250 rpm in YPD
media (1% yeast extract, 2% peptone, 2% glucose) for routine
handling. For selection or maintenance of transformants, 200 µg/mL G418
(KSE Scientific, Durham, NC), 50 µg/mL hygromycin (InvivoGen, San Diego,
CA) or 100 µg/mL nourseothricin (Gold Biotechnology, St. Louis, MO) was
supplemented as necessary. TAL production media include YPD, YP2D (1%
yeast extract, 2% peptone, 4% glucose), YPX (YP plus 2% xylose), YPDX
(YP plus 1.4% glucose, 0.6% xylose), YPG (YP plus 2% glycerol), YPS
(YP plus 2% sucrose), YP-NaAc (YP plus 2% sodium acetate) and SC
(synthetic complete medium: 1.7 g/L yeast nitrogen base, 5 g/L ammonium
sulfate, 0.78 g/L complete synthetic mixture, 2% glucose).
LB broth, bacteriological grade agar, yeast extract, peptone, yeast
nitrogen base, ammonium sulfate, and D-xylose were obtained from Difco
(BD, Sparks, MD), while complete synthetic medium was purchased from MP
Biomedicals (Solon, OH). TAL standard was purchased from Aldrich
Chemical Co. (Milwaukee, WI). All restriction endonucleases, Q5 DNA
polymerase, and Gibson Assembly Cloning Kit were purchased from New
England Biolabs (Ipswich, MA). The QIAprep spin mini-prep kit was
purchased from Qiagen (Valencia, CA), the Wizard Genomic DNA
Purification Kit was purchased from Promega (Madison, WI), whereas
Zymoclean Gel DNA Recovery Kit and Zymoprep Yeast Plasmid Miniprep Kits
were purchased from Zymo Research (Irvine, CA). All other chemicals and
consumables were purchased from Sigma (St. Louis, MO), VWR (Radnor, PA),
and Fisher Scientific (Pittsburgh, PA). Sequences for key primers, N20
of SgRNA, and gene targets (Coradetti et al., 2018) were summarized in
Table S1 and Table S2. Primers were synthesized by Integrated DNA
Technologies (IDT, Coralville, IA), while heterologous genes were codon
optimized by GeneOptimizer or the JGI BOOST tool and synthesized by
GeneArt (Invitrogen, CA) or Twist Bioscience (San Francisco, CA). gRNAs
were designed using the CRISPRdirect (https://crispr.dbcls.jp/) or the
Benchling gRNA tool. DNA sequencing was performed by ACGT, Inc.
(Wheeling, IL). Plasmid mapping and sequencing alignments were carried
out using SnapGene software (GSL Biotech, available at snapgene.com).
Plasmid construction
Plasmids for 2-PS expression . The codon-optimized 2-PS genes were
synthesized with two homologous ends to Mfe I and Spe I
digested pGI2 (Abbott et al., 2013; Zhang et al., 2016) backbone, which
contains nourseothricin resistance (NAT R) for
yeast, and then assembled with pTEF1 promoter and T35Sterminator for gene expression by Gibson assembly in E. coli(Gibson et al., 2009).
Plasmids for overexpression of metabolic gene targets . The
plasmid pRTG2-X (X represents gene expression targets) for gene targets
expression was constructed based on a previously developed plasmid pRTN,
which contains the E. coli genetic elements of pUC19 (pMB1
origin, ampicillin resistance), the S. cerevisiae genetic
elements of pRS426 (2µ origin and URA3 selection marker), the
strong R. toruloides p17 or pANT promoter, the
target gene, the T35S terminator, and a R. toruloides G418
resistance (G418 R) cassette from NM9 (Schultz
et al., 2019) using DNA assembler (Shao et al., 2009). The multiple gene
expression plasmid pRTHyg-X was pieced together from NM8 (Schultz et
al., 2019) for hygromycin resistance (Hyg R),pANT promoter and Tncbt for gene expression.
Plasmids for gene target knockout . The previously constructed
plasmid pRTH-X (X represents gene knockout targets) was used for gRNA
cloning and expression, which contains the E. coli genetic
elements (pMB1 origin, ampicillin resistance), the S. cerevisiaegenetic elements of pRS426 (2µ origin and URA3 ), a gRNA
expression cassette with the IFO0880 5S rRNA, tRNATyr,
N20 (targeting the first 10% ORF), the S. cerevisiae SUP4
terminator, and a R. toruloides Hyg R cassette
from pZPK-PGPD-HYG-Tnos (Lin et al., 2014). For multiple gene knockout,
the plasmid pRT-Cas9-SgRNA-Hyg was constructed from pRTH-X with an
integrated Cas9 expression cassette.
Yeast transformation
Most of the linear fragments for R . toruloidestransformation were generated by PCR amplification of the genes or gRNA
expression cassettes, together with the selection markers using the
primers ZPK F/R, or gRNA F/R (Table S1), respectively, whereas the
fragments with sizes larger than 8 kb (e.g., ACC1-G418/Hyg ) were
excised from the plasmids by restriction enzyme digestion. Fragments
were then cleaned using DNA Clean & Concentrator-5 Kit (Zymo Research)
before transforming to R. toruloides .
R. toruloides was transformed using heat shock as previously
described (Otoupal et al., 2019; Schultz et al., 2021). Briefly, a
single colony was picked and cultured overnight at 30 °C in 3 mL YPD
medium supplemented with an appropriate antibiotic if required. The
overnight culture was transferred to 50 mL fresh YPD with an
OD600 of 0.2 and cultured for another 4 hours at 30 °C
to an OD600 of approximately 1.0. Cells were collected
by centrifugation, washed twice with sterile water and once with 100 mM
LiAc (pH 7.6) (Sigma Aldrich, St. Louis, MO), and then resuspended in a
transformation mixture of 240 µL PEG3350 (Sigma Aldrich, St. Louis, MO),
36 µL 1 M lithium acetate, 50 µL of 2 mg/mL salmon sperm DNA (Sigma
Aldrich, St. Louis, MO), and 1-2 µg of linear DNA dissolved in 34 µL of
water. The cells were incubated with 200 rpm shaking in the mixture for
30 min at 30 °C. Then, 34 µL of dimethyl sulfoxide (DMSO) was added to
the mixture, which was briefly vortexed, and heat shocked at 42 °C for
20 min. The cells were pelleted, washed with YPD, resuspended in 2 mL
YPD, and recovered overnight with shaking. Cells were then collected and
plated on YPD solid medium supplemented with the appropriate
antibiotic(s). To verify the integrated fragments, genomic DNA was
extracted using the Wizard Genomic DNA Purification Kit (Promega), and
the target locus was PCR amplified for sequencing.
Culture tube or shake flask fermentationSingle colonies were picked and cultured for 24 hours at 30 °C in 3 mL
YPD liquid medium supplemented with appropriate antibiotics in 14 mL
culture tubes (VWR) as seed culture. Fermentations were inoculated
from seed culture to media with alternative carbon sources at an
initial OD600 of 0.2 and grown for an additional 72
hours prior to sample preparation. For acetate spike, filter
sterilized 20x sodium acetate (NaAc) was added to media at 12 h.
Samples were collected by centrifuge and diluted 20 times for high
performance liquid chromatography (HPLC) analysis. Since a deficient
cell growth and tiny amounts of TAL were obtained in YP (1% yeast
extract and 2% peptone) medium, yield was calculated based on the
produced TAL over all carbons of sugars and/or acetate.
Fed-batch fermentation
For fed-batch fermentation in bioreactors, single colonies of R.
toruloides I12-ACL1 and I12-ACL1 -ACC1 were used to
inoculate shake flask cultures with each containing 50 mL of YPAD medium
supplemented with appropriate antibiotics, as described in section 2.1.
Cells in the flask cultures were grown at 30 °C and 250 rpm until the
OD600 reached 2-5. The seed culture from each flask was
transferred to a 1-L bioreactor (Biostat B-DCU, Sartorius, Germany) with
0.7 L fermentation medium, which contained 15 g/L yeast extract, 15 g/L
peptone, 10 g/L
(NH4)2SO4, 6 g/L
KH2PO4, 2 g/L
Na2HPO4, 1 mL/L Antifoam 204
(Sigma-Aldrich), 1.5 mg/L Thiamin·HCl, 1.2 g/L MgSO4, 2
mL/L trace metals (100X), 50 g/L glucose, and appropriate antibiotics
(same as described in section 2.1). The trace metal (100X) solution
contained 10 g/L citric acid, 1.5 g/L
CaCl2·2H2O. 10 g/L
FeSO4·7H2O, 0.39 g/L
ZnSO4·7H2O, 0.38 g/L
CuSO4·5H2O, 0.2 g/L
CoCl2·6H2O, and 0.3 g/L
MnCl2·4H2O. The dissolved oxygen level
was maintained at 20% of air saturation, and the temperature was set at
30 °C. The pH value was controlled at 6.0 using glacial acetic acid and
10 M KOH. When the residual glucose concentration decreased to nearly 0
g/L (as indicated by a sharp decreasing in agitation speed and an
increase in pH value), continuous feeding of glucose (from a 600 g/L
stock solution) was used to maintain its residual concentrations within
20 g/L. Sodium acetate (460 g/L) was fed to the fermenter in pulse at
different time points: 10 mL at 36, 48, 60, 72, and 96 hours, and 8 mL
at 108, 120, 132, 144, 156, and 168 hours, respectively. The fed-batch
fermentation was conducted in biological duplicates.
For the fed-batch fermentation experiments with oilcane, the original
oilcane feed solution containing a total sugar of 152 g/L (68.9 g/L
glucose, 61.6 g/L fructose, and 21.5 g/L sucrose) was concentrated to
about 450 g/L total sugar by evaporation through the boiling at
atmospheric pressure. The concentrated oilcane feed solution was further
autoclaved at 121 °C for 30 min before it was used to provide initial
sugars in the medium and to feed sugars during the fed-batch
fermentation. The initial medium contained a total oilcane sugar of 50
g/L and all other medium components, as described previously for the
glucose fed-batch fermentation. The oilcane feeding started when the
initial sugars were depleted, as indicated by a sharp decrease in
agitation speed and an increase in pH value. Oilcane was fed to control
the residual glucose concentrations within 0~10 g/L. All
other fermentation conditions, including acetate feeding, were same as
previously described for the fed-batch fermentation experiments with
glucose.
Analytical methods
Samples were prepared by diluting in methanol to the linear range,
vortex mixing, and centrifuging at 16,000 g for 5 min to remove
cells. After being filtered by a 0.2 μm filter, the supernatant was
injected into the HPLC for TAL, sugars, and acetate analyses. (1)TAL characterization . The analytical HPLC was carried out on an
Agilent 1260 Infinity series instrument equipped with a diode array
detector (DAD) using a Phenomenex Kinetex® 5 µm EVO
C18 100 Å LC column (150 × 4.6 mm; Phenomenex, USA). The solvent system
comprises solvent A (water supplemented with 0.1% trifluoroacetic acid)
and B (acetonitrile supplemented with 0.1% trifluoroacetic acid). The
elution process runs the following program: 2% B to 7% B (linear
gradient, 0–5 min), 7% B to 95% B (5–6 min), 95% B (isocratic
elution, 6–8 min), 95% B to 2% B (8–9 min), 2% B (isocratic
elution, 9–11 min). Full wavelength scanning (UV/Vis) and Liquid
Chromatography-Mass Spectrometry (LC-MS) were performed to determine the
specific absorbance and molecular weight of the target products using
>98.0% purity TAL as a reference. LC-MS analysis was
running on a Waters Synapt G2-Si ESI/LC-MS (Milford, MA), equipped with
ESI positive ion mode (Bruker, Amazon SL Ion Trap) and a Kinetex 2.6-μm
XB-C18 100 Å (Phenomenex). (2) Sugar and acetate analyses.Glucose, xylose, glycerol, sucrose, and acetate consumptions were
measured using an Agilent 1260 Infinity HPLC (Santa Clara, CA), equipped
with RezexTM ROA‐Organic Acid H+(8%) column (Phenomenex Inc., Torrance, CA) and a refractive index
detector (RID). The column and detector were run at 50 °C and 0.6 mL/min
of 0.005 N H2SO4 was used as the mobile
phase (J.-J. Liu et al., 2019).
Results and discussion
R. toruloides can serve as a TAL producer
High lipid production in oleaginous organisms like R. toruloidessuggests a great potential for these organisms to synthesize alternative
acetyl-CoA-derived products, specifically type III polyketide, TAL
(Markham et al., 2018; Park et al., 2018; Wen et al., 2020). It was
reported that R. toruloides could grow normally under harsh
conditions (Lyu et al., 2021) or cultures with non-native products,
including fatty alcohols (Liu et al., 2020), fatty acid ethyl esters
(Zhang et al., 2021), and limonene (Liu et al., 2021). The product
tolerance assay also showed that R. toruloides possessed a
similar growth profile in YPD and YPD with 5 g/L TAL but longer lag and
log phases in YPD with 7 g/L TAL supplementation (Fig. S1). Therefore,R. toruloides can be potentially engineered to produce high
titers of TAL without significantly detrimental growth effects.
As the 2-pyrone synthase (2-PS) gene tested for TAL biosynthesis in
yeast and bacteria was mainly from Gerbera hybrida (GhPS, Uniprot
ID: P48391), we sought to explore additional 2-PS genes from a range of
alternative organisms and characterize their function in R .toruloides . To assist the selection of 2-PS genes, the Enzyme
Function Initiative-Enzyme Similarity Tool (EFI-EST) was used to create
the sequence-similarity network (SSN) based on the InterPro family
IPR011141 (Type-III polyketide synthase) (Gerlt et al., 2015).GhPS and three additional 2-PS genes from previously unexplored
species, Vitis vinifera (Protein ID: CK203_022254),Sphaceloma murrayae (Protein ID: CAC42_3419), andAspergillus oryzae (Protein ID: AO090701000566) were
codon-optimized using the most frequently used codon (Table S2),
synthesized, and cloned to pGI2 under the pTEF1 promoter. The
PCR-amplified 2-PS -NAT cassettes were transformed and
randomly genome-integrated via NHEJ to R. toruloides .
However, of these genes, only GhPS produced TAL (Fig. S2). The
strain I12 with one copy GhPS expression produced 2.0 ± 0.1 g/L
TAL in YPD medium at 72 h in a culture tube, which was close to that
obtained from the Y. lipolytica strain (2.1 g/L) with four copies
of GhPS expression in a defined synthetic medium (Markham et al.,
2018). Although codon optimization algorithms and genome integration
loci may affect the GhPS expression, it indicates R.
toruloides can serve as a promising platform for TAL production.
TAL production using various substrates
To evaluate the effects of substrates on TAL production, we chose the
commonly used carbon sources, including xylose (X), glycerol (G),
sucrose (S), complete synthetic medium (SC), and glucose/xylose mixed
sugar (DX) (Fig. 1A). The results showed that YPD was a more preferred
medium than SC, with a 4-fold higher TAL titer; compared to glucose,
glycerol and glucose/xylose produced 5%~10% higher
TAL, while xylose and sucrose decreased TAL production. We also observed
deficient cell growth and residual sugars in YPX, YPS, and SC media
(Table S3), indicating a positive correlation between cell growth and
TAL titer.
It has been demonstrated that acetate feeding was beneficial to
acetyl-CoA supply and TAL biosynthesis in S. cerevisiae andY. lipolytica (H. Liu et al., 2019; Markham et al., 2018; Sun et
al., 2021). We therefore supplemented 0.5%, 1%, and 2% NaAc to YPD
with the 12 h R. toruloides cell culture and observed significant
improvements, 67%~80% higher TAL production under
these spiking conditions, representing a similar titer to that of YP2D
(3.6 g/L TAL from YP-4% glucose at 72 h) and ~30% of
the theoretical yield calculated from both glucose (2%) and acetate
(0.5%) in culture tube (Fig. 1B).
To explore the potential role of acetate during TAL biosynthesis, we
provided 2% NaAc as an alternative carbon source. However, the TAL
produced from YP-NaAc was only 42% of that produced from YPD (Fig. 1B).
The residual amounts of NaAc were also measured for the above-mentioned
NaAc media, and only YPD+0.5%NaAc showed depletion of acetate after
fermentation while a portion of acetate left in YPD+1%NaAc,
YPD+2%NaAc, and YP-NaAc (Table S3). The acetate consumption indicated
that acetate may not only act as a substrate for TAL production, but
also be associated with the redox and regulatory mechanism, which has
been elaborated in Y. lipolytica (Markham et al., 2018).
Single gene target engineering to improve TAL production
It is generally recognized that malonyl-CoA is the limiting precursor
for polyketide synthase (Xu et al., 2011; Zha et al., 2009). Therefore,
we overexpressed the endogenous acetyl-CoA-carboxylase (ACC1) to test
whether the conversion of acetyl-CoA to malonyl-CoA would facilitate TAL
synthesis (Fig. 2). As shown in Fig. 3A, overexpression of ACC1did not markedly enhance TAL production with only 6% improvement in
YP2D at 120 h compared to that of the starting strain I12. To further
drive the condensation of
acetyl-CoA
and malonyl-CoA, we introduced a second copy of GhPS gene via
genome integration and achieved 4.8 g/L TAL at 120 h,
~11% higher than that of I12 (Fig. 3A). Based on these
results, we deduced that the limiting precursor for TAL overproduction
was acetyl-CoA instead of malonyl-CoA. Therefore, we sought to increase
the flux of acetyl-CoA by enhancing its biosynthetic pathways and
disrupting its competing pathways.
(1) Enhancing acetyl-CoA biosynthetic pathways . We explored three
distinct metabolic engineering strategies and characterized the roles of
associated gene targets in TAL production (Fig. 3). First, we
investigated the pyruvate dehydrogenase (PDH) complex pathway and
overexpressed its subunits, E1 and E3 (LPD1 ) in I12 strain.
Fermentation showed that both E1 and E3 overexpression improved TAL
production by ~16%, which reached ~5.3
g/L at 120 h (Fig. 3A and Table S4). It is known that the PDH complex is
located in the mitochondrial matrix in eukaryotes, and its
compartmentalization is mediated via mitochondrial targeting sequence
(MTS). However, the improved TAL production indicated that the tested
subunits of E1 and E3 may not contain a fully featured MTS, or there may
be leaky expression of these two subunits in the cytoplasm, which was
similar to that of the overexpression of PDH complex in S.
cerevisiae (Lian et al., 2014) and Y. lipoytica (Markham et al.,
2018). As we failed to construct the mutants of other subunits of PDH,
overexpression of the complete PDH or a cytoplastic PDH complex (i.e.,E. coli cytoPDH) (Cardenas & Da Silva, 2016; Kozak et al., 2014)
can be a potential strategy to increase the acetyl-CoA level.
Second, we evaluated the pyruvate dehydrogenase bypass pathway, which
converts pyruvate to acetyl-CoA through a three-step reaction
sequentially catalyzed by pyruvate decarboxylase (PDC), acetaldehyde
dehydrogenase (ALD), and acetyl-CoA synthetase (ACS). The corresponding
genes, PDC1 , ALD5 , and ACS1 were individually
overexpressed in I12 (Fig. 2). As shown in Fig. 3A and Table S4,
overexpression of PDC1 and ALD5 improved TAL production by
13% and 21%, reaching 5.1 g/L and 5.5 g/L in YP2D at 120 h,
respectively. However, no TAL improvement was achieved by ACS1overexpression, which is similar to the study in S. cerevisiaewhere ACS1 overexpression did not improve n -butanol
production because of low activity or post-translational deactivation
(Lian et al., 2014).
Third, we explored the citrate route, a pathway that generates cytosolic
acetyl-CoA from citrate and was reported to be present only in
oleaginous yeasts (Pomraning et al., 2019; Vorapreeda et al., 2012; Zhu
et al., 2012). The pathway gene ACL1 , encoding ATP-citrate lyase
has been overexpressed to increase lipid production in Y.
lipolytica (Blazeck et al., 2014; Wang et al., 2015). A multi-omic
analysis of R. toruloides also revealed that ACL1 was
expressed at extremely high level during lipogenesis stage (Zhu et al.,
2012). Therefore, the endogenous ACL1 was overexpressed in I12
strain (Fig. 2), and the TAL production was dramatically improved by
45%, to 6.6 g/L in YP2D in a test tube at 120 h (Fig. 3A and Table S4),
which was ~35% of the theoretical yield.
In addition, we overexpressed metabolic targets that could indirectly
increase metabolic flux of acetyl-CoA, including AMPD1 (encoding
AMP deaminase) (Zhang et al., 2019), ME1 (encoding malic enzyme),PEX10 (encoding peroxisomal matrix protein), and YLACL1(ACL1 from Y. lipolytica and sequence included in Table S2)
(Blazeck et al., 2014) (Fig. 2). The results showed that PEX10and YLACL1 overexpression increased TAL titer by 12% and 11%,
respectively, whereas AMPD1 decreased TAL titer and ME1had no effect on TAL production (Fig. 3A). This suggests that
up-regulation of β-oxidation by enhancing peroxisome biogenesis throughPEX10 overexpression is an alternative way to recycle acetyl-CoA
for TAL production in R. toruloides .
(2) Disrupting acetyl-CoA competing pathways. Removing acetyl-CoA
consuming pathways was demonstrated as an effective way to increase the
availability of acetyl-CoA. In yeast, the glyoxylate shunt allows
acetyl-CoA to be converted into a C4 carbon without carbon loss (Dolan
& Welch, 2018). Therefore, we performed the inhibition of two key
reactions of the glyoxylate cycle, namely peroxisomal citrate synthase,
encoded by CIT2 , and cytosolic malate synthase, encoded byMLS1 (Chen et al., 2013), by a previously developed CRISPR/Cas9
method (Schultz et al., 2019). As shown in Fig. 3B, compared with
I12-Cas9 strain, the deletion of CIT2 and MLS1improved TAL production by 14% and 20%, respectively.
In addition, we investigated the effects of disrupting other gene
targets, including two acyltransferases (encoded byDGA1 /LRO1 ), pyruvate carboxylase (encoded by PYC1 ),
serine esterase or patatin-domain-containing protein (encoded by NTE1),
and mitochondrial NAD+ transporter (encoded byYIA6 ). Among them, DGA1 and LRO1 are involved in
TAG formation in Y . lipolytica (Athenstaedt, 2011), andPYC1 , NTE1 , and YIA6 were reported to improve TAL
production in S . cerevisiae (Cardenas & Da Silva, 2014).
The fermentation showed that the deletion of DGA1 and LRO1improved TAL titer by 11% and 19%, respectively, while the deletion ofNTE1 , YIA6 , and PYC1 had a marginal effect on TAL
production (Fig. 3B), which is inconsistent with the observation inS . cerevisiae .
Multiple gene target engineering to improve TAL production
To further investigate the effects of multiple gene targets on TAL
production in a combinatorial manner, we selected the top targets that
improved TAL production more than 12%, i.e., ACL1 , ALD5 ,MLS△ , LRO1△ , PDH-E3 , PDH-E1 , CIT2△,PDC1 , and PEX10 for the second round of metabolic
engineering based on I12-ACL1 strain. In addition, we includedACC1 as its overexpression may result in improved malonyl-CoA
concentration in an acetyl-CoA enhanced strain, I12-ACL1 . We
successfully obtained the mutant strains that overexpressed ACL1 ,ALD5 , PDH-E3 , PDH-E1 , PDC1 , PEX10 ,
and ACC1 through random genome integration using HYG selection,
and the optimal combination was ACL1 -ACC1 , which produced
6.9 g/L TAL in YP2D at 120 h, representing a 29% improvement compared
with I12-ACL1 strain (Fig. 3C and Table S4). Unfortunately, we
failed to obtain the correct mutants with MLS1 , LRO1 ,CIT2 deletion after transforming linear fragments containingCas9 -SgRNA -Hyg into I12-ACL1 strain.
Although a decent number of colonies were growing on YPD+HYG plates,
which meant the Hyg expression cassette was integrated into
genome, none of colonies had the expected genome mutation orindel after sequencing-based genotyping. Compared to the high
genome editing (gene knockout) efficiency through transformingSgRNA-Hyg to I12-Cas9 strain, the low editing efficiency
in I12-ACL1 may be caused by the co-expression of Cas9 andSgRNA in a single fragment ofCas9 -SgRNA -Hyg , which could result in the toxicity
or lethality to the host cells. Therefore, a two-step transformation of
individual Cas9 and SgRNA could be beneficial for genome
editing in I12-ACL1 , and future efforts will be made to develop
other antibiotic or auxotrophic selection markers in R .toruloides , or introduce recyclable expression platforms, such as
a replicable episomal plasmid (Schultz et al., 2021) or a Cre/loxP
site-specific recombination system (Díaz et al., 2018).
Scale-up TAL production in bioreactor
To evaluate the possibility of scaling up fermentation, fed-batch
fermentation was performed using strains I12-ACL1 and
I12-ACL1 -ACC1 , which showed high TAL titers in culture
tube fermentations. Before bioreactor fermentation, we performed shake
flask cultures of I12-ACL1 and I12-ACL1 -ACC1 in
YP2D with 0.5% or 1% sodium acetate spike. The results showed that the
cell OD600 and TAL production with 5 g/L sodium acetate
addition were higher than that with 10 g/L sodium acetate (Fig. S3), and
I12-ACL1 -ACC1 produced 7.1 g/L TAL at 96 h, which was 40%
more than that of I12-ACL1 in YP2D-0.5% NaAc. The yield of TAL
production in YP2D-0.5% NaAc for I12-ACL1 -ACC1 reached
35% of the theoretical yield, which was similar to culture tube
fermentation of I12-ACL1 -ACC1 in YP2D. However, it took 24
hours less fermentation time, indicating a higher oxygen concentration
may benefit TAL production.
For bioreactor fermentation, R. toruloides strain
I12-ACL1 -ACC1 was set up for scale-up using fed-batch
cultures in medium containing yeast extract, peptone, glucose, and other
trace metals, and produced 28 g/L of TAL from glucose-based medium at
118 h, representing a high volumetric productivity of 0.24 g/L/h (Fig.
4A and 4B, Table S5). The highest yield of 0.074 g TAL /g carbon source
(glucose and acetate) for I12-ACL1 -ACC1 was also achieved
at 118 h, which was 16% of the theoretical yield. Furthermore, to
demonstrate the feasibility of fermentation using low-cost feedstocks
and to take advantage of the capability of R. toruloides to
assimilate a diverse range of substrates, including monosaccharides,
oligosaccharides, and organic acids, we performed fed-batch fermentation
using oilcane juice, a first-generation feedstock which is comprised of
152 g/L total sugars. As shown in Fig. 4C, I12-ACL1 -ACC1strain grew well in the oilcane juice, suggesting they may have high
tolerance towards the inhibitors present in the oilcane juice. The titer
and yield of TAL were 23 g/L and 0.089 g/g carbon sources (glucose,
fructose, sucrose, and acetate), representing 19% of the theoretical
yield (Fig. 4D and Table S5), while the productivity was 0.19 g/L/h
before 120 h. Although the yields in glucose-and oilcane juice-based
fed-batch fermentation were relatively low, the volumetric
productivities in both conditions were much higher than that in culture
tube (0.058 g/L/h) or shake flask (0.074 g/L/h), and even higher than
that reported in Y . lipolytica (0.12 g/L/h) (Markham et
al., 2018). Meanwhile, the fed-batch fermentation provided us more
knowledge of improving TAL production by optimizing fermentation
conditions, including feeding rates, dissolved oxygen/pH control (Sun et
al., 2021), and in situ product separation (Lee et al., 2016).
Conclusion
In this study, the codon-optimized 2-pyrone synthase (GhPS ) was
introduced into R. toruloides for TAL biosynthesis, and the
resultant strain I12 produced ~2 g/L TAL in culture
tube. The dramatic improvement of TAL production by acetate addition
suggests that acetate can not only serve as a substrate but also
stimulate TAL production. It was found that ACL1 , a citrate route
enzyme, was a superior gene target to accumulate acetyl-CoA flux, and
its overexpression improved TAL titer by 45% compared to I12 strain,
which was 35% of the theoretical yield. The concurrent expression ofACL1 and ACC1 further improved TAL by 29% in culture
tube, and the scale-up bioreactor fermentation achieved 28 g/L or 23 g/L
TAL from glucose or low-cost oilcane juice with acetate spike,
respectively. This work demonstrates that R. toruloidesrepresents a promising microbial cell factory for production of
polyketides and other acetyl-CoA-derived chemicals.