Abstract

   Malonic acid (MA) is a high‐value‐added chemical with significant
   applications in the polymers, pharmaceutical, and food industries.
   Microbial production of MA presents enzyme inefficiencies, competitive
   metabolic pathways, and dispersive carbon flux, which collectively
   limit its biosynthesis. Here, the non‐conventional oleaginous yeast
   Yarrowia lipolytica is genetically engineered to enhance MA production.
   Initially, the malonyl‐CoA pathway, comprising a malonyl‐CoA hydrolase
   from Saccharomyces cerevisiae, is confirmed as the most efficient for
   MA production in Y. lipolytica. To further enhance MA production, two
   novel malonyl‐CoA hydrolases exhibiting higher activity than the
   hydrolase from S. cerevisiae, are identified from Y. lipolytica and
   Fusarium oxysporum, respectively. The introduction of the malonyl‐CoA
   hydrolase from F. oxysporum increases the MA titer to 6.3 g L^−1.
   Subsequently, advanced metabolic engineering strategies are performed
   to ensure a sufficient flux of the precursors acetyl‐CoA and
   malonyl‐CoA for MA production, resulting in a production of 13.8 g L^−1
   MA in shaking‐flasks. Finally, by employing the fermentation conditions
   and feeding strategies, a maximum concentration of 63.6 g L^−1 of MA is
   achieved at 156 h with a productivity of 0.41 g L^−1 h^−1 in fed‐batch
   fermentation. This study provides a new way for engineering Y.
   lipolytica to enhance MA production at high titer.

   Keywords: acetyl‐CoA, Malonic acid (MA), malonyl‐CoA hydrolase,
   malonyl‐CoA, Yarrowia lipolytica
     __________________________________________________________________

   The non‐conventional oleaginous yeast Y. lipolytica has been
   genetically engineered to produce malonic acid via malonyl‐CoA pathway
   through introducing an efficient malonyl‐CoA hydrolase. Advanced
   metabolic engineering and optimization of fermentation conditions
   resulted in a malonic acid production yield of 19.1 g L^−1 in
   shaking‐flasks. Further optimization through fed‐batch fermentation
   techniques led to a maximum malonic acid concentration of 63.6 g L^−1.

   graphic file with name ADVS-12-2411665-g004.jpg

1. Introduction

   Malonic acid (MA) is an organic dicarboxylic acid with a wide range of
   applications in food, pharmaceuticals, manufacturing, and chemical
   industries. As a platform chemical, MA serves as a precursor for
   numerous flavors, fragrances, and pharmaceuticals, including cinnamic
   acid, 3,4,5‐trimethoxycinnamic acid, and γ‐nonanolactone.^[ [32]^1 ^]
   Additionally, MA can also be used in manufacturing industries,
   particularly in electronics,^[ [33]^2 ^] specialty solvents, and
   polymer cross‐linking.^[ [34]^3 ^] For these applications, MA has been
   listed as one of the top 30 chemicals that can be produced from biomass
   by the United States Department of Energy.^[ [35]^4 ^] Currently, MA is
   predominantly produced industrially by chemical synthesis, specifically
   through the hydrolysis of diethyl malonate and cyanoacetic acid.^[
   [36]^5 ^] However, the hydrolysis of diethyl malonate is prone to
   reversible reactions, MA undergoes decarboxylation, decomposing into
   acetic acid, water and carbon dioxide by heat under high temperature
   conditions, which results in low product yield. Conversely, the
   hydrolysis of cyanoacetic acid is a complex process that generates
   impurities, thereby reducing the purity of MA. Therefore, there is an
   urgent need for the development of a sustainable, efficient and
   environmentally friendly method for the production of MA.

   Biological production of MA has been demonstrated in the microorganisms
   of Escherichia Coli, Myceliophthora thermophila, and Saccharomyces
   cerevisiae.^[ [37]^6 ^] At present, MA can be synthesized via three
   intermediates: β‐alanine,^[ [38]^6a ^] oxaloacetate,^[ [39]^6c ^] and
   malonyl‐CoA.^[ [40]^6b ^] The β‐alanine pathway was constructed to
   produce MA from β‐alanine in E. coli by introducing the β‐alanine
   pyruvate transaminase encoded by pa0132 gene from Pseudomonas
   aeruginosa and overexpressing the E. coli succinate semialdehyde
   dehydrogenase encoded by yneI gene, resulting in 3.60 g L^−1 by
   fed‐batch fermentation.^[ [41]^6a ^] Then, a novel MA synthetic pathway
   was designed and constructed in M. thermophila using oxaloacetate as a
   precursor. This pathway involved the conversion of oxaloacetate to
   malonate‐semialdehyde via oxaloacetate dehydrogenase (Mdc), followed by
   the reduction of malonate‐semialdehyde to MA by the dehydrogenase YneI,
   which produced only 42.5 mg L^−1 MA.^[ [42]^6c ^] Given the inherent
   robustness of the budding yeast S. cerevisiae, such as its resistance
   to acidic conditions and lake of phage contamination, the β‐alanine
   pathway was first ported from E. coli to S. cerevisiae to facilitate
   the production of MA in our previous study.^[ [43]^7 ^] However, the
   maximum titer of MA achieved was only 91.5 mg L^−1, which was
   significantly lower than that observed in E. coli. It has been reported
   that the native gene encoding 3‐hydroxyisobutyryl‐CoA hydrolase in S.
   cerevisiae could be mutated to exhibit malonyl‐CoA hydrolase activity
   and catalyze the conversion of malonyl‐CoA to MA.^[ [44]^6b ^] To
   enhance the production of MA, we next constructed the malonyl‐CoA
   pathway by targeting the mitochondrial 3‐hydroxyisobutyryl‐CoA
   hydrolase gene EHD3 from S. cerevisiae to the cytoplasm and mutating
   its active sites to obtain malonyl‐CoA hydrolase activity.^[ [45]^8 ^]
   This genetic modification, combined with the efforts of improving the
   precursor supply of malonyl‐CoA and optimizing the fermentation
   conditions, the MA titer was increased to 1.6 g L^−1 after the
   fed‐batch fermentation.^[ [46]^8 ^] However, this MA titer remains
   significantly lower than that achieved via the β‐alanine pathway E.
   coli in the previous study,^[ [47]^6a ^] indicating that the activity
   of malonyl‐CoA hydrolase might be not high enough for MA production. In
   addition, using S. cerevisiae as a production host usually challenged
   with ethanol accumulation,^[ [48]^9 ^] leading to the scattered carbon
   flux and limiting MA production. Therefore, employing a more efficient
   malonyl‐CoA hydrolase and an attractive host with abundant malonyl‐CoA
   flux could potentially enhance MA production to a higher level.

   Yarrowia lipolytica is a “generally regarded as safe” (GRAS) yeast,^[
   [49]^10 ^] which has several advantages, including the ability to
   metabolize various carbon sources, excellent acid tolerance, elevated
   levels of acetyl‐CoA and malonyl‐CoA, a broad pH tolerance range, the
   capacity to achieve high cell densities and being unaffected by glucose
   repression.^[ [50]^11 ^] Recently, the advancements in genetic tools
   have highlighted the significant potential of Y. lipolytica to produce
   various malonyl‐CoA‐derived products.^[ [51]^12 ^] Y. lipolytica has
   been effectively engineered and optimized as a microbial cell factory
   for the production of lipids,^[ [52]^13 ^] natural product^[ [53]^14 ^]
   and terpenoids,^[ [54]^15 ^] such as squalene,^[ [55]^16 ^]
   farnesene,^[ [56]^17 ^] germacrene A,^[ [57]^18 ^] and carotenoids.^[
   [58]^19 ^] This is largely attributed to the robust activity of its
   tricarboxylic acid (TCA) cycle and acetyl‐CoA metabolism. As a
   prototypical oleaginous yeast, Y. lipolytica exhibits exceptional lipid
   accumulation capabilities, achieving a lipid titer of 72.7 g L^−1 and
   an oil content of 81.4% in bioreactor settings at an industrial
   scale.^[ [59]^20 ^] Furthermore, the biosynthetic pathway for polydatin
   has been successfully established in Y. lipolytica, yielding 6.88
   g L^−1, which represents the highest reported level of polydatin
   production.^[ [60]^21 ^] Additionally, the synthesis of another
   malonyl‐CoA derivative, 3‐hydroxypropionic acid (3‐HP), has been
   efficiently accomplished, with yields reaching 1.128 g L^−1 in shake
   flask fermentation and 16.23 g L^−1 in fed‐batch fermentation using the
   recombinant strain Po1f‐NC‐14.^[ [61]^22 ^] These achievements are
   closely linked to the high supply of acetyl‐CoA and malonyl‐CoA
   precursors of Y. lipolytica.^[ [62]^23 ^]

   In this study, we aim to develop a more efficient Y. lipolytica cell
   factory for MA production. The efficiencies of three MA synthesis
   pathways of malonyl‐CoA pathway, malonyl‐CoA and malonate‐semialdehyde
   pathway, oxaloacetate and malonate‐semialdehyde pathway are evaluated.
   The malonyl‐CoA pathway is identified as the most effective pathway for
   MA production. Subsequently, two novel malonyl‐CoA hydrolases
   exhibiting higher activities are identified from Y. lipolytica and
   Fusarium oxysporum, respectively, and utilized for MA production. To
   enhance the precursor supply of acetyl‐CoA and malonyl‐CoA, their
   competing pathways are further inhibited and the key enzymes are
   overexpressed. Ultimately, through the combined efforts of fermentation
   optimization and fed‐batch fermentation, an MA titer of 63.6 g L^−1 is
   achieved. This represents the highest titer reported to date and
   constitutes a significant breakthrough in the biosynthesis of MA.

2. Results

2.1. Investigating Optimal Biosynthetic Pathway for MA Production in Y.
lipolytica

   To produce MA in Y. lipolytica, a malonyl‐CoA pathway and two
   malonate‐semialdehyde pathways were constructed (Figure [63]1a). First,
   the malonyl‐CoA pathway was established by overexpressing the
   malonyl‐CoA hydrolase derived from the 3‐hydroxyisobutyryl‐CoA
   hydrolase Ehd3** of S. cerevisiae, incorporating two specific
   mutations, F121I and E124S.^[ [64]^8 ^] It has been reported that
   3‐hydroxypropionic acid (3‐HP) can be effectively synthesized from
   malonate‐semialdehyde.^[ [65]^22 ^] Given the synthesis of MA via the
   malonate‐semialdehyde pathway has not been investigated in yeast, we
   focus on two malonate‐semialdehyde pathways by using oxaloacetate or
   malonyl‐CoA as a precursor, respectively. Then, the oxaloacetate and
   malonate‐semialdehyde pathway (Mdc pathway) was constructed in Y.
   lipolytica by introducing the oxaloacetate decarboxylase (Mdc) from O.
   parapolymorpha and the malonate‐semialdehyde dehydrogenase Yne1 from E.
   coli. To construct the malonyl‐CoA and malonate‐semialdehyde pathway
   (McrC pathway), the C‐terminal fragment (McrC, amino acids 550–1219) of
   malonyl‐CoA reductase (Mcr) from C. aurantiacus which can reduce
   malonyl‐CoA to malonate‐semialdehyde,^[ [66]^24 ^] and the
   malonate‐semialdehyde dehydrogenase Yne1 from E. coli, were both
   overexpressed.

Figure 1.

   Figure 1
   [67]Open in a new tab

   Biosynthesis of MA by the yeast Y. lipolytica. a) The design of three
   distinct biosynthetic pathways for MA production. b) Comparison of the
   MA production by three pathways. The asterisks show statistically
   significant differences relative to the YMA‐1 strain. c) Identification
   of MA production by liquid chromatography‐mass spectrometry (LC‐MS). d)
   Evaluation of the MA production levels of the 20 screened colonies. The
   asterisks show statistically significant differences from ScEhd3**
   single‐copy number integrated strain. e) Analysis of the growth, MA
   production, pH value, and glucose consumption of the strain YMA‐6 in
   shake‐flask fermentation. The data were presented as mean values ± SD
   from three independent biological replicates (n = 3). The error bars
   represent the standard deviation (s.d.). Statistical significance was
   evaluated using one‐way analysis of variance (ANOVA). The asterisks of
   *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively.

   The malonyl‐CoA hydrolase of Ehd3** from S. cerevisiae, the
   oxaloacetate decarboxylase Mdc from O. parapolymorpha, the malonyl‐CoA
   reductase McrC from C. aurantiacus as well as the malonate‐semialdehyde
   dehydrogenase Yne1 from E. coli was cloned into the single‐copy number
   integrated plasmid of pINA1269 and expressed using the hp4d promoter
   and XPR2 terminator, respectively. These plasmids were subsequently
   linearized and integrated to the genome of Po1f strain. Following shake
   flask fermentation, the YMA‐1 strain harboring the malonyl‐CoA pathway,
   achieved a maximum MA titer of 1.0 g L^−1 (Figure [68]1b,c).
   Interestingly, the YMA‐2 strain expressing only the oxaloacetate
   decarboxylase Mdc and the YMA‐3 strain expressing only the malonyl‐CoA
   reductase McrC produced 0.3 and 0.5 g L^−1 MA, respectively
   (Figure [69]1b). This observation suggests that the endogenous
   succinate‐semialdehyde dehydrogenase (YALI0_F26191g) of Y. lipolytica
   exhibits malonate‐semialdehyde dehydrogenase activity, catalyzing the
   formation of MA from malonate‐semialdehyde similarly to Uga2 in S.
   cerevisiae.^[ [70]^6a ^] To explore whether it is due to the low levels
   of malonate‐semialdehyde dehydrogenase in Y. lipolytica, the
   malonate‐semialdehyde dehydrogenase Yne1 from E. coli was further
   overexpressed in YMA‐2 and YMA‐3 strain to obtain the strains YMA‐4 and
   YMA‐5, respectively. However, the production of MA was increased to
   only 0.43 and 0.56 g L^−1 in the YMA‐4 and YMA‐5 strains, respectively
   (Figure [71]1b). Compared to the malonyl‐CoA pathway, the lower
   cytoplasmic oxaloacetic acid might have resulted in the Mdc pathway
   producing a lower titer of MA.^[ [72]^6c ^] Despite the overexpression
   of YneI, YMA‐5 strain exhibited only a 20% increase over YMA‐3 strain,
   suggesting that McrC expression may be a rate‐limiting step, as
   previously reported.^[ [73]^24 ^]

   Compared to the two malonate‐semialdehyde pathways, malonyl‐CoA pathway
   demonstrated superior efficacy for MA production. To improve MA
   production by increasing the copy numbers of the malonyl‐CoA hydrolase
   ScEhd3**, the ScEHD3** gene was randomly integrated into the genome of
   Po1f strain, taking advantage of the higher non‐homologous
   recombination efficiency of the Y. lipolytica. After integration, 20
   transformants with higher MA production than YMA‐1 were screened out
   (Figure [74]1d). In the shake flask fermentation, an initial glucose
   concentration of 50 g L^−1 was identified as the most efficient
   concentration for MA production (Figure [75]S1, Supporting
   Information). The maximum achieved MA titer was increased to 3.0
   g L^−1, representing a 16‐fold increase compared to MA production in S.
   cerevisiae (Figure [76]1e). Throughout the fermentation process, MA
   accumulation led to a gradual decrease in the pH of the fermentation
   broth. At the end of the fermentation, the pH decreased to a low level
   which might inhibit the cell growth, resulting in residual glucose
   remaining in the broth. This strain was subsequently named YMA‐6 and
   selected for further studies.

2.2. Characterizing Efficient Malonyl‐CoA Hydrolases to Facilitate MA
Production

   It has been reported that Y. lipolytica exhibits a high flux of
   malonyl‐CoA.^[ [77]^23 ^] However, in addition to being used as a
   precursor, malonyl‐CoA is integral to numerous cellular metabolic
   pathways, including lipid and fatty acid synthesis.^[ [78]^13 ^] The
   malonyl‐CoA hydrolase must compete with other metabolic pathways for
   precursor malonyl‐CoA during the production of MA, making the activity
   of malonyl‐CoA hydrolase crucial for MA production. To identify more
   efficient malonyl‐CoA hydrolases for the conversion of malonyl‐CoA to
   MA, a preliminary screening of fungal malonyl‐CoA hydrolases was
   performed using a phylogenetic tree based on the amino acid sequence of
   3‐hydroxyisobutyryl‐CoA hydrolases (Ehd3s) available in NCBI and
   UniPort databases. The selection criteria were based on the principle
   that all Ehd3s possess the conserved amino acids F121 and E124, and
   exhibit less than 50% amino acid homology with ScEhd3 protein (Figure
   [79]2a; Figure [80]S2 Supporting Information). Five different Ehd3s,
   including YlEhd3 from Y. lipolytica, CaEhd3 from Candida albicans,
   AnEhd3 from Aspergillus niger, LsEhd3 from Lachnellula suecica and
   FoEhd3 from F. oxysporum, were subsequently selected and mutated to
   evaluate their malonyl‐CoA hydrolase activities and MA‐producing
   efficiencies.

Figure 2.

   Figure 2
   [81]Open in a new tab

   Bioinformatics‐guided screening of efficient malonyl‐CoA hydrolases for
   MA production. a) Phylogenetic analysis of different Ehd3s. b)
   Comparison of the enzyme activities of six malonyl‐CoA hydrolases. The
   asterisks show statistically significant differences from ScEhd3**. c)
   Comparison of the MA titer produced by six different malonyl‐CoA
   hydrolases. The asterisks show statistically significant differences
   from YMA‐1 strain. d,e) Visualization of the best docked model with
   YlEhd3**, FoEhd3** (receptor) and malonyl‐CoA (ligand), respectively.
   f) The MA production levels of 20 screened FoEHD3**‐integrated
   colonies. The asterisks show statistically significant differences from
   YMA‐6 strain. g) Growth, MA, pH, and glucose consumption of the strain
   YMA‐7 in shake‐flask fermentation. The data were presented as mean
   values ± SD from three independent biological replicates (n = 3). The
   error bars represent the standard deviation (s.d.). Statistical
   significance was evaluated using one‐way analysis of variance (ANOVA).
   The asterisks of *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001,
   respectively.

   Initially, five mutated Ehd3 variants, including, YlEhd3**
   (ScEhd3^F116I‐E119S), CaEhd3**(CaEhd3^F123I‐E126S), AnEhd3**
   (AnEhd3^F118I‐E121S), LsEhd3** (LsEhd3^F 122I‐E125S) and FoEhd3**
   (FoEhd3^F118I‐E121S), were successfully expressed and purified with a
   6×His tag for subsequent enzyme activity assays, using ScEhd3** as a
   positive control. It was showed that YlEhd3** and FoEhd3** exhibited
   the relatively highest malonyl‐CoA hydrolase activities, which is
   1.4‐fold of ScEhd3**(Figure [82]2b). Next, to further evaluate the
   capabilities of these five malonyl‐CoA hydrolases, they were integrated
   to the genome of Po1f strain with a single‐copy, respectively.
   Following shake flask fermentation, the strain expressing FoEhd3**
   produced the highest MA with a titer of 1.45 g L^−1, while the YlEhd3**
   integrative strain produced the second highest MA titer of 1.38 g L^−1,
   which were both higher than that of YMA‐1 strain using ScEhd3**
   (Figure [83]2c). To further analyze the effects of the two malonyl‐CoA
   hydrolases derived from Y. lipolytica and F. oxysporum, molecular
   docking studies were performed to predict the interactions between the
   two malonyl‐CoA hydrolases (receptor) and malonyl‐CoA (ligand). The
   optimal docking model was visualized using the PyMOL viewer
   (Figure [84]2d,e). It was showed that the affinity of YlEhd3** and
   malonyl‐CoA was −6.25 kcal/mol and that of FoEhd3** was −5.22 kcal/mol,
   indicating that the two malonyl‐CoA hydrolases of FoEhd3** and YlEhd3**
   possess significant potential for MA production.

   To further increase MA production, we performed multicopy integration
   of FoEhd3** or YlEhd3** into the genome of strain YMA‐6, respectively.
   Consequently, a strain of YMA‐7 was screened out with the highest MA at
   6.3 g L^−1 from the FoEhd3**‐integrated strain (Figure [85]2f), which
   was 2.1 times higher than that achieved by YMA‐6 strain. However, no
   transformants exhibiting significantly higher MA titer than 6.3 g L^−1
   were identified among the YlEhd3**‐integrated strains (Figure [86]S3,
   Supporting Information). Therefore, YMA‐7 strain was selected for
   further studies.

2.3. Improving the Supplies of Acetyl‐CoA and Malonyl‐CoA to Enhance MA
Production

   To increase the malonyl‐CoA flux for MA production, we first tried to
   improve the supply of its precursor of acetyl‐CoA by redirecting the
   carbon metabolic flux from lipid synthesis to acetyl‐CoA by metabolic
   engineering (Figure [87]3a). It has been previously found that blocking
   the flow of triacylglycerol (TAG) precursors by disrupting
   diacylglycerol (DGA) can increase the supply of acetyl‐CoA in Y.
   lipolytica,^[ [88]^14 ^] which is an effective strategy to increase the
   titer of malonyl‐CoA derivatives. Therefore, the DGA1 and DGA2 genes
   were deleted in strain YMA‐8 to generate strains YMA‐9 and YMA‐10,
   respectively (Figure [89]3b,c). As expected, this genetic modification
   resulted in a 21.5% and 13.9% increase in MA production for strains
   YMA‐9 and YMA‐10, respectively, without significant changes in the
   biomass (Figure [90]3c). In addition, we found that the intracellular
   lipids produced by YMA‐9 and YMA‐10 strains were reduced by 23.4% and
   17.6% compared to the YMA‐8 strain, respectively (Figure [91]3d). These
   findings indicated that the deletion of DGA1 and DGA2 genes decreased
   the metabolic flux toward fatty acids synthesis, thus increasing the
   availability of acetyl‐CoA. This observation might further increase the
   flux from acetyl‐CoA to malonyl‐CoA, thereby facilitating MA
   production.

Figure 3.

   Figure 3
   [92]Open in a new tab

   Metabolic engineering for enhancing MA production. a) Schematic diagram
   of the metabolic engineering strategies for improving MA production. b)
   Comparison of MA titers in strains with deleted DGA1 and overexpressed
   POX1‐6, MFE1, POT1 and FAA1 in YMA‐8 strain. The asterisks show
   statistically significant differences from YMA‐9 strain. c) The MA
   titer of the strains with deleted DGA1 and DGA2, and combined with
   overexpressed POX1 and/or FAA1. d) Comparison of lipids in the
   indicated engineering strains. The asterisks show statistically
   significant differences from YMA‐8 strain. e) Comparison of MA titers
   in engineered YMA‐21 strain with overexpressed YlAcc1,
   YlAcc1^S667A‐S1178A and ScAcc1^S667A‐S1178A, respectively. The
   asterisks show statistically significant differences from YMA‐21
   strain. The data were presented as mean values ± SD from three
   independent biological replicates (n = 3). The error bars represent the
   standard deviation (s.d.). Statistical significance was evaluated using
   one‐way analysis of variance (ANOVA). The asterisks of *, ** and ***
   denote p < 0.05, p < 0.01 and p < 0.001, respectively.

   Another effective strategy for enhancing MA production is to promote
   the degradation of storage fatty acids through upregulating the
   β‐oxidation pathway to recycle acetyl‐CoA. In Y. lipolytica, the
   initial step of fatty acid β‐oxidation is catalyzed by six distinct
   acyl‐CoA oxidases (Pox1‐6), while the second and third steps are
   mediated by a multifunctional enzyme (Mfe1). The final step is
   catalyzed by 3‐ketoacyl‐CoA‐thiolase (Pot1).^[ [93]^25 ^]
   Overexpression of β‐oxidation‐related genes can accelerate β‐oxidation,
   thereby increasing the acetyl‐CoA supply.^[ [94]^26 ^] In addition,
   four distinct fatty acid acyl‐CoA synthases are responsible for
   activating fatty acids of varying chain lengths prior to their entry
   into peroxisomes in S.cerevisiae. Conversely, only one acyl‐CoA
   synthase (Faa1) has been identified in the cytoplasm in Y.
   lipolytica.^[ [95]^27 ^] Thus, we tried to improve the β‐oxidation
   pathway and the turnover of acyl‐CoA to acetyl‐CoA by overexpressing
   nine genes, namely POX1‐6, MFE1, POT1 and FAA1 in YMA‐8 strain,
   respectively. The results showed that the titer of MA was increased by
   21.6% and 15.6% in the resulting strain of YMA‐11 and YMA‐21, which
   overexpressed POX1 and FAA1, respectively (Figure [96]3b). In contrast,
   the overexpression of POT1 has no significant influence on MA
   production, although it has been reported that its overexpression
   showed significant effect in other studies.^[ [97]^16 , [98]^17 ^]
   Combining all the findings, both of POX1 and FAA1 genes were
   overexpressed by integrating them into the DGA1 and DGA2 loci,
   respectively, to create the YMA‐21 strain. The MA production of YMA‐21
   strain was increased by 63.3% to 10.2 g L^−1 compared with that
   achieved in YMA‐8 strain. Additionally, the intracellular lipids level
   was significantly reduced by 36.3% (Figure [99]3d). These findings
   indicate that this ‘restrain‐pull’ strategy is effective in redirecting
   the metabolic flux from lipid to MA production.

   Although an increase in acetyl‐CoA supply could improve MA production,
   considering the key enzyme of acetyl‐CoA carboxylase (Acc1) which is
   responsible for converting acetyl‐CoA to malonyl‐CoA, was directly
   phosphorylated and inactivated by Snf1 kinase in S. cerevisiae.^[
   [100]^28 ^] and resulted in a deficiency of malonyl‐CoA, we further
   investigated the role of acetyl‐CoA carboxylase in MA production in Y.
   lipolytica. In S. cerevisiae, the two phosphorylation sites (S659 and
   S1157) of Acc1 were mutated from Ser to Ala to inhibit the
   phosphorylation by Snf1.^[ [101]^29 ^] However, there are fewer studies
   on the phosphorylation site of Acc1 in Y. lipolytica. To address this,
   we compared the amino acid sequence of YlAcc1 with that of ScAcc1 for
   homology and found that the amino acids S667 and S1178 are relatively
   conserved with ScAcc1 (S659 and S1157) (Figure [102]S4, Supporting
   Information), suggesting that these residues may be the potential
   phosphorylation sites. To verify our hypothesis, the wild‐type YlAcc1
   and its mutant YlAcc1^S667A‐S1178A (YlAcc1**) were both integrated into
   the MA‐engineered YMA‐21 strain to construct YMA‐22 and YMA‐23 strain,
   respectively. It was found that expressing YlAcc1** in YMA‐23 strain
   increased the titer of MA by 34.8% to 13.8 g L^−1 compared with YMA‐21
   strain, which was higher than the 12.7 g L^−1 of YMA‐22 strain
   expressing the wild‐type YlAcc1. Surprisingly, the expression of
   ScAcc1^S659A‐S1157A (ScAcc1**) mutant did not significantly increase MA
   production of YMA‐24 strain (Figure [103]3e). These findings suggested
   that the YlAcc1 might be also negatively regulated by Snf1 kinase
   through phosphorylation at S667 and S1178, which was similar to the
   regulation observed in S. cerevisiae.

2.4. Optimizing the Culture Conditions to Enhance MA Production

   In addition to glucose, glycerol is frequently employed as the sole
   carbon source for Y. lipolytica. It was shown that the supplementation
   of glycerol resulted in an increase in the pools of acetyl‐CoA and G3P,
   thereby providing sufficient precursors for the MVA pathway and lipid
   accumulation.^[ [104]^30 ^] In addition, acetyl‐CoA can be supplied
   through acetate, which is an economical substrate. To determine the
   optimal growth conditions for the engineered strains, we conducted
   shake flask fermentations utilizing glucose, glycerol, and acetate as
   carbon sources. The results indicated that glycerol resulted in a
   higher titer of MA compared to glucose, although it was not supportive
   of strain growth. Conversely, acetate proved to be detrimental to the
   growth of the engineering strain, yielding the lowest titer of MA
   (Figure [105]4a). Consequently, glycerol was selected as the carbon
   source for subsequent investigations.

Figure 4.

   Figure 4
   [106]Open in a new tab

   Fermentation optimization of the engineered YMA‐23 strain. a) MA
   production by YMA‐23 strain with glucose, glycerol, and acetate. The
   asterisks show statistically significant differences from glucose as
   carbon source. b) MA production by YMA‐23 strain with different
   inorganic ions. The asterisks show statistically significant
   differences from YPG medium. c,d) MA production with variable
   concentrations of Mn^2+ and Zn^2+ by YMA‐23 strain, respectively. The
   asterisks show statistically significant differences from YPG medium.
   e, f) Growth of YMA‐23 strain with variable concentrations of Mn^2+ and
   Zn^2+, respectively. The data were presented as mean values ± SD from
   three independent biological replicates (n = 3). The error bars
   represent the standard deviation (s.d.). Statistical significance was
   evaluated using one‐way analysis of variance (ANOVA). The asterisks of
   *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively.

   A large number of inorganic ions, including NH[4] ^+, HPO[4] ^2−,
   Mg^2+, Ca^2+, Mn^2+and Zn^2+ were found to be essential for microbial
   growth, metabolic regulation and enzyme activities. In the process of
   microbial growth and metabolism, NH[4] ^+ could be utilized as a
   nitrogen source, while phosphorus was an essential element of ATP, ADP,
   cell membrane, protein and nucleic acid.^[ [107]^31 ^] Metal ions can
   effectively regulate the metabolic pathways and enzyme activities
   during the fermentation process primarily because of their significant
   roles as cofactors for a large number of key enzymes.^[ [108]^32 ^] To
   investigate whether these inorganic ions could improve MA production,
   we supplemented the culture medium with 5 mm NH[4]Cl, 5 mm Na[2]HPO[4],
   0.5 mm MnCl[2], 8 mm ZnCl[2], 100 mm MgCl[2], and 50 mm CaCl[2] to
   study their effects on MA production using YMA‐23 strain
   (Figure [109]4a).^[ [110]^8 ^] After shake flask fermentation, the
   titers of MA were all lower than that of the control when HPO[4] ^2−,
   Mg^2+, and Ca^2+ were added to the culture medium. In addition, the MA
   level was almost the same as that of the control after fermenting with
   5 mm NH[4]Cl, indicating that A^+ had no significant effect on MA
   production. Surprisingly, the final titers of MA reached to 18.5 and
   16.5 g L^−1 when the YMA‐23 strain were fermented with 0.5 mM MnCl[2]
   or 8 mM ZnCl[2], which was increased by 34.1% and 19.7%, respectively
   (Figure [111]4b). These results indicated that Mn^2+ and Zn^2+ were
   favorable to MA production. Next, the concentration of MnCl[2] and
   ZnCl[2] were optimized, revealing that 5 mM MnCl[2] and 4 mM ZnCl[2]
   were most effective for MA production. In detail, the titer of MA
   increased by 38.4% to 19.1 g L^−1 with the addition of 5 mM MnCl[2] and
   by 26.7% to 17.4 g L^−1 with the addition of 4 mM ZnCl[2] to the
   culture during shake flask fermentation (Figure [112]4c,d). However, it
   is noteworthy that the combined addition of 5 mM MnCl[2] and 4 mM
   ZnCl[2] did not result in an additive effect, suggesting the
   possibility of a shared mechanism of action (Figure [113]4e,f).

   To further investigate the mechanisms by which Mn^2+and Zn^2+ improve
   MA production, we next analyzed the activity of malonyl‐CoA hydrolase
   in response to different concentrations of Mn^2+and Zn^2+. Our findings
   indicate that the elevated concentrations (>100 µM) of both Mn^2+and
   Zn^2+ inhibited the activity of malonyl‐CoA hydrolase. The optimal
   concentrations for Mn^2+and Zn^2+ were determined to be 10 and 1 µM,
   respectively. In details, the activity of malonyl‐CoA hydrolase was
   increased by 24.4% in response to 10 µm Mn^2+ and by 66.7% in response
   to 1 µM Zn^2+ (Figure [114]S5, Supporting Information). This
   observation might be attributed to the fact that the enzymatic assays
   were conducted in vitro using the purified malonyl‐CoA hydrolase, which
   exhibits specific optimal concentrations for Mn^2+and Zn^2+. Upon the
   addition of varying concentrations of MnCl[2] and ZnCl[2] to the
   fermentation culture, the intracellular levels of Mn^2+and Zn^2+ were
   maintained within a specific range,^[ [115]^33 ^] subsequently
   activating malonyl‐CoA hydrolase and promoting the production of MA.

   We further employed RNA‐sequencing (RNA‐seq) analysis to investigate
   the ameliorative effects of Mn^2+ and Zn^2+ ions on MA production. In
   comparison to standard fermentation without Mn^2+ and Zn^2+ ions, 472
   differentially expressed genes (DEGs) were identified in response to 5
   mM Mn^2+, comprising 192 up‐regulated and 280 down‐regulated DEGs
   (Figure [116]S6a, Supporting Information). While 335 DEGs were
   identified in response to 4 mm Zn^2+, including 110 up‐regulated and
   225 down‐regulated DEGs (Figure [117]S6b, Supporting Information).
   Notably, over 80% of the DEGs in response to Mn^2+ and Zn^2+ were found
   to be identical, which elucidates why the combined presence of Mn^2+
   and Zn^2+ ions did not significantly increase MA titer in shake flask
   fermentation. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
   analysis revealed that the most significant DEGs were associated with
   metabolic pathways, biosynthesis of secondary metabolites, membrane
   metabolism and carbon metabolism (Figure [118]S6c,d, Supporting
   Information). These findings suggested that the improved elevated
   metabolism levels and membrane viability were conducive to increase MA
   production. Given that the supply of acetyl‐CoA is tightly regulated by
   lipid metabolism in Y. lipolytica, we analyzed the gene expression
   levels of genes involved in lipid metabolism when the YMA‐23 strain was
   fermented with either 5 mm Mn^2+or 4 mm Mn^2+. Among the genes involved
   in DGA metabolism and the degradation of storage fatty acids, only the
   expression levels of FAA1 and POT1 were observed to be upregulated in
   the presence of Mn^2+ and Zn^2+ ions (Figure [119]5a,b), which might
   potentially facilitate the degradation of fatty acid. Correspondingly,
   the lipid yield was increased by 7.2% and decreased 10.3% upon the
   addition of 5 mm Mn^2+or 4 mm Mn^2+, respectively, to the culture
   medium (Figure [120]5c). These findings support our hypothesis that
   Mn^2+ and Zn^2+ ions can enhance the production of MA by regulation
   fatty acid metabolism and subsequently increasing the supply of
   acetyl‐CoA through the upregulation of FAA1 and POT1 expression.

Figure 5.

   Figure 5
   [121]Open in a new tab

   Mechanism analysis of Mn^2+ and Zn^2+ ions. a) Transcriptional changes
   of the genes involved MA synthesis pathway. b) Relative expression
   levels of FAA1 and POT1. c) Comparison of lipids of YMA‐23 strain in
   the medium with Mn^2+ and Zn^2+. The number indicates the ratio of
   expression levels (Log[2]FC), red and blue numbers indicate the
   significantly up‐regulation and down‐regulation levels, respectively.
   The box‐shaped and none‐box‐shaped numbers indicate the transcriptome
   results of samples Mn^2+ and Zn^2+, respectively. G‐6‐P,
   Glucose‐6‐phosphate; F‐6‐P, Fructose‐6‐phosphate; GAP,
   Glyceraldehyde‐3phosphate; PYR, Pyruvate; DAG, Diacylglycerol; TAG,
   Triacylglycerols; FFA, Free fatty acid. The data were presented as mean
   values ± SD from three independent biological replicates (n = 3). The
   error bars represent the standard deviation (s.d.).

2.5. MA Fermentation in Scale‐Up Bioreactors

   To evaluate the potential productivity of MA by YMA‐23 strain and to
   further enhance MA production, a 5‐L fed‐batch fermentation was
   performed. Four distinct feeding strategies were designed using glucose
   or glycerol as the carbon source and maintaining a pH of 6, which
   proved to be the most suitable for the MA production (Figure [122]S7,
   Supporting Information). In the first strategy, the initial glucose
   concentration was set at 50 g L^−1, and it was supplemented to a final
   concentration of 50 g L^−1 in a single addition once the glucose was
   depleted. The MA titer reached 45.1 g L^−1 at 144 h, with a
   productivity of 0.31 g L^−1 h^−1 (Figure [123]6a). This MA production
   level was more than twofold higher than that achieved in shake flask
   fermentation. In addition, the engineered YMA‐23 strain exhibited a
   rapid growth rate, achieving its maximum biomass of 220 (OD[600]) only
   after 50 h's fermentation. Given that glycerol was found to be more
   favorable for MA production in shake flask fermentation, it was chosen
   as the carbon source for the second feeding strategy. The titer of MA
   reached 54.3 g L^−1 at 156 h, with a productivity of 0.35 g L^−1 h^−1,
   even though the highest biomass only reached 150 (OD[600],
   Figure [124]6b). Furthermore, in shake flask fermentation, it was found
   that the addition of 5 mM MnCl[2] and 4 mM ZnCl[2] could enhance MA
   production, with MnCl[2] showing a particularly significant effect.
   Then, fed‐batch fermentation was carried out using glycerol as a carbon
   source with the addition of 5 mM MnCl[2] to the culture medium. The
   fed‐batch fermentation results showed that YMA‐23 was able to produce
   59.3 g L^−1 at 156 h with a productivity of 0.38 g L^−1 h^−1
   (Figure [125]6c). In the final strategy, following the depletion of
   glycerol in the initial media, a constant‐rate (0.28 mL/min) feeding
   strategy was implemented to ensure that the glycerol concentration in
   the fermentation broth remained below 5 g L^−1.^[ [126]^34 ^] This
   approach resulted in a production of 63.6 g L^−1 MA at 156 h by YMA‐23
   strain, with an enhanced productivity of 0.41 g L^−1 h^−1
   (Figure [127]6d), which is the highest titer of MA reported. The
   analysis of the fermentation broth revealed that the accumulation of
   citric acid among the organic acids, achieving concentrations of ≈10
   g L^−1, whereas succinic acid and malic acid were detected at
   concentrations below 5 g L^−1 (Figure [128]S8, Supporting Information).
   These findings suggest that the constant‐rate feeding strategy enhances
   glycerol utilization while minimizing the synthesis of by‐products in
   comparison to the fed‐batch feeding approach.

Figure 6.

   Figure 6
   [129]Open in a new tab

   Fed‐batch fermentation of YMA‐23 strain in a 5 L bioreactor. a,b)
   Growth, glucose or glycerol consumption and MA production of the YMA‐23
   strain under the condition of supplementing the glucose or glycerol to
   a final concentration of 50 g L^−1 in a single addition once the
   glucose or glycerol was depleted, respectively. c) Growth, glycerol
   consumption, and MA production of the YMA‐23 strain under the condition
   of pulse feeding of glycerol and Mn^2+. d) Growth, glycerol
   consumption, and MA production of the YMA‐23 strain under the condition
   of limiting the glycerol concentration in the fermentation broth to
   below 5 g L^−1 with 5 mm Mn^2+. The values and error bars reflected the
   mean ± standard deviations (SD) of three biological replicates (n = 3).
   The error bars represent the standard deviation (s.d.).

3. Discussion

   With the ability to grow on various carbon sources including waste
   cooking oil,^[ [130]^35 ^] excellent acid tolerance, and being
   unaffected by glucose repression,^[ [131]^36 ^] Y. lipolytica has been
   recognized as an ideal strain for biosynthesis of high value‐added
   products based on its abundant acetyl‐CoA and malonyl‐CoA precursors.^[
   [132]^37 ^] In this study, an effective malonyl‐CoA pathway was
   constructed to produce MA in Y. lipolytica by introducing the
   malonyl‐CoA hydrolase from S. cerevisiae and F. oxysporum. Enhancing
   the fluxes of acetyl‐CoA and malonyl‐CoA enabled the engineered strain
   to produce a high level of MA both in the shake flasks and the
   scaled‐up fermentation. Compared with other MA‐producing method, this
   engineered strain could synthesize MA with higher titer and
   productivity.

   Microbial production of MA has been successfully performed in E. coli,
   M. thermophila and S. cerevisiae via three intermediates of
   β‐alanine,^[ [133]^6a ^] malonate‐semialdehyde,^[ [134]^6c ^] and
   malonyl‐CoA,^[ [135]^8 ^] respectively. The β‐alanine pathway requires
   the involvement of more than six enzymes to convert glucose to MA. This
   process is further complicated by competition for carbon sources from
   other metabolic pathways and the low activity of semialdehyde
   dehydrogenase (YneI), which results in the accumulation of the
   byproduct of β‐alanine. These factors collectively contribute to a low
   titer of MA.^[ [136]^6a ^] Our previous study has demonstrated that the
   β‐alanine pathway is less efficient than the malonyl‐CoA pathway in S.
   cerevisiae for MA production.^[ [137]^7 , [138]^8 ^] However, the
   cytoplasmic levels of malonyl‐CoA S. cerevisiae might insufficient for
   optimal MA production. Consequently, Y. lipolytica might serve as a
   more suitable host for MA production via malonyl‐CoA pathway.
   Currently, no studies have reported the MA production by the two
   malonate‐semialdehyde pathways in yeast, the Mdc pathway and the McrC
   pathway. The Mdc pathway is shorter than the β‐alanine pathway,
   however, the poor acid tolerance of M. thermophila limits its utility
   as a host for synthesizing MA through MDC pathway.^[ [139]^6c ^]
   Conversely, the Mdc pathway may perform better in Y. lipolytica due to
   its excellent acid tolerance. In addition, the McrC pathway, another
   malonate‐semialdehyde pathway, has significant advantages in the
   production of 3‐HP.^[ [140]^22 , [141]^24 ^] To develop an effective
   synthesis pathway for MA, we evaluated the efficiencies of the
   malonyl‐CoA pathway and two malonate‐semialdehyde pathways of Mdc
   pathway, and McrC pathway for MA production in Y. lipolytica. The
   malonyl‐CoA pathway was constructed by introducing malonyl‐CoA
   hydrolase derived from the mutated 3‐hydroxyisobutyryl‐CoA hydrolase
   ScEhd3** for producing MA in S. cerevisiae in our previously study, the
   highest titer of MA was only 13.6 mg L^−1 after shake flask
   fermentation before engineering the MA synthetic pathway.^[ [142]^8 ^]
   By expressing the malonyl‐CoA hydrolase from ScEhd3** in the Y.
   lipolytica Po1f strain, the engineered YMA‐1 strain produced 1.0 g L^−1
   MA (Figure [143]1b), which was a 73.5‐fold increase compared to that
   produced by the same malonyl‐CoA pathway in S. cerevisiae. This titer
   was also higher than those produced by the two malonate‐semialdehyde
   pathways, namely the Mdc pathway and McrC pathway. Future studies aimed
   to increase MA production via malonate‐semialdehyde pathway should
   focus on identifying more efficient enzymes, specifically the
   oxaloacetate decarboxylase, malonyl‐CoA reductase and
   malonate‐semialdehyde dehydrogenase. These findings indicated that the
   enrichment of MA synthesis precursors, especially acetyl‐CoA and
   malonyl‐CoA in Y. lipolytica, making this non‐traditional yeast an
   ideal host for MA production via the malonyl‐CoA pathway.

   At present, only the mutation of 3‐hydroxyisobutyryl‐CoA hydrolase
   (ScEhd3**) in S. cerevisiae has been reported to have the malonyl‐CoA
   hydrolase activity. In this study, the malonyl‐CoA pathway was
   constructed by overexpressing the malonyl‐CoA hydrolase ScEhd3** in Y.
   lipolytica, resulting in a production yield of 3.0 g L^−1 following
   shake flask fermentation (Figure [144]1e). However, the conversion rate
   was still very low, given the utilization of 50 g L^−1 glucose. These
   findings indicated that the activity of malonyl‐CoA hydrolysis is
   crucial for efficient MA production. To identify more efficient
   malonyl‐CoA hydrolases for the conversion of malonyl‐CoA to MA, a
   preliminary screening for malonyl‐CoA hydrolases from fungal origin was
   performed using a phylogenetic tree based on the amino acids of
   3‐hydroxyisobutyryl‐CoA hydrolase ScEhd3 and its conserved amino acids
   for malonyl‐CoA hydrolase (F121I and E124S) available in NCBI and
   UniPort databases. Two new malonyl‐CoA hydrolases from Y. lipolytica
   and F. oxysporum with higher malonyl‐CoA hydrolase activities than
   ScEhd3** were identified. The MA titer was increased to 6.3 g L^−1 by
   overexpressing FoEhd3** (Figure [145]2g). To increase the production of
   MA, further study might be needed to explore more efficient malonyl‐CoA
   hydrolases from other origins including plants, animals as well as
   bacteria.

   In addition to malonyl‐CoA hydrolase activity, the malonyl‐CoA supply
   is another limiting step for MA production, the intracellular levels of
   which is tightly regulated by the accumulation of its precursor of
   acetyl‐CoA. In Y. lipolytica, more acetyl‐CoA and malonyl‐CoA are used
   for lipid synthesis, as wildtype strains can accumulate lipids up to
   70% of dry biomass.^[ [146]^38 ^] We employ a ‘restrain–pull’ strategy
   effectively switching the carbon metabolic flux from lipid to
   acetyl‐CoA by deleting two diacylglycerol acyltransferases of Dga1 and
   Dga2 to decrease the consumption of acetyl‐CoA, overexpressing the
   enzymes involved in β‐oxidation pathway and acyl‐CoA synthase Faa1 to
   increase the acetyl‐CoA supply. The titer of MA was significantly
   increased to 10.2 g L^−1 through reducing the synthesis of lipids
   (Figure [147]3c,d). The regulatory mechanism of the acetyl‐CoA
   carboxylase (Acc1) in Y. lipolytica has not been well studied yet,
   although it has been reported that the activity of ScAcc1 could be
   tightly regulated by Snf1 kinase by direct phosphorylation.^[ [148]^28
   ^] Here, by comparing the amino acid sequence of ScAcc1 and YlAcc1, we
   found that YlAcc1 also shared the predicted phosphorylation sites (S667
   and S1178) of Snf1 kinase. The MA production was increased 34.8% by
   overexpressing the mutated YlAcc1^S667A‐S1178A (Figure [149]3e),
   indicating that similar regulation mechanism might be exited for YlAcc1
   as the ScAcc1 by protein kinase of Snf1.

   In fed‐batch fermentation, despite the improved MA level using glycerol
   as a carbon source, the highest biomass of YMA‐23 reached only ≈ 150
   (OD[600]), which was just 68.2% of that using glucose as a carbon
   source (Figure [150]5a,b). The addition of glycerol resulted in the
   elevation of the acetyl‐CoA pools, facilitating the flux of carbon
   metabolism to MA thereby competing for some carbon sources, which may
   have resulted in the low biomass.^[ [151]^30b ^] Owing to the Crabtree
   effect, S. cerevisiae produces a large amount of ethanol in the
   presence of oxygen and excess glucose, leading to a loss of carbon for
   the biosynthesis of non‐ethanol chemicals.^[ [152]^39 ^] In contrast,
   Y. lipolytica is not inhibited by high sugar concentrations because of
   its Crabtree‐negative characteristic, and its rate of sugar consumption
   is much higher than that of other microorganisms.^[ [153]^35a ^]
   Therefore, the initial concentrations of the carbon sources were
   usually set to a relatively high level.^[ [154]^40 ^] Currently,
   microbial cell factories utilizing Y. lipolytica as host cells exhibit
   generally low product conversion rates. A significant proportion of
   carbon sources is diverted toward lipid synthesis and other pathways,
   resulting in wasted carbon sources,^[ [155]^41 ^] which limits the
   industrial application of Y. lipolytica as a host strain. To maximize
   the utilization of different carbon sources, enhancing the activity of
   key enzymes within the synthetic pathway emerges as the most effective
   strategy. Given that lipids constitute the primary by‐products of Y.
   lipolytica, promoting lipid degradation and facilitating their
   conversion into desired products is also a viable approach.
   Furthermore, coupling the TCA cycle with the crucial intermediate
   acetyl coenzyme A via the citric acid cycle can further enhance carbon
   source utilization. Organelle engineering holds the potential to
   localize the production of target compounds, thereby minimizing
   substrate loss and improving the carbon conversion rate.^[ [156]^16 ,
   [157]^42 ^] Additionally, transporter engineering can address the
   limiting factors such as product inhibition, thereby enhancing overall
   process efficiency.^[ [158]^43 ^]

   In conclusion, an efficient MA‐producing strain was constructed by the
   selection of different MA synthetic pathways and identification of the
   effective malonyl‐CoA hydrolases in this study. To improve the supply
   of acetyl‐CoA and malonyl‐CoA, the strain was metabolically engineered
   to improve carbon metabolic flux from lipid to acetyl‐CoA by blocking
   the flow of TAG, enhancing the β‐oxidation pathway as well as
   eliminating the inhibition of Acc1 via mutation of its two possible
   phosphorylation sites. Finally, the titer of MA was increased to 63.6
   g L^−1 by using glycerol as carbon source and supplementing MnCl[2] in
   the culture medium in fed‐batch fermentation. This work will facilitate
   the realization of the industrial production of MA and provide a
   reference for the production of other malonyl‐CoA derivatives in the Y.
   lipolytica.

4. Experimental Section

Strains, Plasmids, and Culture Medium

   E. coli DH5α was employed for the construction and propagation of
   plasmids. The bacterial strains were cultured at 37°C with agitation at
   250 rpm in Luria–Bertani (LB) liquid medium (10 g L^−1 NaCl, 10 g L^−1
   Tryptone, and 5 g L^−1 Yeast extract) or incubated at 37°C on LB agar
   plates. When necessary, ampicillin was incorporated into the medium at
   a final concentration of 100 mg L^−1. The wild‐type Y. lipolytica Po1f
   strain, sourced from the ARS Culture Collection (NRRL), was selected as
   the foundational strain for further construction. Y. lipolytica strains
   were cultured in yeast extract peptone dextrose (YPD) liquid medium (10
   g L^−1 Yeast extract, 20 g L^−1 Tryptone and 20 g L^−1 Glucose),
   synthetic dropout (SD) medium (1.7 g L^−1 yeast nitrogen base, 5 g L^−1
   ammonium sulfate, and 20 g L^−1 glucose) or incubated at 30°C on plates
   supplemented with 2 g L^−1 of agar. Detailed descriptions of all
   recombinant plasmids and strains employed in this study are provided in
   Table [159]S1 (Supporting Information).

DNA Manipulation

   Six different EHD3**s including ScEHD3** from S. cerevisiae, YlEHD3**
   from Y. lipolytica, CaEHD3** from C. albicans, AnEHD3** from A. niger,
   LsEHD3** from L. suecica and FoEHD3** from F. oxysporum, the
   oxaloacetate decarboxylase gene MDC from O. parapolymorpha, the
   malonate‐semialdehyde dehydrogenase gene Yne1 from E. coli, the
   malonyl‐CoA reductase gene McrC from C. aurantiacus, as well as the
   acetyl‐CoA carboxylase gene ACC1^S659A‐S1157A from S. cerevisiae were
   first codon‐optimized and synthesized by Genewiz (Suzhou, China). Other
   native promoters, genes, and terminators were amplified from the
   genomic DNA of Po1f strain. The detailed DNA sequence information of
   EHD3** genes used in this study was listed in Table [160]S2 (Supporting
   Information). The EHD3** genes were first cloned into the single‐copy
   number integrated plasmids pINA1269 and expressed using the hp4d
   promoter and XPR2 terminator. The plasmids were then linearized and
   integrated into the genome of Po1f strain. In Y. lipolytica,
   non‐homologous end‐joining (NHEJ) was dominant over homologous
   recombination (HR).^[ [161]^44 ^] To construct YMA‐6 strain, ScEHD3**
   gene was first amplified and cloned into of pKi‐1 plasmid. The
   resulting plasmid was used as a template for PCR to obtain the
   integration fragment of P[ut8]‐ScEHD3**‐T[CYC1]‐LEU2, which was then
   introduced into Po1f strain and randomly integrated into the genome by
   endogenous NHEJ repair.^[ [162]^45 ^] YMA‐6 strain was subsequently
   obtained through screening. Following this, the FoEHD3** gene was
   amplified and assembled with restriction enzymes‐digested expression
   vectors pKi‐2. The integration fragments P[TEFin]‐FoEHD3**‐T[CYC1]‐URA3
   was assembled by fusion PCR and then randomly integrated into the YMA‐6
   genome. YMA‐7 strain was also obtained by screening.

   Efforts to improve gene targeting efficiency have focused on abolishing
   the NHEJ pathway by disrupting either ku70.^[ [163]^46 ^] To achieve
   rational metabolic modification of the engineered YMA‐8 strain, the
   Ku70 gene was deleted in YMA‐7 strain. Genes of POX1, POX2, POX3, POX4,
   POX5, POX6, MFE1, POT1, FAA1, and ACC1 were amplified and subsequently
   assembled with restriction enzyme‐digested expression vectors pKi‐2.
   The integration fragments were then amplified via PCR and transformed
   into the related host strain. Following this, the Cre protein was
   transiently expressed using plasmid pYLXP1, which facilitated the
   removal of the screening tag.^[ [164]^47 ^] The primers used in this
   study is listed in Table [165]S3 (Supporting Information).

Screening and Structural Analysis of Malonyl‐CoA Hydrolases

   The amino acid sequences annotated as 3‐hydroxyisobutyryl‐CoA hydrolase
   were acquired from NCBI or UniProt databases and subsequently aligned
   using ClustalW. The maximum likelihood phylogenetic trees were then
   generated using MEGA‐11 software based on the LG model,^[ [166]^48 ^]
   with the strength of the nodes determined with 100 bootstrap
   replicates. Molecular docking of Ehd3** (S/H) (receptor) and
   malonyl‐CoA (ligand) was performed using the default AutoDock vina
   setting to calculate the interaction between mutant and malonyl‐CoA and
   the docking model was visualized using PyMOL.^[ [167]^49 ^]

RNA‐seq Analysis

   To quantify the relative expression levels of the indicated genes,
   yeast cells were first cultivated overnight in YPD medium, and cultures
   (2%) were then inoculated into 50 mL of fresh YPD medium and cultured
   to an OD[600] of 0.6–0.8. The cells were then inoculated with or
   without (as the control group) 5 mm MnCl[2] or 4 mm ZnCl[2] for an
   additional 24 h. Cells were harvested and the total RNA was extracted
   using hot phenol method.^[ [168]^50 ^] Transcriptome sequencing
   analysis was performed by Vazyme Biotech Co., Ltd. The identification
   of gene expression differences between the different samples was based
   on |log2Ratio| ≥ 1 and q‐value ≤ 0.05 as the standard. Functional
   annotation of the genes with significant differential expression was
   carried out using the Kyoto Encyclo‐pedia of Genes and Genomes (KEGG)
   database. The pathway enrichment analysis tool OmicShare was used to
   classify genes at the level of KEGG_B_class
   ([169]https://www.omicshare.com/tools/Home/Soft /pathwaygsea).^[
   [170]^51 ^]

Shake Flask Fermentations

   Single colonies of recombinant strains were inoculated into 5 mL YPD
   media, and cultivated overnight (16–18 h) at 30°C and 250 rpm. The
   culture was then transferred to a 250 mL shake flask containing 50 mL
   Y[10]P[20]D[50] (10 g L^−1 Yeast extract, 20 g L^−1 Tryptone and 50
   g L^−1 Glucose) with an initial OD[600] = 0.4, and cultivated at 30°C
   with shaking at 250 rpm for 3∼5 days. Glycerol at a concentration of 50
   g L^−1 or 50 g L^−1 sodium acetate was utilized as a substitute for
   glucose in YPD to optimize the carbon source, respectively. The culture
   medium was supplemented with 5 mm NH[4]Cl, 5 mm Na[2]HPO[4], 0.5 mm
   MnCl[2], 8 mm ZnCl[2], 100 mm MgCl[2], and 50 mm CaCl[2] to study their
   effects on MA production. The culture medium was further optimized by
   the addition of Mn^2+ ion at concentrations of 0.5, 1.0, 2.5, 7.5, and
   10 mm, and Zn^2+ ion at concentrations of 0.5, 1.0, 2.0, 4.0, 8.0, and
   12.0 mm, respectively.

Enzyme Activity Assay

   The six linearized plasmids of p1269‐ScEHD3**‐6×his,
   p1269‐YlEHD3**‐6×his, p1269‐CaEHD3**‐6×his, p1269‐AnEHD3**‐6×his,
   p1269‐LsEHD3**‐6×his and p1269‐FoEHD3**‐6×his were first transformed
   into Po1f strain, respectively. Cells were first cultivated overnight
   at 30°C in 20 mL of SD‐LEU medium, which were then transferred into 200
   mL of SD‐LEU medium and cultivated for an additional 12 h. The six
   different Ehd3** proteins were purified and used for analyzing the
   malonyl‐CoA hydrolase activity using 5,5ʹ‐dithiobis‐(2‐nitrobenzoic
   acid) (DTNB) by the method described in our previous study.^[ [171]^8 ,
   [172]^52 ^]

Quantification of Metabolite in Media

   Fermentation samples were centrifuged at 10,000 rpm, and the
   supernatant was collected for analysis via high‐performance liquid
   chromatography (HPLC) or liquid chromatography–mass spectrometry
   (LC‐MS). For HPLC analysis, the supernatant was filtered through a 0.22
   µm membrane and analyzed using an Agilent Technologies 1260 Infinity II
   HPLC system, equipped with an HPX‐87H ion‐exclusion column (300 mm ×
   7.8 mm; BioRad, CA). The mobile phase consisted of 5 mm H[2]SO[4] with
   a flow rate at 0.6 mL min^−1, and the column temperature was maintained
   at 50°C. Compounds were detected from 20 µL injections using a
   refractive index detector. LC‐MS was analyzed using QTRAP® 5500 LC/MS
   system, equipped with an HSS T3 column (1.8 µm, 2.1 × 100 mm, Waters,
   CA). The mobile phase consisted of ammonium acetate and Acetonitrile
   with a flow rate at 0.25 mL min^−1, and the column temperature was
   maintained at 40°C. Compounds were detected from 2 µL injections.

Lipid Extraction and Quantification

   The lipids synthesized by Y. lipolytica, including palmitate (C16:0),
   palmitoleate (C16:1), stearate (C18:0), oleate (C18:1), linoleate
   (C18:2) and long‐chain fatty acid (LCFA), were quantified using a Gas
   Chromatography coupled to a Flame Ionization Detector (GC‐FID). Samples
   of 1 mL cell culture were centrifuged at 16,000 rpm for 10 min, after
   which the supernatant was discarded. Subsequently, 0.5 mL of a 0.5 m
   sodium hydroxide‐methanol solution and 100 µL methyl tridecanoate
   (Sigma‐Aldrich) as internal standards. The samples were vortexed for 1
   h to ensure the transesterification of lipids into fatty acid methyl
   esters (FAMEs). The FAMEs were extracted by adding hexane after
   neutralization with 98% sulfuric acid.^[ [173]^53 ^] The separation of
   the FAME species was performed using an Agilent HP‐INNOWax capillary
   column. The injection volume was set to 1 µL with a split ratio of 10,
   and the injection temperature was maintained at 260°C. The column
   temperature was held constant at 200°C, and helium was used as the
   carrier gas at a flow rate of 1.5 mL min^−1. The flame ionization
   detector (FID) was set at a temperature of 260°C, with helium make up
   gas, hydrogen, and air flow rates at 25, 30, and 300 mL min^−1,
   respectively.^[ [174]^54 ^]

Bioreactor Fermentations

   Fed‐batch fermentations were performed in a 5 L bioreactor (New
   Brunswick Bioflo115 system). The initial fermentation was completed
   with 2 L medium containing 50 g L^−1 glucose or 50 g L^−1 glycerol, 20
   g L^−1 peptone, and 10 g L^−1 yeast extract. The temperature was
   maintained at 30°C, while the dissolved oxygen was controlled at 35%
   using an agitation cascade ranging from 200 to 600 rpm. Air was sparged
   into the fermenter at 4 L min^−1. The pH was maintained by feeding the
   ammonia solution, while the foam was prevented by the addition of
   antifoam 204 (Sigma‐Aldrich). Samples were taken every 12 h to measure
   OD[600], glucose concentration, and MA titer. Four distinct feeding
   strategies were employed for fed‐batch fermentation. In the initial
   strategy, glucose was utilized as the carbon source and was
   supplemented to reach a final concentration of 50 g L^−1 in a single
   addition once depletion occurred, using YPD medium composed of 10
   g L^−1 yeast extract, 20 g L^−1 peptone, and 500 g L^−1 glucose. The
   second and third strategies involved the use of glycerol as the carbon
   source, with or without the addition of 5 mM MnCl[2], respectively.
   Glycerol was supplemented to a final concentration of 50 g L^−1 in a
   single addition upon depletion, using YPG medium consisting of 10
   g L^−1 yeast extract, 20 g L^−1 peptone, and 500 g L^−1 glycerol. In
   the final strategy, after the depletion of glycerol in the initial
   medium, a constant‐rate feeding approach (0.28 mL min^−1) was employed
   to maintain the glycerol concentration in the fermentation broth below
   5 g L^−1.^[ [175]^34 ^]

Statistical Analysis

   All data were presented as mean values ± SD from three independent
   biological replicates (n = 3). The significant differences were
   analyzed by GraphPad Prism 9.0 Project software using one‐way analysis
   of variance (ANOVA). A p‐value of <0.05 was considered statistically
   significant.

Conflict of Interest

   The authors declare no competing interest.

Author Contributions

   Q.Y. and Y.Z. designed the experiments. Q.Y. performed the experiments.
   Q.Y. and Y.Z. analyzed the results. Q.Y. and Y.Z. prepared and revised
   the manuscript. M.T. performed experiments for plasmid construction.
   Q.Y., Y.Z., M.T., P.D., and Y.D. provided overall guidance on the
   project and participated in revising the manuscript. Y.D. conceived and
   directed the project. All authors have given approval to the final
   version of the manuscript.

Supporting information

   Supporting Information
   [176]ADVS-12-2411665-s001.doc^ (3.8MB, doc)

Acknowledgements