Efficient Catalytic Conversion of Acetate to Citric Acid and Itaconic Acid by Engineered Yarrowia lipolytica

Abstract

The bioconversion of agricultural and industrial wastes is considered a green and sustainable alternative method for producing high-value biochemicals. As a major catalytic product of greenhouse gases and a by-product in the fermentation and lignocellulose processing industries, acetate is a promising bioconversion raw material. In this work, endogenous and heterologous enzymes were manipulated in Yarrowia lipolytica to achieve the conversion of acetate to high-value citric acid and itaconic acid, respectively. After the combinational expression of the key enzymes in the acetate metabolic pathway, the citric acid synthesis pathway, and the mitochondrial transport system, acetate could be efficiently converted to citric acid. Coupled with the down-regulation of fatty acid synthase expression in the competitive pathway, more acetyl-CoA flowed into the synthesis of citric acid, and the titer reached 15.11 g/L with a productivity of 0.51 g/g acetate by the engineered Y. lipolytica, which is comparable to the results using glucose as the substrate. On this basis, the heterologous cis-aconitate decarboxylase from Aspergillus terreus was introduced into the engineered Y. lipolytica to achieve the catalytic synthesis of itaconic acid from acetate. Combined with investigating the effects of multiple enzymes in the synthesis pathway, the titer of itaconic acid reached 1.87 g/L with a yield of 0.43 g/g DCW by the final engineered strain, which is the highest reported titer of itaconic acid derived from acetate by engineered microbes in shake flasks. It is demonstrated that acetate has the potential to replace traditional starch-based raw materials for the synthesis of high-value organic acids and our work lays a foundation for the rational utilization of industrial wastes and the catalytic products of greenhouse gases.

1. Introduction

The traditional production of high-value chemicals mainly relies on petrochemical raw materials. With the depletion of petrochemical resources and the environmental pollution caused by the chemical synthesis method, we hope to develop a green and sustainable way to replace the chemical conversion route [1]. It is considered a viable alternative to produce high-value biochemicals via microbial cell factories using renewable feedstocks [2]. Citric acid is a commonly used food acidifier and preservation agent in the food industry, which can reduce the heat resistance of microorganisms and inhibit their growth to prevent food decomposition [3]. As a preferable additive, citric acid also has broad application in the production of chelating agents and detergents, animal feeds, lubricants, and plasticizers [4]. In addition, citric acid is a precursor for the microbial synthesis of itaconic acid via the functions of aconitase (ACO) and cis-aconitate decarboxylase (CADA) [5]. Itaconic acid, as one of the top twelve platform compounds, is an important unsaturated five-carbon dicarboxylic acid [6,7]. It can be used to produce a variety of derivatives, including diols, isomeric lactones, cyclic ethers, and so on. In particular, as a component of resins, elastomers, plastics, and adhesives in the polymer industry, itaconic acid has a potential market demand as high as 3.2 million tons [8]. Currently, the production of citric acid and itaconic acid mainly relies on the fermentation of starch-based raw materials by filamentous fungi (Aspergillus niger and Aspergillus terreus) [9]. However, the cultivation of filamentous fungi has some issues including difficulties in morphology control, laborious handling of spores, high oxygen demand, low reproducibility of fermentation, and complex genetic background [3,5].

With the development of synthetic biology, some cultivable chassis have been engineered to produce citric acid and itaconic acid including Escherichia coli, Saccharomyces cerevisiae, Yarrowia lipolytica, and so on [10]. Among them, Y. lipolytica, as a nonconventional, oleaginous yeast, exhibits potential in the synthesis of citric acid and itaconic acid due to its abundant intracellular acetyl-CoA content, exceptional acid tolerance, and broad substrate assimilation capacity [11,12]. Yuzbasheva and Kamzolova et al. enhanced the synthesis pathway of citric acid in Y. lipolytica and led to a titer of up to 100 g/L using glucose and glycerol as carbon sources, respectively [13,14]. On this basis, Blazeck et al. [15] introduced the itaconic acid synthesis pathway in Y. lipolytica and the titer of itaconic acid reached 4.6 g/L from glucose [15]. However, the current production of citric acid and itaconic acid mainly depends on fermented sugars from starch-based sources, which exacerbates the contradictions and conflicts among natural resources, especially water and land resources [16]. The development of industrial and agricultural wastes as raw materials is thus regarded as a potential way to address this problem and recently received considerable attention [17,18].

Acetate, as a common two-carbon monocarboxylic acid, is generated in many industries as a by-product. For example, acetate is the by-product of the deacetylation of hemicellulose and the content can reach 10 g/L [19]. In the industrial fermentation field, acetate is also one of the major by-products due to the metabolic overflow of central carbon flux in microbial cell factories [20,21,22]. The acetate-rich wastewater not only causes significant environmental pollution but also results in a waste of resources. In addition, carbon dioxide is the main cause of the greenhouse effect, and several studies have focused on the conversion of greenhouse gases to acetate using biological and bio-electrocatalytic methods [22]. Many microorganisms can convert greenhouse gases such as carbon dioxide, hydrogen, and carbon monoxide into acetic acid via the natural Wood–Ljungdahl pathway [23]. With the recent development of bio-electrocatalytic technology, the conversion of carbon dioxide to acetate has been further improved. Marshall et al. reported a production of 10.5 g/L acetate from the fixation of carbon dioxide using microbial electrocatalysis [24]. Therefore, the rational utilization of acetic acid not only helps to promote the reuse of industrial wastes but also lays a foundation for the conversion of greenhouse gas-derived products.

There have been numerous studies on the conversion of acetate to high-value chemicals via engineered microbial cell factories. For example, Niu et al. manipulated the succinate synthesis and acetate utilization pathways in the wild-type Escherichia coli MG1655 strain, and the final engineered E. coli accumulated 11.23 mM succinate from 50 mM acetate [25]. Lai et al. reported synthesizing 3-hydroxypropionic acid by the engineered E. coli using acetate as the substrate bacteria, and the production of 3-hydroxypropionic acid reached 11.2 g/L [26]. Acetate is thus a potential alternative to traditional starch-based feedstocks. However, there are few studies investigating the production of citric acid and itaconic acid from acetate. Only Noh et al. constructed an engineered E. coli for the production of itaconic acid using acetate as the substrate and achieved a yield of 0.879 g/L in a shake flask [27]. It was relatively low-yield due to the weak acid tolerance and low conversion rate of acetate of the engineered E. coli. Therefore, based on the advantages of high acid tolerance and rapid acetate metabolism, Y. lipolytica was selected as the chassis in our work, and multiple strategies were applied to improve the conversion efficiency of acetate to citric acid and itaconic acid, including the enhancement of target product synthesis pathways, the acetate assimilation pathway, transport proteins, as well as the down-regulation of the acetyl-CoA competitive pathway (Figure 1). Our work demonstrates the feasibility of acetate as an alternative carbon source and provides an available platform for the cost-effective production of citric acid, itaconic acid, as well as other acetyl-CoA-based biochemicals from acetate.

2. Results and Discussion

2.1. Enhancement of the Endogenous Enzyme Expression to Achieve Citric Acid Production from Acetate

Y. lipolytica is one of the natural citric acid producers [28]. Compared with the conversion of glucose, the pathway for the conversion of acetate to acetyl-CoA is shorter. It is considered a metabolic shortcut for synthesizing acetyl-CoA-derived products, including citric acid [12]. To investigate its ability to synthesize citric acid from acetate, we first cultivated the engineered strain harboring the plasmid PYLXP’ in an acetate-YNB (CSM-Leu) medium. It was shown that 5.18 g/L citric acid was produced by the strain with a yield of 0.91 g/g DCW after being cultivated for 120 h (Figure 2A), which demonstrates that Y. lipolytica can convert acetate to citric acid. Citrate synthase (CAS) is the key enzyme that catalyzes the condensation of acetyl-CoA and oxaloacetate to generate citric acid. To enhance the synthesis of citric acid, the endogenous CAS was assembled into the plasmid PYLXP’ to overexpress CAS in the engineered strain. The citric acid production increased to 9.06 g/L in the acetate-YNB (CSM-Leu) medium with a yield of 1.68 g/g DCW (Figure 2A), which is 71% higher than that of the original strain. It is suggested that the overexpression of CAS is advantageous for citric acid synthesis and does not affect the growth of the cells. On this basis, the utilization of acetate was further enhanced by the overexpressing acetyl-CoA synthase (ACS), which is responsible for the conversion of acetate to acetyl-CoA. In previous studies, it has been reported that the overexpression of ACS from E. coli can significantly promote acetate assimilation and improve the acetyl-CoA levels in yeast [29]. Therefore, the endogenous ACS and the ACS from E. coli were assembled into the plasmid PYLXP’-ylCAS, respectively, to explore the effect on the synthesis of citric acid. Interestingly, the introduction of the ACS from E. coli did not show a significant promotion of citric acid synthesis (Figure 2A). When the endogenous ACS was overexpressed, the accumulation of citric acid increased to 10.63 g/L with a yield of 1.96 g/g DCW, which is about two-fold higher than that of the original strain. It demonstrates that the endogenous ACS can function better than that of E. coli.

Citric acid is one of the intermediate metabolites in the TCA cycle, and it is suspected that the enhancement of citric acid transportation from mitochondria to cytoplasm can avoid feedback inhibition, thus promoting its production. MTT is a mitochondrial dicarboxylate transporter protein, and it has been demonstrated that MTT can transport various dicarboxylates. Steiger et al. investigated the function of MTT and demonstrated that it could transport dicarboxylic acids such as citric acid, cis-aconitic acid, and itaconic acid [30,31]. Therefore, the endogenous MTT was further overexpressed to investigate its effect on the synthesis of citric acid. The plasmid PYLXP’-ylCAS-ylACS-ylMTT was constructed and transformed into Y. lipolytica to obtain the engineered strain HLYaLiCA1, which could produce 11.77 g/L citric acid with a yield of 2.32 g/g DCW, about 2.27 times higher than that of the original strain (Figure 2A). It is demonstrated that the overexpression of endogenous MTT can promote the citric acid transportation from mitochondria to the cytoplasm in Y. lipolytica, thereby enhancing citric acid production.

Afterward, the engineered strain HLYaLiCA1 was cultivated in shake flasks to explore the synthesis of citric acid and the consumption of acetate in detail. As shown in Figure 2B, acetate was completely consumed after 96 h, while the accumulation of citric acid reached the highest level with a production of 13.88 g/L with a yield of 3.15 g/g DCW. It is indicated that the engineered strain HLYaLiCA1 can rapidly utilize acetate and has a high conversion efficiency of acetate. At present, when glucose is used as the substrate, the productivity of citric acid is 0.5 g/g glucose by the engineered Y. lipolytic [13]. In our study, the productivity of citric acid was 0.47 g/g acetate by the engineered strain HLYaLiCA1, which is comparable to the result using glucose as the substrate. It demonstrates the potential of acetate as the substrate for the production of citric acid as well as its derivatives.

2.2. Manipulation of Multiple Enzymes to Construct Itaconic Acid Synthesis Pathway

Although there is no natural synthesis pathway for itaconic acid in Y. lipolytica, it has a preferable ability in the synthesis of the precursor citric acid, which makes Y. lipolytica an available host to achieve itaconic acid production. The cis-aconitic acid decarboxylase (CADA) catalyzing the conversion of cis-aconitic acid to itaconic acid is the essential enzyme in the synthesis pathway [32]. The CADA in the natural itaconic acid-producing fungus A. terreus shows high activity, and a large number of studies have expressed it in commonly used hosts to achieve itaconic acid production. Herein, the CADA from A. terreus was assembled into the plasmid PYLXP’ and introduced into Y. lipolytica to evaluate its function. After being cultivated for 120 h in the glucose-YNB (CSM-Leu) medium, the engineered strain only produced 0.11 g/L itaconic acid with a DCW of 10.8 g/L (Figure 3A). The aconitase (ACO) is responsible for the conversion of citric acid to cis-aconitic acid, and it has been shown that overexpression of ACO can promote the conversion of citric acid to itaconic acid [33]. Zhao et al. [34] overexpressed CADA in Y. lipolytica Po1f and 0.363 g/L itaconic acid was produced using glucose as the substrate, while the co-expression of ACO only led to the titer of itaconic acid increasing to 0.389 g/L. They found that the heterologously expressed CADA was located in the cytoplasm, while cis-aconitic acid was mainly produced in mitochondria. A. terreus endogenous mitochondrial tricarboxylate transporter MTT was thus introduced into the engineered Y. lipolytica, which was proven to significantly promote itaconic acid production and increase the titer up to 5.21 g/L [34]. Therefore, the endogenous ACO and MTT were co-overexpressed with CADA to explore their effects on the synthesis of itaconic acid in our work, respectively. After being cultivated for 120 h in the glucose-YNB (CSM-Leu) medium, it was shown that the production of itaconic acid was increased to 0.28 g/L with a yield of 29.48 mg/g DCW upon the overexpression of ACO, while 0.32 g/L itaconic acid was produced with a yield of 37.52 mg/g DCW by the engineered strain overexpressing MTT (Figure 3A). It is suggested that the overexpression of both enzymes did promote the synthesis of itaconic acid. Subsequently, the endogenous ACO and MTT were simultaneously assembled into the plasmid PYLXP’-AtCADA to achieve combinational overexpression (Figure 3A). Unexpectedly, it did not show a significant promotion effect, and the production of itaconic acid was 0.34 g/L with a yield of 35.18 mg/g DCW, which is similar to the results achieved by separately overexpressing MTT. Our result is different from that in the reports [34], and it is speculated to be caused by the differences in the genetic background of the hosts. In addition, ACO is a multifunctional enzyme that can not only catalyze the conversion of citric acid to cis-aconitic acid but also catalyze the synthesis of isocitric acid from cis-aconitic acid [13]. It is speculated that the overexpression of ACO might lead to a partial carbon flow into TCA and impact the production efficiency of itaconic acid.

2.3. Achieving Itaconic Acid Production from Acetate by the Engineered Strain

Even though the itaconic acid synthesis pathway was enhanced, it still had a lower production compared to the results from the engineered Y. lipolytica in previous work [34]. One of the reasons is possibly the genetic differences between different types of Y. lipolytica, which leads to the production capacity varying greatly [15,34]. In addition, it was found that the titer of citric acid reached 3.2 g/L, which is suspected to be due to the fact that the synthesis of citric acid was limited in the glucose-YNB (CSM-Leu) medium and negatively affected the synthesis of itaconic acid. According to our results above, acetate is an available carbon source for the efficient accumulation of citric acid, which is thus considered a potential substrate for itaconic acid synthesis.

The engineered Y. lipolytica harboring the plasmid PYLXP’-AtCADA-ylMTT was thus applied to investigate its production capacity using acetate as the carbon source. As shown in Figure 3B, the itaconic acid production was about 0.77 g/L with a yield of 144.44 mg/g DCW after being cultivated for 120 h in the acetate-YNB (CSM-Leu) medium, which is about 2.48-fold higher than the results using glucose as the carbon source. It is demonstrated that the engineered strain can efficiently utilize acetate for itaconic acid synthesis. Given that citric acid is the precursor for the itaconic acid synthesis and a preferable citric acid production is achieved from acetate by the engineered strain HLYaLiCA1, we thus combined the citric acid synthesis pathway with the itaconic acid synthesis pathway by assembling the endogenous CAS and ACS into the plasmid PYLXP’-AtCADA-ylMTT. The final engineered strain HLYaLiITA1 harboring the plasmid PYLXP’-AtCADA-ylMTT-ylCAS-ylACS was able to produce 1.21 g/L itaconic acid with a yield of 237.38 mg/g DCW using acetate as the carbon source (Figure 3B), which is about a 1.57-fold increase compared to the strain only overexpressing CADA and MTT. It is indicated that the combination of citric acid synthesis is beneficial for the production of itaconic acid.

The engineered strain HLYaLiITA1 was cultivated in shake flasks to further explore the synthesis of itaconic acid and the consumption of acetate in detail. As shown in Figure 4, acetate was almost consumed at 96 h, and the accumulation of the precursor citric acid reached the highest at about 9.88 g/L. As the carbon source acetate was completely depleted, citric acid served as the alternative substrate for cell growth and itaconic acid synthesis. Therefore, the DCW and the production of itaconic acid continued increasing. After being cultivated for 144 h, the titer of itaconic acid reached 1.31 g/L with a yield of 0.24 g/g DCW and the citric acid content decreased to 5.43 g/L (Figure 4). It is indicated that acetate is a preferred carbon source for itaconic acid production by the engineered strain HLYaLiITA1, but citric acid was the main by-product in this process. It is suspected that the activity of CADA is insufficient in the synthesis of itaconic acid and more work is needed to understand the expression process and improve the function of CADA in Y. lipolytica.

2.4. Down-Regulation of Fatty Acid Synthase in Acetyl-CoA Competitive Pathway to Promote the Synthesis of Target Products

Acetyl-CoA is a metabolic link connecting cell growth and the synthesis of various products [35,36]. Lipids are the signature metabolites of oleaginous yeasts and the pathway is a relatively active metabolic branch of intracellular acetyl-CoA in Y. lipolytica. Down-regulating the lipids synthesis pathway helps to reduce the loss of central carbon flux, thereby improving the synthesis of the target product [37]. The fatty acid synthase (FAS) is the critical enzyme for lipid synthesis in Y. lipolytica, while cerulenin has been proven to significantly inhibit FAS activity [38]. However, cerulenin is a very expensive small molecule, and its addition will increase production costs. Lv et al. applied a CRISPRi system to dynamically regulate the fatty acid synthase (FAS) by combinational expression of gRNA controlled by the fatty acid-responsive promoter pPOX and dCas protein in Y. lipolytica, which led to the yield of the target product naringenin increasing by 29.9% [39]. Accordingly, our work utilized the same strategy to down-regulate the activity of FAS by integrating gRNA and dCas into the genome of strain Y. lipolytica Po1fk so as to obtain a new chassis Y. lipolytica Po1fk-FAS. On this basis, the plasmid PYLXP’-CAS-ACS-MTT was transformed into the new chassis to obtain the engineered strain HLYALICA2 to explore the capability of citric acid synthesis. As shown in Figure 5A, the titer of citric acid reached 15.11 g/L with a yield of 3.65 g/g DCW after the cultivation of HLYALICA2 for 96 h in the acetate-YNB (CSM-Leu) medium, which increased by approximately 8% and 16% compared to that of the strain HLYALICA1. It is indicated that down-regulating the activity of FAS can effectively reduce the consumption of acetyl-CoA and allow more acetyl-CoA to flow toward the citric acid synthesis pathway. The highest citric acid production was reported at about 49.79 g/L by Y. lipolytica using glucose as the substrate with a yield of 3.11 g/g DCW [13]. Our results show that the yield of the engineered strain HLYALICA2 using acetic acid as a substrate is higher than that in the report using glucose as a substrate. Meanwhile, the productivity of citric acid reached 0.51 g/g acetate, which is similar to results from glucose reported in previous work (0.5 g/g) [13]. These results indicate the superiority of the new chassis in the production of citric acid and also demonstrate the great potential of acetate in the production of citric acid.

Subsequently, the plasmid PYLXP’-CADA-MTT-CAS-ACS was also transformed into the new chassis strain Y. lipolytica Po1fk-FAS to obtain the engineered strain HLYALIIA2 to explore its capability in the synthesis of itaconic acid. It is shown that the titer of itaconic acid reached 1.87 g/L with a yield of 0.43 g/g DCW after 132 h cultivation using acetate as the sole carbon source (Figure 5B), which led to increases of about 43% and 79% compared to the strain HLYALICA1. It is indicated that the down-regulation of FAS not only increases citric acid accumulation but also promotes the synthesis of itaconic acid. At present, there is relatively little research on the synthesis of itaconic acid from acetate. Noh et al. introduced the itaconic acid synthesis pathway in E. coli with a high tolerance to high concentrations of acetate (10 g/L) and enhanced the synthesis of itaconic acid by regulation of the precursor supply pathway, acetate assimilation pathway, and glyoxylate bypass. The final tier of itaconic acid reached 0.879 g/L with a yield of 0.32 g/g DCW after 36 h cultivation of the engineered strain in shake flasks [27]. In our work, the titer of itaconic acid was much higher than that in the engineered E. coli, and it is also the highest reported titer of itaconic acid derived from acetate by engineered microbes in shake flasks, which is attributed to the advantages of the chassis Y. lipolytica in the utilization of acetate and the synthesis of citric acid as well as its derivatives. It also demonstrated the great potential of oleaginous yeast in the synthesis of itaconic acid from acetate.

However, we also found that cell growth was limited in the acetate-YNB (CSM-Leu) medium, and the DCW of the engineered strain HLYaLiIA2 was only 4.34 g/L in shake flask cultivation, which is much lower than that in the glucose-YNB (CSM-Leu) medium [13]. This might be due to the simple metabolic pathway of acetate which leads to limited ATP synthesis and insufficient energy supply. Moreover, acetate specifically inhibits the activity of some enzymes and interferes with cellular metabolism, thus affecting cell growth [20,40]. Therefore, although acetate has been demonstrated as a potential substrate for the synthesis of acetyl-CoA-based products, the inhibition mechanism of acetate on cells should be focused on in future work and various strategies can be developed to improve cell growth, including screening more available hosts with a preferable acetate tolerance, modifying the enzymes via rational design to improve their activity in acetate, constructing a multi-carbon-source co-utilization system to improve acetate conversion rate, and so on.

3. Materials and Methods

3.1. Strains and Culture Conditions

E. coli DH5α (purchased from Tiangen Biochemical Technology (Beijing, China) Co.) was used for plasmid construction, preparation, and storage. Y. lipolytica Po1fk is a derivative of Y. lipolytica Po1f (ATCC MYA-2613, MatA, Leu-, Ura-, ΔAEP, ΔAXP, Suc+) by removing the Ku70 gene, which was used as the chassis in this work [39]. All the strains and plasmids are listed in

Table 1. Strains and plasmids used in this work.

Plasmid or Strain	Relevant Properties or Genotype	Source
Plasmid
PYLXP’	-	[41]
PYLXP’-ylCAS	PYLXP’ carrying CAS from Y. lipolytica	This study
PYLXP’-ylCAS-ylACS	PYLXP’ carrying CAS and ACS from Y. lipolytica	This study
PYLXP’-ylCAS-EcACS	PYLXP’ carrying CAS from Y. lipolytica and ACS from E. coli	This study
PYLXP’-ylCAS-ylACS-ylMTT	PYLXP’ carrying CAS, ACS, MTT from Y. lipolytica	This study
PYLXP’-AtCADA	PYLXP’ carrying CADA from A. terreus	This study
PYLXP’-AtCADA-ylMTT	PYLXP’ carrying CADA from A. terreus, MTT from Y. lipolytica	This study
PYLXP’-AtCADA-ylACO	PYLXP’ carrying CADA from A. terreus, ACO from Y. lipolytica	This study
PYLXP’-AtCADA-ylMTT-ylCAS -ylACS	PYLXP’ carrying CADA from A. terreus, ACS, MTT, CAS from Y. lipolytica	This study
pΔleu2loxP-gRNA(FAS1-FAS2)-dCas9	pΔleu2loxP carrying gRNAs (FAS1and FAS2) and dCas9	[39]
Strains
E. coli DH5α	φ80,lacZΔM15,Δ (lacZYA-argF) U169, endA1, recA1,hsdR17(rk−,mk+), supE44, λ−, thi-1, gyrA96, relA1, phoA	Tian Gen Biotech Co., Ltd., Beijing, China
Y. lipolytica Po1fk	MatA, Leu-, Ura-, ΔAEP, ΔAXP, Suc+, ΔKu70	[42]
Y. lipolytica Po1fk-FAS	Y. lipolytica Po1fk with the integration of gRNAs (FAS1and FAS2) and dCas9 circuits at leu2 site	This study
HLYaLiCA1	Y. lipolytica Po1fk with vector PYLXP’-ylCAS-ylACS-ylMTT	This study
HLYaLiCA2	Y. lipolytica Po1fk-FAS with vector PYLXP’-ylCAS-ylACS-ylMTT	This study
HLYaLiITA1	Y. lipolytica Po1fk with vector PYLXP’-AtCADA-ylMTT-ylCAS -ylACS	This study
HLYaLiITA2	Y. lipolytica Po1fk-FAS with vector PYLXP’-AtCADA-ylMTT-ylCAS -ylACS	This study

.

An LB medium and agar plates containing 100 μg/mL ampicillin were used to cultivate the E. coli. A yeast-rich medium (YPD) was used for the cultivation of Y. lipolytica, consisting of 20 g/L peptone (Oxoid, Basingstoke, UK), 10 g/L yeast extract (Oxoid), and 20 g/L glucose (Oxoid), and supplemented with 15 g/L agar (Solarbio) for the preparation of YPD plates. A YNB medium with a carbon/nitrogen ratio of 10:1 for seed culture consisted of 1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate) (Oxoid), 5 g/L ammonium sulfate (Sigma-Aldrich, Burlington, MA, USA), 0.69 g/L CSM-Leu (Oxoid), and 20 g/L glucose. A total of 15 g/L agar (Solarbio, Beijing, China) was added to prepare the YNB plates for the screening of engineered yeast colonies. A YNB fermentation medium with a carbon/nitrogen ratio of 80:1 consisted of 1.7 g/L yeast nitrogen base (without amino acids and ammonium sulfate), 1.1 g/L ammonium sulfate, 0.69 g/L CSM-Leu, and 40 g/L glucose. When acetate was used as the carbon source, glucose in the YNB fermentation medium was replaced by 41 g/L sodium acetate, and the carbon/nitrogen ratio was maintained at 80:1 by changing the content of ammonium sulfate to 0.825 g/L. In the fermentation process, the pH of the medium was controlled to about 6.8 by 6 mol/L HCl, which was adjusted every 12 h.

3.2. Plasmid Construction and Transformation

The genes of acetyl-CoA synthase (ylACS, EcACS), cis-aconitase (ylACO), citrate synthase (ylCAS), and mitochondrial dicarboxylate transporter protein (ylMTT) were amplified from the genome of Y. lipolytica and E. coli using primers (Supplementary Materials, Table S1). The cis-aconitate decarboxylase (AtCADA) gene from A. terreus was codon-optimized and synthesized by Genewiz. The vector PYLXP’ was used as the backbone plasmid, and the gene was assembled into the downstream of the TEF-intron promoter at the KpnI and SnaBI sites using NovoRec plus One step PCR Cloning Kit (purchased from Novoprotein Scientific Inc., Shanghai, China). After sequence verification by Genewiz, the plasmids were digested using the restriction enzymes AvrII, NheI, NotI, ClaI, and SalI, and the purified DNA fragments were assembled according to the YaliBricks subcloning method [41]. All restriction enzymes were purchased from New England Biolabs, and the Plasmid Mini Kit, Gel Extraction Kit, and Cycle-Pure Kit were purchased from OMEGA.

Upon the verification of all assembled plasmids by gel digestion, these plasmids were transformed into Y. lipolytica Po1fk or Y. lipolytica Po1fk-Fas using the lithium acetate/single-stranded carrier DNA/PEG method [43]. Y. lipolytica colonies were picked from YNB plates and inoculated into the YNB seed medium, which was cultivated for 48 h at 30 °C, 250 rpm. For test tube cultivation, a 100 μL seed broth was inoculated into a 50 mL test tube containing 5 mL fermentation medium and was incubated for 120 h at 30 °C, 250 rpm. For shake flask cultivation, a 600 μL seed broth was inoculated into a 250 mL shake flask containing 30 mL fermentation medium and was incubated for 168 h at 30 °C, 250 rpm.

3.3. Analytical Methods

Cell growth was monitored by measuring the optical density at 600 nm (OD600), which could also be converted to dry cell weight (DCW) according to the calibration curve DCW: OD600 = 0.33:1 (g/L). After being centrifuged at 13,200× g for 5 min, the supernatant of the fermentation broth was analyzed for the concentrations of citric acid, itaconic acid, glucose, and acetic acid using the Aminex HPX-87H organic acid column (25 cm × 0.4 cm, Bio-Rad, Hercules, CA, USA). Citric acid, itaconic acid, and acetic acid were detected by a UV detector at a wavelength of 210 nm, while glucose was detected by a refractive index detector. The column was eluted with 5 mM H₂SO₄ with a flow rate of 0.6 mL/min at a column temperature of 50 °C, and the temperature of the refractive index detector was also set to 50 °C.

4. Conclusions

Acetate is a by-product of fermentation and lignocellulose industries, as well as one of the catalytic products of greenhouse gases. In this study, Y. lipolytica was engineered to produce high-value citric acid and itaconic acid using acetate as the alternative carbon source. After manipulation of the acetate metabolic pathway, citric acid synthesis pathway, and the mitochondrial transporter protein, the engineered strain HLYaLiCA1 was capable of efficiently converting acetate to citric acid, and the citric acid production reached 13.88 g/L with a productivity of 0.47 g/g acetate. Furthermore, a new chassis Y. lipolytica Po1fk-FAS was obtained via integrating the CRISPRi system into the genome to down-regulate the enzyme FAS in the acetyl-CoA competitive pathway. The titer of citric acid was further increased to 15.11 g/L with a yield of 3.65 g/g DCW, which is comparable to the results using glucose as the substrate by Y. lipolytica. On this basis, the itaconic acid synthesis pathway was introduced by heterologously expressing CADA from A. terreus. Compared to glucose, acetate had a significant promotion effect in the synthesis of itaconic acid. The engineered strain HLYaLiITA2 was able to produce 1.87 g/L itaconic acid with a yield of 0.43 g/g DCW, which is the highest reported titer of itaconic acid derived from acetate by engineered microbes in shake flasks. Our work demonstrates the potential of acetate as a substitute for traditional starch-based raw materials in the synthesis of high-value organic acids by Y. lipolytica and lays the foundation for further research on the synthesis of other biochemicals using acetate as the carbon source.