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Squalene is a triterpenoid serving as an ingredient of various products in the food, cosmetic, pharmaceutical industries. The oleaginous yeast Yarrowia lipolytica offers enormous potential as a microbial chassis for the production of terpenoids, such as carotenoid, limonene, linalool, and farnesene, as the yeast provides ample storage space for hydrophobic products. Here, we present a metabolic design that allows the enhanced accumulation of squalene in Y. lipolytica. First, we improved squalene accumulation in Y. lipolytica by overexpressing the genes (ERG and HMG) coding for the mevalonate pathway enzymes. Second, we increased the production of lipid where squalene is accumulated by overexpressing DGA1 (encoding diacylglycerol acyltransferase) and deleting PEX10 (for peroxisomal membrane E3 ubiquitin ligase). Third, we deleted URE2 (coding for a transcriptional regulator in charge of nitrogen catabolite repression [NCR]) to induce lipid accumulation regardless of the carbon-to-nitrogen ratio in culture media. The resulting engineered Y. lipolytica exhibited a 115-fold higher squalene content (22.0 mg/g dry cell weight) than the parental strain. These results suggest that the biological function of Ure2p in Y. lipolytica is similar to that in Saccharomyces cerevisiae, and its deletion can be utilized to enhance the production of hydrophobic target products in oleaginous yeast strains. IMPORTANCE This study demonstrated a novel strategy for increasing squalene production in Y. lipolytica. URE2, a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response, was identified and demonstrated correlation to squalene production. The data suggest that double deletion of PEX10 and URE2 can serve as a positive synergistic effect to help yeast cells in boosting squalene production. This discovery can be combined with other strategies to engineer cell factories to efficiently produce terpenoid in the future.

As an alternative terpenoid producer, non-conventional oleaginous yeast Yarrowia lipolytica was chosen for limonene production. Y. lipolytica can give high biomass yield and metabolize a broad range of substrates such as glycerol, alkanes, fatty acid, fats, and oils. As previously reported, optimization of limonene synthesis pathway and mevalonate (MVA) pathway leads to the accumulation of 112-fold higher limonene as compared to an initial strain. In this study, we introduced an additional copy of limonene synthesis gene (LS), which resulted in an increase of limonene production. This engineered strain was used to carry out further optimization study. Amongst all the carbon sources tested, the highest level of limonene production was obtained from glycerol, and citrate was selected as an auxiliary carbon source. In fed-batch fermentation with an optimized medium, the engineered strain was found to produce 165.3 mg/L limonene, which corresponds to the highest yield till date for the production of limonene in Y. lipolytica.

Sophorolipids are important biosurfactants produced by the wild-type strain of Candida bombicola ATCC 22214, with antimicrobial, antiviral, and anticancer activities. Our previous study showed that a mutant strain lacking multifunctional enzyme type 2 displayed extremely low sophorolipid production capacity compared to the wild-type strain. In the present study, we aimed to reveal the possible reasons for this deficiency through a series of analytical methods. First, comparative metabolic profiling analysis between the wild-type and mutant strains was performed. Multivariate statistical analysis, including principal component analysis and orthogonal partial least squares discriminant analysis, showed that nine potential metabolites were closely associated with the metabolic differences between strains. Second, metabolic flux analysis revealed that an inadequate supply of intracellular acetyl-CoA in the mutant strain could significantly affect the biosynthesis of sophorolipids. In addition, supplementation of citric acid recovered the sophorolipid production in the mutant strain by approximately 83%, suggesting that citrate metabolism plays an important role in sophorolipid biosynthesis. These findings provide novel theoretical insights for further improving the production of sophorolipids in C. bombicola.

Lycopene has been broadly studied in recent decades due to its health benefits including cancer prevention, anti-atherogenic and anti-obesity effects, and modulation of the immune system. To obtain efficient synthesis of lycopene, extensive researches have been conducted in various microbial cells, including Yarrowia lipolytica, to heterologously produce lycopene using various genetic and metabolic engineering methods. In this study, the effects of copy numbers of lycopene synthesis genes, a variety of key central metabolic genes (especially AMP deaminase-encoding gene AMPD), and 5-L fermenter cultivation on lycopene production in Y. lipolytica were investigated and the engineered strains with significantly enhanced lycopene content (46–60 mg/g DCW) were achieved. It is therefore possible to make use of the obtained strains to meet the industrial demand of lycopene production on the basis of further genetic and process optimization.

Background The filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression. Results We found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E. Conclusions The findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP–PLC–calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.

Given the grave concerns over increasing consumption of petroleum resources and dramatic environmental changes arising from carbon dioxide emissions worldwide, microbial biosynthesis of fatty acid ethyl ester (FAEE) biofuels as renewable and sustainable replacements for petroleum-based fuels has attracted much attention. As one of the most important microbial chassis, the nonconventional oleaginous yeast Yarrowia lipolytica has emerged as a paradigm organism for the production of several advanced biofuels and chemicals. Here, we report the engineering of Y. lipolytica for use as an efficient dual biocatalytic system for in situ and one-pot production of FAEEs from renewable feedstock. Compared to glucose with 5.7% (w/w) conversion rate to FAEEs, sunflower seed oil in the culture medium was efficiently used to generate FAEEs with 84% (w/w) conversion rate to FAEEs by the engineered Y. lipolytica strain GQY20 that demonstrates an optimized intercellular heterologous FAEE synthesis pathway. In particular, the titer of extracellular FAEEs from sunflower seed oil reached 9.9 g/L, 10.9-fold higher than that with glucose as a carbon source. An efficient dual biocatalytic system combining ex vivo and strengthened in vitro FAEE production routes was constructed by overexpression of a lipase (Lip2) variant in the background strain GQY20, which further increased FAEEs levels to 13.5 g/L. Notably, deleting the ethanol metabolism pathway had minimal impact on FAEE production. Finally, waste cooking oil, a low-cost oil-based substance, was used as a carbon source for FAEE production in the Y. lipolytica dual biocatalytic system, resulting in production of 12.5 g/L FAEEs. Thus, the developed system represents a promising green and sustainable process for efficient biodiesel production. KEY POINTS: • FAEEs were produced by engineered Yarrowia lipolytica. • A Lip2 variant was overexpressed in the yeast to create a dual biocatalytic system. • Waste cooking oil as a substrate resulted in a high titer of 12.5 g/L FAEEs.

Shao-Chun Chen,1,2,* Jing-Wei Liu,1,2,* Xing-Zhong Wu,3 Wen-Ling Cao,4 Feng Wang,5 Jin-Mei Huang,3 Yan Han,1,2 Xiao-Yu Zhu,1,2 Bang-Yong Zhu,6 Quan Gan,6 Xiao-Zheng Tang,7 Xing Shen,8 Xiao-Lin Qin,3 Yu-Qi Yu,3 He-Ping Zheng,3 Yue-Ping Yin1,2 1Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, People’s Republic of China; 2National Center for STD Control, Chinese Center for Disease Control and Prevention, Nanjing, People’s Republic of China; 3Clinical Laboratory, Guangdong Provincial Dermatology Hospital, Guangzhou, Guangdong, People’s Republic of China; 4Clinical Laboratory, Guangzhou Dermatology Hospital, Guangzhou, Guangdong, People’s Republic of China; 5Shenzhen Center for Chronic Diseases Control, Shenzhen, Guangdong, People’s Republic of China; 6Clinical Laboratory, Guangxi Provincial Dermatology Hospital, Nanning, Guangxi, People’s Republic of China; 7Clinical Laboratory, Hainan Provincial Dermatology Hospital, Haikou, Hainan, People’s Republic of China; 8Zhuhai Center for Chronic Diseases Control, Zhuhai, Guangdong, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yue-Ping YinNational Center for STD Control,Chinese Center for Disease Control and Prevention, 12 Jiangwangmiao Street, Nanjing 210042, People’s Republic of ChinaTel +86-25-85478024Email yinyp@ncstdlc.orgHe-Ping ZhengGuangdong Provincial Dermatology Hospital, 2 Lujing Road, Guangzhou 510091, People’s Republic of ChinaEmail zhhpf@hotmail.comIntroduction: Antimicrobial resistance (AMR) of Neisseria gonorrhoeae (N. gonorrhoeae) becomes a grave public health problem in the world. A strengthened Antimicrobial Resistance Surveillance Program is needed to track the trend of AMR development. However, the lack of a proper antimicrobial susceptibility test (AST) method is a barrier to expand the AMR surveillance in China. Traditional agar dilution (AD) method is laborious and E-test strips have no approval license for clinical use. Herein, a Chinese group modified the microdilution (MD) method for clinical ASTs. The objective of this study is to compare the MD method with the AD method for N. gonorrhoeae AST.Materials and Methods: A total of 166 clinical isolates were tested for antimicrobial susceptibility of ceftriaxone, spectinomycin, azithromycin, ciprofloxacin, tetracycline, and penicillin using MD and AD method simultaneously. Results of MD method were read manually or automatically. Rates of essential agreement (EA), category agreement (CA), minor error, and very major error were compared.Results: The total EAs (compared with results read manually) of penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, and azithromycin were 90.4%, 97.0%, 85.5%, 100.0%, 94%, and 72.3%; and CAs were 82.5%, 94.0%, 100%, 100%, 95.2%, and 94%, respectively.Conclusion: We conclude that the MD method might be an alternative for clinical AST of N. gonorrhoeae in China. In particular, MD method has the potency of accurate differentiation of isolates resistant to ceftriaxone or azithromycin, which were empirically recommended for gonococcal treatment, but its quality remained suboptimal, and further improvement is needed for clinical use.Keywords: Neisseria gonorrhoeae, antimicrobial susceptibility test

Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica. The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica. A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

Limonene, a valuable cyclic monoterpene, has been broadly studied in recent decades due to its wide application in the food, cosmetics and pharmaceutical industries. Engineering of the yeast Yarrowia lipolytica for fermentation of renewable biomass lignocellulosic hydrolysate may reduce the cost and improve the economics of bioconversion for the production of limonene. The aim of this study was to engineer Y. lipolytica to produce limonene from xylose and low-cost lignocellulosic feedstock. The heterologous genes XR and XDH and native gene XK encoding xylose assimilation enzymes, along with the heterologous genes tNDPS1 and tLS encoding orthogonal limonene biosynthetic enzymes, were introduced into the Po1f strain to facilitate xylose fermentation to limonene. The initially developed strain produced 0.44 mg/L of limonene in 72 h with 20 g/L of xylose. Overexpression of genes from the mevalonate pathway, including HMG1 and ERG12, significantly increased limonene production from xylose to ∼9.00 mg/L in 72 h. Furthermore, limonene production peaked at 20.57 mg/L with 50% hydrolysate after 72 h when detoxified lignocellulosic hydrolysate was used. This study is the first to report limonene production by yeast from lignocellulosic feedstock, and these results indicate the initial steps toward economical and sustainable production of isoprenoids from renewable biomass by engineered Y. lipolytica.

Squalene, a valuable acyclic triterpene, can be used as a chemical commodity for pharmacology, flavor, and biofuel industries. Microbial production of squalene has been of great interest due to its limited availability, and increasing prices extracted from animal and plant tissues. Here we report genetic perturbations that synergistically improve squalene production in Saccharomyces cerevisiae. As reported previously, overexpression of a truncated HMG-CoA reductase 1 (tHMG1) led to the accumulation 20-fold higher squalene than a parental strain. In order to further increase squalene accumulation in the tHMG1 overexpressing yeast, we introduced genetic perturbations-known to increase lipid contents in yeast-to enhance squalene accumulation as lipid body is a potential storage of squalene. Specifically, DGA1 coding for diacylglycerol acyltranferase was overexpressed to enhance lipid biosynthesis, and POX1 and PXA2 coding for acyl-CoA oxidase and a subunit of peroxisomal ABC transporter were deleted to reduce lipid β-oxidation. Simultaneous overexpression of tHMG1 and DGA1 coding for rate-limiting enzymes in the mevalonate and lipid biosynthesis pathways led to over 250-fold higher squalene accumulation than a control strain. However, deletion of POX1 and PXA2 in the tHMG1 overexpressing yeast did not improve squalene accumulation additionally. Fed-batch fermentation of the tHMG1 and DGA1 co-overexpressing yeast strain resulted in the production of squalene at a titer of 445.6 mg/L in a nitrogen-limited minimal medium. This report demonstrates that increasing storage capacity for hydrophobic compounds can enhance squalene production, suggesting that increasing lipid content is an effective strategy to overproduce a hydrophobic molecule in yeast.

As a novel chemical production platform, controllable and inducible modules in Synechococcus elongatus plus the ability of working in diurnal conditions are necessary. To the endeavors, inducible promoters, such as PTrc, have been refined from Escherichia coli, but the inducer isopropyl-β-D-thiogalactoside may cause several side-effects. Meanwhile, to promote the efficiency, photomixotrophic cultivation has been applied in S. elongatus with the additional organic carbon sources. In this study, we developed L-arabinose based modules consisted of both the PBAD inducible promoter and the metabolism of L-arabinose in S. elongatus, since L-arabinose is an ideal heterologous feedstock for its availability and economic and environmental benefits. As expected, we achieved homogeneous and linear expression of the exogenous reporter through the PBAD promoter, and the biomass increased in diurnal light condition via introducing L-arabinose metabolism pathway. Moreover, the combined AraBAD based toolkit containing both the PBAD inducible module and the L-arabinose metabolism module could obtain gene expression and metabolic robustness improvement in S. elongatus. With the only additive L-arabinose, the novel strategy may generate a win-win scenario for both regulation and metabolism for autotrophic bio-production platforms.

Carotenoids, as potent antioxidant compounds, have gained extensive attention, especially in human health. In this study, the combination of CRISPR/Cas9 integration strategy and fermenter cultivation was utilized to obtain efficient β-carotene-producing Yarrowia lipolytica cell factories for potential industrial application. The introduction of the genes of Mucor circinelloides, encoding phytoene dehydrogenase (carB) and bifunctional phytoene synthase/lycopene cyclase (carRP), contributed to the heterologous production of β-carotene in Y. lipolytica XK2. Furthermore, β-carotene production was efficiently enhanced by increasing the copy numbers of the carB and carRP genes and overexpressing of GGS1, ERG13, and HMG, the genes related to the mevalonate (MVA) pathway. Thus, the optimized strain overexpressed a total of eight genes, including three copies of carRP, two copies of carB, and single copies of GGS1, HMG, and ERG13. As a consequence, strain Y. lipolytica XK19 accumulated approximately 408 mg/L β-carotene in shake flask cultures, a twenty-four-fold increase compared to the parental strain Y. lipolytica XK2. 4.5 g/L β-carotene was obtained in a 5-L fermenter through a combination of genetic engineering and culture optimization, suggesting a great capacity and flexibility of Y. lipolytica in the production of carotenoids.

Firstly, the parameter analysis of the double-cylinder permanent-magnet eddy current coupler is carried out by using Maxwell finite element analysis software. Considering the theoretical and practical situation, the relationship between the main structural parameters and the transfer torque is determined. Then, in order to reduce the calculation cost of the subsequent optimization, BP neural network and support vector machine are used to establish the nonlinear model between the main structural parameters and the transfer torque. Comparing the two methods, it is found that the SVM method is superior to the BP method in predicting performance and running time. Finally, a test system is built to verify the accuracy of the SVM nonlinear model.

Jing-Wei Liu,1,2 Wen-Qi Xu,1,2 Xiao-Yu Zhu,1,2 Xiu-Qin Dai,1,2 Shao-Chun Chen,1,2 Yan Han,1,2 Jun Liu,3 Xiang-Sheng Chen,1,2 Yue-Ping Yin1,21Institute of Dermatology and Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, People’s Republic of China; 2National Center for Sexually Transmitted Diseases Control, Chinese Center for Disease Control and Prevention, Nanjing, People’s Republic of China; 3SYNAPSE Center, Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USAPurpose: Gentamicin is a promising antimicrobial for the treatment of gonorrhea. The study aimed to evaluate gentamicin minimum inhibitory concentrations (MICs) of Neisseria gonorrhoeae isolates in China.Methods: In this study, the agar dilution method was used to determine the MICs of 470 isolates collected in 2016 to four effective antimicrobials (gentamicin, azithromycin, ceftriaxone, and spectinomycin).Results: Gentamicin MICs ranged from 1 to 8mg/L. No isolate was resistant to gentamicin. Of seven isolates simultaneously resistant to azithromycin and ceftriaxone, 6 isolates demonstrated MICs of 4mg/L or less to gentamicin. No cross relationships were found between MICs of gentamicinand susceptibility profiles of azithromycin, ceftriaxone, and spectinomycin.Conclusion: The in vitro results suggest that gentamicin can be a promising treatment option for gonococcal infections in China. Clinical trials to evaluate the therapeutic efficacy of gentamicin are required.Keywords: Neisseria gonorrhoeae, agar dilution method, gentamicin, antimicrobial surveillance

Ansamitocin P-3 (AP-3), a 19-membered polyketide macrocyclic lactam, has potent antitumor activity. Our previous study showed that a relatively low organic nitrogen concentration in culture medium could significantly improve AP-3 production of Actinosynnema pretiosum. In the present study, we aimed to reveal the possible reasons for this improvement through metabolomic and gene transcriptional analytical methods. At the same time, a metabolic pathway profile based on metabolome data and pathway correlation information was performed to obtain a systematic view of the metabolic network modulations of A. pretiosum. Orthogonal partial least squares discriminant analysis showed that nine and eleven key metabolites directly associated with AP-3 production at growth phase and ansamitocin production phase, respectively. In-depth pathway analysis results highlighted that low organic nitrogen availability had significant impacts on central carbon metabolism and amino acid metabolic pathways of A. pretiosum and these metabolic responses were found to be beneficial to precursor supply and ansamitocin biosynthesis. Furthermore, real-time PCR results showed that the transcription of genes involved in precursor and ansamitocin biosynthetic pathways were remarkably upregulated under low organic nitrogen condition thus directing increased carbon flux toward ansamitocin biosynthesis. More importantly, the metabolic pathway analysis demonstrated a competitive relationship between fatty acid and AP-3 biosynthesis could significantly affect the accumulation of AP-3. Our findings provided new knowledge on the organic nitrogen metabolism and ansamitocin biosynthetic precursor in A. pretiosum and identified several important rate-limiting steps involved in ansamitocin biosynthesis thus providing a theoretical basis of further improvement in AP-3 production.

Background Ansamitocin P-3 (AP-3), produced by Actinosynnema pretiosum, has extraordinary antitumor activity and has been used as toxic “warhead” in antibody-conjugated drugs. However, AP-3 production is limited by its low yield and thus the high cost of fermentation. In this study, we aimed to reduce fermentation costs by utilizing the low-cost substrates and improve the AP-3 production, simultaneously. Result In this study, low-cost substrates used for ansamitocins production were selected via single-factor experiments and then the concentrations of these suitable carbon and nitrogen sources were optimized by response surface method (RSM). Cane molasses, glycerol, and cold-pressed soybean powder were determined to be the most suitable economical carbon and nitrogen sources for AP-3 accumulation. The AP-3 titer in shaking flasks reached 111.9 mg/L using the optimized medium containing cane molasses (63.22 g/L), glycerol (22.91 g/L), and cold-pressed soybean powder (3.29 g/L). In addition, appropriate supplementation with isobutanol, soybean oil, and vitamin B1 resulted in a marked enhancement in AP-3 production, to 141 mg/L. Meanwhile, the cost of this optimized fermentation medium was around 50% lower than those of two other high-yielding media. Conclusion Our findings provided practical and economically competitive cultivation conditions for the production of a value-added antitumor agent and may thus extend the applications of ansamitocin P-3.

Additional file 1. Additional Table S1 and Figure S1.

Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y. lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated β-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y. lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.

As a bioactive triterpenoid, squalene is widely used in the food industry, cosmetics, and pharmacology. Squalene’s major commercial sources are the liver oil of deep-sea sharks and plant oils. In this study, we focused on the enhancement of squalene biosynthesis in Yarrowia lipolytica, with particular attention to the engineering of acetyl-CoA metabolism based on genome-scale metabolic reaction network analysis. Although the overexpression of the rate-limiting endogenous ylHMG1 (3-hydroxy-3-methylglutaryl-CoA reductase gene) could improve squalene synthesis by 3.2-fold over that by the control strain, the availability of the key intracellular precursor, acetyl-CoA, was found to play a more significant role in elevating squalene production. Analysis of metabolic networks with the newly constructed genome-scale metabolic model of Y. lipolytica iYL_2.0 showed that the acetyl-CoA pool size could be increased by redirecting carbon flux of pyruvate dehydrogenation towards the ligation of acetate and CoA or the cleavage of citrate to form oxaloacetate and acetyl-CoA. The overexpression of either acetyl-CoA synthetase gene from Salmonella enterica (acs*) or the endogenous ATP citrate lyase gene (ylACL1) resulted in a more than 50% increase in the cytosolic acetyl-CoA level. Moreover, iterative chromosomal integration of the ylHMG1, asc*, and ylACL1 genes resulted in a significant improvement in squalene production (16.4-fold increase in squalene content over that in the control strain). We also found that supplementation with 10 mM citrate in a flask culture further enhanced squalene production to 10 mg/g DCW. The information obtained in this study demonstrates that rationally engineering acetyl-CoA metabolism to ensure the supply of this key metabolic precursor is an efficient strategy for the enhancement of squalene biosynthesis.