Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

De novo synthesis of nervonic acid and optimization of metabolic regulation by Yarrowia lipolytica.

Nervonic acid, a natural fatty acid compound and also a core component of nerve fibers and nerve cells, has been widely used to prevent and treat related diseases of the brain nervous system. At present, fatty acids and their derivatives are mainly obtained by natural extraction or chemical synthesis which are limited by natural resources and production costs. In this study, the de novo synthetic pathway of nervonic acid was constructed in Yarrowia lipolytica by means of synthetic biology, and the yield of nervonic acid was further improved by metabolic engineering and fermentation optimization. Specially, heterologous elongases and desaturases derived from different organism were successfully expressed and evaluated for their potential for the production of nervonic acid in Y. lipolytica. Meanwhile, we overexpressed the genes involved in the lipid metabolism to increase the nervonic acid titer to 111.6 mg/L. In addition, the potential of adding oil as auxiliary carbon sources for nervonic acid production by the engineered Y. lipolytica was analyzed. The results indicated that supplementation with colleseed oil as an auxiliary carbon source can be beneficial for the nervonic acid productivity, which led to the highest concentration of 185.0 mg/L in this work. To summarize, this study describes that the Y. lipolytica can be used as a promising platform for the production of nervonic acid and other very long-chain fatty acids.

Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica.

Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.

Enhancing Trans-Nerolidol Productivity in Yarrowia lipolytica by Improving Precursor Supply and Optimizing Nerolidol Synthase Activity.

The low enzymatic capability of terpene synthases and the limited availability of precursors often hinder the productivity of terpenes in microbial hosts. Herein, a systematic approach combining protein engineering and pathway compartmentation was exploited in Yarrowia lipolytica for the high-efficient production of trans-nerolidol, a sesquiterpene with various commercial applications. Through the single-gene overexpression, the reaction catalyzed by nerolidol synthase (FaNES1) was identified as another rate-limiting step. An optimized FaNES1G498Q was then designed by rational protein engineering using homology modeling and docking studies. Additionally, further improvement of trans-nerolidol production was observed as enhancing the expression of an endogenous carnitine acetyltransferase (CAT2) putatively responsible for acetyl-CoA shuttling between peroxisome and cytosol. To harness the peroxisomal acetyl-CoA pool, a parallel peroxisomal pathway starting with acetyl-CoA to trans-nerolidol was engineered. Finally, the highest reported titer of 11.1 g/L trans-nerolidol in the Y. lipolytica platform was achieved in 5 L fed-batch fermentation with the carbon restriction approach.

Integrated pathway engineering and transcriptome analysis for improved astaxanthin biosynthesis in Yarrowia lipolytica.

Astaxanthin is a high value carotenoid with a broad range of commercial applications due to its superior antioxidant properties. In this study, ß-carotene-producing Yarrowia lipolytica XK17 constructed in the lab was employed for astaxanthin biosynthesis. The catalytic effects of ß-carotene ketolase CrtW and ß-carotene hydroxylase CrtZ from various species were investigated. The PspCrtW from Paracoccus sp. and HpCrtZ# from Haematococcus pluvialis were confirmed to be the best combination in converting ß-carotene. Several key bottlenecks in biomass and astaxanthin biosynthesis were effectively eliminated by optimizing the expression of the above enzymes and restoring uracil/leucine biosynthesis. In addition, the effects of astaxanthin biosynthesis on cell metabolism were investigated by integrated analysis of pathway modification and transcriptome information. After further optimization, strain DN30 was able to synthesize up to 730.3 mg/L astaxanthin in laboratory 5-L fermenter. This study provides a good metabolic strategy and a sustainable development platform for high-value carotenoid production.

Metabolic engineering Yarrowia lipolytica for a dual biocatalytic system to produce fatty acid ethyl esters from renewable feedstock in situ and in one pot.

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.

Increased Accumulation of Squalene in Engineered Yarrowia lipolytica through Deletion of PEX10 and URE2.

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.

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica.

OBJECTIVE: 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. RESULTS: 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. CONCLUSIONS: 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.

Biosynthesis of α-Pinene by Genetically Engineered Yarrowia lipolytica from Low-Cost Renewable Feedstocks.

α-Pinene, an important biologically active natural monoterpene, has been widely used in fragrances, medicines, and fine chemicals, especially, in high-density renewable fuels such as jet fuel. The development of an α-pinene production platform in a highly modifiable microbe from renewable substitute feedstocks could lead to a green, economical avenue, and sustainable biotechnological process for the biosynthesis of α-pinene. Here, we report engineering of an orthogonal biosynthetic pathway for efficient production of α-pinene in oleaginous yeast Yarrowia lipolytica that resulted in an α-pinene titer of 19.6 mg/L when using glucose as the sole carbon source, a significant 218-fold improvement than the initial titer. In addition, the potential of using waste cooking oil and lignocellulosic hydrolysate as carbon sources for α-pinene production from the engineered Y. lipolytica strains was analyzed. The results indicated that α-pinene titers of 33.8 and 36.1 mg/L were successfully obtained in waste cooking oil and lignocellulosic hydrolysate medium, thereby representing the highest titer reported to date in yeast. To our knowledge, this is also the first report related to microbial production of α-pinene from waste cooking oil and lignocellulosic hydrolysate.

Yarrowia lipolytica as a Metabolic Engineering Platform for the Production of Very-Long-Chain Wax Esters.

The oleaginous yeast Yarrowia lipolytica is an attractive cell factory platform strain and can be used for sustainable production of high-value oleochemical products. Wax esters (WEs) have a good lubricating property and are usually used as a base for the production of advanced lubricants and emollient oils. In this study, we reported the metabolic engineering of Y. lipolytica to heterologously biosynthesize high-content very-long-chain fatty acids (VLCFAs) and fatty alcohols and efficiently esterify them to obtain very-long-chain WEs. Co-expression of fatty acid elongases from different sources in Y. lipolytica could yield VLCFAs with carbon chain lengths up to 24. Combining with optimization of the central metabolic modules could further enhance the biosynthesis of VLCFAs. Furthermore, through the screening of heterologous fatty acyl reductases (FARs), we enabled high-level production of fatty alcohols. Genomic integration and heterologous expression of wax synthase (WS) and FAR in a VLCFA-producing Y. lipolytica strain yielded 95-650 mg/L WEs with carbon chain lengths from 32 to 44. Scaled-up fermentation in 5 L laboratory bioreactors significantly increased the production of WEs to 2.0 g/L, the highest content so far in yeasts. This study contributes to the further efficient biosynthesis of VLCFAs and their derivatives.

Engineering oleaginous yeast Yarrowia lipolytica for enhanced limonene production from xylose and lignocellulosic hydrolysate.

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.

Pathway engineering and medium optimization for α-farnesene biosynthesis in oleaginous yeast Yarrowia lipolytica.

Farnesene is a typical sesquiterpene with applications as fragrance, flavor and precursor for the synthesis of vitamin E/K1. In this study, a series of strategies were employed to facilitate α-farnesene accumulation in Yarrowia lipolytica. Among them, the promoter optimization of OptFSLERG20, Sc-tHMG1 and IDI resulted in more than 62 % increase in α-farnesene production. Together with the overexpression of Yl-HMGR and ERG19, α-farnesene content was significantly improved by more than 3.5 times. The best metabolic engineered strain obtained was therefore used for a uniform design in shake flasks to determine the optimal medium compositions. Furthermore, a maximum α-farnesene production of approximately 2.57 g/L (34 mg/g DCW) was obtained in fed-batch fermentation where glycerol was supplemented as the feeding carbon source when initial glucose was depleted. This study has laid a good foundation for the development of Y. lipolytica as a promising chassis microbial cell for heterologous biosynthesis of α-farnesene and other sesquiterpenes.

Metabolomics analysis of Actinosynnema pretiosum with improved AP-3 production by enhancing UDP-glucose biosynthesis.

Ansamitocin P-3 (AP-3) shows strong anticancer effects and has used as a payload for antibody-drug conjugates. Our previous study have shown that although genetically engineered Actinosynnema pretiosum strains with enhanced UDP-glucose (UDPG) biosynthesis displayed improved AP-3 production compared to the wild-type strain, the increase in yield was far from meeting the industrial demand. In this study, comparative metabolomics analysis complemented with quantitative real-time PCR analysis was performed for the wild-type strain and two mutants (OpgmOugp, ΔzwfΔgnd) to identify possible metabolic bottlenecks and non-intuitive targets for further enhancement of AP-3 production. We observed that enhancing intracellular UDPG availability facilitated the accumulation of intracellular N-demethyl-AP-3 and AP-3, where the transporting of them outside the cell still needs to be developed. We also found that the UDPG biosynthesis was closely associated with the availability of fructose in the medium and a suitable fructose feeding strategy could promote the further improvement of AP-3 titer. In addition, pathway abundance analysis revealed that undesired fatty acid accumulation and down-regulation of amino acid metabolism may be unfavorable for ansamitocin biosynthesis in later stage of production. These results indicate that genetic modification of the UDPG biosynthetic pathways may have pleiotropic effects on AP-3 production. Efforts must be made to eliminate these newly identified metabolic bottlenecks to boost AP-3 production in A. pretiosum.

Metabolomic change and pathway profiling reveal enhanced ansamitocin P-3 production in Actinosynnema pretiosum with low organic nitrogen availability in culture medium.

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.

Metabolic engineering of ß-carotene biosynthesis in Yarrowia lipolytica.

OBJECTIVE: 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 ß-carotene-producing Yarrowia lipolytica cell factories for potential industrial application. RESULTS: 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 ß-carotene in Y. lipolytica XK2. Furthermore, ß-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 ß-carotene in shake flask cultures, a twenty-four-fold increase compared to the parental strain Y. lipolytica XK2. CONCLUSIONS: 4.5 g/L ß-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.

Multicopy integrants of crt genes and co-expression of AMP deaminase improve lycopene production in Yarrowia lipolytica.

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.

Enhanced squalene biosynthesis in Yarrowia lipolytica based on metabolically engineered acetyl-CoA metabolism.

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.

Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization.

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 ß-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.

Improved squalene production through increasing lipid contents in Saccharomyces cerevisiae.

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 ß-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.

AraBAD Based Toolkit for Gene Expression and Metabolic Robustness Improvement in Synechococcus elongatus.

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-ß-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.