Effect of low frequency alternating magnetic field for erythritol production in Yarrowia lipolytica.

Numerous works have reported that magnetic fields serve as signals capable of influencing microbial metabolism. However, little is known about the effect of magnetic field on erythritol production by the model microorganism Yarrowia lipolytica (Y. lipolytica). Therefore, we investigated the effect of low-frequency alternating magnetic fields (LF-AMF) with different magnetic field intensities (0-1.5 mT) and different magnetic field treatment times (1-10 days) on the production of erythritol by Y. lipolytica -JZ204. The optimal treatment condition was 0.5 mT for 8 days. As a result, a maximal erythritol yield was achieved 63.74 g/L, the biomass was reached 37 g/L, and the specific erythritol yield per unit of biomass was 1.7227 g/g, which were 60.72%, 32.09%, and 24.85% higher than the control, respectively. We investigated the internal mechanism of magnetic fields impact by using transcriptomics and RT-qPCR technology. This study demonstrated the effectiveness of LF-AMF in enhancing erythritol production by Y. lipolytica JZ-204, providing insights for the application of magnetic field in assisting microbial fermentation and improving the synthesis of beneficial products.

Engineering Yarrowia lipolytica for Efficient Synthesis of Geranylgeraniol.

Geranylgeraniol (GGOH) is a crucial component in fragrances and essential oils, and a valuable precursor of vitamin E. It is primarily extracted from the oleoresin of Bixa orellana, but is challenged by long plant growth cycles, severe environmental pollution, and low extraction efficiency. Chemically synthesized GGOH typically comprises a mix of isomers, making the separation process both challenging and costly. Advancements in synthetic biology have enabled the construction of microbial cell factories for GGOH production. In this study, Yarrowia lipolytica was engineered to efficiently synthesize GGOH by expressing heterologous phosphatase genes, enhancing precursor supplies of farnesyl diphosphate, geranylgeranyl pyrophosphate, and acetyl-CoA, and downregulating the squalene synthesis pathway by promoter engineering. Additionally, optimizing fermentation conditions and reducing reactive oxygen species significantly increased the GGOH titer to 3346.47 mg/L in a shake flask. To the best of our knowledge, this is the highest reported GGOH titer in shaking flasks to date, setting a new benchmark for terpenoid production.

An Interplay between Transcription Factors and Recombinant Protein Synthesis in Yarrowia lipolytica at Transcriptional and Functional Levels-The Global View.

Transcriptional regulatory networks (TRNs) associated with recombinant protein (rProt) synthesis in Yarrowia lipolytica are still under-described. Yet, it is foreseen that skillful manipulation with TRNs would enable global fine-tuning of the host strain's metabolism towards a high-level-producing phenotype. Our previous studies investigated the transcriptomes of Y. lipolytica strains overproducing biochemically different rProts and the functional impact of transcription factors (TFs) overexpression (OE) on rProt synthesis capacity in this species. Hence, much knowledge has been accumulated and deposited in public repositories. In this study, we combined both biological datasets and enriched them with further experimental data to investigate an interplay between TFs and rProts synthesis in Y. lipolytica at transcriptional and functional levels. Technically, the RNAseq datasets were extracted and re-analyzed for the TFs' expression profiles. Of the 140 TFs in Y. lipolytica, 87 TF-encoding genes were significantly deregulated in at least one of the strains. The expression profiles were juxtaposed against the rProt amounts from 125 strains co-overexpressing TF and rProt. In addition, several strains bearing knock-outs (KOs) in the TF loci were analyzed to get more insight into their actual involvement in rProt synthesis. Different profiles of the TFs' transcriptional deregulation and the impact of their OE or KO on rProts synthesis were observed, and new engineering targets were pointed.

Reprogramming Yarrowia lipolytica metabolism for efficient synthesis of itaconic acid from flask to semipilot scale.

Itaconic acid is an emerging platform chemical with extensive applications. Itaconic acid is currently produced by Aspergillus terreus through biological fermentation. However, A. terreus is a fungal pathogen that needs additional morphology controls, making itaconic acid production on industrial scale problematic. Here, we reprogrammed the Generally Recognized As Safe (GRAS) yeast Yarrowia lipolytica for competitive itaconic acid production. After preventing carbon sink into lipid accumulation, we evaluated itaconic acid production both inside and outside the mitochondria while fine-tuning its biosynthetic pathway. We then mimicked the regulation of nitrogen limitation in nitrogen-replete conditions by down-regulating NAD+-dependent isocitrate dehydrogenase through weak promoters, RNA interference, or CRISPR interference. Ultimately, we optimized fermentation parameters for fed-batch cultivations and produced itaconic acid titers of 130.1 grams per liter in 1-liter bioreactors and 94.8 grams per liter in a 50-liter bioreactor on semipilot scale. Our findings provide effective approaches to harness the GRAS microorganism Y. lipolytica for competitive industrial-scale production of itaconic acid.

Comparative transcriptomics analysis-guided metabolic engineering of Yarrowia lipolytica for improved erythritol and fructooligosaccharides production.

Currently, fructooligosaccharides (FOS) are converted from sucrose by purified enzymes or fungal cells, but these methods are costly and time-consuming. Here, the optimal fermentation conditions for strain E326 were determined through fermentation optimization: initial glucose 200 g/L, NaCl 25 g/L, inoculum volume 20 %, dissolved oxygen 20-30 %, pH 3, and glucose feeding concentration 100 g/L, which increased erythritol titer by 1.5 times. The co-expression of HGT1 and APC11 genes alleviated the erythritol synthesis stagnation, shorten the fermentation time by 16.7 %, and increased the erythritol productivity by 17.2 %. The episomal plasmids based on yeast mitochondrial replication origins (mtORIs) were constructed to surface display fructosyltransferase, effectively utilizing waste yeast cells generated during erythritol fermentation. Under the conditions of 60℃ and pH 6, the FOS yield reached 65 %, which to our best of knowledge is so-far the highest yield of FOS obtained. These findings will contribute to the industrial production of erythritol and FOS.

Screening of broad-host expression promoters for shuttle expression vectors in non-conventional yeasts and bacteria.

BACKGROUND: Non-conventional yeasts and bacteria gain significance in synthetic biology for their unique metabolic capabilities in converting low-cost renewable feedstocks into valuable products. Improving metabolic pathways and increasing bioproduct yields remain dependent on the strategically use of various promoters in these microbes. The development of broad-spectrum promoter libraries with varying strengths for different hosts is attractive for biosynthetic engineers. RESULTS: In this study, five Yarrowia lipolytica constitutive promoters (yl.hp4d, yl.FBA1in, yl.TEF1, yl.TDH1, yl.EXP1) and five Kluyveromyces marxianus constitutive promoters (km.PDC1, km.FBA1, km.TEF1, km.TDH3, km.ENO1) were selected to construct promoter-reporter vectors, utilizing α-amylase and red fluorescent protein (RFP) as reporter genes. The promoters' strengths were systematically characterized across Y. lipolytica, K. marxianus, Pichia pastoris, Escherichia coli, and Corynebacterium glutamicum. We discovered that five K. marxianus promoters can all express genes in Y. lipolytica and that five Y. lipolytica promoters can all express genes in K. marxianus with variable expression strengths. Significantly, the yl.TEF1 and km.TEF1 yeast promoters exhibited their adaptability in P. pastoris, E. coli, and C. glutamicum. In yeast P. pastoris, the yl.TEF1 promoter exhibited substantial expression of both amylase and RFP. In bacteria E. coli and C. glutamicum, the eukaryotic km.TEF1 promoter demonstrated robust expression of RFP. Significantly, in E. coli, The RFP expression strength of the km.TEF1 promoter reached ∼20% of the T7 promoter. CONCLUSION: Non-conventional yeast promoters with diverse and cross-domain applicability have great potential for developing innovative and dynamic regulated systems that can effectively manage carbon flux and enhance target bioproduct synthesis across diverse microbial hosts.

Using Euf1 transcription factor as a titrator of erythritol-inducible promoters in Yarrowia lipolytica; insight into the structure, splicing, and regulation mechanism.

Controllable regulatory elements, like inducible, titratable promoters, are highly desired in synthetic biology toolboxes. A set of previously developed erythritol-inducible promoters along with an engineered Yarrowia lipolytica host strain were shown to be a very potent expression platform. In this study, we push the previously encountered limits of the synthetic promoters' titratability (by the number of upstream motifs) by using a compatible transcription factor, Euf1, as the promoter titrator. Overexpression of spliced EUF1 turned out to be very efficient in promoting expression from the compatible promoter, however, the erythritol-inducible character of the promoter was then lost. Analysis of the EUF1's splicing pattern suggests that the intron removal is promoted in the presence of erythritol, but is not dependent on it. The 3D structures of spliced versus unspliced Euf1 were modeled, and ligand-binding strength was calculated and compared. Furthermore, the EUF1-dependent expression profile under different chemical stimulants was investigated. Depletion of carbon source was identified as the significant factor upregulating the expression from the Euf1-dependent promoter (2-10-fold). Considering these findings and transcriptomics data, a new mechanism of the Euf1-regulated promoter action is proposed, involving a 'catabolite repression' transcription factor-Adr1, both acting on the same ERY-inducible promoter.

Efficient expression of OUC-Sb-lip2 in Yarrowia lipolytica and its comprehensive utilization in the enrichment of DHA and EPA from fish oil.

Lipases are widely used in the modification of functional lipids, particularly in the enrichment of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). In this study, a lipase named OUC-Sb-lip2 was expressed in Yarrowia lipolytica, achieving a promising enzyme activity of 472.6 U/mL by optimizing the culture medium, notably through olive oil supplementation. A significant proportion (58.8%) of the lipase activity was located in the cells, whereas 41.2% was secreted into the supernatant. Both whole-cell and immobilized OUC-Sb-lip2 were used to enrich DHA and EPA from fish oil. The whole-cell approach increased the DHA and EPA contents to 2.59 and 2.55 times that of the original oil, respectively. Similarly, the immobilized OUC-Sb-lip2 resulted in a 2.00-fold increase in DHA and an 1.99-fold increase in EPA after a 6-h hydrolysis period. Whole cell and the immobilized OUC-Sb-lip2 retained 48.7% and 52.7% of their activity after six cycles of reuse, respectively.

The PAH1-encoded phosphatidate phosphatase of Yarrowia lipolytica differentially affects gene expression and lipid biosynthesis.

Yarrowia lipolytica is a model oleaginous yeast with a strong capacity for lipid accumulation, yet its lipid metabolic pathways and regulatory mechanisms remain largely unexplored. The PAH1-encoded phosphatidate (PA) phosphatase governs lipid biosynthesis by its enzymatic activity and regulating the transcription of genes involved in phospholipid biosynthesis. In this work, we examined the effect of the loss of Pah1 (i.e., pah1Δ) on cell metabolism in cells growing in low- and high-glucose media. Multi-omics analyses revealed the global effect of the pah1Δ mutation on lipid and central carbon metabolism. Lipidomics analyses showed that the pah1Δ mutation caused a massive decrease in the masses of triacylglycerol (TAG) and diacylglycerol (DAG), and these effects were independent of glucose concentration in the media. Conversely, phospholipid levels declined in low-glucose media but increased in high-glucose media. The loss of Pah1 affected the expression of genes involved in key pathways of glucose metabolism, such as glycolysis, citric acid cycle, oxidative phosphorylation, and the pentose phosphate pathway, and these effects were more pronounced in high-glucose media. In lipid biosynthesis, the genes catalyzing phosphatidylcholine (PC) synthesis from phosphatidylethanolamine (PE) were upregulated within the CDP-DAG pathway. In contrast, PC synthesis through the Kennedy pathway was downregulated. The ethanolamine branch of the Kennedy pathway that synthesizes PE was also upregulated in pah1Δ. Interestingly, we noted a massive increase in the levels of lysophospholipids, consistent with the upregulation of genes involved in lipid turnover. Overall, this work identified novel regulatory roles of Pah1 in lipid biosynthesis and gene expression.

Production of γ-aminobutyric acid by immobilization of two Yarrowia lipolytica glutamate decarboxylases on electrospun nanofibrous membrane.

This study harnesses glutamate decarboxylase (GAD) from Yarrowia lipolytica to improve the biosynthesis of γ-aminobutyric acid (GABA), focusing on boosting the enzyme's catalytic efficiency and stability by immobilizing it on nanofibrous membranes. Through recombinant DNA techniques, two GAD genes, YlGAD1 and YlGAD2, were cloned from Yarrowia lipolytica and then expressed in Escherichia coli. Compared to their soluble forms, the immobilized enzymes exhibited significant improvements in thermal and pH stability and increased resistance to chemical denaturants. The immobilization notably enhanced substrate affinity, as evidenced by reduced Km values and increased kcat values, indicating heightened catalytic efficiency. Additionally, the immobilized YlGAD1 and YlGAD2 enzymes showed substantial reusability, maintaining 50% and 40% of their activity, respectively, after six consecutive cycles. These results underscore the feasibility of employing immobilized YlGAD enzymes for cost-effective and environmentally sustainable GABA production. This investigation not only affirms the utility of YlGADs in GABA synthesis but also underscores the advantages of enzyme immobilization in industrial settings, paving the way for scalable biotechnological processes.

Metabolic engineering of the oleaginous yeast Yarrowia lipolytica for 2-phenylethanol overproduction.

The rose fragrance molecule 2-phenylethanol (2-PE) has huge market demand in the cosmetics, food and pharmaceutical industries. However, current 2-PE synthesis methods do not meet the efficiency market requirement. In this study, CRISPR-Cas9-related metabolic engineering strategies were applied to Yarrowia lipolytica for the de novo biosynthesis of 2-PE. Initially, overexpressing exogenous feedback-resistant EcAROGfbr and EcPheAfbr increased 2-PE production to 276.3 mg/L. Subsequently, the ylARO10 and ylPAR4 from endogenous genes were enhanced with the multi-copies to increase the titer to 605 mg/L. Knockout of ylTYR1 and enhancement of shikimate pathway by removing the precursor metabolic bottleneck and overexpressing the genes ylTKT, ylARO1, and ylPHA2 resulted in a significant increase of the 2-PE titer to 2.4 g/L at 84 h, with the yield of 0.06 g/gglu, which is the highest yield for de novo synthesis in yeast. This study provides a valuable precedent for the efficient biosynthesis of shikimate pathway derivatives.

Protein rational design and modification of erythrose reductase for the improvement of erythritol production in Yarrowia lipolytica.

Erythritol is a natural non-caloric sweetener, which is produced by fermentation and extensively applied in food, medicine and chemical industries. The final step of the erythritol synthesis pathway is involved in erythritol reductase, whose activity and NADPH-dependent become the limiting node of erythritol production efficiency. Herein, we implemented a strategy combining molecular docking and thermal stability screening to construct an ER mutant library. And we successfully obtained a double mutant ERK26N/V295M (ER*) whose catalytic activity was 1.48 times that of wild-type ER. Through structural analysis and MD analysis, we found that the catalytic pocket and the enzyme stability of ER* were both improved. We overexpressed ER* in the engineered strain ΔKU70 to obtain the strain YLE-1. YLE-1 can produce 39.47 g/L of erythritol within 144 h, representing a 35% increase compared to the unmodified strain, and a 10% increase compared to the strain overexpressing wild-type ER. Considering the essentiality of NADPH supply, we further co-expressed ER* with two genes from the oxidative phase of PPP, ZWF1 and GND1. This resulted in the construction of YLE-3, which exhibited a significant increase in production, producing 47.85 g/L of erythritol within 144 h, representing a 63.90% increase compared to the original chassis strain. The productivity and the yield of the engineered strain YLE-3 were 0.33 g/L/h and 0.48 g/g glycerol, respectively. This work provided an ER mutation with excellent performance, and also proved the importance of cofactors in the process of erythritol synthesis, which will promote the industrial production of erythritol by metabolic engineering of Y. lipolytica.

Mar1, a high mobility group box protein, regulates n-alkane adsorption and cell morphology of the dimorphic yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica possesses an excellent ability to utilize n-alkane as a sole carbon and energy source. Although there are detailed studies on the enzymes that catalyze the reactions in the metabolic processes of n-alkane in Y. lipolytica, the molecular mechanism underlying the incorporation of n-alkane into the cells remains to be elucidated. Because Y. lipolytica adsorbs n-alkane, we postulated that Y. lipolytica incorporates n-alkane through direct interaction with it. We isolated and characterized mutants defective in adsorption to n-hexadecane. One of the mutants harbored a nonsense mutation in MAR1 (Morphology and n-alkane Adsorption Regulator 1) encoding a protein containing a high mobility group box. The deletion mutant of MAR1 exhibited defects in adsorption to n-hexadecane and filamentous growth on solid media, whereas the strain that overexpressed MAR1 exhibited hyperfilamentous growth. Fluorescence microscopic observations suggested that Mar1 localizes in the nucleus. RNA-sequencing analysis revealed the alteration of the transcript levels of several genes, including those encoding transcription factors and cell surface proteins, by the deletion of MAR1. These findings suggest that MAR1 is involved in the transcriptional regulation of the genes required for n-alkane adsorption and cell morphology transition.IMPORTANCEYarrowia lipolytica, a dimorphic yeast capable of assimilating n-alkane as a carbon and energy source, has been extensively studied as a promising host for bioconversion of n-alkane into useful chemicals and bioremediation of soil and water contaminated by petroleum. While the metabolic pathway of n-alkane in this yeast and the enzymes involved in this pathway have been well characterized, the molecular mechanism to incorporate n-alkane into the cells is yet to be fully understood. Due to the ability of Y. lipolytica to adsorb n-alkane, it has been hypothesized that Y. lipolytica incorporates n-alkane through direct interaction with it. In this study, we identified a gene, MAR1, which plays a crucial role in the transcriptional regulation of the genes necessary for the adsorption to n-alkane and the transition of the cell morphology in Y. lipolytica. Our findings provide valuable insights that could lead to advanced applications of Y. lipolytica in n-alkane bioconversion and bioremediation.

Sustainable succinic acid production from lignocellulosic hydrolysates by engineered strains of Yarrowia lipolytica at low pH.

Succinic acid (SA) is a valuable C4 platform chemical with diverse applications. Lignocellulosic biomass represents an abundant and renewable carbon resource for microbial production of SA. However, the presence of toxic compounds in pretreated lignocellulosic hydrolysates poses challenges to cell metabolism, leading to inefficient SA production. Here, engineered Yarrowia lipolytica Hi-SA2 was shown to utilize glucose and xylose from corncob hydrolysate to produce 32.6 g/L SA in shaking flasks. The high concentration of undetoxified hydrolysates significantly inhibited yeast growth and SA biosynthesis, with furfural identified as the key inhibitor. Through overexpressing glutathione synthetase encoding gene YlGsh2, the tolerance of engineered strain to furfural and toxic hydrolysate was significantly improved. In a 5-L bioreactor, Hi-SA2-YlGsh2 strain produced 45.34 g/L SA within 32 h, with a final pH of 3.28. This study provides a sustainable process for bio-based SA production, highlighting the efficient SA synthesis from lignocellulosic biomass through low pH fermentation.

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica.

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

Recent Advances and Multiple Strategies of Monoterpenoid Overproduction in Saccharomyces cerevisiae and Yarrowia lipolytica.

Monoterpenoids are an important subclass of terpenoids that play important roles in the energy, cosmetics, pharmaceuticals, and fragrances fields. With the development of biotechnology, microbial synthesis of monoterpenoids has received great attention. Yeasts such Saccharomyces cerevisiae and Yarrowia lipolytica are emerging as potential hosts for monoterpenoids production because of unique advantages including rapid growth cycles, mature gene editing tools, and clear genetic background. Recently, advancements in metabolic engineering and fermentation engineering have significantly enhanced the accumulation of monoterpenoids in cell factories. First, this review introduces the biosynthetic pathway of monoterpenoids and comprehensively summarizes the latest production strategies, which encompass enhancing precursor flux, modulating the expression of rate-limited enzymes, suppressing competitive pathway flux, mitigating cytotoxicity, optimizing substrate utilization, and refining the fermentation process. Subsequently, this review introduces four representative monoterpenoids. Finally, we outline the future prospects for efficient construction cell factories tailored for the production of monoterpenoids and other terpenoids.

The bioproduction of astaxanthin: A comprehensive review on the microbial synthesis and downstream extraction.

Astaxanthin is a valuable orange-red carotenoid with wide applications in agriculture, food, cosmetics, pharmaceuticals and nutraceuticals areas. At present, the biological synthesis of astaxanthin mainly relies on Haematococcus pluvialis and Xanthophyllomyces dendrorhous. With the rapid development of synthetic biology, more recombinant microbial hosts have been genetically constructed for astaxanthin production including Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica. As multiple genes (15) were involved in the astaxanthin synthesis, it is particularly important to adopt different strategies to balance the metabolic flow towards the astaxanthin synthesis. Furthermore, astaxanthin is a fat-soluble compound stored intracellularly, hence efficient extraction methods are also essential for the economical production of astaxanthin. Several efficient and green extraction methods of astaxanthin have been reported in recent years, including the superfluid extraction, ionic liquid extraction and microwave-assisted extraction. Accordingly, this review will comprehensively introduce the advances on the astaxanthin production and extraction by using different microbial hosts and strategies to improve the astaxanthin synthesis and extraction efficiency.

Broadening the application of Yarrowia lipolytica synthetic biology tools to explore the potential of Yarrowia clade diversity.

Yeasts have established themselves as prominent microbial cell factories, and the availability of synthetic biology tools has led to breakthroughs in the rapid development of industrial chassis strains. The selection of a suitable microbial host is critical in metabolic engineering applications, but it has been largely limited to a few well-defined strains. However, there is growing consideration for evaluating strain diversity, as a wide range of specific traits and phenotypes have been reported even within a specific yeast genus or species. Moreover, with the advent of synthetic biology tools, non-type strains can now be easily and swiftly reshaped. The yeast Yarrowia lipolytica has been extensively studied for various applications such as fuels, chemicals, and food. Additionally, other members of the Yarrowia clade are currently being evaluated for their industrial potential. In this study, we demonstrate the versatility of synthetic biology tools originally developed for Y. lipolytica by repurposing them for engineering other yeasts belonging to the Yarrowia clade. Leveraging the Golden Gate Y. lipolytica tool kit, we successfully expressed fluorescent proteins as well as the carotenoid pathway in at least five members of the clade, serving as proof of concept. This research lays the foundation for conducting more comprehensive investigations into the uncharacterized strains within the Yarrowia clade and exploring their potential applications in biotechnology.

Engineering Yarrowia lipolytica for sustainable Cis-13, 16-docosadienoic acid production.

Cis-13, 16-docosadienoic acid (DDA) is an omega-6 polyunsaturated fatty acid with great potential for application in medicine and health. Using microbial cell factories for DDA production is considered a viable alternative to extracting DDA from plant seeds. In this study, using Yarrowia lipolytica Po1f (Δku70) as a chassis, firstly, the adaptation of three elongases in Po1f (Δku70) were explored. Secondly, the DDA biosynthetic pathway was redesigned, resulting in a DDA content of 0.046 % of total fatty acids (TFAs). Thirdly, through the "push-pull" strategy, the DDA content increased to 0.078 % of TFAs. By enhancing the supply of acetyl-CoA, the DDA production in the engineered strain YL-7 reached 0.391 % of the TFAs (3.19 mg/L). Through optimizing the fermentation conditions, the DDA titer of YL-7 reached 29.34 mg/L. This research achieves the sustainable biological production of DDA in Y. lipolytica.

Multidimensional combinatorial screening for high-level production of erythritol in Yarrowia lipolytica.

Yarrowia lipolytica was successfully engineered to synthesize erythritol from crude glycerol, a cheap by-product of biodiesel production, but the yield remained low. Here, a biosensor-guided adaptive evolution screening platform was constructed to obtain mutant strains which could efficiently utilize crude glycerol to produce erythritol. Erythrose reductase D46A (M1) was identified as a key mutant through whole-genome sequencing of the strain G12, which exhibited higher catalytic activity (1.6-fold of the wild-type). M1 was further modified to obtain a combinatorial mutant with 4.1-fold enhancement of catalytic activity. Finally, the metabolic network was reconfigured to redirect carbon fluxes toward erythritol synthesis. The erythritol titer of the engineered strain G31 reached 220.5 g/L with a productivity of 1.8 g/L/h in a 5-L bioreactor. The study provides valuable guidance for biosensor-based ultra-high-throughput screening strategies in Y. lipolytica, as well as presenting a new paradigm for the sustainable valorization of crude glycerol.