Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica.

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO2-derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.

Production of the antidepressant orcinol glucoside in Yarrowia lipolytica with yields over 6,400-fold higher than plant extraction.

Orcinol glucoside (OG), mainly found in the rhizome of the traditional Chinese herb Curculigo orchioides Gaertn, is noted for its antidepressant effects. In this study, an efficient screening pipeline was established for identifying the highly active orcinol synthase (ORS) and UDP-dependent glycosyltransferase (UGT) involved in the biosynthesis of OG by combining transcriptome analysis, structure-based virtual screening, and in vitro enzyme activity assays. By enhancing the downstream pathway, metabolic engineering and fermentation optimization, the OG production in Yarrowia lipolytica was improved 100-fold, resulting in a final yield of 43.46 g/L (0.84 g/g DCW), which is almost 6,400-fold higher than the extraction yield from C. orchioides roots. This study provides a reference for rapid identification of functional genes and high-yield production of natural products.

Roles of the transcriptional regulators Fts1, YlNrg1, YlTup1, and YlSsn6 in the repression of the yeast-to-filament transition in the dimorphic yeast Yarrowia lipolytica.

In dimorphic fungi, the yeast-to-filament transition critical for cell survival under nutrient starvation is controlled by both activators and repressors. However, very few filamentation repressors are known. Here we report that, in the dimorphic yeast Yarrowia lipolytica, the conserved transcription factor YlNrg1 plays a minor role whereas Fts1, a newly identified Zn(II)2 Cys6 zinc cluster transcription factor, plays a key role in filamentation repression. FTS1 deletion caused hyperfilamentation whereas Fts1 overexpression drastically reduced filamentation. The expression of FTS1 is downregulated substantially during the yeast-to-filament transition. Transcriptome sequencing revealed that Fts1 represses 401 genes, including the filamentation-activating transcription factor genes MHY1, YlAZF1, and YlWOR4 and key cell wall protein genes. Tup1-Ssn6, a general transcriptional corepressor, is involved in the repression of many cellular functions in fungi. We show that both YlTup1 and YlSsn6 strongly repress filamentation in Y. lipolytica. YlTup1 and YlSsn6 together repress 1383 genes, including a large number of transcription factor and cell wall protein genes, which overlap substantially with Fts1-repressed genes. Fts1 interacts with both YlTup1 and YlSsn6, and LexA-Fts1 fusion represses a lexAop-promoter-lacZ reporter in a Tup1-Ssn6-dependent manner. Our findings suggest that Fts1 functions as a transcriptional repressor, directing the repression of target genes through the Tup1-Ssn6 corepressor.

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.

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.

High titer production of gastrodin enabled by systematic refactoring of yeast genome and an antisense-transcriptional regulation toolkit.

Gastrodin, a phenolic glycoside, is a prominent component of Gastrodia elata, which is renowned for its sedative, hypnotic, anticonvulsant, and neuroprotective activities. Engineering heterologous production of plant natural products in microbial host represents a safe, cost-effective, and scalable alternative to plant extraction. Here, we present the construction of an engineered Yarrowia lipolytica yeast that achieves a high-titer production of gastrodin. We systematically refactored the yeast genome by enhancing the flux of the shikimate pathway and optimizing the glucosyl transfer system. We introduced more than five dozen of genetic modifications onto the yeast genome, including enzyme screening, alleviation of rate-limiting steps, promoter selection, genomic integration site optimization, downregulation of competing pathways, and elimination of gastrodin degradation. Meanwhile, we developed a Copper-induced Antisense-Transcriptional Regulation (CATR) tool. The developed CATR toolkit achieved dynamic repression and activation of violacein synthesis through the addition of copper in Y. lipolytica. This strategy was further used to dynamically regulate the pyruvate kinase node to effectively redirect glycolytic flux towards the shikimate pathway while maintaining cell growth at proper rate. Taken together, these efforts resulted in 9477.1 mg/L of gastrodin in shaking flaks and 13.4 g/L of gastrodin with a yield of 0.149 g/g glucose in a 5-L bioreactor, highlighting the potential for large-scale and sustainable production of gastrodin from microbial fermentation.

YaliCMulti and YaliHMulti: Stable, efficient multi-copy integration tools for engineering Yarrowia lipolytica.

Yarrowia lipolytica is widely used in biotechnology to produce recombinant proteins, food ingredients and diverse natural products. However, unstable expression of plasmids, difficult and time-consuming integration of single and low-copy-number plasmids hampers the construction of efficient production pathways and application to industrial production. Here, by exploiting sequence diversity in the long terminal repeats (LTRs) of retrotransposons and ribosomal DNA (rDNA) sequences, a set of vectors and methods that can recycle multiple and high-copy-number plasmids was developed that can achieve stable integration of long-pathway genes in Y. lipolytica. By combining these sequences, amino acids and antibiotic tags with the Cre-LoxP system, a series of multi-copy site integration recyclable vectors were constructed and assessed using the green fluorescent protein (HrGFP) reporter system. Furthermore, by combining the consensus sequence with the vector backbone of a rapidly degrading selective marker and a weak promoter, multiple integrated high-copy-number vectors were obtained and high levels of stable HrGFP expression were achieved. To validate the universality of the tools, simple integration of essential biosynthesis modules was explored, and 7.3 g/L of L-ergothioneine and 8.3 g/L of (2S)-naringenin were achieved in a 5 L fermenter, the highest titres reported to date for Y. lipolytica. These novel multi-copy genome integration strategies provide convenient and effective tools for further metabolic engineering of Y. lipolytica.

Engineering Yarrowia lipolytica for sustainable ricinoleic acid production: A pathway to free fatty acid synthesis.

Ricinoleic acid (C18:1-OH, RA) is a valuable hydroxy fatty acid with versatile applications. The current industrial source of RA relies on the hydrolysis of castor bean oil. However, the coexistence of the toxic compound ricin and the unstable supply of this plant have led to an exploration of promising alternatives: generating RA in heterologous plants or microorganisms. In this study, we engineered the oleaginous yeast Yarrowia lipolytica to produce RA in the form of free fatty acids (FFA). First, we overexpressed fungal Δ12 oleate hydroxylase gene (CpFAH12) from Claviceps purpurea while deleting genes related to fatty acid degradation (MEF1 and PEX10) and oleic acid desaturation (FAD2). Since Δ12 oleate hydroxylase converts oleic acid (C18:1) located at the sn-2 position of phosphatidylcholine (PC), we next focused on increasing the PC pool containing oleic acid. This objective was achieved thorough implementing metabolic engineering strategies designed to enhance the biosynthesis of PC and C18 fatty acids. To increase the PC pool, we redirected the flux towards phospholipid biosynthesis by deleting phosphatidic acid phosphatase genes (PAH1 and APP1) and diacylglycerol acyltransferase gene (DGA1), involved in the production of diacylglycerol and triacylglycerol, respectively. Furthermore, the PC biosynthesis via the CDP-DAG pathway was enhanced through the overexpression of CDS1, PSD1, CHO2, and OPI3 genes. Subsequently, to increase the oleic acid content within PC, we overexpressed the heterologous fatty acid elongase gene (MaC16E) involved in the conversion of C16 to C18 fatty acids. As RA production titer escalated, the produced RA was mainly found in the FFA form, leading to cell growth inhibition. The growth inhibition was mitigated by inducing RA secretion via Triton X-100 treatment, a process that simultaneously amplified RA production by redirecting flux towards RA synthesis. The final engineered strain JHYL-R146 produced 2.061 g/L of free RA in a medium treated with 5% Triton X-100, constituting 74% of the total FFAs produced. Generating free RA offers the added benefit of bypassing the hydrolysis stage required when employing castor bean oil as an RA source. This achievement represents the highest level of RA synthesis from glucose reported thus far, underscoring the potential of Y. lipolytica as a host for sustainable RA production.

Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside.

Flavonoids are a diverse set of natural products with promising bioactivities including anti-inflammatory, anti-cancer, and neuroprotective properties. Previously, the oleaginous host Yarrowia lipolytica has been engineered to produce high titers of the base flavonoid naringenin. Here, we leverage this host along with a set of E. coli bioconversion strains to produce the flavone apigenin and its glycosylated derivative isovitexin, two potential nutraceutical and pharmaceutical candidates. Through downstream strain selection, co-culture optimization, media composition, and mutant isolation, we were able to produce168 mg/L of apigenin, representing a 46% conversion rate of 2-(R/S)-naringenin to apigenin. This apigenin platform was modularly extended to produce isovitexin by addition of a second bioconversion strain. Together, these results demonstrate the promise of microbial production and modular bioconversion to access diversified flavonoids.

Advances in the metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica for the production of ß-carotene.

ß-Carotene is one kind of the most important carotenoids. The major functions of ß-carotene include the antioxidant and anti-cardiovascular properties, which make it a growing market. Recently, the use of metabolic engineering to construct microbial cell factories to synthesize ß-carotene has become the latest model for its industrial production. Among these cell factories, yeasts including Saccharomyces cerevisiae and Yarrowia lipolytica have attracted the most attention because of the: security, mature genetic manipulation tools, high flux toward carotenoids using the native mevalonate pathway and robustness for large-scale fermentation. In this review, the latest strategies for ß-carotene biosynthesis, including protein engineering, promoters engineering and morphological engineering are summarized in detail. Finally, perspectives for future engineering approaches are proposed to improve ß-carotene production.

Inhibition of hyphal formation together with biochar addition promotes erythritol production by Yarrowia lipolytica.

This study aimed to investigate the effect of hyphal formation in Yarrowia lipolytica and biochar addition on erythritol production by submerged fermentation. Hyphal formation significantly inhibited erythritol production by Y. lipolytica. Transcriptome analysis suggested that the impaired erythritol synthesis of hyphal cells was associated with the differential expression of genes involved in amino acid metabolism, lipid metabolism, and cell wall stability. Deletion of RAS2 responsible for yeast-to-hypha transition and EYD1 included in erythritol degradation blocked hyphal formation and improved erythritol production. Biochar prepared from corncob, sugarcane bagasse (SB), corn straw, peanut shell, coconut shell, and walnut shell (WS) had a positive effect on erythritol production, of which WS pyrolyzed at 500°C (WSc) performed the best in flask fermentation. In a 3.7 L bioreactor, 220.20 ± 10 g/L erythritol with a productivity of 2.30 ± 0.10 g/L/h was obtained in the presence of 1.4% (w/v) WSc and 0.7% SBc (SB pyrolyzed at 500°C) within 96 h. These results suggest that inhibition of hyphal formation together with biochar addition is an efficient way to promote erythritol production.

'Small volume-big problem': culturing Yarrowia lipolytica in high-throughput micro-formats.

With the current progress in the 'design' and 'build' stages of the 'design-build-test-learn' cycle, many synthetic biology projects become 'test-limited'. Advances in the parallelization of microbes cultivations are of great aid, however, for many species down-scaling leaves a metabolic footprint. Yarrowia lipolytica is one such demanding yeast species, for which scaling-down inevitably leads to perturbations in phenotype development. Strictly aerobic metabolism, propensity for filamentation and adhesion to hydrophobic surfaces, spontaneous flocculation, and high acidification of media are just several characteristics that make the transfer of the micro-scale protocols developed for the other microbial species very challenging in this case. It is well recognized that without additional 'personalized' optimization, either MTP-based or single-cell-based protocols are useless for accurate studies of Y. lipolytica phenotypes. This review summarizes the progress in the scaling-down and parallelization of Y. lipolytica cultures, highlighting the challenges that occur most frequently and strategies for their overcoming. The problem of Y. lipolytica cultures down-scaling is illustrated by calculating the costs of micro-cultivations, and determining the unintentionally introduced, thus uncontrolled, variables. The key research into culturing Y. lipolytica in various MTP formats and micro- and pico-bioreactors is discussed. Own recently developed and carefully pre-optimized high-throughput cultivation protocol is presented, alongside the details from the optimization stage. We hope that this work will serve as a practical guide for those working with Y. lipolytica high-throughput screens.

'Mother(Nature) knows best' - hijacking nature-designed transcriptional programs for enhancing stress resistance and protein production in Yarrowia lipolytica; presentation of YaliFunTome database.

BACKGROUND: In the era of rationally designed synthetic biology, heterologous metabolites production, and other counter-nature engineering of cellular metabolism, we took a step back and recalled that 'Mother(-Nature) knows best'. While still aiming at synthetic, non-natural outcomes of generating an 'over-production phenotype' we dug into the pre-designed transcriptional programs evolved in our host organism-Yarrowia lipolytica, hoping that some of these fine-tuned orchestrated programs could be hijacked and used. Having an interest in the practical outcomes of the research, we targeted industrially-relevant functionalities-stress resistance and enhanced synthesis of proteins, and gauged them over extensive experimental design's completion. RESULTS: Technically, the problem was addressed by screening a broad library of over 120 Y. lipolytica strains under 72 combinations of variables through a carefully pre-optimized high-throughput cultivation protocol, which enabled actual phenotype development. The abundance of the transcription program elicitors-transcription factors (TFs), was secured by their overexpression, while challenging the strains with the multitude of conditions was inflicted to impact their activation stratus. The data were subjected to mathematical modeling to increase their informativeness. The amount of the gathered data prompted us to present them in the form of a searchable catalog - the YaliFunTome database ( https://sparrow.up.poznan.pl/tsdatabase/ )-to facilitate the withdrawal of biological sense from numerical data. We succeeded in the identification of TFs that act as omni-boosters of protein synthesis, enhance resistance to limited oxygen availability, and improve protein synthesis capacity under inorganic nitrogen provision. CONCLUSIONS: All potential users are invited to browse YaliFunTome in the search for homologous TFs and the TF-driven phenotypes of interest.

Transcriptomic profiling of an evolved Yarrowia lipolytica strain: tackling hexanoic acid fermentation to increase lipid production from short-chain fatty acids.

BACKGROUND: Short-chain fatty acids (SCFAs) are cost-effective carbon sources for an affordable production of lipids. Hexanoic acid, the acid with the longest carbon chain in the SCFAs pool, is produced in anaerobic fermentation of organic residues and its use is very challenging, even inhibiting oleaginous yeasts growth. RESULTS: In this investigation, an adaptive laboratory evolution (ALE) was performed to improve Yarrowia lipolytica ACA DC 50109 tolerance to high hexanoic acid concentrations. Following ALE, the transcriptomic analysis revealed several genetic adaptations that improved the assimilation of this carbon source in the evolved strain compared to the wild type (WT). Indeed, the evolved strain presented a high expression of the up-regulated gene YALI0 E16016g, which codes for FAT1 and is related to lipid droplets formation and responsible for mobilizing long-chain acids within the cell. Strikingly, acetic acid and other carbohydrate transporters were over-expressed in the WT strain. CONCLUSIONS: A more tolerant yeast strain able to attain higher lipid content under the presence of high concentrations of hexanoic acid has been obtained. Results provided novel information regarding the assimilation of hexanoic acid in yeasts.

Transcriptome analysis reveals multiple targets of erythritol-related transcription factor EUF1 in unconventional yeast Yarrowia Lipolytica.

BACKGROUND: Erythritol is a four-carbon polyol with an unclear role in metabolism of some unconventional yeasts. Its production has been linked to the osmotic stress response, but the mechanism of stress protection remains unclear. Additionally, erythritol can be used as a carbon source. In the yeast Yarrowia lipolytica, its assimilation is activated by the transcription factor Euf1. The study investigates whether this factor can link erythritol to other processes in the cell. RESULTS: The research was performed on two closely related strains of Y. lipolytica: MK1 and K1, where strain K1 has no functional Euf1. Cultures were carried out in erythritol-containing and erythritol-free media. Transcriptome analysis revealed the effect of Euf1 on the regulation of more than 150 genes. Some of these could be easily connected with different aspects of erythritol assimilation, such as: utilization pathway, a new potential isoform of transketolase, or polyol transporters. However, many of the upregulated genes have never been linked to metabolism of erythritol. The most prominent examples are the degradation pathway of branched-chain amino acids and the glyoxylate cycle. The high transcription of genes affected by Euf1 is still dependent on the erythritol concentration in the medium. Moreover, almost all up-regulated genes have an ATGCA motif in the promoter sequence. CONCLUSIONS: These findings may be particularly relevant given the increasing use of erythritol-induced promoters in genetic engineering of Y. lipolytica. Moreover, use of this yeast in biotechnological processes often takes place under osmotic stress conditions. Erythritol might be produce as a by-product, thus better understanding of its influence on cell metabolism could facilitate processes optimization.

The mechanisms of yeast extracellular metabolites in stimulating microbial degradation of trichloroethylene: Physiological characteristics and omics analysis.

The biodegradation of Trichloroethylene (TCE) is limited by low microbial metabolic capacity but can be enhanced through biostimulation strategies. This study explored the physiological effects and potential molecular mechanisms of the yeast Yarrowia lipolytica extracellular metabolites (YEMs) on the degradation of TCE by Acinetobacter LT1. Results indicated that YEMs stimulated the efficiency of strain LT1 by 50.28%. At the physiological level, YEMs exhibited protective effects on cell morphology, reduced oxidative stress, lessened membrane damage, and enhanced energy production and conversion. Analysis of omics results revealed that the regulation of various metabolic pathways by YEMs improved the degradation of TCE. Furthermore, RT-qPCR showed that the genes encoding YhhW protein in TCE stress and YEMs stimulation groups were 1.72 and 3.22 times the control group, respectively. Molecular docking results showed that the conformation of YhhW after binding to TCE changed into a more active form, which enhanced enzyme activity. Therefore, it is speculated that YhhW is the primary degradative enzyme involved in the process of YEMs stimulating strain LT1 to degrade TCE. These results reveal how YEMs induce strain LT1 to enhance TCE degradation.

Development of genetic markers in Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica represents a potential microbial cell factory for the recombinant production of various valuable products. Currently, the commonly used selection markers for transformation in Y. lipolytica are limited, and successive genetic manipulations are often restricted by the number of available selection markers. In our study, we developed a dominant marker, dsdA, which encodes a D-serine deaminase for genetic manipulation in Y. lipolytica. In Y. lipolytica, this marker confers the ability to use D-serine as a nitrogen source. In addition, the selection conditions of several infrequently used dominant markers including bleoR (zeocin resistance), kanMX (G418 resistance), and guaB (mycophenolic acid resistance) were also analyzed. Our results demonstrated that these selection markers can be used for the genetic manipulation of Y. lipolytica and their selection conditions were different for various strains. Ultimately, the selection markers tested here will be useful to expand the genetic toolbox of Y. lipolytica. KEY POINTS: • The dsdA from Escherichia coli was developed as a dominant marker. • The applicability of several resistance markers in Y. lipolytica was determined. • We introduced the Cre/mutant lox system for marker recycling.

Adaptive responses of erythritol-producing Yarrowia lipolytica to thermal stress after evolution.

Elucidation of the thermotolerance mechanism of erythritol-producing Yarrowia lipolytica is of great significance to breed robust industrial strains and reduce cost. This study aimed to breed thermotolerant Y. lipolytica and investigate the mechanism underlying the thermotolerant phenotype. Yarrowia lipolytica HT34, Yarrowia lipolytica HT36, and Yarrowia lipolytica HT385 that were capable of growing at 34 °C, 36 °C, and 38.5 °C, respectively, were obtained within 150 days (352 generations) by adaptive laboratory evolution (ALE) integrated with 60Co-γ radiation and ultraviolet ray radiation. Comparative genomics analysis showed that genes involved in signal transduction, transcription, and translation regulation were mutated during adaptive evolution. Further, we demonstrated that thermal stress increased the expression of genes related to DNA replication and repair, ceramide and steroid synthesis, and the degradation of branched amino acid (BCAA) and free fatty acid (FFA), while inhibiting the expression of genes involved in glycolysis and the citrate cycle. Erythritol production in thermotolerant strains was remarkably inhibited, which might result from the differential expression of genes involved in erythritol metabolism. Exogenous addition of BCAA and soybean oil promoted the growth of HT385, highlighting the importance of BCAA and FFA in thermal stress response. Additionally, overexpression of 11 out of the 18 upregulated genes individually enabled Yarrowia lipolytica CA20 to grow at 34 °C, of which genes A000121, A003183, and A005690 had a better effect. Collectively, this study provides novel insights into the adaptation mechanism of Y. lipolytica to thermal stress, which will be conducive to the construction of thermotolerant erythritol-producing strains. KEY POINTS: • ALE combined with mutagenesis is efficient for breeding thermotolerant Y. lipolytica • Genes encoding global regulators are mutated during thermal adaptive evolution • Ceramide and BCAA are critical molecules for cells to tolerate thermal stress.

Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology.

We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of ∼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology.

Expansion of YALIcloneHR toolkit for Yarrowia lipolytica combined with Golden Gate and CRISPR technology.

Metabolic Engineering of yeast is a critical approach to improving the production capacity of cell factories. To obtain genetically stable recombinant strains, the exogenous DNA is preferred to be integrated into the genome. Previously, we developed a Golden Gate toolkit YALIcloneNHEJ, which could be used as an efficient modular cloning toolkit for the random integration of multigene pathways through the innate non-homologous end-joining repair mechanisms of Yarrowia lipolytica. We expanded the toolkit by designing additional building blocks of homologous arms and using CRISPR technology. The reconstructed toolkit was thus entitled YALIcloneHR and designed for gene-specific knockout and integration. To verify the effectiveness of the system, the gene PEX10 was selected as the target for the knockout. This system was subsequently applied for the arachidonic acid production, and the reconstructed strain can accumulate 4.8% of arachidonic acid. The toolkit will expand gene editing technology in Y. lipolytica, which would help produce other chemicals derived from acetyl-CoA in the future.