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.

A comprehensive review on the recent advances for 5-aminolevulinic acid production by the engineered bacteria.

5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid essential for synthesizing tetrapyrrole compounds, including heme, chlorophyll, cytochrome, and vitamin B12. As a plant growth regulator, 5-ALA is extensively used in agriculture to enhance crop yield and quality. The complexity and low yield of chemical synthesis methods have led to significant interest in the microbial synthesis of 5-ALA. Advanced strategies, including the: enhancement of precursor and cofactor supply, compartmentalization of key enzymes, product transporters engineering, by-product formation reduction, and biosensor-based dynamic regulation, have been implemented in bacteria for 5-ALA production, significantly advancing its industrialization. This article offers a comprehensive review of recent developments in 5-ALA production using engineered bacteria and presents new insights to propel the field forward.

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.

Dual cytoplasmic-peroxisomal engineering for high-yield production of sesquiterpene α-humulene in Yarrowia lipolytica.

The sesquiterpene α-humulene is an important plant natural product, which has been used in the pharmaceutical industry due to its anti-inflammatory and anticancer activities. Although phytoextraction and chemical synthesis have previously been applied in α-humulene production, the low efficiency and high costs limit the development. In this study, Yarrowia lipolytica was engineered as the robust cell factory for sustainable α-humulene production. First, a chassis with high α-humulene output in the cytoplasm was constructed by integrating α-humulene synthases with high catalytic activity, optimizing the flux of mevalonate and acetyl-CoA pathways. Subsequently, the strategy of dual cytoplasmic-peroxisomal engineering was adopted in Y. lipolytica; the best strain GQ3006 generated by introducing 31 copies of 12 different genes could produce 2280.3± 38.2 mg/l (98.7 ± 4.2 mg/g dry cell weight) α-humulene, a 100-fold improvement relative to the baseline strain. To further improve the titer, a novel strategy for downregulation of squalene biosynthesis based on Cu2+ -repressible promoters was firstly established, which significantly improved the α-humulene titer by 54.2% to 3516.6 ± 34.3 mg/l. Finally, the engineered strain could produce 21.7 g/l α-humulene in a 5-L bioreactor, 6.8-fold higher than the highest α-humulene titer reported before this study. Overall, system metabolic engineering strategies used in this study provide a valuable reference for the highly sustainable production of terpenoids in Y. lipolytica.

High-yield α-humulene production in Yarrowia lipolytica from waste cooking oil based on transcriptome analysis and metabolic engineering.

BACKGROUND: α-Humulene is an important biologically active sesquiterpene, whose heterologous production in microorganisms is a promising alternative biotechnological process to plant extraction and chemical synthesis. In addition, the reduction of production expenses is also an extremely critical factor in the sustainable and industrial production of α-humulene. In order to meet the requirements of industrialization, finding renewable substitute feedstocks such as low cost or waste substrates for terpenoids production remains an area of active research. RESULTS: In this study, we investigated the feasibility of peroxisome-engineering strain to utilize waste cooking oil (WCO) for high production of α-humulene while reducing the cost. Subsequently, transcriptome analysis revealed differences in gene expression levels with different carbon sources. The results showed that single or combination regulations of target genes identified by transcriptome were effective to enhance the α-humulene titer. Finally, the engineered strain could produce 5.9 g/L α-humulene in a 5-L bioreactor. CONCLUSION: To the best of our knowledge, this is the first report that converted WCO to α-humulene in peroxisome-engineering strain. These findings provide valuable insights into the high-level production of α-humulene in Y. lipolytica and its utilization in WCO bioconversion.

Recent advances in the application of multiplex genome editing in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a widely used microorganism and a greatly popular cell factory for the production of various chemicals. In order to improve the yield of target chemicals, it is often necessary to increase the copy numbers of key genes or engineer the related metabolic pathways, which traditionally required time-consuming repetitive rounds of gene editing. With the development of gene-editing technologies such as meganucleases, TALENs, and the CRISPR/Cas system, multiplex genome editing has entered a period of rapid development to speed up cell factory optimization. Multi-copy insertion and removing bottlenecks in biosynthetic pathways can be achieved through gene integration and knockout, for which multiplexing can be accomplished by targeting repetitive sequences and multiple sites, respectively. Importantly, the development of the CRISPR/Cas system has greatly increased the speed and efficiency of multiplex editing. In this review, the various multiplex genome editing technologies in S. cerevisiae were summarized, and the principles, advantages, and the disadvantages were analyzed and discussed. Finally, the practical applications and future prospects of multiplex genome editing were discussed. KEY POINTS: • The development of multiplex genome editing in S. cerevisiae was summarized. • The pros and cons of various multiplex genome editing technologies are discussed. • Further prospects on the improvement of multiplex genome editing are proposed.

Application of the CRISPR/Cas system for genome editing in microalgae.

Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.

Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering.

In recent years, eukaryotic microorganisms have been widely applied to offer many solutions for everyday life and have come to play important roles in agriculture, food, health care, and the fine-chemicals industry. However, the complex genetic background and low homologous recombination efficiency have hampered the implementation of large-scale and high-throughput gene editing in many eukaryotic microorganisms. The low efficiency of homologous recombination (HR) not only makes the modification process labor-intensive but also completely precludes the application of many otherwise very useful genome editing techniques. Thus, increasing the efficiency of HR is clearly an enabling technology for basic research and gene editing in eukaryotic microorganisms. In this review, we summarize the current strategies for enhancing the efficiency of HR in eukaryotic microorganisms (particularly yeasts and filamentous fungi), list some small molecules and candidate genes associated with homologous and non-homologous recombination, and briefly discuss the further development prospects of these strategies.

Advancing metabolic engineering of Yarrowia lipolytica using the CRISPR/Cas system.

The oleaginous yeast Yarrowia lipolytica is widely used for the production of both bulk and fine chemicals, including organic acids, fatty acid-derived biofuels and chemicals, polyunsaturated fatty acids, single-cell proteins, terpenoids, and other valuable products. Consequently, it is becoming increasingly popular for metabolic engineering applications. Multiple gene manipulation tools including URA blast, Cre/LoxP, and transcription activator-like effector nucleases (TALENs) have been developed for metabolic engineering in Y. lipolytica. However, the low efficiency and time-consuming procedures involved in these methods hamper further research. The emergence of the CRISPR/Cas system offers a potential solution for these problems due to its high efficiency, ease of operation, and time savings, which can significantly accelerate the genomic engineering of Y. lipolytica. In this review, we summarize the research progress on the development of CRISPR/Cas systems for Y. lipolytica, including Cas9 proteins and sgRNA expression strategies, as well as gene knock-out/knock-in and repression/activation applications. Finally, the most promising and tantalizing future prospects in this area are highlighted.

CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art.

In recent years, a variety of genetic tools have been developed and applied to various filamentous fungi, which are widely applied in agriculture and the food industry. However, the low efficiency of gene targeting has for many years hampered studies on functional genomics in this important group of microorganisms. The emergence of CRISPR/Cas9 genome-editing technology has sparked a revolution in genetic research due to its high efficiency, versatility, and easy operation and opened the door for the discovery and exploitation of many new natural products. Although the application of the CRISPR/Cas9 system in filamentous fungi is still in its infancy compared to its common use in E. coli, yeasts, and mammals, the deep development of this system will certainly drive the exploitation of fungal diversity. In this review, we summarize the research progress on CRISPR/Cas9 systems in filamentous fungi and finally highlight further prospects in this area.

Advances in Flavonoid and Derivative Biosynthesis: Systematic Strategies for the Construction of Yeast Cell Factories.

Flavonoids, a significant group of natural polyphenolic compounds, possess a broad spectrum of pharmacological effects. Recent advances in the systematic metabolic engineering of yeast cell factories (YCFs) provide new opportunities for enhanced flavonoid production. Herein, we outline the latest research progress on typical flavonoid products in YCFs. Advanced engineering strategies involved in flavonoid biosynthesis are discussed in detail, including enhancing precursor supply, cofactor engineering, optimizing core pathways, eliminating competitive pathways, relieving transport limitations, and dynamic regulation. Additionally, we highlight the existing problems in the biosynthesis of flavonoid glucosides in yeast, such as endogenous degradation of flavonoid glycosides, substrate promiscuity of UDP-glycosyltransferases, and an insufficient supply of UDP-sugars, with summaries on the corresponding solutions. Discussions also cover other typical postmodifications like prenylation and methylation, and the recent biosynthesis of complex flavonoid compounds in yeast. Finally, a series of advanced technologies are envisioned, i.e., semirational enzyme engineering, ML/DL algorithn, and systems biology, with the aspiration of achieving large-scale industrial production of flavonoid compounds in the future.

Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi.

Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in F. fujikuroi, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5'-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.

Improving the productivity of gibberellic acid by combining small-molecule compounds-based targeting technology and transcriptomics analysis in Fusarium fujikuroi.

Gibberellic acid (GA3), produced industrially by Fusarium fujikuroi, stands as a crucial plant growth regulator extensively employed in the agriculture filed while limited understanding of the global metabolic network hinders researchers from conducting rapid targeted modifications. In this study, a small-molecule compounds-based targeting technology was developed to increase GA3 production. Firstly, various small molecules were used to target key nodes of different pathways and the result displayed that supplement of terbinafine improved significantly GA3 accumulation, which reached to 1.08 g/L. Subsequently, lipid and squalene biosynthesis pathway were identified as the key pathways influencing GA3 biosynthesis by transcriptomic analysis. Thus, the strategies including in vivo metabolic engineering modification and in vitro supplementation of lipid substrates were adopted, both contributed to an enhanced GA3 yield. Finally, the engineered strain demonstrated the ability to achieve a GA3 yield of 3.24 g/L in 5 L bioreactor when utilizing WCO as carbon source and feed.

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.

Advances in multi-enzyme co-localization strategies for the construction of microbial cell factory.

Biomanufacturing, driven by technologies such as synthetic biology, offers significant potential to advance the bioeconomy and promote sustainable development. It is anticipated to transform traditional manufacturing and become a key industry in future strategies. Cell factories are the core of biomanufacturing. The advancement of synthetic biology and growing market demand have led to the production of a greater variety of natural products and increasingly complex metabolic pathways. However, this progress also presents challenges, notably the conflict between natural product production and chassis cell growth. This conflict results in low productivity and yield, adverse side effects, metabolic imbalances, and growth retardation. Enzyme co-localization strategies have emerged as a promising solution. This article reviews recent progress and applications of these strategies in constructing cell factories for efficient natural product production. It comprehensively describes the applications of enzyme-based compartmentalization, metabolic pathway-based compartmentalization, and synthetic organelle-based compartmentalization in improving product titers. The article also explores future research directions and the prospects of combining multiple strategies with advanced technologies.

Improving Gibberellin GA3 Production with the Construction of a Genome-Scale Metabolic Model of Fusarium fujikuroi.

Liquid fermentation is the primary method for GA3 production usingFusarium fujikuroi. However, production capacity is limited due to unknown metabolic pathways. To address this, we constructed a genome-scale metabolic model (iCY1235) with 1753 reactions, 1979 metabolites, and 1235 genes to understand the GA3 regulation mechanisms. The model was validated by analyzing growth rates under different glucose uptake rates and identifying essential genes. We used the model to optimize fermentation conditions, including carbon sources and dissolved oxygen. Through the OptForce algorithm, we identified 20 reactions as targets. Overexpressing FFUJ_02053 and FFUJ_14337 resulted in a 37.5 and 75% increase in GA3 titers, respectively. These targets enhance carbon flux toward GA3 production. Our model holds promise for guiding the metabolic engineering of F. fujikuroi to achieve targeted overproduction. In summary, our study utilizes the iCY1235 model to understand GA3 regulation, optimize fermentation conditions, and identify specific targets for enhancing GA3 production through metabolic engineering.

High-Level Production of Patchoulol in Yarrowia lipolytica via Systematic Engineering Strategies.

Patchoulol is an important sesquiterpene alcohol with a strong and lasting odor, which has led to prominent applications in perfumes and cosmetics. In this study, systematic metabolic engineering strategies were adopted to create an efficient yeast cell factory for patchoulol overproduction. First, a baseline strain was constructed by selecting a highly active patchoulol synthase. Subsequently, the mevalonate precursor pool was expanded to boost patchoulol synthesis. Moreover, a method for downregulating squalene synthesis based on Cu2+-repressible promoter was optimized, which significantly improved the patchoulol titer by 100.9% to 124 mg/L. In addition, a protein fusion strategy resulted in a final titer of 235 mg/L in shake flasks. Finally, 2.864 g/L patchoulol could be produced in a 5 L bioreactor, representing a remarkable 1684-fold increase compared to the baseline strain. To our knowledge, this is the highest patchoulol titer reported so far.

Engineering the Lipid and Fatty Acid Metabolism in Yarrowia lipolytica for Sustainable Production of High Oleic Oils.

Oleic acid is widely applied in the chemical, material, nutritional, and pharmaceutical industries. However, the current production of oleic acid via high oleic plant oils is limited by the long growth cycle and climatic constraints. Moreover, the global demand for high oleic plant oils, especially the palm oil, has emerged as the driver of tropical deforestation causing tropical rainforest destruction, climate change, and biodiversity loss. In the present study, an alternative and sustainable strategy for high oleic oil production was established by reprogramming the metabolism of the oleaginous yeast Yarrowia lipolytica using a two-layer "push-pull-block" strategy. Specifically, the fatty acid synthesis pathway was first engineered to increase oleic acid proportion by altering the fatty acid profiles. Then, the content of storage oils containing oleic acid was boosted by engineering the synthesis and degradation pathways of triacylglycerides. The strain resulting from this two-layer engineering strategy produced the highest titer of high oleic microbial oil reaching 56 g/L with 84% oleic acid in fed-batch fermentation, representing a remarkable improvement of a 110-fold oil titer and 2.24-fold oleic acid proportion compared with the starting strain. This alternative and sustainable method for high oleic oil production shows the potential of substitute planting.

Engineering Yarrowia lipolytica to produce nutritional fatty acids: Current status and future perspectives.

Due to their vital physiological functions, nutritional fatty acids have great potential as nutraceutical food supplements for preventing an array of diseases such as inflammation, depression, arthritis, osteoporosis, diabetes and cancer. Microbial biosynthesis of fatty acids follows the trend of sustainable development, as it enables green, environmentally friendly and efficient production. As a natural oleaginous yeast, Yarrowia lipolytica is especially well-suited for the production of fatty acids. Moreover, it has a variety of genetic engineering tools and novel metabolic engineering strategies that make it a robust workhorse for the production of an array of value-added products. In this review, we summarize recent advances in metabolic engineering strategies for accumulating nutritional fatty acids in Y. lipolytica, including conjugated fatty acids and polyunsaturated fatty acids. In addition, the future prospects of nutritional fatty acid production using the Y. lipolytica platform are discussed in light of the current progress, challenges, and trends in this field. Finally, guidelines for future studies are also emphasized.

Engineering Yarrowia lipolytica to Produce Tailored Chain-Length Fatty Acids and Their Derivatives.

Microbial production of value-added chemicals derived from fatty acids is a sustainable alternative to petroleum-derived chemicals and unsustainable lipids from animals and plants. Fatty acids with different carbon chain lengths including short- (C20), with either even or odd number of carbons, have significantly different characteristics and wide applications in energy, material, medicine, and nutrition. Tailoring chain-length specificity of these compounds using metabolic engineering would be of high interest. Yarrowia lipolytica, as an oleaginous yeast, is a superior industrial chassis for the production of tailored chain-length fatty acids and their derivatives due to its hyper-oil-producing capability. In this Review, we cover metabolic engineering approaches that can lead to fatty acid chain length control in this microorganism. These approaches involve the manipulation of the fatty acid synthase, the thioesterase, the ß-oxidation pathway, the elongation and desaturation pathway, the polyketide synthase-like polyunsaturated fatty acid synthase pathway, and the odd-chain fatty acids synthesis pathway. Finally, we also discuss alternative strategies that can be used in the future to tailored chain-length control.