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.

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.

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.

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.

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.

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.

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 Development of Advanced Biotechnology in the Oleaginous Fungi for Arachidonic Acid Production.

Arachidonic acid is an essential ω-6 polyunsaturated fatty acid, which plays a significant role in cardiovascular health and neurological development, leading to its wide use in the food and pharmaceutical industries. Traditionally, ARA is obtained from deep-sea fish oil. However, this source is limited by season and is depleting the already threatened global fish stocks. With the rapid development of synthetic biology in recent years, oleaginous fungi have gradually attracted increasing attention as promising microbial sources for large-scale ARA production. Numerous advanced technologies including metabolic engineering, dynamic regulation of fermentation conditions, and multiomics analysis were successfully adapted to increase ARA synthesis. This review summarizes recent advances in the bioengineering of oleaginous fungi for ARA production. Finally, perspectives for future engineering approaches are proposed to further improve the titer yield and productivity of ARA.

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.

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.

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.

Advances in Metabolic Engineering Paving the Way for the Efficient Biosynthesis of Terpenes in Yeasts.

Terpenes are a large class of secondary metabolites with diverse structures and functions that are commonly used as valuable raw materials in food, cosmetics, and medicine. With the development of metabolic engineering and emerging synthetic biology tools, these important terpene compounds can be sustainably produced using different microbial chassis. Currently, yeasts such as Saccharomyces cerevisiae and Yarrowia lipolytica have received extensive attention as potential hosts for the production of terpenes due to their clear genetic background and endogenous mevalonate pathway. In this review, we summarize the natural terpene biosynthesis pathways and various engineering strategies, including enzyme engineering, pathway engineering, and cellular engineering, to further improve the terpene productivity and strain stability in these two widely used yeasts. In addition, the future prospects of yeast-based terpene production are discussed in light of the current progress, challenges, and trends in this field. Finally, guidelines for future studies are also emphasized.

Advances in synthetic biology tools paving the way for the biomanufacturing of unusual fatty acids using the Yarrowia lipolytica chassis.

Unusual fatty acids with special carbon chain length or functional groups have been extensively used in the chemical, material, nutraceutical, and pharmaceutical industries. The traditional sources of these valuable fatty acids mainly include animal or plant extraction and chemical synthesis, which are unsustainable and may cause considerable environmental issues. The advancement of synthetic biology tools has facilitated the microbial production of lipids enriched in these fatty acids. The oleaginous yeast Yarrowia lipolytica is considered an attractive industrial host suitable for the production of advanced unusual fatty acids due to its high intrinsic lipogenesis ability. In this review, we introduce the most cutting-edge developments in synthetic biology tools for Y. lipolytica, as well as the recent progress in harnessing these tools to engineer the Y. lipolytica chassis to overaccumulate various unusual fatty acids, including odd-chain fatty acids, conjugated fatty acids, polyunsaturated fatty acids, cyclopropane fatty acids, methyl-branched fatty acids, hydroxylated fatty acids, and medium chain fatty acids. In addition, the future prospects of the unusual fatty acids 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 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.

Advancing Yarrowia lipolytica as a superior biomanufacturing platform by tuning gene expression using promoter engineering.

Yarrowia lipolytica is recognized as an excellent non-conventional yeast in the field of biomanufacturing, where it is used as a host to produce oleochemicals, terpenes, organic acids, polyols and recombinant proteins. Consequently, metabolic engineering of this yeast is becoming increasingly popular to advance it as a superior biomanufacturing platform, of which promoters are the most basic elements for tuning gene expression. Endogenous promoters of Yarrowia lipolytica were reviewed, which are the basis for promoter engineering. The engineering strategies, such as hybrid promoter engineering, intron enhancement promoter engineering, and transcription factor-based inducible promoter engineering are described. Additionally, the applications of Yarrowia lipolytica promoter engineering to rationally reconstruct biosynthetic gene clusters and improve the genome-editing efficiency of the CRISPR-Cas systems were reviewed. Finally, research needs and future directions for promoter engineering are also discussed in this review.

CRISPR-Based Construction of a BL21 (DE3)-Derived Variant Strain Library to Rapidly Improve Recombinant Protein Production.

Escherichia coli BL21 (DE3) is the most widely used host for recombinant protein expression. However, not every protein can be highly expressed in BL21 (DE3), so individual optimization strategies are often required for different proteins, which is time-consuming and difficult to apply rapidly for industrial production. Constructing more hosts is a good choice to enrich protein expression selection. The expression level of T7 RNAP is the core control node of the pET expression system, so regulating its expression level is an effective way of improving the production of difficult-to-express proteins. Various BL21 (DE3)-derived variant hosts with different translation levels of T7 RNAP could be obtained by changing the ribosomal binding site (RBS) sequences of T7 RNAP in a genome. Here, a BL21 (DE3)-derived variant strain library with different RBS sequences of T7 RNAP was constructed using a base editor and CRISPR-Cas9. Notably, the CRISPR-Cas9 system combined with degenerate primers enabled the construction of an RBS library with 87.5% of the theoretical coverage in single editing, which is more convenient and efficient than the use of a base editor. The expression level of a target gene in the variant strain library ranged from 28 to 220% of the parental strain. Furthermore, a high-throughput host-screening platform for recombinant protein production was constructed, which enabled us to obtain the best expression host for certain target proteins in only 3 days. As a proof of concept, the production of all eight difficult-to-express proteins was greatly improved, including autolytic protein, membrane proteins, antimicrobial peptides, and hardly soluble proteins. Among them, the expression of glucose dehydrogenase in the best host exhibited a 298-fold increase compared to the parental strain. This strategy is simple and effective, requires no advanced equipment, and can be carried out in any laboratory.