Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

Metabolic engineering of Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids.

Odd-numbered fatty acids (FAs) have been widely used in nutrition, agriculture, and chemical industries. Recently, some studies showed that they could be produced from bacteria or yeast, but the products are almost exclusively odd-numbered long-chain FAs. Here we report the design and construction of two biosynthetic pathways in Saccharomyces cerevisiae for de novo production of odd-numbered medium-chain fatty acids (OMFAs) via ricinoleic acid and 10-hydroxystearic acid, respectively. The production of OMFAs was enabled by introducing a hydroxy fatty acid cleavage pathway, including an alcohol dehydrogenase from Micrococcus luteus, a Baeyer-Villiger monooxygenase from Pseudomonas putida, and a lipase from Pseudomonas fluorescens. These OMFA biosynthetic pathways were optimized by eliminating the rate-limiting step, generating heptanoic acid, 11-hydroxyundec-9-enoic acid, nonanoic acid, and 9-hydroxynonanoic acid at 7.83 mg/L, 9.68 mg/L, 9.43 mg/L and 13.48 mg/L, respectively. This work demonstrates the biological production of OMFAs in a sustainable manner in S. cerevisiae.

Multiplexed CRISPR-Based Nucleic Acid Detection Using a Single Cas Protein.

Thanks to its ease, speed, and sensitivity, CRISPR-based nucleic acid detection has been increasingly explored for molecular diagnostics. However, one of its major limitations is lack of multiplexing capability because the detection relies on the trans-cleavage activity of the Cas protein, which necessitates the use of multiple orthogonal Cas proteins for multiplex detection. Here we report the development of a multiplexed CRISPR-based nucleic acid detection system with single-nucleotide resolution using a single Cas protein (Cas12a). This method, termed as CRISPR-TMSD, integrates the toehold-mediated strand displacement (TMSD) reaction, and the cis-cleavage activity of the Cas protein and multiplexed detection are achieved using a single Cas protein owing to the use of target-specific reporters. A set of computational simulation toolkits was used to design the TMSD reporter, allowing for highly sensitive and specific identification of target sequences. In combination with the recombinase polymerase amplification (RPA), the detection limit can reach as low as 1 copy/µL. As proof of concept, CRISPR-TMSD was subsequently used to detect an oncogenic gene and SARS-CoV-2 RNA with a single-nucleotide resolution. This work represents a conceptually new strategy for designing a CRISPR-based diagnostic system and has great potential to expand the application of CRISPR-based diagnostics.

Engineering propionyl-CoA pools for de novo biosynthesis of odd-chain fatty acids in microbial cell factories.

Odd-chain fatty acids (OcFAs) and their derivatives have attracted great interest due to their wide applications in the food, pharmaceutical and petrochemical industries. Microorganisms can naturally de novo produce fatty acids (FAs), where mainly, even-chain with acetyl-CoA instead of odd-chain with propionyl-CoA is used as the primer. Usually, the absence of the precursor propionyl-CoA is considered the main reason that limits the efficient production of OcFAs. It is thus crucial to explore/evaluate/identify promising propionyl-CoA biosynthetic pathways to achieve large-scale biosynthesis of OcFAs. This review discusses the latest advances in microbial metabolism engineering toward producing propionyl-CoA and considers future research directions and challenges toward optimized production of OcFAs.

Microbial production of odd-chain fatty acids.

Odd-chain fatty acids (OcFAs) and their derivatives have attracted much attention due to their beneficial physiological effects and their potential to be alternatives to advanced fuels. However, cells naturally produce even-chain fatty acids (EcFAs) with negligible OcFAs. In the process of biosynthesis of fatty acids (FAs), the acetyl-CoA serves as the starter unit for EcFAs, and propionyl-CoA works as the starter unit for OcFAs. The lack of sufficient propionyl-CoA, the precursor, is usually regarded as the main restriction for large-scale bioproduction of OcFAs. In recent years, synthetic biology strategies have been used to modify several microorganisms to produce more propionyl-CoA that would enable an efficient biosynthesis of OcFAs. This review discusses several reported and potential metabolic pathways for propionyl-CoA biosynthesis, followed by advances in engineering several cell factories for OcFAs production. Finally, trends and challenges of synthetic biology driven OcFAs production are discussed.

De novo bio-production of odd-chain fatty acids in Saccharomyces cerevisiae through a synthetic pathway via 3-hydroxypropionic acid.

Odd-chain fatty acids (OCFAs) and their derivatives have attracted increasing attention due to their wide applications in the chemical, fuel, and pharmaceutical industry. However, most natural fatty acids are even-chained, and OCFAs are rare. In this work, a novel pathway was designed and established for de novo synthesis of OCFAs via 3-hydroxypropionic acid (3-HP) as the intermediate in Saccharomyces cerevisiae. First, the OCFAs biosynthesis pathway from 3-HP was confirmed, followed by an optimization of the precursor 3-HP. After combining these strategies, a de novo production of OCFAs at 74.8 mg/L was achieved, and the percentage of OCFAs in total lipids reached 20.3%, reaching the highest ratio of de novo-produced OCFAs. Of the OCFAs produced by the engineered strain, heptadecenoic acid (C17:1) and heptadecanoic acid (C17:0) accounted for 12.1% and 7.6% in total lipid content, respectively. This work provides a new and promising pathway for the de novo bio-production of OCFAs.

Directed evolution of a wax ester synthase for production of fatty acid ethyl esters in Saccharomyces cerevisiae.

Wax ester synthases (WSs) utilize a fatty alcohol and a fatty acyl-coenzyme A (activated fatty acid) to synthesize the corresponding wax ester. There is much interest in developing novel cell factories that can produce shorter esters, e.g., fatty acid ethyl esters (FAEEs), with properties similar to biodiesel in order to use these as transportation fuels. However, ethanol is a poor substrate for WSs, and this may limit the biosynthesis of FAEEs. Here, we implemented a random mutagenesis approach to enhance the catalytic efficiency of a WS from Marinobacter hydrocarbonoclasticus (MhWS2, encoded by the ws2 gene). Our selection system was based on FAEE formation serving as a detoxification mechanism for excessive oleate, where high WS activity was essential for a storage-lipid free yeast to survive. A random mutagenesis library of ws2 was used to transform the storage-lipid free yeast, and mutants could be selected by plating the transformants on oleate containing plates. The variants encoding WS with improved activity were sequenced, and an identified point mutation translated into the residue substitution at position A344 was discovered to substantially increase the selectivity of MhWS2 toward ethanol and other shorter alcohols. Structural modeling indicated that an A344T substitution might affect the alcohol selectivity due to change of both steric effects and polarity changes near the active site. This work not only provides a new WS variant with altered selectivity to shorter alcohols but also presents a new high-throughput selection system to isolate WSs with a desired selectivity. KEY POINTS: • The work provides WS variants with altered substrate preference for shorter alcohols • A novel method was developed for directed evolution of WS of desired selectivity.

Ultra-specific nucleic acid testing by target-activated nucleases.

Specific and sensitive detection of nucleic acids is essential to clinical diagnostics and biotechnological applications. Currently, amplification steps are necessary for most detection methods due to the low concentration of nucleic acid targets in real samples. Although amplification renders high sensitivity, poor specificity is prevalent because of the lack of highly accurate precise strategies, resulting in significant false positives and false negatives. Nucleases exhibit high catalytic activity for nucleic acid cleavage which is regulated in a programmable manner. This review focuses on the latest progress in nucleic acid testing methods based on the target-activated nucleases. It summarizes the property of enzymes such as CRISPR/Cas, Argonautes, and some gene-editing irrelevant nucleases, which have been leveraged to create highly specific and sensitive nucleic acid testing tools. We elaborate on recent advances in the field of nuclease-mediated DNA recognition techniques for nucleic acid detection, and discuss its future applications and challenges in molecular diagnostics.

Characterization of cross-species transcription and splicing from Penicillium to Saccharomyces cerevisiae.

Heterologous expression of eukaryotic gene clusters in yeast has been widely used for producing high-value chemicals and bioactive secondary metabolites. However, eukaryotic transcription cis-elements are still undercharacterized, and the cross-species expression mechanism remains poorly understood. Here we used the whole expression unit (including original promoter, terminator, and open reading frame with introns) of orotidine 5'-monophosphate decarboxylases from 14 Penicillium species as a showcase, and analyzed their cross-species expression in Saccharomyces cerevisiae. We found that pyrG promoters from the Penicillium species could drive URA3 expression in yeast, and that inefficient cross-species splicing of Penicillium introns might result in weak cross-species expression. Thus, this study demonstrates cross-species expression from Penicillium to yeast, and sheds light on the opportunities and challenges of cross-species expression of fungi expression units and gene clusters in yeast without refactoring for novel natural product discovery.

Ligand fishing with cellular membrane-coated cellulose filter paper: a new method for screening of potential active compounds from natural products.

Ligand fishing is a widely used approach for screening active compounds from natural products. Recently, cell membrane (CM) as affinity ligand has been applied in ligand fishing, including cell membrane chromatography (CMC) and CM-coated magnetic bead. However, these methods possess many weaknesses, including complicated preparation processes and time-consuming operation. In this study, cheap and easily available cellulose filter paper (CFP) was selected as carrier of CM and used to fabricate a novel CM-coated CFP (CMCFP) for the first time. The type of CFP was optimized according to the amount of immobilized protein, and the immobilization of CM onto CFP by the insertion and self-fusion process was verified by confocal imaging. The CMCFP exhibited good selectivity and stability and was used for fishing potentially active compounds from extracts of Angelica dahurica. Three potentially active compounds, including bergapten, pabulenol, and imperatorin, were fished out and identified. The traditional Chinese medicine systems pharmacology database and analysis platform was used to build an active compound-target protein network, and accordingly, the gamma-aminobutyric acid receptor subunit alpha-1 (GABRA1) was deduced as potential target of CM for the active compounds of Angelica dahurica. Molecular docking was performed to evaluate the interaction between active compounds and GABRA1, and bergapten was speculated as a new potentially active compound. Compared with other methods, the fishing assay based on CMCFP was more effective, simpler, and cheaper.

In vivo biosensors: mechanisms, development, and applications.

In vivo biosensors can recognize and respond to specific cellular stimuli. In recent years, biosensors have been increasingly used in metabolic engineering and synthetic biology, because they can be implemented in synthetic circuits to control the expression of reporter genes in response to specific cellular stimuli, such as a certain metabolite or a change in pH. There are many types of natural sensing devices, which can be generally divided into two main categories: protein-based and nucleic acid-based. Both can be obtained either by directly mining from natural genetic components or by engineering the existing genetic components for novel specificity or improved characteristics. A wide range of new technologies have enabled rapid engineering and discovery of new biosensors, which are paving the way for a new era of biotechnological progress. Here, we review recent advances in the design, optimization, and applications of in vivo biosensors in the field of metabolic engineering and synthetic biology.

A highly efficient single-step, markerless strategy for multi-copy chromosomal integration of large biochemical pathways in Saccharomyces cerevisiae.

Despite recent advances in genome editing capabilities for the model organism Saccharomyces cerevisiae, the chromosomal integration of large biochemical pathways for stable industrial production remains challenging. In this work, we developed a simple platform for high-efficiency, single-step, markerless, multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. In this Di-CRISPR (delta integration CRISPR-Cas) platform based on the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas), we specifically designed guide RNA sequences to target multiple delta sites in the yeast genome. The generation of double stranded breaks at the delta sites allowed simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With our newly developed Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. The simplicity and high efficiency of the Di-CRISPR platform could provide a superior alternative to high copy plasmids and would render this platform an invaluable tool for genome editing and metabolic engineering in S. cerevisiae.

Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica.

Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae.

Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration.

Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE (L641P) ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 (S1157A,S659A) ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE (L641P) , ACC1 (S1157A,S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and ß-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae.

Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters.

In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast.

Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway.

BACKGROUND: Due to an increasing demand of transportation fuels, a lower availability of cheap crude oil and a lack of sustainability of fossil fuels, a gradual shift from petroleum based fuels towards alternative and renewable fuel resources will be required in the near future. Fatty acid ethyl esters (FAEEs) have properties similar to current crude diesel and could therefore form an important contribution to the development of sustainable transportation fuels in future. It is important to develop novel cell factories for efficient production of FAEEs and their precursors. RESULTS: Here, a Saccharomyces cerevisiae cell factory expressing a heterologous wax ester synthase (ws2) from Marinobacter hydrocarbonoclasticus was used to produce FAEEs from ethanol and acyl-coenzyme A (acyl-CoA). The production of acyl-CoA requires large amounts of NADPH and acetyl-CoA. Therefore, two metabolic engineering strategies for improved provision of NADPH and acetyl-CoA were evaluated. First, the ethanol degradation pathway was employed to re-channel carbon flow towards the synthesis of acetyl-CoA. Therefore, ADH2 and ALD6 encoding, respectively, alcohol dehydrogenase and acetaldehyde dehydrogenase were overexpressed together with the heterologous gene acsSEL641P encoding acetyl-CoA synthetase. The co-overexpression of ADH2, ALD6 and acsSEL641P with ws2 resulted in 408 ± 270 µg FAEE gCDW-1, a 3-fold improvement. Secondly, for the expression of the PHK pathway two genes, xpkA and ack, both descending from Aspergillus nidulans, were co-expressed together with ws2 to catalyze, respectively, the conversion of xylulose-5-phosphate to acetyl phosphate and glyceraldehyde-3-phosphate and acetyl phosphate to acetate. Alternatively, ack was substituted with pta from Bacillus subtilis, encoding phosphotransacetylase for the conversion of acetyl phosphate to acetyl-CoA. Both PHK pathways were additionally expressed in a strain with multiple chromosomally integrated ws2 gene, which resulted in respectively 5100 ± 509 and 4670 ± 379 µg FAEE gCDW-1, an up to 1.7-fold improvement. CONCLUSION: Two different strategies for engineering of the central carbon metabolism for efficient provision of acetyl-CoA and NADPH required for fatty acid biosynthesis and hence FAEE production were evaluated and it was found that both the ethanol degradation pathway as well as the phosphoketolase pathway improve the yield of FAEEs.

Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast.

CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.

De Novo Genome Sequencing and Assembly of Rhodosporidium toruloides Strain Δdao1e.

Rhodosporidium toruloides is an important industrial strain with high oil production. Here, we report the de novo sequencing of R. toruloides strain Δdao1e, which was derived from R. toruloides strain ATCC 10657, by combining sequencing results from the Illumina and PacBio Sequel platforms.

Development and Perspective of Rhodotorula toruloides as an Efficient Cell Factory.

Rhodotorula toruloides is receiving significant attention as a novel cell factory because of its high production of lipids and carotenoids, fast growth and high cell density, as well as the ability to utilize a wide variety of substrates. These attractive traits of R. toruloides make it possible to become a low-cost producer that can be engineered for the production of various fuels and chemicals. However, the lack of understanding and genetic engineering tools impedes its metabolic engineering applications. A number of research efforts have been devoted to filling these gaps. This review focuses on recent developments in genetic engineering tools, advances in systems biology for improved understandings, and emerging engineered strains for metabolic engineering applications. Finally, future trends and barriers in developing R. toruloides as a cell factory are also discussed.

De Novo Biosynthesis of Free Vaccenic Acid with a Low Content of Oleic Acid in Saccharomyces cerevisiae.

Omega-7 (ω-7) fatty acids have potential application in the fields of nutraceutical, agricultural, and food industry. The natural ω-7 fatty acids are currently from plants or vegetable oils, which are unsustainable and limited by the availability of plant sources. Here, we developed an innovative biosynthetic route to produce vaccenic acid (C18:1 ω-7) while minimizing oleic acid (C18:1 ω-9) content in Saccharomyces cerevisiae. We have engineered S. cerevisiaeto produce C18:1 ω-7 by expressing a fatty acid elongase from Rattus norvegicus. To reduce the content of C18:1 ω-9, the endogenous desaturase Ole1 was replaced by the desaturase, which has specific activity on palmitoyl-coenzyme A (C16:0-CoA). Finally, the production of free C18:1 ω-7 was improved by optimizing the source of cytochrome b5 and overexpressing endoplasmic reticulum chaperones. After combining these strategies, the yield of C18:1 ω-7 was increased from 0 to 9.3 mg/g DCW and C18:1 ω-9 was decreased from 25.2 mg/g DCW to 1.6 mg/g DCW. This work shows a de novo synthetic pathway to produce the highest amount of free C18:1 ω-7 with a low content of C18:1 ω-9 in S. cerevisiae.