DELLA proteins recruit the Mediator complex subunit MED15 to coactivate transcription in land plants.

DELLA proteins are negative regulators of the gibberellin response pathway in angiosperms, acting as central hubs that interact with hundreds of transcription factors (TFs) and regulators to modulate their activities. While the mechanism of TF sequestration by DELLAs to prevent DNA binding to downstream targets has been extensively documented, the mechanism that allows them to act as coactivators remains to be understood. Here, we demonstrate that DELLAs directly recruit the Mediator complex to specific loci in Arabidopsis, facilitating transcription. This recruitment involves DELLA amino-terminal domain and the conserved MED15 KIX domain. Accordingly, partial loss of MED15 function mainly disrupted processes known to rely on DELLA coactivation capacity, including cytokinin-dependent regulation of meristem function and skotomorphogenic response, gibberellin metabolism feedback, and flavonol production. We have also found that the single DELLA protein in the liverwort Marchantia polymorpha is capable of recruiting MpMED15 subunits, contributing to transcriptional coactivation. The conservation of Mediator-dependent transcriptional coactivation by DELLA between Arabidopsis and Marchantia implies that this mechanism is intrinsic to the emergence of DELLA in the last common ancestor of land plants.

Yeast Platforms for Production and Screening of Bioactive Derivatives of Rauwolscine.

Monoterpene indole alkaloids (MIAs) make up a highly bioactive class of metabolites produced by a range of tropical and subtropical plants. The corynanthe-type MIAs are a stereochemically complex subclass with therapeutic potential against a large number of indications including cancer, psychotic disorders, and erectile dysfunction. Here, we report yeast-based cell factories capable of de novo production of corynanthe-type MIAs rauwolscine, yohimbine, tetrahydroalstonine, and corynanthine. From this, we demonstrate regioselective biosynthesis of 4 fluorinated derivatives of these compounds and de novo biosynthesis of 7-chlororauwolscine by coexpression of a halogenase with the biosynthetic pathway. Finally, we capitalize on the ability of these cell factories to produce derivatives of these bioactive scaffolds to establish a proof-of-principle drug discovery pipeline in which the corynanthe-type MIAs are screened for bioactivity on human drug targets, expressed in yeast. In doing so, we identify antagonistic and agonistic behavior against the human adrenergic G protein-coupled receptors ADRA2A and ADRA2B, and the serotonergic receptor 5HT4b, respectively. This study thus demonstrates a proto-drug discovery pipeline for bioactive plant-inspired small molecules based on one-pot biocatalysis of natural and new-to-nature corynanthe-type MIAs in yeast.

A molecular toolkit of cross-feeding strains for engineering synthetic yeast communities.

Engineered microbial consortia often have enhanced system performance and robustness compared with single-strain biomanufacturing production platforms. However, few tools are available for generating co-cultures of the model and key industrial host Saccharomyces cerevisiae. Here we engineer auxotrophic and overexpression yeast strains that can be used to create co-cultures through exchange of essential metabolites. Using these strains as modules, we engineered two- and three-member consortia using different cross-feeding architectures. Through a combination of ensemble modelling and experimentation, we explored how cellular (for example, metabolite production strength) and environmental (for example, initial population ratio, population density and extracellular supplementation) factors govern population dynamics in these systems. We tested the use of the toolkit in a division of labour biomanufacturing case study and show that it enables enhanced and tuneable antioxidant resveratrol production. We expect this toolkit to become a useful resource for a variety of applications in synthetic ecology and biomanufacturing.

Exploring engineering strategies that enhance de novo production of exotic cyclopropane fatty acids in Saccharomyces cerevisiae.

Cycloalkanes have broad applications as specialty fuels, lubricants, and pharmaceuticals but are not currently available from renewable sources, whereas, production of microbial cycloalkanes such as cyclopropane fatty acids (CFA) has bottlenecks. Here, a systematic investigation was undertaken into the biosynthesis of CFA in Saccharomyces cerevisiae heterologously expressing bacterial CFA synthase. The enzyme catalyzes formation of a 3-membered ring in unsaturated fatty acids. Monounsaturated fatty acids in phospholipids (PL) are the site of CFA synthesis; precursor cis-Δ9 C16 and C18 fatty acids were enhanced through OLE1 and SAM2 overexpression which enhanced CFA in PL. CFA turnover from PL to storage in triacylglycerols (TAG) was achieved by phospholipase PBL2 overexpression and acyl-CoA synthase to increase flux to TAG. Consequently, CFA storage as TAG reached 12 mg g-1 DCW, improved 3-fold over the base strain and >22% of TAG was CFA. Our research improves understanding of cycloalkane biosynthesis in yeast and offers insights into processing of other exotic fatty acids.

Spontaneously established syntrophic yeast communities improve bioproduction.

Nutritional codependence (syntrophy) has underexplored potential to improve biotechnological processes by using cooperating cell types. So far, design of yeast syntrophic communities has required extensive genetic manipulation, as the co-inoculation of most eukaryotic microbial auxotrophs does not result in cooperative growth. Here we employ high-throughput phenotypic screening to systematically test pairwise combinations of auxotrophic Saccharomyces cerevisiae deletion mutants. Although most coculture pairs do not enter syntrophic growth, we identify 49 pairs that spontaneously form syntrophic, synergistic communities. We characterized the stability and growth dynamics of nine cocultures and demonstrated that a pair of tryptophan auxotrophs grow by exchanging a pathway intermediate rather than end products. We then introduced a malonic semialdehyde biosynthesis pathway split between different pairs of auxotrophs, which resulted in increased production. Our results report the spontaneous formation of stable syntrophy in S. cerevisiae auxotrophs and illustrate the biotechnological potential of dividing labor in a cooperating intraspecies community.

Valorisation of Biomass Waste for Sustainable Bioenergy and Biofuel Production.

Although the rapid development of industrialisation has brought great benefits to our societies, waste accumulation and energy depletion have inevitably grown to be critical issues in recent decades [...].

Modular Metabolic Engineering and Synthetic Coculture Strategies for the Production of Aromatic Compounds in Yeast.

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

Enhanced production of D-pantothenic acid in Corynebacterium glutamicum using an efficient CRISPR-Cpf1 genome editing method.

BACKGROUND: Corynebacterium glutamicum has industrial track records for producing a variety of valuable products such as amino acids. Although CRISPR-based genome editing technologies have undergone immense developments in recent years, the suicide-plasmid-based approaches are still predominant for C. glutamicum genome manipulation. It is crucial to develop a simple and efficient CRISPR genome editing method for C. glutamicum. RESULTS: In this study, we developed a RecombinAtion Prior to Induced Double-strand-break (RAPID) genome editing technology for C. glutamicum, as Cpf1 cleavage was found to disrupt RecET-mediated homologous recombination (HR) of the donor template into the genome. The RAPID toolbox enabled highly efficient gene deletion and insertion, and notably, a linear DNA template was sufficient for gene deletion. Due to the simplified procedure and iterative operation ability, this methodology could be widely applied in C. glutamicum genetic manipulations. As a proof of concept, a high-yield D-pantothenic acid (vitamin B5)-producing strain was constructed, which, to the best of our knowledge, achieved the highest reported titer of 18.62 g/L from glucose only. CONCLUSIONS: We developed a RecET-assisted CRISPR-Cpf1 genome editing technology for C. glutamicum that harnessed CRISPR-induced DSBs as a counterselection. This method is of great importance to C. glutamicum genome editing in terms of its practical applications, which also guides the development of CRISPR genome editing tools for other microorganisms.

Perception Research of Artificial Intelligence in Environmental Public Health Physiotherapy Nursing for the Elderly.

This perceptual study focuses on developing artificial intelligence for elderly care design. It analyses and discusses the role of artificial intelligence in elderly care and its application to physiotherapy care. Artificial intelligence, as an emerging disruptive technology, is releasing the enormous energy accumulated in the technological and industrial revolutions, profoundly transforming how humans produce, live, and think about the world. Economic development and social progress are significantly impacted by it, and it has a great deal of practicality and broad application scope. Although there is a basic consensus in the 18 public understanding of AI, there is still some ambiguity and misunderstanding, knowing what is happening without understanding why. As a result, it is necessary to systematize and gain a comprehensive understanding of this concept and its associated practices.

Metabolic Engineering Strategies for Improved Lipid Production and Cellular Physiological Responses in Yeast Saccharomyces cerevisiae.

Microbial lipids have been a hot topic in the field of metabolic engineering and synthetic biology due to their increased market and important applications in biofuels, oleochemicals, cosmetics, etc. This review first compares the popular hosts for lipid production and explains the four modules for lipid synthesis in yeast, including the fatty acid biosynthesis module, lipid accumulation module, lipid sequestration module, and fatty acid modification module. This is followed by a summary of metabolic engineering strategies that could be used for enhancing each module for lipid production. In addition, the efforts being invested in improving the production of value-added fatty acids in engineered yeast, such as cyclopropane fatty acid, ricinoleic acid, gamma linoleic acid, EPA, and DHA, are included. A discussion is further made on the potential relationships between lipid pathway engineering and consequential changes in cellular physiological properties, such as cell membrane integrity, intracellular reactive oxygen species level, and mitochondrial membrane potential. Finally, with the rapid development of synthetic biology tools, such as CRISPR genome editing tools and machine learning models, this review proposes some future trends that could be employed to engineer yeast with enhanced intracellular lipid production while not compromising much of its cellular health.

Engineering Biology of Yeast for Advanced Biomanufacturing.

Advanced biomanufacturing has been widely involved in people's daily life, such as the production of molecules used as pharmaceuticals, in foods and beverages, and in bio-fuels [...].

Metabolic engineering strategies to enable microbial utilization of C1 feedstocks.

One-carbon (C1) substrates are preferred feedstocks for the biomanufacturing industry and have recently gained attention owing to their natural abundance, low production cost and availability as industrial by-products. However, native pathways to utilize these substrates are absent in most biotechnologically relevant microorganisms. Recent advances in synthetic biology, genome engineering and laboratory evolution are enabling the first steps towards the creation of synthetic C1-utilizing microorganisms. Here, we briefly review the native metabolism of methane, methanol, CO2, CO and formate, and how these C1-utilizing pathways can be engineered into heterologous hosts. In addition, this review analyses the potential, the challenges and the perspectives of C1-based biomanufacturing.

Humidity Control Strategies for Solid-State Fermentation: Capillary Water Supply by Water-Retention Materials and Negative-Pressure Auto-controlled Irrigation.

Solid-state fermentation (SSF) has regained interest owing to its advantages in solid waste treatment and fermentation industries. However, heterogeneous heat and mass transfer are often caused by the absence of free water and noticeable water loss from microbial utilization and moisture evaporation in SSF. It is necessary to explore more effective ways to solve issues of water loss and water supplement in SSF based on online capillary water monitoring, because capillary water is the dominant form of water that is present and lost in substrate. Two novel capillary-water supply strategies were proposed, established and evaluated using three selected reference strains, including water-retention materials and negative-pressure auto-controlled irrigation (NPACI). This study employed superabsorbent polymer, a kind of water-retention material to enhance enzyme productivity with the most significant increase of 2.47 times. Moreover, the combination of NPACI and 0.1% superabsorbent polymers increased productivity by 2.80-fold, together with lowered gradients of temperature, moisture and products. Furthermore, a modified liquid-supply SSF was constructed through successful capillary water control by proposed humidity control strategies. This modified SSF system could address the shortcomings of inhomogeneous culture of traditional SSF.

Flow-cytometry-based physiological characterisation and transcriptome analyses reveal a mechanism for reduced cell viability in yeast engineered for increased lipid content.

BACKGROUND: Yeast has been the focus of development of cell biofactories for the production of lipids and interest in the field has been driven by the need for sustainably sourced lipids for use in a broad range of industrial applications. Previously, we reported a metabolic engineering strategy for enhanced lipid production in yeast which delivered high per-cell lipid but with low cell growth and compromised physiology. To investigate the relationship between lipid engineering and cellular physiological responses and to identify further metabolic engineering targets, we analysed transcriptomes and measured cell physiology parameters in engineered strains. RESULTS: In the engineering strategy, the central carbon pathway was reprogrammed to provide more precursors for lipid production and lipid accumulation and sequestration steps were enhanced through the expression of heterologous genes. Genes coding for enzymes within the pentose phosphate, beta-oxidation pathways, ATP and NADPH biosynthesis had lower transcript levels in engineered cells. Meanwhile, flow-cytometry analysis of fluorescent-dye stained cells showed the highest reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δψm) in cells with the highest lipid content, supporting the known relationship between mitochondrial activity and ROS generation. High intracellular ROS and low membrane integrity were not ameliorated by application of antioxidants. CONCLUSIONS: The limited intracellular energy supplies and the unbalanced redox environment could be regarded as targets for further lipid engineering, similarly for native lipid accumulation genes that were upregulated. Thus, lipid pathway engineering has an important effect on the central carbon pathway, directing these towards lipid production and sacrificing the precursors, energy and cofactor supply to satisfy homeostatic metabolic requirements.

Enhanced Production of High-Value Cyclopropane Fatty Acid in Yeast Engineered for Increased Lipid Synthesis and Accumulation.

The unique strained ring structure in cyclopropane fatty acids (CFA) conveys oxidative stability and lubricity to lipids. These attributes are highly valuable for industrial applications such as cosmetics and specialist lubrication but there is currently no commercial source of the lipid. Here, built on recently engineered strains of Saccharomyces cerevisiae, the authors have developed an efficient strategy for CFA production. Expression of the Escherichia coli cyclopropane fatty acid synthetase (Ec.CFAS) in the engineered yeast resulted in formation of cis-9,10-methylene-hexadecanoic and octadecanoic acids in both the phospholipid (PL) and triacylglycerol (TAG) fractions. CFA concentration in TAG of engineered yeast is 12 mg CFA g-1 DCW (fourfold above the strain expressing CFAS only). The yield of CFA increases from 13.2 to 68.3 mg L-1 , the highest reported in yeast, using a two-stage bioprocess strategy that separated cell growth from the lipid modification stage. Strategies for further improvement of this valuable lipid are proposed.

Raman spectroscopy as a tool for tracking cyclopropane fatty acids in genetically engineered Saccharomyces cerevisiae.

Cyclopropane fatty acids (CFAs) are a group of lipids with unique physical and chemical properties between those of saturated and monounsaturated fatty acids. The distinctive physicochemical characteristics of CFAs (e.g. oxidative stability, self-polymerization at high temperatures, etc.) results from the presence of a cyclopropane ring within their structure making them highly useful in industrial applications. CFAs are present in several species of plants and bacteria and are typically detected with standard lipid profiling techniques, such as gas or liquid chromatography. In this work we investigated several strains of S. cerevisiae, genetically modified to introduce the production of CFAs, in comparison to control strain using confocal Raman spectroscopy (CRS). The aim of our work was to demonstrate the potential of CRS not only to detect changes introduced due to the CFAs presence, but also to track CFAs within the cells. We present for the first time Raman and IR spectra of CFA standard (cis-9,10-methyleneoctadecanoic acid), completed with quantum chemical calculations and band assignment. We identified marker bands of CFA (e.g. 2992, 1222, 942 cm-1) attributed to the vibrations of the cyclopropyl ring. Furthermore, we analysed lipid bodies (LBs) from modified and control yeast using CRS imaging and identified multiple changes in size, number and composition of LBs from engineered strains. We observed a significant reduction in the degree of unsaturation of LBs using the ratio of bands located at 1660 cm-1 (ν(C[double bond, length as m-dash]C)) and 1448 cm-1 (δ(CH2)) in the modified cell lines. In addition, we were able to detect the presence of CFAs in LBs, using the established marker bands. CRS shows tremendous potential as technique to identify CFAs in lipid bodies providing a new way to track lipid production in genetically modified single yeast cells.

Metabolic engineering of lipid pathways in Saccharomyces cerevisiae and staged bioprocess for enhanced lipid production and cellular physiology.

Microbially produced lipids have attracted attention for their environmental benefits and commercial value. We have combined lipid pathway engineering in Saccharomyces cerevisiae yeast with bioprocess design to improve productivity and explore barriers to enhanced lipid production. Initially, individual gene expression was tested for impact on yeast growth and lipid production. Then, two base strains were prepared for enhanced lipid accumulation and stabilization steps by combining DGAT1, ΔTgl3 with or without Atclo1, which increased lipid content ~ 1.8-fold but reduced cell viability. Next, fatty acid (FA) biosynthesis genes Ald6-SEACSL641P alone or with ACC1** were co-expressed in base strains, which significantly improved lipid content (8.0% DCW, 2.6-fold than control), but severely reduced yeast growth and cell viability. Finally, a designed two-stage process convincingly ameliorated the negative effects, resulting in normal cell growth, very high lipid productivity (307 mg/L, 4.6-fold above control) and improved cell viability.

Single cell assessment of yeast metabolic engineering for enhanced lipid production using Raman and AFM-IR imaging.

BACKGROUND: Biodiesel is a valuable renewable fuel made from derivatized fatty acids produced in plants, animals, and oleaginous microbes. Of the latter, yeasts are of special interest due to their wide use in biotechnology, ability to synthesize fatty acids and store large amounts of triacylglycerols while utilizing non-food carbon sources. While yeast efficiently produce lipids, genetic modification and indeed, lipid pathway metabolic engineering, is usually required for cost-effective production. Traditionally, gas chromatography (GC) is used to measure fatty acid production and to track the success of a metabolic engineering strategy in a microbial culture; here we have employed vibrational spectroscopy approaches at population and single cell level of engineered yeast while simultaneously investigating metabolite levels in subcellular structures. RESULTS: Firstly, a strong correlation (r2 > 0.99) was established between Fourier transform infrared (FTIR) lipid in intact cells and GC analysis of fatty acid methyl esters in the differently engineered strains. Confocal Raman spectroscopy of individual cells carrying genetic modifications to enhance fatty acid synthesis and lipid accumulation revealed changes to the lipid body (LB), the storage organelle for lipids in yeast, with their number increasing markedly (up to tenfold higher); LB size was almost double in the strain that also expressed a LB stabilizing gene but considerable variation was also noted between cells. Raman spectroscopy revealed a clear trend toward reduced unsaturated fatty acid content in lipids of cells carrying more complex metabolic engineering. Atomic force microscopy-infrared spectroscopy (AFM-IR) analysis of individual cells indicated large differences in subcellular constituents between strains: cells of the most highly engineered strain had elevated lipid and much reduced carbohydrate in their cytoplasm compared with unmodified cells. CONCLUSIONS: Vibrational spectroscopy analysis allowed the simultaneous measurement of strain variability in metabolite production and impact on cellular structures as a result of different gene introductions or knockouts, within a lipid metabolic engineering strategy and these inform the next steps in comprehensive lipid engineering. Additionally, single cell spectroscopic analysis measures heterogeneity in metabolite production across microbial cultures under genetic modification, an emerging issue for efficient biotechnological production.

Functional assessment of plant and microalgal lipid pathway genes in yeast to enhance microbial industrial oil production.

As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks.

A novel combined pretreatment of ball milling and microwave irradiation for enhancing enzymatic hydrolysis of microcrystalline cellulose.

Microcrystalline cellulose (MCC) was performed as a mode substrate to investigate its potential ability of bioconversion in a novel combined pretreatment of ball milling (BM) and/or microwave irradiation (MWI). The variation of structure characteristics of MCC before/after pretreatment were investigated, including crystallinity index (CrI), size of crystal (S(C)), specific surface area (SSA) and degree of polymerization (DP). Their correlation with the rate of enzymatic hydrolysis was differentiated by an optimized equation which indicated the rate of hydrolysis was much more sensitive to CrI than SSA and DP. To achieve the same or higher glucose yield of BM for 3h and 6h, BM for 1h with MWI for 20min could save 54.8% and 77.40% energy consumption, respectively. Moreover, chemicals were not required in this process. It is concluded that the combination of BM and short time MWI is an environment-friendly, economical and effective approach to treat biomass.