Abstract Agrobacterium -mediated transformation (AMT) is a critical method for genetic manipulation of non-model fungi, yet it remains a laborious and inefficient technique. When cocultured in acetosyringone-supplemented induction medium, Agrobacterium transfers DNA directly into yeast cells using its virulence machinery. Membrane filters are commonly used to support the co-culture of yeast and Agrobacterium on agar plates, however some reports demonstrate that these filters are unnecessary for specific yeast species. Here we confirm across diverse budding yeasts that membrane filters are not necessary for effective AMT. Concentrating the cells via centrifugation and “spotting” the cell pellet directly onto the induction medium proved effective. This reduces hands-on time to 15 minutes and eliminates filter cost. In the oleaginous yeast, Rhodotorula toruloides, this simplified method increases transformation efficiency by 66% to 2,500 transformants per 10 6 recipient cells. We further optimized the Agrobacterium : Rhodotorula cell ratio and culture resuspension volume to achieve more than 200,000 CFU per transformation representing a 2-3 fold improvement over previously implemented protocols. This spot-plating method was successfully applied to seven yeast species, including one for which genetic transformation has not previously been reported, Botryozyma nematodophila . This approach highlights the broad applicability of the spot-plating method across diverse yeast systems. Furthermore, this method could facilitate high-throughput transformation workflows that are critical for genome-scale functional studies.

Agrobacterium is not only a costly plant pathogen but is also an essential tool for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and complex transcriptional regulation make identification of the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here, we use a bottom-up synthetic approach to systematically engineer the tumor-inducing plasmid (pTi), wherein the majority of virulence machinery is encoded. Using a validated toolkit to control Agrobacterium gene expression in planta, we perform a quantitative dissection of AMT to investigate the contributions of critical vir-genes at different expression levels. We construct a synthetic pTi capable of transient plant and stable fungal transformation and characterize bottlenecks and solutions for complex polygenic synthetic pTi designs. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, laying the foundation for future engineering to establish full synthetic control over the critical process of AMT.

Putrescine plays a significant role in green food production and agriculture by promoting plant growth and enhancing crop quality. Its application reduces the reliance on chemical fertilizers and pesticides, thereby supporting the advancement of sustainable agricultural practices. This study achieved efficient production of putrescine in Serratia marcescens. S. marcescens has been extensively used to synthesize antimicrobial substances and express proteins, but its application has been limited by the lack of efficient genome-editing tools. This study presents a CRISPR-Cas9-based tool for gene editing in S. marcescens. A dual-plasmid system was constructed, incorporating an editing template into the plasmid pEdit with target-specific sgRNA. A stationary phase promoter was used to express Cas9 from Streptococcus pyogenes protein, avoiding the need for additional inducers and ensuring efficient one-step gene knockout and integration. The tool demonstrated over 80% editing efficiency across various S. marcescens strains and enabled successful single-base mutations. Using this tool, we enhanced putrescine production in S. marcescens HBQA7, optimizing the expression of ornithine decarboxylase from Clostridium aceticum DSM1496 with the P2 promoter and identifying the optimal integration site. Putrescine production reached 8.46 g/L within 48 h. This study significantly advances S. marcescens gene editing and metabolic engineering.

Iron-coupled fungal denitrification provides promising strategies for oligotrophic water remediation, although its molecular drivers remain unclear. This study applied scrap iron filings (SIFs) to enhance aerobic denitrification in three fungi (Penicillium sp. N8, Leptobacillium leptobactrum N9, Aspergillus sp. DH4) at C/N = 2, achieving 38.44-62.95% higher nitrate degradation with optimized nitrogen flux. Kinetics model suggested that culture conditions changes influenced competition between biotic and abiotic reduction in the SIFs-enhanced group. Transcriptomic analysis revealed that SIFs triggered fungal metabolic reprogramming to sustain survival and denitrification. Sugar transport channels were upregulated to enhance organic substrate transport (4-6 upregulated subunits) and activate the Embden-Meyerhof-Parnas pathway and tricarboxylic acid cycle to produce sufficient energy. Concurrently, glutamine synthetases facilitate ammonia assimilation into biomass, supporting long-term fungal viability. SIFs addition also stimulated the divalent iron transporters (0.10-0.97-fold) and facilitated iron transport to synthesize more iron-containing protein active sites in reinforcing electron transport chain and nitrogen metabolism. Crucially, SIFs optimized the allocation of electrons to denitrifying enzymes, minimizing nitrite accumulation. These adaptive mechanisms, including enhanced substrate utilization and metabolic coordination, address survival and denitrification challenges in oligotrophic conditions. This study elucidated fungal denitrification strategies under iron additions, providing a mechanistic foundation for optimizing sustainable nitrogen removal technologies.

Oleaginous yeast are remarkably versatile organisms, distinguished by their natural capacities to accumulate high levels of neutral lipids and broad substrate range. With recent growing interests in engineering non-model organisms as superior biomanufacturing platforms, oleaginous yeasts have emerged as promising chassis for oleochemicals, terpenoids, organic acids, and other valuable products. Advancement in systems biology along with genetic tool development have significantly expanded our understanding of the metabolism in these species and enabled engineering efforts to produce biofuels and bioproducts from diverse feedstocks. This review examines the latest technical advances in oleaginous yeast research toward sustainable biomanufacturing. We cover recent developments in systems biology-enabled metabolism understanding, genetic tools, feedstock utilization, and strain engineering approaches for the production of various valuable chemicals.

The oleaginous yeast Rhodosporidiumtoruloides has been exploited for many bioproducts, including several terpenes, owing to its oleaginous nature and biomass inhibitor tolerance. Here, we built upon previous (E)-α-bisabolene work by iteratively stacking the complete mevalonate pathway from Saccharomyces cerevisiae onto a multicopy bisabolene synthase parent strain. Metabolomics and proteomics verified heterologous pathway expression and identified metabolic bottlenecks at three intermediate steps, with candidate feedback-resistant mevalonate kinases screening improving titers 15%. Subtle differences in codon optimization, and preliminary attenuation of competing flux toward lipids resulted in 6-fold, 7-fold higher titers relative to controls, respectively. Media optimization led to modest improvements, with zinc identified as the most promising at 10% titer improvement. Ultimately, high-performance strains were cultivated with corn-stover biomass hydrolysate in microtiter plates at 300 g/L total sugar, achieving 20.8 g/L bisabolene, the highest reported titer in the literature. A 2 L glucose minimal medium bioreactor achieved 19.3 g/L bisabolene and a literature-high productivity of 0.11 g/L/h.

Rhodosporidium toruloides has gained increasing interests as a promising non-model host organism to produce a wide range of bioproducts from lignocellulosic biomass. Increasing the bioproduct titers, rates, and yields remains a challenge, largely due to a lack of robust and well-characterized genetic tools in this host. Here we developed an inducible flippase (FLP) and flippase recognition target (FRT) system that enables genetic manipulations without the need for additional selection markers. Synthetic inducible promoters were established, enabling regulation of FLP expression and efficient antibiotic marker removal. Leveraging this system, we engineered a strain to optimize 3-hydroxypropionic acid (3HP) production. Over four rounds of iterative genomic editing to resolve pathway bottlenecks, we achieved a 3HP titer of 69.4 g/L in fed-batch fermentation - the highest level reported in yeast from lignocellulosic hydrolysates. The engineered high 3HP producing strain offers a robust platform for sustainable bio-based chemical production from lignocellulosic feedstocks.

Industrialization drives economic and technological advances, with chemical synthesis and extraction playing pivotal roles in enabling large-scale chemical production. However, the rapid expansion of industrialization has resulted in pervasive pollution, posing significant threats to human health and ecological systems. This pollution is characterized by the widespread presence of synthetic pollutants, detectable across diverse environments, underscoring the urgent need for robust monitoring and targeted remediation. Herein, we review recent advances in biosensor development for pollutant monitoring and microbial bioremediation for environmental restoration. Furthermore, we discuss the engineering of microorganisms to degrade and convert diverse feedstocks, including agriculture residues and waste streams, into valuable biofuels, biomaterials, and natural products. Collectively, these approaches support the economy by minimizing waste, reducing pollution, and promoting manufacturing.

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomics and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4 g/L 3HP production. This represents one of the highest 3HP titers reported in yeast from lignocellulosic feedstocks. This work establishes R. toruloides as a host for 3HP production from lignocellulosic hydrolysate at high titers, and paves the way for further strain and process optimization towards enabling industrial production of 3HP in the future.

EDITORIAL article Front. Microbiol., 15 February 2023Sec. Systems Microbiology Volume 13 - 2022 | https://doi.org/10.3389/fmicb.2022.1109221

Members of Agrobacterium are costly plant pathogens while also essential tools for plant transformation. Though Agrobacterium -mediated transformation (AMT) has been heavily studied, its polygenic nature and its complex transcriptional regulation make identifying the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here we use a bottom-up synthetic approach to systematically refactor the tumor-inducing plasmid, wherein the majority of AMT machine components are encoded, into a minimal set of genes capable of plant and fungal transformation that is both controllable and orthogonal to its environment. We demonstrate that engineered vectors can be transferred to new heterologous bacteria, enabling them to transform plants. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, and may lead to the development of strains of bacteria more capable of transforming recalcitrant plant species of societal importance.

PDF file - 90KB, Supplementary Figure 3 Confirmation of the surface expression of LSECtin on stable B16-mock and B16-LSECtin transfectants by flow cytometry.

Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R. toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R. toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R. toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.

Abstract Background Lignocellulosic resources are promising feedstocks for the manufacture of bio-based products and bioenergy. However, the inherent recalcitrance of biomass to conversion into simple sugars currently hinders the deployment of advanced bioproducts at large scale. Lignin is a primary contributor to biomass recalcitrance as it protects cell wall polysaccharides from degradation and can inhibit hydrolytic enzymes via non-productive adsorption. Several engineering strategies have been designed to reduce lignin or modify its monomeric composition. For example, expression of bacterial 3-dehydroshikimate dehydratase (QsuB) in poplar trees resulted in a reduction in lignin due to redirection of metabolic flux toward 3,4-dihydroxybenzoate at the expense of lignin. This reduction was accompanied with remarkable changes in the pools of aromatic compounds that accumulate in the biomass. Results The impact of these modifications on downstream biomass deconstruction and conversion into advanced bioproducts was evaluated in the current study. Using ionic liquid pretreatment followed by enzymatic saccharification, biomass from engineered trees released more glucose and xylose compared to wild-type control trees under optimum conditions. Fermentation of the resulting hydrolysates using Rhodosporidium toruloides strains engineered to produce α-bisabolene, epi-isozizaene, and fatty alcohols showed no negative impact on cell growth and yielded higher titers of bioproducts (as much as + 58%) in the case of QsuB transgenics trees. Conclusion Our data show that low-recalcitrant poplar biomass obtained with the QsuB technology has the potential to improve the production of advanced bioproducts.

Plants and microbes share common metabolic pathways for producing a range of bioproducts that are potentially foundational to the future bioeconomy. However, in planta accumulation and microbial production of bioproducts have never been systematically compared on an economic basis to identify optimal routes of production. A detailed technoeconomic analysis of four exemplar compounds (4-hydroxybenzoic acid [4-HBA], catechol, muconic acid, and 2-pyrone-4,6-dicarboxylic acid [PDC]) is conducted with the highest reported yields and accumulation rates to identify economically advantaged platforms and breakeven targets for plants and microbes. The results indicate that in planta mass accumulation ranging from 0.1 to 0.3 dry weight % (dwt%) can achieve costs comparable to microbial routes operating at 40 to 55% of maximum theoretical yields. These yields and accumulation rates are sufficient to be cost competitive if the products are sold at market prices consistent with specialty chemicals ($20 to $50/kg). Prices consistent with commodity chemicals will require an order-of-magnitude-greater accumulation rate for plants and/or yields nearing theoretical maxima for microbial production platforms. This comparative analysis revealed that the demonstrated accumulation rates of 4-HBA (3.2 dwt%) and PDC (3.0 dwt%) in engineered plants vastly outperform microbial routes, even if microbial platforms were to reach theoretical maximum yields. Their recovery and sale as part of a lignocellulosic biorefinery could enable biofuel prices to be competitive with petroleum. Muconic acid and catechol, in contrast, are currently more attractive when produced microbially using a sugar feedstock. Ultimately, both platforms can play an important role in replacing fossil-derived products.

Fatty alcohols (FOHs) are important feedstocks in the chemical industry to produce detergents, cosmetics, and lubricants. Microbial production of FOHs has become an attractive alternative to production in plants and animals due to growing energy demands and environmental concerns. However, inhibition of cell growth caused by intracellular FOH accumulation is one major issue that limits FOH titers in microbial hosts. In addition, identification of FOH-specific exporters remains a challenge and previous studies towards this end are limited. To alleviate the toxicity issue, we exploited nonionic surfactants to promote the export of FOHs in Rhodosporidium toruloides, an oleaginous yeast that is considered an attractive next-generation host for the production of fatty acid-derived chemicals. Our results showed FOH export efficiency was dramatically improved and the growth inhibition was alleviated in the presence of small amounts of tergitol and other surfactants. As a result, FOH titers increase by 4.3-fold at bench scale to 352.6 mg/L. With further process optimization in a 2-L bioreactor, the titer was further increased to 1.6 g/L. The method we show here can potentially be applied to other microbial hosts and may facilitate the commercialization of microbial FOH production.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metabolic models can estimate intrinsic product yields for microbial factories, but such frameworks struggle to predict cell performance (including product titer or rate) under suboptimal metabolism and complex bioprocess conditions. On the other hand, machine learning, complementary to metabolic modeling necessitates large amounts of data. Building such a database for metabolic engineering designs requires significant manpower and is prone to human errors and bias. We propose an approach to integrate data-driven methods with genome scale metabolic model for assessment of microbial bio-production (yield, titer and rate). Using engineered E. coli as an example, we manually extracted and curated a data set comprising about 1200 experimentally realized cell factories from ~100 papers. We furthermore augmented the key design features (e.g., genetic modifications and bioprocess variables) extracted from literature with additional features derived from running the genome-scale metabolic model iML1515 simulations with constraints that match the experimental data. Then, data augmentation and ensemble learning (e.g., support vector machines, gradient boosted trees, and neural networks in a stacked regressor model) are employed to alleviate the challenges of sparse, non-standardized, and incomplete data sets, while multiple correspondence analysis/principal component analysis are used to rank influential factors on bio-production. The hybrid framework demonstrates a reasonably high cross-validation accuracy for prediction of E.coli factory performance metrics under presumed bioprocess and pathway conditions (Pearson correlation coefficients between 0.8 and 0.93 on new data not seen by the model).