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The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) functions in the shikimate pathway which is responsible for the production of aromatic amino acids and precursors of other essential secondary metabolites in all plant species. EPSPS is also the molecular target of the herbicide glyphosate. While some plant EPSPS variants have been characterized with reduced glyphosate sensitivity and have been used in biotechnology, the glyphosate insensitivity typically comes with a cost to catalytic efficiency. Thus, there exists a need to generate additional EPSPS variants that maintain both high catalytic efficiency and high glyphosate tolerance. Here, we create a synthetic yeast system to rapidly study and evolve heterologous EPSP synthases for these dual traits. Using known EPSPS variants, we first validate that our synthetic yeast system is capable of recapitulating growth characteristics observed in plants grown in varying levels of glyphosate. Next, we demonstrate that variants from mutagenesis libraries with distinct phenotypic traits can be isolated depending on the selection criteria applied. By applying strong dual-trait selection pressure, we identify a notable EPSPS mutant after just a single round of evolution that displays robust glyphosate tolerance (K i of nearly 1 mM) and improved enzymatic efficiency over the starting point (~2.5 fold). Finally, we show the crystal structure of corn EPSPS and the top resulting mutants and demonstrate that certain mutants have the potential to outperform previously reported glyphosate-resistant EPSPS mutants, such as T102I and P106S (denoted as TIPS), in whole-plant testing. Altogether, this platform helps explore the trade-off between glyphosate resistance and enzymatic efficiency., Competing Interests: Competing interests statement:A patent application has been filed relating to the mutants and selection system developed in this study. C.T.L. and S.G. are employees of Bayer Crop Science, a manufacturer of crop protection products. This is listed in the conflict of interest statement.

Electroactive organisms contribute to metal cycling, pollutant removal, and other redox-driven environmental processes. Studying this phenomenon in high-throughput is challenging since extracellular reduction cannot easily be traced back to its cell of origin within a mixed population. Here, we describe the development of a microdroplet emulsion system to enrich EET-capable organisms. We validated our system using the model electroactive organism S. oneidensis and describe the tooling of a benchtop microfluidic system for oxygen-limited processes. We demonstrated enrichment of EET-capable phenotypes from a mixed wild-type and EET-knockout population. As a proof-of-concept application, bacteria were collected from iron sedimentation from Town Lake (Austin, TX) and subjected to microdroplet enrichment. We observed an increase in EET-capable organisms in the sorted population that was distinct when compared to a population enriched in a bulk culture more closely akin to traditional techniques for discovering EET-capable bacteria. Finally, two bacterial species, C. sakazakii and V. fessus not previously shown to be electroactive, were further cultured and characterized for their ability to reduce channel conductance in an organic electrochemical transistor (OECT) and to reduce soluble Fe(III). We characterized two bacterial species not previously shown to exhibit electrogenic behavior. Our results demonstrate the utility of a microdroplet emulsions for identifying putative EET-capable bacteria and how this technology can be leveraged in tandem with existing methods., Competing Interests: Competing Interests The authors declare no competing interests.

Plastic waste is an environmental challenge, but also presents a biotechnological opportunity as a unique carbon substrate. With modern biotechnological tools, it is possible to enable both recycling and upcycling. To realize a plastics bioeconomy, significant intrinsic barriers must be overcome using a combination of enzyme, strain, and process engineering. This article highlights advances, challenges, and opportunities for a variety of common plastics., (© 2024. The Author(s).)

Halogen-containing molecules are ubiquitous in modern society and present unique chemical possibilities. As a whole, de novo fermentation and synthetic pathway construction for these molecules remain relatively underexplored and could unlock molecules with exciting new applications in industries ranging from textiles to agrochemicals to pharmaceuticals. Here, we report a mix-and-match co-culture platform to de novo generate a large array of halogenated tryptophan derivatives in Escherichia coli from glucose. First, we engineer E. coli to produce between 300 and 700 mg/L of six different halogenated tryptophan precursors. Second, we harness the native promiscuity of multiple downstream enzymes to access unexplored regions of metabolism. Finally, through modular co-culture fermentations, we demonstrate a plug-and-play bioproduction platform, culminating in the generation of 26 distinct halogenated molecules produced de novo including precursors to prodrugs 4-chloro- and 4-bromo-kynurenine and new-to-nature halogenated beta carbolines., (© 2024. The Author(s).)

Metabolic engineering and synthetic biology endeavors benefit from promoters that perform consistently (or robustly) with respect to cellular growth phase (exponential and stationary) and fermentation scale (microtiter plates, tubes, flasks, and bioreactors). However, nearly all endogenous promoters (especially in Saccharomyces cerevisiae ) do not perform in this manner. In this work, a hybrid promoter engineering strategy is leveraged to create novel synthetic promoters with robustness across these conditions. Using a multi-dimensional RNA-seq dataset, promoters with specific phase dependencies were identified. Fragments enriched with functional transcription factors were identified using MEME suite. These motif-containing fragments could impart activity dependence in the opposing condition. Specifically, we obtain two new promoters with high and consistent expression across both phases by increasing the exponential phase activity of the starting stationary-phase scaffold by 38 and 23-fold respectively. Further, we show that these promoters function consistently across various laboratory growth scales over time in a microtiter plate and in flasks. Overall, this work presents and validates a new strategy for engineering promoters in S. cerevisiae with high levels of expression that are robust to cellular growth phase and the scale of the culture., Competing Interests: HA is an Associate Editor for Synthetic and Systems Biotechnology and was not involved in the editorial review or the decision to publish this article. All authors declare that there are no competing interests., (© 2024 The Authors.)

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC 50  = 20 μM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations., (© 2024. The Author(s).)

Biological catalysts are emerging with the capability to depolymerize a wide variety of plastics. Improving and discovering these catalysts has leveraged a range of tools, including microbial ecology studies, high-throughput selections, and computationally guided mutational studies. In this review, we discuss the prospects for biological solutions to plastic recycling and upcycling with a focus on major advances in polyethylene terephthalate depolymerization, expanding the range of polymers with known biological catalysts, and the utilization of derived products. We highlight several recent improvements in enzymes and reaction properties, the discovery of a wide variety of novel plastic-depolymerizing biocatalysts, and how depolymerization products can be utilized in recycling and upcycling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Plant-derived phenylpropanoids, in particular phenylpropenes, have diverse industrial applications ranging from flavors and fragrances to polymers and pharmaceuticals. Heterologous biosynthesis of these products has the potential to address low, seasonally dependent yields hindering ease of widespread manufacturing. However, previous efforts have been hindered by the inherent pathway promiscuity and the microbial toxicity of key pathway intermediates. Here, in this study, we establish the propensity of a tripartite microbial co-culture to overcome these limitations and demonstrate to our knowledge the first reported de novo phenylpropene production from simple sugar starting materials. After initially designing the system to accumulate eugenol, the platform modularity and downstream enzyme promiscuity was leveraged to quickly create avenues for hydroxychavicol and chavicol production. The consortia was found to be compatible with Engineered Living Material production platforms that allow for reusable, cold-chain-independent distributed manufacturing. This work lays the foundation for further deployment of modular microbial approaches to produce plant secondary metabolites., (© 2023. The Author(s).)

More than 200 million tons of plant oils and animal fats are produced annually worldwide from oil, crops, and the rendered animal fat industry. Triacylglycerol, an abundant energy-dense compound, is the major form of lipid in oils and fats. While oils or fats are very important raw materials and functional ingredients for food or related products, a significant portion is currently diverted to or recovered as waste. To significantly increase the value of waste oils or fats and expand their applications with a minimal environmental footprint, microbial biomanufacturing is presented as an effective strategy for adding value. Though both bacteria and yeast can be engineered to use oils or fats as the biomanufacturing feedstocks, the yeast Yarrowia lipolytica is presented as one of the most attractive platforms. Y. lipolytica is oleaginous, generally regarded as safe, demonstrated as a promising industrial producer, and has unique capabilities for efficient catabolism and bioconversion of lipid substrates. This review summarizes the major challenges and opportunities for Y. lipolytica as a new biomanufacturing platform for the production of value-added products from oils and fats. This review also discusses relevant cellular and metabolic engineering strategies such as fatty acid transport, fatty acid catabolism and bioconversion, redox balances and energy yield, cell morphology and stress response, and bioreaction engineering. Finally, this review highlights specific product classes including long-chain diacids, wax esters, terpenes, and carotenoids with unique synthesis opportunities from oils and fats in Y. lipolytica., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Engineered living materials (ELMs) fabricated by encapsulating microbes in hydrogels have great potential as bioreactors for sustained bioproduction. While long-term metabolic activity has been demonstrated in these systems, the capacity and dynamics of gene expression over time is not well understood. Thus, we investigate the long-term gene expression dynamics in microbial ELMs constructed using different microbes and hydrogel matrices. Through direct gene expression measurements of engineered E. coli in F127-bisurethane methacrylate (F127-BUM) hydrogels, we show that inducible, input-responsive genetic programs in ELMs can be activated multiple times and maintained for multiple weeks. Interestingly, the encapsulated bacteria sustain inducible gene expression almost 10 times longer than free-floating, planktonic cells. These ELMs exhibit dynamic responsiveness to repeated induction cycles, with up to 97% of the initial gene expression capacity retained following a subsequent induction event. We demonstrate multi-week bioproduction cycling by implementing inducible CRISPR transcriptional activation (CRISPRa) programs that regulate the expression of enzymes in a pteridine biosynthesis pathway. ELMs fabricated from engineered S. cerevisiae in bovine serum albumin (BSA) - polyethylene glycol diacrylate (PEGDA) hydrogels were programmed to express two different proteins, each under the control of a different chemical inducer. We observed scheduled bioproduction switching between betaxanthin pigment molecules and proteinase A in S. cerevisiae ELMs over the course of 27 days under continuous cultivation. Overall, these results suggest that the capacity for long-term genetic expression may be a general property of microbial ELMs. This work establishes approaches for implementing dynamic, input-responsive genetic programs to tailor ELM functions for a wide range of advanced applications., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: James M. Carothers is an advisor to Wayfinder Biosciences., (© 2023 Published by Elsevier Ltd.)

Since 2011, a massive influx of pelagic brown algae Sargassum has invaded coastlines causing environmental and economic disaster. Valorizing this plentiful macroalgae can present much needed economic relief to the areas affected. Here the production of biodiesel and a high-value alginate stream using Sargassum biomass collected from the coast of Quintana Roo, Mexico is reported. Biomass was pretreated via AEA (Alginate Extraction Autohydrolysis) and enzymatic saccharification via fungal Solid State Fermentation, releasing 7 g/L total sugars. The sugar mixture was fermented using engineered Yarrowia lipolytica resulting in 0.35 g/L total lipid titer at the lab tube scale. Additionally, the capability of extracting 0.3875 g/g DW of a high-value, purified alginate stream from this material is demonstrated. The findings presented here are promising and suggest an opportunity for the optimization and scale up of a biodiesel production biorefinery for utilization of Sargassum seaweeds during seasons of high invasion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Production of biofuels and biochemicals from xylose using yeast cell factory is of great interest for lignocellulosic biorefinery. Our previous studies revealed that a natural yeast isolate Saccharomyces cerevisiae YB-2625 has superior xylose-fermenting ability. Through integrative omics analysis, NGG1 , which encodes a transcription regulator as well as a subunit of chromatin modifying histone acetyltransferase complexes was revealed to regulate xylose metabolism. Deletion of NGG1 in S. cerevisiae YRH396h, which is the haploid version of the recombinant yeast using S. cerevisiae YB-2625 as the host strain, improved xylose consumption by 28.6%. Comparative transcriptome analysis revealed that NGG1 deletion down-regulated genes related to mitochondrial function, TCA cycle, ATP biosynthesis, respiration, as well as NADH generation. In addition, the NGG1 deletion mutant also showed transcriptional changes in amino acid biosynthesis genes. Further analysis of intracellular amino acid content confirmed the effect of NGG1 on amino acid accumulation during xylose utilization. Our results indicated that NGG1 is one of the core nodes for coordinated regulation of carbon and nitrogen metabolism in the recombinant S. cerevisiae. This work reveals novel function of Ngg1p in yeast metabolism and provides basis for developing robust yeast strains to produce ethanol and biochemicals using lignocellulosic biomass., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cheng, Wang, Sun, Tang, Bai, Alper and Zhao.)

The growing burden of waste disposal coupled with natural resource scarcity has renewed interest in the remediation, valorization, and/or repurposing of waste. Traditional approaches such as composting, anaerobic digestion, use in fertilizers or animal feed, or incineration for energy production extract very little value out of these waste streams. In contrast, waste valorization into fuels and other biochemicals via microbial fermentation is an area of growing interest. In this review, we discuss microbial valorization of nonconventional, aqueous waste streams such as food processing effluents, wastewater streams, and other industrial wastes. We categorize these waste streams as carbohydrate-rich food wastes, lipid-rich wastes, and other industrial wastes. Recent advances in microbial valorization of these nonconventional waste streams are highlighted, along with a discussion of the specific challenges and opportunities associated with impurities, nitrogen content, toxicity, and low productivity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Plastic waste poses an ecological challenge 1-3 and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling 4 . Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste 5 , and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products 6-10 . Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics 11 . Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives 12 between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

A variety of yeast species have been considered ideal hosts for metabolic engineering to produce value-added chemicals, including the model organism Saccharomyces cerevisiae , as well as non-conventional yeasts including Yarrowia lipolytica , Kluyveromyces marxianus , and Pichia pastoris . However, the metabolic capacity of these microbes is not simply dictated or implied by genus or species alone. Within the same species, yeast strains can display distinct variations in their phenotypes and metabolism, which affect the performance of introduced pathways and the production of interesting compounds. Moreover, it is unclear how this metabolic potential corresponds to function upon rewiring these organisms. These reports thus point out a new consideration for successful metabolic engineering, specifically: what are the best strains to utilize and how does one achieve effective metabolic engineering? Understanding such questions will accelerate the host selection and optimization process for generating yeast cell factories. In this review, we survey recent advances in studying yeast strain variations and utilizing non-type strains in pathway production and metabolic engineering applications. Additionally, we highlight the importance of employing portable methods for metabolic rewiring to best access this metabolic diversity. Finally, we conclude by highlighting the importance of considering strain diversity in metabolic engineering applications.

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis., (© 2021. The Author(s).)

Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes ( Escherichia coli , Saccharomyces cerevisiae , and Yarrowia lipolytica ), biosensors from two organisms ( E. coli and S. cerevisiae ), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion., Competing Interests: Competing interest statement: X.L. and F.F.C. are employees of Sphere Fluidics Limited.

Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing., (© 2021. The Author(s).)

Since long ago, pelagic Sargassum mats have been known to be abundant in the Sargasso Sea, where they provide habitat to diverse organisms. However, over the last few years, massive amounts of pelagic Sargassum have reached the coast of several countries in the Caribbean and West Africa, causing economic and environmental problems. Aiming for lessening the impacts of the blooms, governments and private companies remove the seaweeds from the shore, but this process results expensive. The valorization of this abundant biomass can render Sargassum tides into an economic opportunity and concurrently solve their associated environmental problems. Despite the diverse fields where algae have found applications and the relevance of this recurrent situation, Sargassum biomass remains without large scale applications. Therefore, this review aims to present the potential uses of these algae, identifying the limitations that must be assessed to effectively valorize this bioresource. Due to the constraints identified for each of the presented applications, it is concluded that a biorefinery approach should be developed to effectively valorize this abundant biomass. However, there is an urgent need for investigations focusing on holopelagic Sargassum to be able to truly valorize this seaweed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Synthetic biology holds great promise for addressing global needs. However, most current developments are not immediately translatable to 'outside-the-lab' scenarios that differ from controlled laboratory settings. Challenges include enabling long-term storage stability as well as operating in resource-limited and off-the-grid scenarios using autonomous function. Here we analyze recent advances in developing synthetic biological platforms for outside-the-lab scenarios with a focus on three major application spaces: bioproduction, biosensing, and closed-loop therapeutic and probiotic delivery. Across the Perspective, we highlight recent advances, areas for further development, possibilities for future applications, and the needs for innovation at the interface of other disciplines.

Food additives and colorants are extensively used in the food industry to improve food quality and safety during processing, storage and packing. Sourcing of these molecules is predominately through three means: extraction from natural sources, chemical synthesis, and bio-production, with the first two being the most utilized. However, growing demands for sustainability, safety and "natural" products have renewed interest in using bio-based production methods. Likewise, the move to more cultured foods and meat alternatives requires the production of new additives and colorants. The production of bio-based food additives and colorants is an interdisciplinary research endeavor and represents a growing trend in future food. To highlight the potential of microbial hosts for food additive and colorant production, we focus on current advances for example molecules based on their utilization stage and bio-production yield as follows: (I) approved and industrially produced with high titers; (II) approved and produced with decent titers (in the g/L range), but requiring further engineering to reduce production costs; (III) approved and produced with very early stage titers (in the mg/L range); and (IV) new/potential candidates that have not been approved but can be sourced through microbes. Promising approaches, as well as current challenges and future directions will also be thoroughly discussed for the bioproduction of these food additives and colorants., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity. As a result, these systems are not easily portable or reusable for on-demand protein production applications. In this study, we employ Bioproduced Proteins On Demand (Bio-POD), a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable, on-demand production and secretion of medium- and high-molecular weight proteins (in this case, SEAP, α-amylase, and anti-HER2). The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration. The hydrogel platform described here is the first hydrogel immobilization using a P. pastoris system to produce recombinant proteins of this breadth. These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production., Competing Interests: None of the authors have any conflicts of interest with the work presented., (© 2021 [The Author/The Authors].)

Lactams, cyclic carboxamide acids, are important building blocks as monomers for the manufacture of polyamides (nylons), with a market of millions of tons per year. Likewise, their non-natural building blocks, straight chain ω-amino acids, also have a wide range of applications as pharmaceuticals, therapeutic agents, and precursors to other platform chemicals. Current industrial lactam production requires petrochemically-derived routes that involve the use of harsh chemicals and reaction conditions. Microbial production provides a more sustainable method for production, from cost effective renewable resources. This review provides an extensive overview of progress toward the microbial production of lactams, particularly 4C butyrolactam, 5C valerolactam and 6C caprolactam, and their ω-amino acid precursors. Additionally, recent advances in the field as well as proposed microbial production pathways will be discussed, as well as future perspectives for the production of these important bulk chemicals., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model of Saccharomyces cerevisiae' s metabolic network. When coupled with a CRISPR-dCas9-based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches., Competing Interests: The authors declare no competing interest.

Background: Resveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction., Results: Here we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibition-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroG fbr ) and chorismate mutase/prephenate dehydrogenase (tyrA fbr ) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1 S659A,S1157A ) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved., Conclusions: This is first study that demonstrates the use of a synthetic E. coli-S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.

Most mono- and co-culture bioprocess applications rely on large-scale suspension fermentation technologies that are not easily portable, reusable, or suitable for on-demand production. Here, we describe a hydrogel system for harnessing the bioactivity of embedded microbes for on-demand small molecule and peptide production in microbial mono-culture and consortia. This platform bypasses the challenges of engineering a multi-organism consortia by utilizing a temperature-responsive, shear-thinning hydrogel to compartmentalize organisms into polymeric hydrogels that control the final consortium composition and dynamics without the need for synthetic control of mutualism. We demonstrate that these hydrogels provide protection from preservation techniques (including lyophilization) and can sustain metabolic function for over 1 year of repeated use. This approach was utilized for the production of four chemical compounds, a peptide antibiotic, and carbohydrate catabolism by using either mono-cultures or co-cultures. The printed microbe-laden hydrogel constructs' efficiency in repeated production phases, both pre- and post-preservation, outperforms liquid culture.

Ionic liquids show promise for deconstruction of lignocellulosic biomass prior to fermentation. Yet, imidazolium ionic liquids (IILs) can be toxic to microbes even at concentrations present after recovery. Here, we show that dominant overexpression of an Ilt1p homolog (encoded by YlILT1/YALI0C04884) from the IIL-tolerant yeast Yarrowia lipolytica confers an improvement in 1-ethyl-3-methylimidazolium acetate tolerance in Saccharomyces cerevisiae compared to the endogenous Ilt1p (ScILT1/YDR090C). We subsequently enhance tolerance in S. cerevisiae through directed evolution of YlILT1 using growth-based selection, leading to identification of mutants that grow in up to 3.5% v/v ionic liquid. Lastly, we demonstrate that strains expressing YlILT1 variants demonstrate improved growth rate and ethanol production in the presence of residual IIL. This shows that dominant overexpression of a heterologous protein (wild type or evolved) from an IIL-tolerant yeast can increase tolerance in S. cerevisiae at concentrations relevant to bioethanol production from IIL-treated biomass.

Although only 6 years old, the CRISPR system has blossomed into a tool for rapid, on-demand genome engineering and gene regulation in Saccharomyces cerevisiae. In this minireview, we discuss fundamental CRISPR technologies, tools to improve the efficiency and capabilities of gene targeting, and cutting-edge techniques to explore gene editing and transcriptional regulation at genome scale using pooled approaches. The focus is on applications to metabolic engineering with topics including development of techniques to edit the genome in multiplex, tools to enable large numbers of genetic modifications using pooled single-guide RNA libraries and efforts to enable programmable transcriptional regulation using endonuclease-null Cas enzymes., (© FEMS 2019.)

Fatty alcohols are important industrial oleochemicals with broad applications and a growing market. Here, we sought to engineer Yarrowia lipolytica to serve as a renewable source of fatty alcohols (specifically hexadecanol, heptadecanol, octadecanol, and oleyl alcohol) directly from glucose. Through screening four fatty acyl-CoA reductase (FAR) enzyme variants across two engineered background strains, we identified that MhFAR enabled the highest production. Further strain engineering, fed-batch flask cultivation, and extractive fermentation improved the fatty alcohol titer to 1.5 g/L. Scale-up of this strain in a 2L bioreactor led to 5.8 g/L total fatty alcohols at an average yield of 36 mg/g glucose with a maximum productivity of 39 mg/L hr. Finally, we utilized this fatty alcohol reductase to generate a customized fatty alcohol, linolenyl alcohol, from α-linolenic acid. Overall, this work demonstrates Y. lipolytica is a robust chassis for diverse fatty alcohol production and highlights the capacity to obtain high titers and yields from a purely minimal media formulation directly from glucose without the need for complex additives., (© 2019 The Authors.)

Due to unsustainable petroleum supply and poor yields from plant and animal sources, there is an increased effort to engineer microbial hosts for renewable chemical production. When compared to microbes such as Escherichia coli, fungal hosts show advantages due to their natural robust tolerance for industrial fermentation. Synthetic biology has focused on implementing heterologous pathways and manipulating native flux towards downstream products to achieve industrial productivity, titers, and yields. This review highlights recent advances in the engineering of yeasts for fuels and other molecules. As the field progresses, strains with improved productivities will begin to compete with the traditional chemical-based industrial approaches., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Metabolic engineering requires fine-tuned gene expression for most pathway optimization applications. To develop a suitable suite of promoters, traditional bacterial promoter engineering efforts have focused on modifications to the core region, especially the -10 and -35 regions, of native promoters. Here, we demonstrate an alternate, unexplored route of promoter engineering through randomization of the UP element of the promoter-a region that contacts the alpha subunit carboxy-terminal domain instead of the sigma subunit of the RNA polymerase holoenzyme. Through this work, we identify five novel UP element sequences through library-based searches in Escherichia coli. The resulting elements were used to activate the E. coli core promoter, rrnD promoter, to levels on par and higher than the prevalent strong bacterial promoter, OXB15. These relative levels of expression activation were transferrable when applied upstream of alternate core promoter sequences, including rrnA and rrnH . This work thus presents and validates a novel strategy for bacterial promoter engineering with transferability across varying core promoters and potential for transferability across bacterial species.

Metabolic engineering allows for the rewiring of basic metabolism to overproduce both native and non-native metabolites. Among these biomolecules, nutraceuticals have received considerable interest due to their health-promoting or disease-preventing properties. Likewise, microbial engineering efforts to produce these value-added nutraceuticals overcome traditional limitations of low yield from extractions and complex chemical syntheses. This review covers current strategies of metabolic engineering employed for the production of a few key nutraceuticals with selecting polyunsaturated fatty acids, polyphenolic compounds, carotenoids and non-proteinogenic amino acids as exemplary molecules. We focus on the use of both mono-culture and co-culture strategies to produce these molecules of interest. In each of these cases, metabolic engineering efforts are enabling rapid production of these molecules.

Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair, genetic recombination, and gene expression. Here, we present CRISPR-PIN, a CRISPR/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae . This approach utilizes a cohesin-dockerin interaction between dCas9 and a perinuclear protein. In doing so, we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery. We demonstrate the utility of this approach for two applications: the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci. In both cases, we obtain results on par with prior reports using traditional, more cumbersome genetic systems. Thus, CRISPR-PIN offers the opportunity for future studies of chromosome biology and gene localization.

Yarrowia lipolytica , known to accumulate lipids intracellularly, lacks the cellulolytic enzymes needed to break down solid biomass directly. This study aimed to evaluate the potential metabolic burden of expressing core cellulolytic enzymes in an engineered high lipid-accumulating strain of Y. lipolytica . Three fungal cellulases, Talaromyces emersonii - Trichoderma reesei chimeric cellobiohydrolase I (chimeric-CBH I), T. reesei cellobiohydrolase II (CBH II), and T. reesei endoglucanase II (EG II) were expressed using three constitutive strong promoters as a single integrative expression block in a recently engineered lipid hyper-accumulating strain of Y. lipolytica (HA1). In yeast extract-peptone-dextrose (YPD) medium, the resulting cellulase co-expressing transformant YL165-1 had the chimeric-CBH I, CBH II, and EG II secretion titers being 26, 17, and 132 mg L -1 , respectively. Cellulase co-expression in YL165-1 in culture media with a moderate C/N ratio of ∼4.5 unexpectedly resulted in a nearly two-fold reduction in cellular lipid accumulation compared to the parental control strain, a sign of cellular metabolic drain. Such metabolic drain was ameliorated when grown in media with a high C/N ratio of 59 having a higher glucose utilization rate that led to approximately twofold more cell mass and threefold more lipid production per liter culture compared to parental control strain, suggesting cross-talk between cellulase and lipid production, both of which involve the endoplasmic reticulum (ER). Most importantly, we found that the chemical chaperone, trimethylamine N-oxide dihydride increased glucose utilization, cell mass and total lipid titer in the transformants, suggesting further amelioration of the metabolic drain. This is the first study examining lipid production in cellulase-expressing Y. lipolytica strains under various C/N ratio media and with a chemical chaperone highlighting the metabolic complexity for developing robust, cellulolytic and lipogenic yeast strains.

Yarrowia lipolytica is an industrial host organism with incredible potential for metabolic engineering. However, the genetic tools and capacities in this host lag behind those of conventional counterparts. In this study, we sought to increase the transformation efficiency of Y. lipolytica by creating a simple protocol using electroporation. Efficiency was increased by optimizing wash buffers, pre-culture growth time, OD600 of competent cells, voltage, competent cell volume, DNA concentration, and recovery time. The outcome of these optimizations led to a simple protocol with maximum linear fragment transformation efficiency of 1.6 × 104 transformants per μg DNA and 2.8 × 104 transformants per μg DNA for episomal plasmid transformation. The protocol presented here is superior to other Y. lipolytica transformation protocols as it requires no lengthy pretreatment and no required carrier DNA to achieve efficiencies on par with, or exceeding, previously reported methods.

Utilization of renewable and low-cost lignocellulosic wastes has received major focus in industrial lactic acid production. The use of high solid loadings in biomass pretreatment potentially offers advantages over low solid loadings including higher lactic acid concentration with decreased production and capital costs. In this study, an isolated Enterococcus faecalis SI with optimal temperature 42 °C was used to produce optically pure L-lactic acid (> 99%) from enzyme-saccharified hydrolysates of acid-impregnated steam explosion (AISE)-treated plywood chips. The L-lactic acid production increased by 10% at 5 L scale compared to the similar fermentation scheme reported by Wee et al. The fermentation with a high solid loading of 20% and 35% (w/v) AISE-pretreated plywood chips had been successfully scaled up to process development unit scale (100 L) and pilot scale (9 m 3 ), respectively. This is the first report of pilot-scale lignocellulosic lactic acid fermentation by E. faecalis with high lactic acid titer (nearly 92 g L -1 ) and yield (0.97 kg kg -1 ). Therefore, large-scale L-lactic acid production by E. faecalis SI shows the potential application for industries.

Competing Interests: The authors declare no conflict of interest.

Traditional synthesis of biodiesel competes with food sources and has limitations with storage, particularly due to limited oxidative stability. Microbial synthesis of lipids provides a platform to produce renewable fuel with improved properties from various renewable carbon sources. Specifically, biodiesel properties can be improved through the introduction of a cyclopropane ring in place of a double bond. In this study, we demonstrate the production of C19 cyclopropanated fatty acids in the oleaginous yeast Yarrowia lipolytica through the heterologous expression of the Escherichia coli cyclopropane fatty acid synthase. Ultimately, we establish a strain capable of 3.03 ± 0.26 g/L C19 cyclopropanated fatty acid production in bioreactor fermentation where this functionalized lipid comprises over 32% of the total lipid pool. This study provides a demonstration of the flexibility of lipid metabolism in Y. lipolytica to produce specialized fatty acids.

Background: Saccharomyces cerevisiae is widely studied for production of biofuels and biochemicals. To improve production efficiency under industrially relevant conditions, coordinated expression of multiple genes by manipulating promoter strengths is an efficient approach. It is known that gene expression is highly dependent on the practically used environmental conditions and is subject to dynamic changes. Therefore, investigating promoter activities of S. cerevisiae under different culture conditions in different time points, especially under stressful conditions is of great importance., Results: In this study, the activities of various promoters in S. cerevisiae under stressful conditions and in the presence of xylose were characterized using yeast enhanced green fluorescent protein (yEGFP) as a reporter. The stresses include toxic levels of acetic acid and furfural, and high temperature, which are related to fermentation of lignocellulosic hydrolysates. In addition to investigating eight native promoters, the synthetic hybrid promoter P 3xC-TEF1 was also evaluated. The results revealed that P TDH3 and the synthetic promoter P 3xC-TEF1 showed the highest strengths under almost all the conditions. Importantly, these two promoters also exhibited high stabilities throughout the cultivation. However, the strengths of P ADH1 and P PGK1 , which are generally regarded as 'constitutive' promoters, decreased significantly under certain conditions, suggesting that cautions should be taken to use such constitutive promoters to drive gene expression under stressful conditions. Interestingly, P HSP12 and P HSP26 were able to response to both high temperature and acetic acid stress. Moreover, P HSP12 also led to moderate yEGFP expression when xylose was used as the sole carbon source, indicating that this promoter could be used for inducing proper gene expression for xylose utilization., Conclusion: The results here revealed dynamic changes of promoter activities in S. cerevisiae throughout batch fermentation in the presence of inhibitors as well as using xylose. These results provide insights in selection of promoters to construct S. cerevisiae strains for efficient bioproduction under practical conditions. Our results also encouraged applications of synthetic promoters with high stability for yeast strain development.

Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, Yarrowia lipolytica ) can overcome such production limitations owing to a natural propensity for high flux through acetyl-CoA. By exploring three distinct metabolic engineering strategies for acetyl-CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a β-oxidation-related overexpression ( PEX10 ) can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials., Competing Interests: The authors declare no conflict of interest.

Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner. However, most industrial applications have focused on only a small subset of elements from the periodic table, centered around carbon biochemistry. This review aims to illustrate the expanse of chemical elements that can currently (and potentially) be integrated into useful products using cellular systems. Specifically, we describe recent advances in expanding the cellular scope to include the halogens, selenium and the metalloids, and a variety of metal incorporations. These examples range from small molecules, heteroatom-linked uncommon elements, and natural products to biomining and nanotechnology applications. Collectively, this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology.

Within the Design-Build-Test Cycle for strain engineering, rapid product detection and selection strategies remain challenging and limit overall throughput. Here we summarize a wide variety of modalities that transduce chemical concentrations into easily measured absorbance, luminescence, and fluorescence signals. Specifically, we cover protein-based biosensors (including transcription factors), nucleic acid-based biosensors, coupled enzyme reactions, bioorthogonal chemistry, and fluorescent and chromogenic dyes and substrates as modalities for detection. We focus on the use of these methods for strain engineering and enzyme discovery and conclude with remarks on the current and future state of biosensor development for application in the metabolic engineering field., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Synthetic biology and metabolic engineering seek to re-engineer microbes into "living foundries" for the production of high value chemicals. Through a "design-build-test" cycle paradigm, massive libraries of genetically engineered microbes can be constructed and tested for metabolite overproduction and secretion. However, library generation capacity outpaces the rate of high-throughput testing and screening. Well plate assays are flexible but with limited throughput, whereas droplet microfluidic techniques are ultrahigh-throughput but require a custom assay for each target. Here we present RNA-aptamers-in-droplets (RAPID), a method that greatly expands the generality of ultrahigh-throughput microfluidic screening. Using aptamers, we transduce extracellular product titer into fluorescence, allowing ultrahigh-throughput screening of millions of variants. We demonstrate the RAPID approach by enhancing production of tyrosine and secretion of a recombinant protein in Saccharomyces cerevisiae by up to 28- and 3-fold, respectively. Aptamers-in-droplets affords a general approach for evolving microbes to synthesize and secrete value-added chemicals.Screening libraries of genetically engineered microbes for secreted products is limited by the available assay throughput. Here the authors combine aptamer-based fluorescent detection with droplet microfluidics to achieve high throughput screening of yeast strains engineered for enhanced tyrosine or streptavidin production.

Directed evolution remains a powerful, highly generalizable approach for improving the performance of biological systems. However, implementations in eukaryotes rely either on in vitro diversity generation or limited mutational capacities. Here we synthetically optimize the retrotransposon Ty1 to enable in vivo generation of mutant libraries up to 1.6 × 10 7  l -1 per round, which is the highest of any in vivo mutational generation approach in yeast. We demonstrate this approach by using in vivo-generated libraries to evolve single enzymes, global transcriptional regulators and multi-gene pathways. When coupled to growth selection, this approach enables in vivo continuous evolution (ICE) of genes and pathways. Through a head-to-head comparison, we find that ICE libraries yield higher-performing variants faster than error-prone PCR-derived libraries. Finally, we demonstrate transferability of ICE to divergent yeasts, including Kluyveromyces lactis and alternative S. cerevisiae strains. Collectively, this work establishes a generic platform for rapid eukaryotic-directed evolution across an array of target cargo.

Polylactic acid (PLA) is an important renewable polymer, but current processes for producing its precursor, lactic acid, suffer from process inefficiencies related to the use of bacterial hosts. Therefore, improving the capacity of Saccharomyces cerevisiae to produce lactic acid is a promising approach to improve industrial production of lactic acid. As one such improvement required, the lactic acid tolerance of yeast must be significantly increased. To enable improved tolerance, we employed an RNAi-mediated genome-wide expression knockdown approach as a means to rapidly identify potential genetic targets. In this approach, several gene knockdown targets were identified which confer increased acid tolerance to S. cerevisiae BY4741, of which knockdown of the ribosome-associated chaperone SSB1 conferred the highest increase (52%). This target was then transferred into a lactic acid-overproducing strain of S. cerevisiae CEN.PK in the form of a knockout and the resulting strain demonstrated up to 33% increased cell growth, 58% increased glucose consumption, and 60% increased L-lactic acid production. As SSB1 contains a close functional homolog SSB2 in yeast, this result was counterintuitive and may point to as-yet-undefined functional differences between SSB1 and SSB2 related to lactic acid production. The final strain produced over 50 g/L of lactic acid in under 60 h of fermentation.

Gene expression control is critical to increase production of recombinant proteins, fine-tune metabolic pathways and reliably express synthetic pathways. The importance of transcriptional control seems to be most important in eukaryotic systems. In this review, we highlight recent developments in the field of transcriptional engineering with an emphasis on the opportunities and challenges. We discuss the engineering of 'parts' that influence transcriptional throughput including promoters, terminators, and transcription factors as well as the genetic context of the expression cassette. While great strides have been made in the area, the robustness of these parts has been largely untested. This review highlights the importance of considering robustness in biological systems and the limitations that current synthetic parts possess., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Synthetic promoters, especially minimally sized, are critical for advancing fungal synthetic biology. Fungal promoters often span hundreds of base pairs, nearly ten times the amount of bacterial counterparts. This size limits large-scale synthetic biology efforts in yeasts. Here we address this shortcoming by establishing a methodical workflow necessary to identify robust minimal core elements that can be linked with minimal upstream activating sequences to develop short, yet strong yeast promoters. Through a series of library-based synthesis, analysis and robustness tests, we create a set of non-homologous, purely synthetic, minimal promoters for yeast. These promoters are comprised of short core elements that are generic and interoperable and 10 bp UAS elements that impart strong, constitutive function. Through this methodology, we are able to generate the shortest fungal promoters to date, which can achieve high levels of both inducible and constitutive expression with up to an 80% reduction in size.