Search

Synthetic biology encompasses many kinds of ideas and techniques with the common theme of creating something novel. The industrially relevant microorganism, Ralstonia eutropha (also known as Cupriavidus necator), has long been a subject of metabolic engineering efforts to either enhance a product it naturally makes (polyhydroxyalkanoate) or produce novel bioproducts (e.g., biofuels and other small molecule compounds). Given the metabolic versatility of R. eutropha and the existence of multiple molecular genetic tools and techniques for the organism, development of a synthetic biology toolkit is underway. This toolkit will allow for novel, user-friendly design that can impart new capabilities to R. eutropha strains to be used for novel application. This article reviews the different synthetic biology techniques currently available for modifying and enhancing bioproduction in R. eutropha. KEY POINTS: • R. eutropha (C. necator) is a versatile organism that has been examined for many applications. • Synthetic biology is being used to design more powerful strains for bioproduction. • A diverse synthetic biology toolkit is being developed to enhance R. eutropha's capabilities., (© 2024. The Author(s).)

Erythritol is a four-carbon sugar alcohol naturally produced by microorganisms as an osmoprotectant. As a new sugar substitute, erythritol has recently been popular on the ingredient market because of its unique nutritional characteristics. Even though the history of erythritol biosynthesis dates from the turn of the twentieth century, scientific advancement has lagged behind other polyols due to the relative complexity of making it. In recent years, biosynthetic methods for erythritol have been rapidly developed due to an increase in market demand, a better understanding of metabolic pathways, and the rapid development of genetic engineering tools. This paper reviews the history of industrial strain development and focuses on the underlying mechanism of high erythritol production by strains gained through screening or mutagenesis. Meanwhile, we highlight the metabolic pathway knowledge of erythritol biosynthesis in microorganisms and summarize the metabolic engineering and research progress on critical genes involved in different stages of the synthetic pathway. Lastly, we talk about the still-contentious issues and promising future research directions that will help break the erythritol production bottleneck and make erythritol production greener and more sustainable.

Synthetic biology can play a major role in the development of sustainable industrial biotechnology processes. However, the development of economically viable production processes is currently hampered by the limited availability of host organisms that can be engineered for a specific production process. To date, standard hosts such as Escherichia coli and Saccharomyces cerevisiae are often used as starting points for process development since parts and tools allowing their engineering are readily available. However, their suboptimal metabolic background or impaired performance at industrial scale for a desired production process, can result in increased costs associated with process development and/or disappointing production titres. Building a universal and portable gene expression system allowing genetic engineering of hosts across the bacterial domain would unlock the bacterial domain for industrial biotechnology applications in a highly standardized manner and, doing so, render industrial biotechnology processes more competitive compared to the current polluting chemical processes. This review gives an overview of a selection of bacterial hosts highly interesting for industrial biotechnology based on both their metabolic and process optimization properties. Moreover, the requirements and progress made so far to enable universal, standardized, and portable gene expression across the bacterial domain is discussed., 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. Published by Elsevier Inc.)

Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Vibrio natriegens is emerging as a promising host for biotechnology which is basically due to the remarkable intrinsic properties such as the exceptionally high growth and substrate consumption rates. The facultatively anaerobic marine bacterium possesses a versatile metabolism, is able to utilize a variety of substrates as carbon and energy sources and is easy to handle in the lab. These features initiated the rapid development of genetic tools and resulted in extensive engineering of production strains in the past years. Although recent examples illustrate the potential of V. natriegens for biotechnology, a comprehensive understanding of the metabolism and its regulation is still lacking but essential to exploit the full potential of this bacterium. In this review, we summarize the current knowledge on the physiological traits and the genomic organization, provide an overview of the available genetic engineering tools and recent advances in metabolic engineering of V. natriegens. Finally, we discuss the obstacles which have to be overcome in order to establish V. natriegens as industrial production host., (© 2021 The Author(s).)

¹³C metabolic flux analysis (¹³C-MFA) enables the precise quantification of intracellular metabolic reaction rates by analyzing the distribution of mass isotopomers of proteinogenic amino acids or intracellular metabolites through ¹³C labeling experiments. ¹³C-MFA has received much attention as it can help systematically understand cellular metabolic characteristics, guide metabolic engineering design and gain mechanistic insights into pathophysiology. This article reviews the advances of ¹³C-MFA in the past 30 years and discusses its potential and future perspective, with a focus on its application in industrial biotechnology and biomedicine.

As an important bulk chemical, benzoic acid is currently manufactured from nonrenewable feedstocks under harsh conditions. Although there are natural pathways for biosynthesis of benzoic acid, they are often inefficient and subjected to complex regulation. Here we develop a nonnatural enzyme cascade to efficiently produce benzoic acid from styrene or biogenic L-phenylalanine under mild conditions. By using a modular approach, two whole-cell catalysts Escherichia coli LZ305 and LZ325 are engineered for coexpressing seven and nine enzymes for production of 133-146 mM benzoic acid (16.2-17.8 g/L aq ) with 88-97% conversion via seven- and nine-step cascade biotransformation of styrene and L-phenylalanine, respectively. The seven-step cascade represents a formal high-yielding biocatalytic oxidative cleavage of styrene, and the nine-step cascade showcases the high efficiency of extended nonnatural enzyme cascades. Moreover, to achieve benzoic acid production directly from low-cost renewable glycerol, a novel coupled fermentation-biotransformation process was developed by integration of fermentative production of L-phenylalanine with in situ biotransformation to give 63-70 mM benzoic acid (7.6-8.6 g/L aq ), which is around 20 times higher than the reported value via a natural pathway. The coupled fermentation-biotransformation process could be generally applicable to microbial production of growth-inhibitory or toxic chemicals in high concentrations., (© 2020 Wiley Periodicals LLC.)

Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production., Competing Interests: Declarations of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Metabolic engineering allows development of microbial strains efficiently producing chemicals and materials, but it requires much time, effort, and cost to make the strains industrially competitive. Systems metabolic engineering, which integrates tools and strategies of systems biology, synthetic biology, and evolutionary engineering with traditional metabolic engineering, has recently been used to facilitate development of high-performance strains. The past decade has witnessed this interdisciplinary strategy continuously being improved toward the development of industrially competitive overproducer strains. In this article, current trends in systems metabolic engineering including tools and strategies are reviewed, focusing on recent developments in selection of host strains, metabolic pathway reconstruction, tolerance enhancement, and metabolic flux optimization. Also, future challenges and prospects are discussed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The goal of strain optimization is to create high-performance strains producing compounds of interest at a high titer, yield, and volumetric productivity. The effectiveness of strain optimization relies on methodologies used to aid optimization of native or novel pathways within cells. Many different factors, including mRNA abundance, protein abundance, and enzyme activity/stability, will contribute to the strain performance, which is not often evident by simply monitoring product titers. To this end, targeted proteomics tools utilizing LC-MS-MS (liquid chromatography coupled with tandem mass spectrometry) have recently been developed and can monitor protein levels at great sensitivities. Here, we describe all relevant aspects when developing proteomics tools for strain optimization.

The non-conventional oleaginous yeast Yarrowia lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Moving our society towards a bioeconomy requires efficient and sustainable microbial production of chemicals and fuels. Rhodotorula (Rhodosporidium) toruloides is a yeast that naturally synthesizes substantial amounts of specialty chemicals and has been recently engineered to (i) enhance its natural production of lipids and carotenoids, and (ii) produce novel industrially relevant compounds. The use of R. toruloides by companies and research groups has exponentially increased in recent years as a result of recent improvements in genetic engineering techniques and the availability of multiomics information on its genome and metabolism. This review focuses on recent engineering approaches in R. toruloides for bioproduction and explores its potential as a biotechnological chassis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Since its discovery 60 years ago, Corynebacterium glutamicum has evolved into a workhorse for industrial biotechnology. Traditionally well known for its remarkable capacity to produce amino acids, this Gram-positive soil bacterium, has become a flexible, efficient production platform for various bulk and fine chemicals, materials, and biofuels. The central turnstile of all these achievements is our excellent understanding of its metabolism and physiology. This knowledge base, together with innovative systems metabolic engineering concepts, which integrate systems and synthetic biology into strain engineering, has upgraded C. glutamicum into one of the most successful industrial microorganisms in the world.

In the modern era of next-generation genomics and Fourth Industrial Revolution, there is a growing demand for translational research that brings about not only impactful research but also potential commercialisation of R- and D-based products. Advancement of metabolic engineering and synthetic biology has put forward a viable and innovative biotechnological platform for bioproduct development especially using microbial chassis. In this chapter, readers will be introduced on the concepts of metabolic engineering, synthetic biology and microbial chassis and the applications of these biological engineering (BioE) components in the advancement of industrial and agricultural biotechnology. Main strategies in employing BioE platform are discussed especially for waste bioconversion and value-added product development. More importantly, this chapter will also discuss current endeavours in integrating systems and synthetic biology for microbial production of natural products by introducing flavonoid biosynthesis genes of Polygonum minus, a medicinally important tropical plant in engineered yeast.

Ketocarotenoids are high-value pigments used commercially across multiple industrial sectors as colorants and supplements. Chemical synthesis using petrochemical-derived precursors remains the production method of choice. Aquaculture is an example where ketocarotenoid supplementation of feed is necessary to achieve product viability. The biosynthesis of ketocarotenoids, such as canthaxanthin, phoenicoxanthin, or astaxanthin in plants is rare. In the present study, complex engineering of the carotenoid pathway has been performed to produce high-value ketocarotenoids in tomato fruit (3.0 mg/g dry weight). The strategy adopted involved pathway extension beyond β-carotene through the expression of the β-carotene hydroxylase ( CrtZ ) and oxyxgenase ( CrtW ) from Brevundimonas sp. in tomato fruit, followed by β-carotene enhancement through the introgression of a lycopene β-cyclase (β- Cyc ) allele from a Solanum galapagense background. Detailed biochemical analysis, carried out using chromatographic, UV/VIS, and MS approaches, identified the predominant carotenoid as fatty acid (C14:0 and C16:0) esters of phoenicoxanthin, present in the S stereoisomer configuration. Under a field-like environment with low resource input, scalability was shown with the potential to deliver 23 kg of ketocarotenoid/hectare. To illustrate the potential of this "generally recognized as safe" material with minimal, low-energy bioprocessing, two independent aquaculture trials were performed. The plant-based feeds developed were more efficient than the synthetic feed to color trout flesh (up to twofold increase in the retention of the main ketocarotenoids in the fish fillets). This achievement has the potential to create a new paradigm in the renewable production of economically competitive feed additives for the aquaculture industry and beyond., Competing Interests: The authors declare no conflict of interest.

Engineering microbes with new properties is an important goal in industrial engineering, to establish biological factories for production of biofuels, commodity chemicals and pharmaceutics. But engineering microbes to produce new compounds with high yield remains a major challenge toward economically viable production. Incorporating several modern approaches, including synthetic and systems biology, metabolic modeling and regulatory rewiring, has proven to significantly advance industrial strain engineering. This review highlights how comparative genomics can also facilitate strain engineering, by identifying novel genes and pathways, regulatory mechanisms and genetic background effects for engineering. We discuss how incorporating comparative genomics into the design-test-learn cycle of strain engineering can provide novel information that complements other engineering strategies., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

It is a common misconception in whole-cell biocatalysis to refer to an enzyme as the biocatalyst, thereby neglecting the structural and metabolic framework provided by the cell. Here, the low whole-cell biocatalyst stability, that is, the stability of specific biocatalyst activity, in a process for the terminal oxyfunctionalization of renewable fatty acid methyl esters was investigated. This reaction, which is difficult to achieve by chemical means, is catalyzed by Escherichia coli featuring the monooxygenase system AlkBGT and the uptake facilitator AlkL from Pseudomonas putida GPo1. Corresponding products, that is, terminal alcohols, aldehydes, and acids, constitute versatile bifunctional building blocks, which are of special interest for polymer synthesis. It could clearly be shown that extensive dodecanoic acid methyl ester uptake mediated by high AlkL levels leads to whole-cell biocatalyst toxification. Thus, cell viability constitutes the primary factor limiting biocatalyst stability and, as a result, process durability. Hence, a compromise had to be found between low biocatalyst activity due to restricted substrate uptake and poor biocatalyst stability due to AlkL-mediated toxification. This was achieved by the fine-tuning of heterologous alkL expression, which, furthermore, enabled the identification of the alkBGT expression level as another critical factor determining biocatalyst stability. Controlled synthesis of AlkL and reduced alkBGT expression finally enabled an increase of product titers by a factor of 4.3 up to 229 g L org -1 in a two-liquid phase bioprocess setup. Clearly, ω-oxyfunctionalization process performance was determined by cell viability and thus biocatalyst stability rather than the maximally achievable specific biocatalyst activity. Biotechnol. Bioeng. 2017;114: 874-884. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Microbial oils are sustainable alternatives to petroleum for the production of chemicals and fuels. Oleaginous yeasts are promising source of oils and Yarrowia lipolytica is the most studied and engineered one. Nonetheless the commercial production of biolipids is so far limited to high value products due to the elevated production and extraction costs. In order to contribute to overcoming these limitations we exploited the possibility of secreting lipids to the culture broth, uncoupling production and biomass formation and facilitating the extraction. We therefore considered two synthetic approaches, Strategy I where fatty acids are produced by enhancing the flux through neutral lipid formation, as typically occurs in eukaryotic systems and Strategy II where the bacterial system to produce free fatty acids is mimicked. The engineered strains, in a coupled fermentation and extraction process using alkanes, secreted the highest titer of lipids described so far, with a content of 120% of DCW., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Synthetic Biology Research Centre (SBRC)-Nottingham (www.sbrc-nottingham.ac.uk) was one of the first three U.K. university-based SBRCs to be funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and Engineering and Physical Sciences Research Council (EPSRC) as part of the recommendations made in the U.K.'s Synthetic Biology Roadmap. It was established in 2014 and builds on the pioneering work of the Clostridia Research Group (CRG) who have previously developed a range of gene tools for the modification of clostridial genomes. The SBRC is primarily focussed on the conversion of single carbon waste gases into platform chemicals with a particular emphasis on the use of the aerobic chassis Cupriavidus necator., (© 2016 The Author(s).)

The genetic, physiological and metabolic diversity of microalgae has driven fundamental research into photosynthesis, flagella structure and function, and eukaryotic evolution. Within the last 10 years these organisms have also been investigated as potential biotechnology platforms, for example to produce high value compounds such as long chain polyunsaturated fatty acids, pigments and antioxidants, and for biodiesel precursors, in particular triacylglycerols (TAGs). Transformation protocols, molecular tools and genome sequences are available for a number of model species including the green alga Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum, although for both species there are bottlenecks to be overcome to allow rapid and predictable genetic manipulation. One approach to do this would be to apply the principles of synthetic biology to microalgae, namely the cycle of Design-Build-Test, which requires more robust, predictable and high throughput methods. In this mini-review we highlight recent progress in the areas of improving transgene expression, genome editing, identification and design of standard genetic elements (parts), and the use of microfluidics to increase throughput. We suggest that combining these approaches will provide the means to establish algal synthetic biology, and that application of standard parts and workflows will avoid parallel development and capitalize on lessons learned from other systems., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Metabolic engineering strategies mark a milestone for the fermentative production of bulk and fine chemicals. Yet, toxic products and volatile reaction intermediates with low solubilities remain challenging. Prominent examples are artificial multistep pathways like the production of perillyl acetate (POHAc) from glucose via limonene. For POHAc, these limitations can be overcome by mixed-culture fermentations. A limonene biosynthesis pathway and cytochrome P450 153A6 (CYP153A6) as regioselective hydroxylase are used in two distinct recombinant E. coli. POHAc formation from glucose in one recombinant cell was hindered by ineffective coupling of limonene synthesis and low rates of oxyfunctionalization. The optimization of P450 gene expression led to the formation of 6.20 ± 0.06 mg gcdw (-1) POHAc in a biphasic batch cultivation with glucose as sole carbon and energy source. Increasing the spatial proximity between limonene synthase and CYP153A6 by a genetic fusion of both enzymes changed the molar limonene/POHAc ratio from 3.2 to 1.6. Spatial separation of limonene biosynthesis from its oxyfunctionalization improved POHAc concentration 3.3-fold to 21.7 mg L(-1) as compared to a biphasic fermentation. Mixed-cultures of E. coli BL21 (DE3) containing the limonene biosynthesis pathway and E. coli MG1655 harboring either CYP153A6, or alternatively a cymene monooxygenase, showed POHAc formation rates of 0.06 or 0.11 U gcdw (-1) , respectively. This concept provides a novel framework for fermentative syntheses involving toxic, volatile, or barely soluble compounds or pathway intermediates., (© 2015 Wiley Periodicals, Inc.)

In recent years, bio-based chemicals have gained traction as a sustainable alternative to petrochemicals. However, despite rapid advances in metabolic engineering and synthetic biology, there remain significant economic and environmental challenges. In order to maximize the impact of research investment in a new bio-based chemical industry, there is a need for assessing the technological, economic, and environmental potentials of combinations of biomass feedstocks, biochemical products, bioprocess technologies, and metabolic engineering approaches in the early phase of development of cell factories. To address this issue, we have developed a comprehensive Multi-scale framework for modeling Sustainable Industrial Chemicals production (MuSIC), which integrates modeling approaches for cellular metabolism, bioreactor design, upstream/downstream processes and economic impact assessment. We demonstrate the use of the MuSIC framework in a case study where two major polymer precursors (1,3-propanediol and 3-hydroxypropionic acid) are produced from two biomass feedstocks (corn-based glucose and soy-based glycerol) through 66 proposed biosynthetic pathways in two host organisms (Escherichia coli and Saccharomyces cerevisiae). The MuSIC framework allows exploration of tradeoffs and interactions between economy-scale objectives (e.g. profit maximization, emission minimization), constraints (e.g. land-use constraints) and process- and cell-scale technology choices (e.g. strain design or oxygenation conditions). We demonstrate that economy-scale assessment can be used to guide specific strain design decisions in metabolic engineering, and that these design decisions can be affected by non-intuitive dependencies across multiple scales., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Recent concerns over the sustainability of petrochemical-based processes for production of desired chemicals have fueled research into alternative modes of production. Metabolic engineering of microbial cell factories such as Saccharomyces cerevisiae and Escherichia coli offers a sustainable and flexible alternative for the production of various molecules. Acetyl-CoA is a key molecule in microbial central carbon metabolism and is involved in a variety of cellular processes. In addition, it functions as a precursor for many molecules of biotechnological relevance. Therefore, much interest exists in engineering the metabolism around the acetyl-CoA pools in cells in order to increase product titers. Here we provide an overview of the acetyl-CoA metabolism in eukaryotic and prokaryotic microbes (with a focus on S. cerevisiae and E. coli), with an emphasis on reactions involved in the production and consumption of acetyl-CoA. In addition, we review various strategies that have been used to increase acetyl-CoA production in these microbes., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

An increasing number of industrial bioprocesses capitalize on living cells by using them as cell factories that convert sugars into chemicals. These processes range from the production of bulk chemicals in yeasts and bacteria to the synthesis of therapeutic proteins in mammalian cell lines. One of the tools in the continuous search for improved performance of such production systems is the development and application of mathematical models. To be of value for industrial biotechnology, mathematical models should be able to assist in the rational design of cell factory properties or in the production processes in which they are utilized. Kinetic models are particularly suitable towards this end because they are capable of representing the complex biochemistry of cells in a more complete way compared to most other types of models. They can, at least in principle, be used to in detail understand, predict, and evaluate the effects of adding, removing, or modifying molecular components of a cell factory and for supporting the design of the bioreactor or fermentation process. However, several challenges still remain before kinetic modeling will reach the degree of maturity required for routine application in industry. Here we review the current status of kinetic cell factory modeling. Emphasis is on modeling methodology concepts, including model network structure, kinetic rate expressions, parameter estimation, optimization methods, identifiability analysis, model reduction, and model validation, but several applications of kinetic models for the improvement of cell factories are also discussed., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up the development of yeast cell factories. We also present an overview of metabolic engineering strategies for developing yeast strains for production of polymer monomers: lactic, succinic, and cis,cis-muconic acids. S. cerevisiae has already firmly established itself as a cell factory in industrial biotechnology and the advances in yeast strain engineering will stimulate development of novel yeast-based processes for chemicals production., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Production of recombinant proteins for use as pharmaceuticals, so-called biopharmaceuticals, is a multi-billion dollar industry. Many different cell factories are used for the production of biopharmaceuticals, but the yeast Saccharomyces cerevisiae is an important cell factory as it is used for production of several large volume products. Insulin and insulin analogs are by far the dominating biopharmaceuticals produced by yeast, and this will increase as the global insulin market is expected to grow from USD12B in 2011 to more than USD32B by 2018. Other important biopharmaceuticals produced by yeast are human serum albumin, hepatitis vaccines and virus like particles used for vaccination against human papillomavirus. Here is given a brief overview of biopharmaceutical production by yeast and it is discussed how the secretory pathway can be engineered to ensure more efficient protein production. The involvement of directed metabolic engineering through the integration of tools from genetic engineering, systems biology and mathematical modeling, is also discussed.

Indigo is widely used in textile industries for denim garments dyeing and is mainly produced by chemical synthesis which, however, raises environmental sustainability issues. Bio-indigo may be produced by fermentation of metabolically engineering bacteria, but current methods are economically incompetent due to low titer and the need for an inducer. To address these problems, we first characterized several synthetic promoters in E. coli and demonstrated the feasibility of inducer-free indigo production from tryptophan using the inducer-free promoter. We next coupled the tryptophan-to-indigo and glucose-to-tryptophan pathways to generate a de novo glucose-to-indigo pathway. By rational design and combinatorial screening, we identified the optimal promoter-gene combinations, which underscored the importance of promoter choice and expression levels of pathway genes. We thus created a new E. coli strain that exploited an indole pathway to enhance the indigo titer to 123 mg/L. We further assessed a panel of heterologous tryptophan synthase homologs and identified a plant indole lyase (TaIGL), which along with modified pathway design, improved the indigo titer to 235 mg/L while reducing the tryptophan byproduct accumulation. The optimal E. coli strain expressed 8 genes essential for rewiring carbon flux from glucose to indole and then to indigo: mFMO, ppsA, tktA, trpD, trpC, TaIGL and feedback-resistant aroG and trpE. Fed-batch fermentation in a 3-L bioreactor with glucose feeding further increased the indigo titer (≈965 mg/L) and total quantity (≈2183 mg) at 72 h. This new synthetic glucose-to-indigo pathway enables high-titer indigo production without the need of inducer and holds promise for bio-indigo production.

CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guide's targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to metabolic engineering targets, including protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens.

Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 μg/L (4.1 ± 0.1 μg/g DCW). Together, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.

Plant natural products (PNPs) hold significant pharmaceutical importance. The sessile nature of plants has led to the evolution of chemical defense mechanisms over millions of years to combat environmental challenges, making it a crucial and essential defense weapon. Despite their importance, the abundance of these bioactive molecules in plants is typically low, and conventional methods are time-consuming for enhancing production. Moreover, there is a pressing need for novel drug leads, exemplified by the shortage of antibiotics and anticancer drugs. Understanding how plants respond to stress and regulate metabolism to produce these molecules presents an opportunity to explore new avenues for discovering compounds that are typically under the detection limit or not naturally produced. Additionally, this knowledge can contribute to the advancement of plant engineering, enabling the development of new chassis for the biomanufacturing of these valuable molecules. In this perspective, we explore the intricate regulation of PNP biosynthesis in plants, and discuss the biotechnology strategies that have been and can be utilized for the discovery and production enhancement of PNPs in plants.

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

Advancements in synthetic biology have facilitated the incorporation of heterologous metabolic pathways into various bacterial chassis, leading to the synthesis of targeted bioproducts. However, total output from heterologous production pathways can suffer from low flux, enzyme promiscuity, formation of toxic intermediates, or intermediate loss to competing reactions, which ultimately hinder their full potential. The self-assembling, easy-to-modify, protein-based bacterial microcompartments (BMCs) offer a sophisticated way to overcome these obstacles by acting as an autonomous catalytic module decoupled from the cell's regulatory and metabolic networks. More than a decade of fundamental research on various types of BMCs, particularly structural studies of shells and their self-assembly, the recruitment of enzymes to BMC shell scaffolds, and the involvement of ancillary proteins such as transporters, regulators, and activating enzymes in the integration of BMCs into the cell's metabolism, has significantly moved the field forward. These advances have enabled bioengineers to design synthetic multi-enzyme BMCs to promote ethanol or hydrogen production, increase cellular polyphosphate levels, and convert glycerol to propanediol or formate to pyruvate. These pioneering efforts demonstrate the enormous potential of synthetic BMCs to encapsulate non-native multi-enzyme biochemical pathways for the synthesis of high-value products.

Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

The industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance, and commodity chemical. Here, we demonstrate TMP production (∼0.8 g L-1) in C. glutamicum type strain ATCC13032 via overexpression of acetolactate synthase and/or α-acetolactate decarboxylase from Lactococcus lactis in CGXII minimal medium supplemented with 40 g L-1 glucose. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v v-1) hydrolysates derived from ionic liquid-pretreated sorghum biomass. A key objective was to take the fully engineered strain developed in this study and interrogate medium parameters that influence the production of TMP, a critical post-strain engineering optimization. Design of experiments in a high-throughput plate format identified glucose, urea, and their ratio as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g L-1 TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol mol-1) from 80 g L-1 glucose and 11.9 g L-1 urea in shake flask batch cultivation.One-sentence summaryCorynebacterium glutamicum was metabolically engineered to produce 2,3,5,6-tetramethylpyrazine followed by a design of experiments approach to optimize medium components for high-titer production.

Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming multi-gene expression, but poor predictability of guide RNA folding can disrupt expression control. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA folding rate into the active structure (rS = 0.8). This parameter also enables forward design of scRNAs, allowing us to design a system of three synthetic CRISPRa promoters that can orthogonally activate (>35-fold) expression of chosen outputs. Through combinatorial activation tuning, we profile a three-dimensional design space expressing two different biosynthetic pathways, demonstrating variable production of pteridine and human milk oligosaccharide products. This RNA design approach aids combinatorial optimization of metabolic pathways and may accelerate routine design of effective multi-gene regulation programs in bacterial hosts.

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

Anthropogenic carbon emissions are driving rapid changes to the earth's climate, disrupting whole ecosystems and endangering the stability of human society. Innovations in engineered microbial fermentation enable the fossil resource-free production of fuels, commodity chemicals, and materials, thereby reducing the carbon emissions associated with these products. Microorganisms have been engineered to catabolize sustainable sources of carbon and energy (i.e., plant biomass, plastic waste, and one-carbon feedstocks) and biosynthesize carbon-neutral or carbon-negative products. These engineering efforts exploit and optimize natural biological pathways or generate unnatural pathways which can biosynthesize chemicals that have not yet been accessed using synthetic chemistry. Recent advances in microbial fermentation seek not only to maximize the titer, rate, and yield of desired products, but also to tailor microbial catabolism to utilize inexpensive feedstocks. Ultimately, these advances aim to lower the cost of bioproduction so that microorganism-derived chemicals can be economically competitive with fossil-derived chemicals.

We have developed an electrical-biological hybrid system wherein an engineered microorganism consumes electrocatalytically produced formate from CO2 to supplement the bioproduction of isobutanol, a valuable fuel chemical. Biological CO2 sequestration is notoriously slow compared to electrochemical CO2 reduction, while electrochemical methods struggle to generate carbon-carbon bonds which readily form in biological systems. A hybrid system provides a promising method for combining the benefits of both biology and electrochemistry. Previously, Escherichia coli was engineered to assimilate formate and CO2 in central metabolism using the reductive glycine pathway. In this work, we have shown that chemical production in E. coli can benefit from single carbon substrates when equipped with the RGP. By installing the RGP and the isobutanol biosynthetic pathway into E. coli and by further genetic modifications, we have generated a strain of E. coli that can consume formate and produce isobutanol at a yield of >100% of theoretical maximum from glucose. Our results demonstrate that carbon produced from electrocatalytically reduced CO2 can bolster chemical production in E. coli. This study shows that E. coli can be engineered towards carbon efficient methods of chemical production.

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.

Valerolactam is a monomer used to manufacture high-value nylon-5 and nylon-6,5. However, the biological production of valerolactam has been limited by the inadequate efficiency of enzymes to cyclize 5-aminovaleric acid to produce valerolactam. In this study, we engineered Corynebacterium glutamicum with a valerolactam biosynthetic pathway consisting of DavAB from Pseudomonas putida to convert L-lysine to 5-aminovaleric acid and β-alanine CoA transferase (Act) from Clostridium propionicum to produce valerolactam from 5-aminovaleric acid. Most of the L-lysine was converted into 5-aminovaleric acid, but promoter optimization and increasing the copy number of Act were insufficient to significantly improve the titer of valerolactam. To eliminate the bottleneck at Act, we designed a dynamic upregulation system (a positive feedback loop based on the valerolactam biosensor ChnR/Pb). We used laboratory evolution to engineer ChnR/Pb to have higher sensitivity and a higher dynamic output range, and the engineered ChnR-B1/Pb-E1 system was used to overexpress the rate-limiting enzymes (Act/ORF26/CaiC) that cyclize 5-aminovaleric acid into valerolactam. In glucose fed-batch culture, we obtained 12.33 g/L valerolactam from the dynamic upregulation of Act, 11.88 g/L using ORF26, and 12.15 g/L using CaiC. Our engineered biosensor (ChnR-B1/Pb-E1 system) was also sensitive to 0.01-100 mM caprolactam, which suggests that this dynamic upregulation system can be used to enhance caprolactam biosynthesis in the future.

Campesterol is a major phytosterol that plays important roles in regulating membrane properties and serves as the precursor to multiple specialized metabolites, such as the phytohormone brassinosteroids. Recently, we established a campesterol-producing yeast strain and extended the bioproduction to 22-hydroxycampesterol and 22-hydroxycampest-4-en-3-one, the precursors to brassinolide. However, there is a trade-off in growth due to the disrupted sterol metabolism. In this study, we enhanced the growth of the campesterol-producing yeast by partially restoring the activity of the sterol acyltransferase and engineering upstream FPP supply. Furthermore, genome sequencing analysis also revealed a pool of genes possibly associated with the altered sterol metabolism. Retro engineering implies an essential role of ASG1, especially the C-terminal asparagine-rich domain of ASG1, in the sterol metabolism of yeast especially under stress. The performance of the campesterol-producing yeast strain was enhanced with the titer of campesterol to 18.4 mg/L, and the stationary OD600 was improved by ∼33% compared to the unoptimized strain. In addition, we examined the activity of a plant cytochrome P450 in the engineered strain, which exhibits more than 9-fold higher activity than when expressed in the wild-type yeast strain. Therefore, the engineered campesterol-producing yeast strain also serves as a robust host for the functional expression of plant membrane proteins.

Corynebacterium glutamicum, a natural glutamate-producing bacterium adopted for industrial production of amino acids, has been extensively explored recently for high-level biosynthesis of amino acid derivatives, bulk chemicals such as organic acids and short-chain alcohols, aromatics, and natural products, including polyphenols and terpenoids. Here, we review the recent advances with a focus on biosystem design principles, metabolic characterization and modeling, omics analysis, utilization of nonmodel feedstock, emerging CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) tools for Corynebacterium strain engineering, biosensors, and novel strains of C. glutamicum. Future research directions for developing C. glutamicum cell factories are also discussed.

BackgroundCyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803).ResultsTo establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers.ConclusionA green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.

BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts.ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter MhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive.ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

Optimization of metabolically engineered organisms requires good understanding of producing balanced level of pathway proteins. Targeted proteomics via selected-reaction monitoring (SRM) has been increasingly used in metabolic engineering studies to detect and quantify sets of proteins with high selectivity, multiplexity, and reproducibility. In combination with metabolomics and other omics tools, targeted proteomics has helped optimize the production of many bio-based chemicals in various metabolic engineering cell factories. In this review, we present recent applications of targeted proteomics in metabolic engineering studies and highlight several successful cases of targeted proteomics in boosting production of commodity and high value chemicals. Additionally, we also discuss challenges and limitations of current targeted proteomics and map opportunities for future research.

Marine macroalgae have huge potential as feedstocks for production of a wide spectrum of chemicals used in biofuels, biomaterials, and bioactive compounds. Harnessing macroalgae in these ways could promote wellbeing for people while mitigating climate change and environmental destruction linked to use of fossil fuels. Microorganisms play pivotal roles in converting macroalgae into valuable products, and metabolic engineering technologies have been developed to extend their native capabilities. This review showcases current achievements in engineering the metabolisms of various microbial chassis to convert red, green, and brown macroalgae into bioproducts. Unique features of macroalgae, such as seasonal variation in carbohydrate content and salinity, provide the next challenges to advancing macroalgae-based biorefineries. Three emerging engineering strategies are discussed here: (1) designing dynamic control of metabolic pathways, (2) engineering strains of halophilic (salt-tolerant) microbes, and (3) developing microbial consortia for conversion. This review illuminates opportunities for future research communities by elucidating current approaches to engineering microbes so they can become cell factories for the utilization of macroalgae feedstocks.