Synthetic Biology and Engineering Open Access

ISSN: 2958-9053 (Online)

2958-9045 (Print)

An Official Journal of Center of Synthetic Biology and Integrated Bioengineering, Westlake University

Synthetic Biology and Engineering (SBE) is an international, peer-reviewed, open access journal dealing with interdisciplinary research of synthetic biology, from living systems to industry translation, published quarterly online by SCIEPublish.

Editors-in-Chief

Articles (37) All articles

Article

03 December 2024

Pathway Engineering of E. coli for Production of Fritschiellaxanthin and Other Carotenoids with α-Carotene Backbone and Their Singlet Oxygen-Quenching Activities

Some photosynthetic organisms are capable of biosynthesizing carotenoids (xanthophylls) with α-carotene backbone, that is, α-carotene-derived carotenoids, such as (3R,3′R,6′R)-3,3′-dihydroxy α-carotene (lutein). Except for lutein, such carotenoids are minor compounds in nature. In this study, α-carotene-derived carotenoids were produced with E. coli. To achieve this, carotenoid biosynthesis genes from the bacterium Pantoea ananatis containing the 4-β-ketolase (crtW) gene with/without the 3-β-hydroxylase (crtZ) gene, in addition to crtEBI genes, and biosynthesis genes (MpLCYb, MpLCYe, and MpCYP97C) from liverwort Marchantia polymorpha, along with the HpIDI gene, were cloned into plasmids. The transformed E. coli cells biosynthesized (3S,3′R,6′R)-3,3′-dihydroxy-4-keto-α-carotene (fritschiellaxanthin (4-ketolutein)), (3′R,6′R)-3′-hydroxy-4-keto-α-carotene (4-keto-α-cryptoxanthin), and (3′R,6′R)-3′-hydroxy-α-carotene (α-cryptoxanthin), as carotenoids that have not been produced by a heterologous microbial system so far. These carotenoids show potent singlet oxygen-quenching activity.

Article

14 November 2024

Sortase A-Mediated Enzyme Assembly on Multimeric Protein for Improving Mevalonate Production

Microorganisms have been extensively studied for their production of valuable chemicals. However, conventional gene fusion approaches often lack versatility and can result in enzyme inactivation. This study explored an alternative strategy for inducing metabolic channeling through sortase A-mediated ligation of metabolic enzymes. Sortase A recognizes specific amino acid sequences and selectively conjugates proteins at these sites. We focused on mevalonate production as a proof-of-concept to enhance the yield by assembling metabolic enzymes on a protein scaffold using sortase A. Although metabolic enzyme complexes were successfully formed using streptavidin as a scaffold, production did not improve. The use of CutA as a scaffold led to a 1.32-fold increase in production compared with that of the strain without the scaffold, demonstrating the efficacy of CutA in mevalonate production. These findings suggest that using sortase A to assemble metabolic enzymes onto a scaffold can effectively enhance microbial bioproduction.

Review

13 November 2024

Recent Advances in Developing Aldehyde-Accumulating Microbes and Future Perspective of Biosynthetic Applications

Aldehydes are a class of compounds that contain carbonyl groups in their side chains and are widely used in industries such as fragrances, flavoring compounds, and pharmaceutical intermediates. In recent years, there has been a substantial rise in the application of microbial synthesis to generate aldehyde compounds and their derivatives. This review will conduct an in-depth analysis of the literature related to the manipulation of microorganisms for aldehyde accumulation and the subsequent generation of aldehyde-derived products using metabolic engineering and synthetic biology principles. Furthermore, the review further highlights the prospects and future potential of employing these aldehyde-accumulating microorganisms to synthesize a diverse range of value-added chemicals.

Article

30 September 2024

Synthetic Biology in Nigeria: The Level of Awareness amongst Stakeholders

Synthetic biology, an emerging field at the intersection of biotechnology and engineering, has seen a global surge in application and awareness, necessitating a comprehensive understanding of its safe potentials to drive the bio-economy. This study aimed to assess the awareness and perceptions of synthetic biology among Nigerian biosciences stakeholders, including researchers, academicians, policymakers and students. The study employed a purposive online survey targeting diverse bioscience individuals and groups across Nigeria’s six geopolitical zones. The study received 107 responses from balanced gender representation with majority within the age group of 3145 years old. The findings revealed a significant knowledge gap, with only 27.1% of respondents familiar with synthetic biology and 23.4% entirely unaware of it. Most respondents associated synthetic biology with biotechnology or genetic engineering and identified its applications to be in agriculture, medicine, environmental sustainability and research. Despite recognizing its benefits, many expressed concerns about safety, ethics, and regulation; notably, 43.9% of the respondents had concerns about synthetic biology with primary focus on safety and ethical implications. As regards the regulation of synthetic biology, the study showed that 80.4% of the respondents supported the need for synthetic biology regulation with few of the respondents (16.8%) aware of existing agency mandated to regulate synthetic biology. The respondents provided valuable insights into the various ways synthetic biology can be advanced in Nigeria which include increased awareness and capacity building, engagement through social media platforms, integration into education curricula and increased funding and investment in the research. The overall sentiment towards synthetic biology was positive, with 81.3% supporting its practice and 76.6% recognizing its positive global impact. However, a significant portion of respondents remained undecided. This study concludes that there is substantial gap in the knowledge of synthetic biology among bioscience stakeholders in Nigeria and the need for a heightened advocacy including continuous conferences and symposiums for the Nigeria bioscience community on the global potentials, concerns and regulation of synthetic biology. This will foster the acceptance of safe and responsible synthetic biology in Nigeria, thereby contributing to the broader national bio-economy development.

Article

26 August 2024

Delivery of Novel Replicating Vectors to Synechococcus sp. PCC 7002 Via Natural Transformation of Plasmid Multimers

In most cyanobacteria, genetic engineering efforts currently rely upon chromosomal integration; a time-consuming process due to their polyploid nature. To enhance strain construction, here we develop and characterize two novel replicating plasmids for use in Synechococcus sp. PCC 7002. Following an initial screen of plasmids comprising seven different origins of replication, two were found capable of replication: one based on the WVO1 broad host range plasmid and the other a shuttle vector derived from pCB2.4 from Synechocystis sp. PCC 6803. These were then used to construct a set of new replicating plasmids, which were shown to be both co-transformable and stably maintained in PCC 7002 at copy numbers between 716 and 0.61.4, respectively. Lastly, we demonstrate the importance of using multimeric plasmids during natural transformation of PCC 7002, with higher order multimers providing a 30-fold increase in transformation efficiency relative to monomeric plasmids. Useful considerations and methods for enhancing multimer content in plasmid samples are also presented.

Review

22 August 2024

Current Application of Modeling and Cell-Free System for Synthetic Gene Circuit Design

The desire to harness nature’s capability of precise gene expression regulation has motivated the pursuit of synthetic gene circuits. However, designing and building novel synthetic gene circuits with predictable dynamics is nontrivial. To facilitate the design, cell-free systems have emerged as an effective alternative testbed to living biological systems in characterizing and prototyping synthetic gene regulatory networks, given its relative simplicity and designability in terms of cellular contents. Meanwhile, as parameterizing and analyzing first principle-based models can shed light on the required kinetic parameter values, thus the specific regulatory components, for the desired dynamics, coupling mathematical modeling with cell-free experiments has become an effective approach in exploring novel synthetic gene circuits. In this mini-review, we provide an overview of current progress on using deterministic first principle-based mathematical modeling in conjunction with cell-free systems, in designing and characterizing novel gene circuits, as well as the standing challenges and issues with this approach.

Review

14 August 2024

Application of Synthetic Biology to the Biosynthesis of Polyketides

Polyketides (PKs) are a large class of secondary metabolites produced by microorganisms and plants, characterized by highly diverse structures and broad biological activities. They have wide market and application prospects in medicine, agriculture, and the food industry. The complex chemical structures and multiple steps of natural polyketides result in yield that cannot be met by purely synthetic methods. With the development of synthetic biology, a number of novel technologies and synthetic strategies have been developed for the efficient synthesis of polyketides. This paper first introduces polyketides from different sources and classifications, then the reconstruction of biosynthetic pathways is described using a “bottom-up” synthetic biology approach. Through methods such as enhancing precursors, relieving feedback inhibition, and dynamic regulation, the efficient production of polyketides is achieved. Finally, the challenges faced by polyketides research and future development directions are discussed.

Article

17 June 2024

Cytosine Deaminase-Assisted Mutator for Genome Evolution in Cupriavidus necator

Cupriavidus necator H16 has been intensively explored for its potential as a versatile microbial cell factory, especially for its CO2 fixation capability over the past few decades. However, rational metabolic engineering remains challenging in the construction of microbial cell factories with complex phenotypes due to the limited understanding of its metabolic regulatory network. To overcome this obstacle, laboratory adaptive evolution emerges as an alternative. In the present study, CAM (cytosine deaminase-assisted mutator) was established for the genome evolution of C. necator, addressing the issue of low mutation rates. By fusing cytosine deaminase with single-stranded binding proteins, CAM introduced genome-wide C-to-T mutations during DNA replication. This innovative approach could boost mutation rates, thereby expediting laboratory adaptive evolution. The applications of CAM were demonstrated in improving cell factory robustness and substrate utilization, with H2O2 resistance and ethylene glycol utilization as illustrative case studies. This genetic tool not only facilitates the development of efficient cell factories but also opens avenues for exploring the intricate phenotype-genotype relationships in C. necator.

Review

24 May 2024

Current Progress on Microbial l-malic Acid Production

As an important intermediate in the tricarboxylic acid (TCA) cycle, l-malic acid (l-MA) is also one of the 12 important platform bulk chemicals with high added value. Owing to its various applications in food, pharmaceuticals, cosmetics and industry, the global l-MA market size is growing year by year. Over the last few decades, increasing concerns regarding fossil fuels depletion and excessive CO2 emissions have led the global commitment to fostering a green economy and sustainable development. Alternatively, the sustainable microbial fermentation of l-MA has gradually attracted more and more attention. Here, this review summarizes the common l-MA biosynthesis pathways and compares the differences between different chassis microorganisms as well. Moreover, regulation strategies of genetic metabolic engineering and fermentation process to boost the l-MA production are summarized, and the research status of l-MA production from the cheaper substrates is also discussed. Finally, the direction of further exploration of industrialized l-MA biosynthesis is proposed, which provides a theoretical guidance on promoting technological innovation in industrial l-MA production.

Review

20 May 2024

Advancements in the Bio-degradation of Plastic Waste into Value-added Chemicals: A Recent Perspective

Plastics are an essential component of modern life, but the plastic waste has caused significant environmental pollution and economic losses. The effective solution to these problems is the biodegradation and high-value conversion of plastic waste. After biodegradation, plastic waste is broken into smaller molecules and eventually transformed into innocuous substances like water, carbon dioxide and biomass. High-value conversion enables plastic waste to be converted into products with higher economic value and environmental friendliness. Based on this, we summarize the biodegradation methods of bioplastics and analyze the shortage of these methods. Subsequently, we summarize the progress of converting the degradation products into value-added chemicals, comprehensively analyze the advantages and disadvantages of these bioconversion process, and propose some strategies to address these disadvantages. Finally, we analyze the significance of establishing a microbial-based conversion process that integrates the degradation and the conversion, and propose some potential strategies.

Commentary

14 September 2023

Synthetic Biology Industry in China: Current State and Future Prospects

In this article, we provided an overview of the current state of the SynBio industry in China with a focus on its research and technology, its main applications, and major players. We also discussed future prospects including the challenges and advantages of the SynBio industry in China.

WeiLuo
YangZhang
JunPeng
LishanZhao

Review

31 October 2023

Metabolic Engineering of Microorganisms Towards the Biomanufacturing of Non-Natural C5 and C6 Chemicals

Five-carbon (C5) and six-carbon (C6) chemicals are essential components in the manufacturing of a variety of pharmaceuticals, fuels, polymers, and other materials. However, the predominant reliance on chemical synthesis methods and unsustainable feedstock sources has placed significant strain on Earth’s finite fossil resources and the environment. To address this challenge and promote sustainability, significant efforts have been undertaken to re-program microorganisms through metabolic engineering and synthetic biology approaches allowing for bio-based manufacturing of these compounds. This review provides a comprehensive overview of the advancements in microbial production of commercially significant non-natural C5 chemicals, including 1-pentanol, 1,5-pentanediol, cadaverine, δ-valerolactam, glutaric acid, glutaconic acid, and 5-hydroxyvaleric acid, as well as C6 chemicals, including cis, cis-muconic acid, adipic acid, 1,6-hexamethylenediamine, 6-aminocaproic acid, β-methyl-δ-valerolactone, 1-hexanol, ε-caprolactone, 6-hydroxyhexanoic acid, and 1,6-hexanediol.

Ashley Tseng
VannaNguyen
YuhengLin

Perspective

29 December 2023

Article

13 March 2023

Design of Oscillatory Networks through Post-Translational Control of Network Components

Many essential functions in biological systems, including cell cycle progression and circadian rhythm regulation, are governed by the periodic behaviors of specific molecules. These periodic behaviors arise from the precise arrangement of components in biomolecular networks that generate oscillatory output signals. The dynamic properties of individual components of these networks, such as maturation delays and degradation rates, often play a key role in determining the network's oscillatory behavior. In this study, we explored the post-translational modulation of network components as a means to generate genetic circuits with oscillatory behaviors and perturb the oscillation features. Specifically, we used the NanoDeg platform—A bifunctional molecule consisting of a target-specific nanobody and a degron tag—to control the degradation rates of the circuit’s components and predicted the effect of NanoDeg-mediated post-translational depletion of a key circuit component on the behavior of a series of proto-oscillating network topologies. We modeled the behavior of two main classes of oscillators, namely relaxation oscillator topologies (the activator-repressor and the Goodwin oscillator) and ring oscillator topologies (repressilators). We identified two main mechanisms by which non-oscillating networks could be induced to oscillate through post-translational modulation of network components: an increase in the separation of timescales of network components and mitigation of the leaky expression of network components. These results are in agreement with previous findings describing the effect of timescale separation and mitigation of leaky expression on oscillatory behaviors. This work thus validates the use of tools to control protein degradation rates as a strategy to modulate existing oscillatory signals and construct oscillatory networks. In addition, this study provides the design rules to implement such an approach based on the control of protein degradation rates using the NanoDeg platform, which does not require genetic manipulation of the network components and can be adapted to virtually any cellular protein. This work also establishes a framework to explore the use of tools for post-translational perturbations of biomolecular networks and generates desired behaviors of the network output.

Brianna E.K. Jayanthi
Shridhar Jayanthi
Laura Segatori

Review

01 September 2023

In Vitro BioTransformation (ivBT): Definitions, Opportunities, and Challenges

Great needs always motivate the birth and development of new disciplines and tools. Here we propose in vitro BioTransformation (ivBT) as a new biomanufacturing platform, between the two dominant platforms—microbial fermentation and enzymatic biocatalysis. ivBT mediated by in vitro synthetic enzymatic biosystems (ivSEBs) is an emerging biomanufacturing platform for the production of biocommodities (i.e., low-value and bulk biochemicals). ivSEB is the in vitro reconstruction of artificial (non-natural) enzymatic pathways with numerous natural enzymes, artificial enzymes, and/or (biomimetic or natural) coenzymes without living cell’s constraints, such as cell duplication, basic metabolisms, complicated regulation, bioenergetics, and so on. The two great needs (i.e., food security and the carbon-neutral renewable energy system) have motivated the birth and development of ivBT. Food security could be addressed by making artificial food from nonfood lignocellulose and artificial photosynthesis for starch synthesis from CO2. The carbon-neutral renewable energy system could be addressed by the construction of the electricity-hydrogen-carbohydrate cycle, where starch could be a high density of hydrogen carrier (up to 14.8% H2 wt/wt) and an electricity storage compound (greater than 3000 Wh/kg). Also, ivBT can make a number of biocommodities, such as inositol, healthy sweeteners (e.g., D-allulose, D-tagatose, D-mannose), advanced biofuels, polymer precursors, organic acids, and so on. The industrial biomanufacturing of the first several biocommodities (e.g., myo-inositol, D-tagatose, D-mannose, and cellulosic starch) would wipe off any prejudice and doubt on ivBT. Huge potential markets of biocommodities with more than tens of trillions of Chinese Yuan would motivate scientists and engineers to address the remaining technical challenges and develop new tools within the next decade.

Yi-HengP. JobZhang
Zhiguang Zhu
ChunYou
Lingling Zhang
KuanqingLiu

Editorial

13 December 2022

Article

10 May 2023

Development of a New 1,2,4-butanetriol Biosynthesis Pathway in an Engineered Homoserine-producing Strain of Escherichia coli

1,2,4-butanetriol (BT) is a compound of high interest with applications in pharmaceutical and materials. In this work, we designed a novel biosynthetic pathway for BT from glucose via a nonessential amino acid homoserine. This non-natural pathway used an engineered phosphoserine transaminase (SerCR42W/R77W) to achieve the deamination of homoserine to 4-hydroxy-2-oxobutanoic acid (HOBA). Three consecutive enzymes including a lactate dehydrogenase, a 4-hydroxybutyrate CoA-transferase and a bifunctional aldehyde/alcohol dehydrogenase are used to catalyze HOBA to BT. To enhance the carbon flux to homoserine, a homoserine-producing Escherichia coli was developed by improving the overexpression of two relevant key genes metL and lysC (V339A). The simultaneous overexpression of the genes encoding these enzymes for the homoserine-derived BT pathway enabled production of 19.6 mg/L BT from glucose in the homoserine-producing E. coli.

Yujun Zhang
Lin Chen
Antu Thomas
An-Ping Zeng

Review

16 February 2023

Increasing Nutritional Value of Cyanobacteria by Engineering Valine, Phenylalanine, and Fatty Acid Production

In 2020, the United Nations estimated that 2.37 billion people globally were without food or unable to eat a healthy balanced diet. The number of people with insufficient nutrition has increased in the short term due to COVID-19 pandemic and longer-term climate change is leading to shifts in arable land and water availability leading to a continued need to develop scalable sources of nutrition. One of the options that can yield high food mass per square foot of land use is the high-density culture of microalgae or other photosynthetic microorganisms. While photosynthetic microorganisms may provide high amounts of biomass with a small land footprint, the nutritional value of unmodified microorganisms may be limited. This mini-review presents the base nutritional value in terms of macro- and micronutrients of several cyanobacteria (Nostoc, Anabaena, Spirulina) in relation to established human nutritional requirements as a starting point for better utilization of cyanobacteria as nutritional supplements. It also discusses synthetic biology approaches that have been implemented in different organisms to increase the production of L-valine, L-phenylalanine, and fatty acids demonstrating some common genetic engineering design approaches and some approaches that are organism-specific.

NickLopez-Riveira
StephenS.Fong

Article

31 May 2023

Nitrogen-controlled Valorization of Xylose-derived Compounds by Metabolically Engineered Corynebacterium glutamicum

The implementation of bioprocesses in an economically feasible and industrial competitive manner requires the optimal allocation of resources for a balanced distribution between biomass formation and product synthesis. The decoupling of growth and production in two-stage bioprocesses, aiming to ensure sufficient growth before the onset of production, is particularly relevant when target products inhibit growth. In order to avoid expensive inducer molecules, continuing process monitoring, elaborate individual process optimization, and strain engineering, we developed and applied nitrogen deprivation-induced expression of genes for product biosynthesis. Two native nitrogen deprivation-inducible promoters were identified and shown to function for dynamic growth-decoupled gene expression or CRISPRi-mediated gene knockdown in C. glutamicum with superior induction factors than the standard IPTG-inducible Ptrc promoter. Valorization of xylose to produce either the sugar acid xylonic acid or the sugar alcohol xylitol from xylose as sole source of carbon and energy was demonstrated. Competitive titers of up to 34 g·L−1 xylonate and 13 g·L−1 xylitol were achieved in two-stage processes. We discussed that the transfer to bioprocesses with C. glutamicum using carbon sources other than xylose appears straightforward in particular regarding production of growth-inhibitory compounds by their growth-decoupled fermentative production.

LynnS.Schwardmann
Marielle Rieks
Volker F.Wendisch

Article

07 February 2023

Production of Highly Modified C30-carotenoids with Singlet Oxygen-quenching Activities, 5-glucosyl-5,6-dihydro-4,4’-diapolycopen-4’-oic Acid, and Its Three Intermediates Using Genes from Planococcus maritimus Strain iso-3

Planococcus maritimus strain iso-3 was previously isolated from intertidal sediment in the North Sea and was found to produce a highly modified C30-carotenoid, methyl-5-glucosyl-5,6-dihydro-4,4’-diapolycopenoate, as the final product. In this study, we analyzed the function of the carotenoid terminal oxidase crtP (renamed cruO) and aldehyde dehydrogenase aldH genes in P. maritimus strain iso-3 and elucidated the carotenoid biosynthetic pathway for this strain at the gene level. We produced four novel C30-carotenoids with potent singlet oxygen-quenching activities, 5-glucosyl-5,6-dihydro-4,4’-diapolycopen-4’-oic acid and its three intermediates, which were obtained using E. coli cells carrying the cruO (and aldH) gene(s) in addition to the known P. maritimus carotenogenic genes.

MoeHagiwara
ChinatsuMaehara
MihoTakemura
NorihikoMisawa
KazutoshiShindo

News

Topic Collection

Molecular Tools in Synthetic Biology

Synthetic Biology in the Manufacturing of Chemicals and Fuels

Synthetic Biology in Therapeutics and Healthcare: Innovations and Applications

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