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 (30) All articles

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.

Article

13 May 2024

Expression of Redox Partner Fusions for Light Driven Cytochrome P450s in the Cyanobacterium Synechocystis sp. PCC. 6803

Cytochrome P450s (P450s) catalyze stereo- and regioselective monooxygenations in the biosynthesis of a wide range of valuable natural compounds. The turnover of P450s requires dedicated electron transfer, usually via a NADPH-dependent reductase. The need for an NADPH-dependent reductase can be circumvented if expressed in photosynthetic organisms by exploiting the photosynthetic reducing power. However, partitioning reducing equivalents towards the P450s needs further optimization. Using our model P450, SbCYP79A1, we have previously shown that by targeting this P450 to the thylakoid membrane, the P450 can obtain its reducing power directly from photosystem I via soluble ferredoxin. Furthermore, we demonstrated using transient expression that fusing a soluble electron carrier to this P450 improves electron partitioning towards the P450 in tobacco. In order to characterize these fusions in a stably transformed organism, we expressed three different redox partner fusions in the cyanobacterium Synechocystis sp. PCC. 6803. We show that biochemical trends observed in the tobacco system are recapitulated in stably transformed Synechocystis sp. PCC. 6803. Overall, the FMN binding domain fusion produces the most oxime per unit of enzyme with and without the presence of the endogenous competing electron sink FNR and NADP+. However, the overall yield of oxime is comparable to the other strains, due to poor steady state levels of the fusion protein. Synechocystis sp. PCC. strains expressing the P450-FMN fusion also display a chlorotic phenotype that can be rescued by switching the nitrogen source from nitrate to ammonia, implying impaired nitrate assimilation. Optimizing electron transport towards the P450 is indeed possible in vivo but also highlights interference with native metabolic processes.

Review

08 April 2024

Tolerance in Solventogenic Clostridia for Enhanced Butanol Production: Genetic Mechanisms and Recent Strain Engineering Advances

Biobutanol is a promising candidate for replacing fossil fuels due to its superior properties compared to ethanol. Solventogenic clostridia can naturally produce biobutanol among other valuable chemicals. Lignocellulosic material stands out as a promising source for biobutanol production, avoiding competition with food production and making use of residues from both agroindustry and forestry activities. However, Clostridium strains are subject to different chemical stressors, including oxygen, self-product inhibition, inhibitors generated during biomass pretreatment and hydrolysis, and others. Recent advances in genetic engineering tools have enabled the metabolic engineering of Clostridium strains to increase their robustness and tolerance to these stressors. This review provides a summary of the various types of inhibitors, the genetic mechanisms related to tolerance, and recent strain engineering efforts for tolerance enhancement. In addition, we offer a valuable perspective on the future research directions in this area.

Article

25 March 2024

Proteomic Analysis of Pleurotus ostreatus Grown on Glucose and Xylose Mixtures in Submerged Fermentation Provides Insights into Differentiated Mycelial Composition

Pleurotus ostreatus, an edible white-rot fungus of great commercial and nutritional value, grows by metabolizing mainly glucose and xylose, the two major sugars in lignocellulosic biomass. In this study, a comparative proteomic analysis of P. ostreatus grown in submerged fermentation on a medium with glucose, xylose and mixtures of them as carbon sources was conducted. In the same conditions, the metabolic response of the fungus was evaluated in the production of the main nutritional components of the fungus such as proteins, lipids, and intracellular and extracellular polysaccharides. The proteomic analysis revealed that glucose and xylose upregulate different clusters of proteins. Glucose mainly up-regulates macromolecule metabolic processes, translation and glycolysis whereas xylose up-regulates, small molecule metabolic processes and tricarboxylic acid cycle (TCA). The mixtures show mostly similarities with the proteome response to glucose, although there are differential responses depending on xylose concentration. The carbon source type found to affect the basic macromolecule metabolic processes, with amino acids biosynthesis to differentiate mostly. An analysis of the upregulated proteins through the STRING database revealed that xylose upregulates mostly proteins related to amino acid biosynthesis. Leucine, Valine and Isoleucine biosynthesis pathways were found to be the most triggered pathway. All the branched-chain amino acids (BCAAs)-related enzymes intensities were gradually increased when xylose concentration was increased in the growth medium. BCAAs play an important role in the human diet so the enhancement of BCAAs biosynthesis pathway for P. ostreatus could convert it to a very remarkable protein substitute in human diet. These findings provide new insights into the proteomic and metabolic response of the fungus to the major sugars of lignocellulosic biomass, which are not well understood until now. 

Article

22 March 2024

Modulation of the MEP Pathway for Overproduction of 13-R-manoyl Oxide in Cyanobacteria

The cyanobacterium Synechocystis sp. PCC 6803 has gained scientific interest for its potential to use solar energy and atmospheric CO2 for the production of high-value chemicals like pharmaceuticals, flavors, and fragrances. Forskolin is a diterpenoid found in the root cork of the plant Plectranthus barbatus and its biosynthetic pathway is initiated by two terpene synthases that convert geranylgeranyl diphosphate (GGDP) into the precursor 13-R-manoyl oxide (13-R-MO). Using the cyanobacterium Synechocystis sp. PCC 6803 as host, we expressed the two terpene synthases resulting in the synthesis of 0.83 mg/L 13-R-MO. Three different geranylgeranyl diphosphate synthases (GGDPSs) were selected for screening; a prokaryotic (Synechococcus sp. JA-3-3Ab (Sj)), a yeast (Saccharomyces cerevisiae (Sc)), and a plant (P. barbatus (Pb)) derived GGDPS. Strains containing the prokaryotic Sj- or the yeast ScGGDPS consistently yielded more 13-R-MO than the base strain. By overexpression of 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) positioned at the entry of the 2-C-methyl-d-erythritol 4-phosphate pathway (MEP) together with the prokaryotic SjGGDPS, the 13-R-MO titer was increased 11-fold to reach 9.7 mg/L by boosting the synthesis of GGDP, the direct substrate for the diterpenoid synthases. We further show that application of a n-dodecane overlay to remove 13-R-MO from the culture medium provided a 2–3 fold increase of the 13-R-MO in a separate cultivation system.

Review

21 February 2024

One-pot Multi-enzyme Cascade Synthesis of Bifunctional Compounds from Vegetable Oils

Green and efficient biocatalytic technology has become a complementary or alternative means of organic synthesis. Chemicals with two functional groups, such as α,ω-dicarboxylic acids, ω-amino fatty acids and ω-hydroxy fatty acids, are widely used in the synthesis of polymers such as polyesters and polyamides. In recent years, the production of biodegradable materials using renewable and abundant vegetable oils as green raw materials has attracted increasing attention, receiving an additional impetus from synthetic biology. This paper presents the recent research progress in the production of bifunctional chemicals with medium chain lengths of C8–C12 using multi-enzyme cascades. Recent studies have developed multilevel optimization strategies to improve the efficiency, economics, and sustainability of multi-enzyme cascades. Cofactor regeneration strategies were developed to avoid large additions of expensive coenzymes. Protein engineering strategies were applied to improve enzyme stability and catalytic performance. In addition, blocking the β-oxidation pathway, improving the efficiency of substrate transport across membranes and increasing cellular robustness are effective optimization strategies for whole-cell catalytic systems. In addition, we discuss the development prospects of producing high value-added fine chemicals from vegetable oils using one-pot multi-enzyme reaction systems.

Review

13 February 2024

Development and Perspective of Production of Terpenoids in Yeast

Terpenoids are a large class of secondary metabolites known for their remarkable diverse biological activities, making them widely utilized in the pharmaceutical, food, cosmetic, biofuel and agricultural fields. However, the current production of terpenoids heavily relies on plant extraction and chemical synthesis, which brings about concerns regarding infield, environmental and ecological issues. With the advancements in metabolic engineering and emerging synthetic biology tools, it is now possible to sustainably produce these high value-added terpenoids using microbial chassis. Among them, yeast has emerged as a promising candidate for the heterologous biosynthesis of terpenoids due to its inherent advantages, including robustness, safety, and the availability of sufficient precursor. This review focuses on the diverse strategies employed to enable terpenoids production in yeasts. These strategies encompass metabolic engineering approaches to optimize the mevalonate pathway, protein engineering techniques to improve terpenoid biosynthesis, the applications of organelles compartmentalization, high throughput screening and global approaches for the development of efficient cell factories. Furthermore, this review discusses the future prospects and challenges associated with yeast-based terpenoid production, while also emphasizing guidelines for future studies in this field.

Article

06 February 2024

Bio-Based Production of Uroporphyrin in Escherichia coli

Uroporphyrin (UP) is a porphyrin compound with medical applications and a key precursor for heme biosynthesis. However, there is no biosynthetic strategy for UP production. In this study, we present a novel bioprocess for enhanced production of UP in engineered Escherichia coli. We first implemented the Shemin/C4 pathway heterologously in an E. coli strain with an enlarged intracellular pool of succinyl-CoA. Using a plasmid with the trc promoter regulating the expression of a synthesized gene operon, the effects of key pathway genes, including hemA, hemB, hemC, and hemD, on UP biosynthesis were characterized. By cultivating the resulting engineered E. coli strains in a batch bioreactor with 30 g/L glycerol under aerobic conditions, up to 901.9 mg/L UP was produced. Most of the synthesized UP was extracellularly secreted with a high purity more than 80 wt%, facilitating its downstream purification. The study paves the way for large-scale bio-based production of UP using synthetic biology and metabolic engineering strategies.

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

Editorial

13 December 2022

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
StephenFong

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

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

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

Review

15 March 2023

Thermoanaerobacter Species: The Promising Candidates for Lignocellulosic Biofuel Production

Thermoanaerobacter species, which have broad substrate range and high operating temperature, can directly utilize lignocellulosic materials for biofuels production. Compared with the mesophilic process, thermophilic process shows greater prospects in consolidated bioprocessing (CBP) due to its relatively higher efficiency of lignocellulose degradation and lower risk of microbial contamination. Additionally, thermophilic conditions can reduce cooling costs, and further facilitate downstream product recovery. This review comprehensively summarizes the advances of Thermoanaerobacter species in lignocellulosic biorefinery, including their performance on substrates utilization, and genetic modification or other strategies for enhanced biofuels production. Furthermore, bottlenecks of sugar co-fermentation, metabolic engineering, and bioprocessing are also discussed.

KaiqunDai
ChunyunQu
HongxinFu
JufangWang

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.

LynnSchwardmann
Marielle Rieks
Volker F.Wendisch

Review

06 April 2023

Coiled Coils as Versatile Modules for Mammalian Cell Regulation

Synthetic biology is a rapidly growing field that allows us to better understand biological processes at the molecular level, and enables therapeutic interventions and biotechnological applications. One of the most powerful tools in synthetic biology is the small, customizable, and modular protein–protein interaction domains, which is used to regulate a wide variety of processes within mammalian cells. Here we review designed coiled coil dimers that represent a set of heterodimerization domains with many advantages. These dimers have been useful for directing the localization of selected proteins within cells, enhancing chemical or light-regulated transcription, creating fast proteolysis-based responsive systems and protein secretion, genome editing, and cell–cell interaction motifs. Additionally, we will discuss how these building blocks are used in diverse applications, such as CAR T cell regulation and genome editing. Finally, we will look at the potential for future advances in synthetic biology using these building modules.

EsteraMerljak
AnjaGolob-Urbanc
TjašaPlaper
RomanJerala

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

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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