Hydroxybenzoic acids (HBAs), including 4-HBA, 3-HBA, and 2-HBA, are valuable platform chemicals for production of commodity materials and fine chemicals. Herein, we employed metabolic engineering techniques to enhance the production of these HBAs in Corynebacterium glutamicum ATCC 13032. Our approach augmented the shikimate pathway and eliminated genes associated with HBA degradation, particularly phenol 2-monooxygenase encoded by cg2966. Increased titers of 3-HBA and 4-HBA were achieved via selection of suitable promoters for 3-hydroxybenzoate synthase and chorismate pyruvate lyase. A tac-M1 promoter was suitable for chorismate pyruvate lyase expression and 8.3 g/L of 4-HBA production was achieved. Efficient production of 2-HBA was enabled by maintaining a balanced expression of isochorismate synthase and isochorismate pyruvate lyase. Consequently, strains KSD5-tacM1-H and KSD5-J2-PE exhibited production levels of 19.2 g/L of 3-HBA and 12.9 g/L of 2-HBA, respectively, using 1 L jar fermenter containing 80 g/L of glucose. Therefore, this engineered strain platform holds significant potential for production of other valuable products derived from chorismate.
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.
As important bio-chemicals, glycerol and 1,3-propanediol (1,3-PDO) have been widely used in numerous fields, e.g., polymers, cosmetics, foods, lubricants, medicines, and so on. Bio-based 1,3-PDO is generally produced from glycerol or glucose by natural or recombinant strains, during which organic acids are always co-produced. In this work, acetic acid was also co-produced when 1,3-PDO was obtained from glucose by a recombinant strain of E. coli MG1655. Usually, a base was added to adjust the fermentation pH, resulting in the accumulation of organic salts and difficulty in the next down streaming process. Herein, a novel strategy was developed to consume the produced acetic acid by cells self in fed-batch self-regulated fermentation. The recombinant E. coli cells produced 48.33 g/L glycerol and 61.27 g/L 1,3-PDO with a total mass yield of 45.6% and without any other byproducts at the end of 5 fed-batch fermentations. The initial buffer pH, glucose concentration, pulse feeding sugar amount, time for a single batch fermentation and reducing acid were investigated by a series of comparative experiments. This fed-batch self-regulated fermentation has potential for the co-production of 1,3-PDO and glycerol, and will highlight the subsequent modification of recombinant E. coli strain by synthetic biology.
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.
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.
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.
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.
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.
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.
