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 7–16 and 0.6–1.4, 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.
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.
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.
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.
As an important
intermediate in the tricarboxylic acid (TCA) cycle,
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.
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.
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.
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.
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.
