Computational tools for nonnatural pathway design: Algorithms, applications, and challenges

Progress and perspective on development of non-model industrial bacteria as chassis cells for biochemical production in the synthetic biology era

Human breast milk (HBM) is composed of various components that are crucial for providing essential nutrients and enhancing infant immune system. Recently, the synthesis of HBM components through microbial fermentation has garnered significant attention due to its potential to reduce production costs, simplify manufacturing processes, and mitigate environmental pollution. However, this approach results in low yield and is difficult to scale up at the industrial level. Therefore, various synthetic biology tools have been used to enhance the efficiency of HBM component synthesis. This review first summarizes several synthetic biology tools that may improve HBM component production. Next, we have summarized HBM component production using microbial cell factories. Finally, we have summarized the challenges and opportunities presented by the construction of cell factories for the synthesis of HBM using synthetic biology tools. This article therefore provides a general guide to the construction of microbial cell factories for HBM components.

Scopoletin is a typical example of coumarins, which can be produced in plants. Scopoletin acts as a precursor for pharmaceutical and health care products, and also possesses promising biological properties, including antibacterial, anti-tubercular, anti-hypertensive, anti-inflammatory, anti-diabetic, and anti-hyperuricemic activity. Despite the potential benefits, the production of scopoletin using traditional extraction processes from plants is unsatisfactory. In recent years, synthetic biology has developed rapidly and enabled the effective construction of microbial cell factories for production of high value-added chemicals. Herein, this review summarizes the progress of scopoletin biosynthesis in artificial microbial cell factories. The two main pathways of scopoletin biosynthesis are summarized firstly. Then, synthetic microbial cell factories are reviewed as an attractive improvement strategy for biosynthesis. Emerging techniques in synthetic biology and metabolic engineering are introduced as innovative tools for the efficient synthesis of scopoletin. This review showcases the potential of biosynthesis of scopoletin in artificial microbial cell factories.

De novo production of 1,3-olein-2-palmitin (OPO) and 1-olein-2-palmitin-3-linolein (OPL) by multiplexed reconstruction of lipid metabolism in yeasts.

Construction of microbial cell factories is a feasible strategy for the sustainable production of chemicals, biofuels, and pharmaceutical molecules. However, the complex metabolism and rigid regulation of microbes hinders the efficient synthesis of the products of interest. Proteomics and metabolomics analyze enzymes and metabolites in terms of systems biology, thus unraveling complex biological systems and providing significant clues for microbial metabolic engineering. Here, the applications of proteomics and metabolomics in microbial metabolic engineering were reviewed, including the construction of genome-scale metabolic models, optimization of microbial product synthesis, guidance of microbial stress tolerance engineering, and prediction of rate-limiting steps. In addition, proteomics and metabolomics could be employed to explore secondary metabolic pathways in plants, to reveal novel genes or pathways for microbial synthesis of natural products. Finally, the development of bio-big data was discussed.

Monitoring intracellular pyruvate is useful for the exploration of fundamental metabolism and for guiding the construction of yeast cell factories for chemical production. Here, we employed a genetically encoded fluorescent Pyronic biosensor to light up the pyruvate metabolic state in the cytoplasm, nucleus, and mitochondria of Saccharomyces cerevisiae BY4741. A strong correlation was observed between the pyruvate fluctuation in mitochondria and cytoplasm when exposed to different metabolites. Further metabolic analysis of pyruvate uptake and glycolytic dynamics showed that glucose and fructose dose-dependently activated cytoplasmic pyruvate levels more effectively than direct exposure to pyruvate. Meanwhile, the Pyronic biosensor could visually distinguish phenotypes of the wild-type S. cerevisiae BY4741 and the pyruvate-hyperproducing S. cerevisiae TAM at a single-cell resolution, having the potential for high-throughput screening. Overall, Pyronic biosensors targeting different suborganelles contribute to mapping and studying the central carbon metabolism in-depth and guide the design and construction of yeast cell factories.

5-Aminolevulinic acid (5-ALA) plays a pivotal role in the biosynthesis of heme and chlorophyll and has garnered great attention for its agricultural applications. This study explores the multifaceted construction of 5-ALA microbial cell factories. Evolutionary analysis-guided screening identified a novel 5-ALA synthase from Sphingobium amiense as the best synthase. An sRNA library facilitated global gene screening that demonstrated that trpC and ilvA repression enhanced 5-ALA production by 74.3% and 102%, respectively. Subsequently, efflux of 5-ALA by the transporter Gdx increased 5-ALA biosynthesis by 25.7%. To mitigate oxidative toxicity, DNA-binding proteins from starved cells were employed, enhancing cell density and 5-ALA titer by 21.1 and 4.1%, respectively. Combining these strategies resulted in an Escherichia coli strain that produced 5-ALA to 1.51 g·L-1 in shake flask experiments and 6.19 g·L-1 through fed-batch fermentation. This study broadens the repertoire of available 5-ALA synthases and transporters and provides a new platform for optimizing 5-ALA bioproduction.

EDITORIAL article Front. Microbiol., 11 January 2016Sec. Microbial Physiology and Metabolism Volume 6 - 2015 | https://doi.org/10.3389/fmicb.2015.01562

Investigation of essential genes, besides contributing to understanding the fundamental principles of life, has numerous practical applications. Essential genes can be exploited as building blocks of a tightly controlled cell 'chassis'. Bacillus subtilis and Escherichia coli K-12 are both well-characterized model bacteria used as hosts for a plethora of biotechnological applications. Determination of the essential genes that constitute the B. subtilis and E. coli minimal genomes is therefore of the highest importance. Recent advances have led to the modification of the original B. subtilis and E. coli essential gene sets identified 10 years ago. Furthermore, significant progress has been made in the area of genome minimization of both model bacteria. This review provides an update, with particular emphasis on the current essential gene sets and their comparison with the original gene sets identified 10 years ago. Special attention is focused on the genome reduction analyses in B. subtilis and E. coli and the construction of minimal cell factories for industrial applications.

Microbial synthesis of glycosaminoglycans and their oligosaccharides.

Three-dimensional bioprinting integrating living cells and bioactive materials enables the fabrication of scaffold structures supporting diverse cellular growth and metabolism. Microalgae are among the most promising microbial platforms for the construction of photosynthetic cell factories, while the current industrial-scale cultivation of microalgae remains predominantly dependent on traditional liquid submerged systems, imposing limitations on commercial viability due to both process and economic constraints. Encapsulation of microalgae within bioactive matrices combined with 3D bioprinting to fabricate customized structures has been explored to address the limitations of submerged cultivation, which are expected to expand microalgal applications and establish new research directions in microalgal biotechnology. This review analyzes both matrices and methods of 3D bioprinting, summarizing the advancement of microalgae-based 3D bioprinting into six main domains including living building materials, biophotovoltaics, photosynthetic biosynthesis, bioremediation, tissue engineering, and food engineering. Lastly, synthetic biology-informed perspectives are provided on future developments of 3D bioprinting technologies and their potential in microalgal research.

The filamentous fungus Aspergillus niger is widely used in the biotechnology industry for the production of chemicals and enzymes. Engineering of this valuable organism to improve its productivity is currently hampered by the lack of efficient genetic tools. Here, a Cre-loxP-based system for gene editing in A. niger was developed and its application in construction of A. niger cell factories to produce various organic acids was explored. Two established inducible systems, the xylanase A gene promoter Pxln and Tet-on system, were examined for driving cre expression and thus selection marker hyh deletion. Under inducing conditions, the efficiency of loxP site-specific recombination in the strain with cre driven by Pxln is about 2%, while cre driven by Tet-on system is about 34% which was used as the platform strain for further genetic engineering. As a proof of application of this system, strains containing different copies of oxaloacetate acetylhydrolase-encoding gene (oahA) were constructed, and the resultant strain S428 showed as high as 3.1-fold increase in oxalic acid production. Furthermore, an efficient malate-producing strain was generated through four-step genetic manipulation (oahA deletion, pyc, mdh3 and C4-dicarboxylate transporter gene c4t318 insertion). The resultant strain S575 achieved a titer 120.38 g/L malic acid with the flask culture, and a titer 201.24 g/L malic acid in fed-batch fermentation. These results demonstrated that this modified Cre-loxP system is a powerful tool for genetic engineering in A. niger, which has the potential to be genetically modified as a viable aciduric platform strain to produce high levels of various organic acids.

Regardless of bacteria or eukaryotic microorganism hosts, improving their ability to express heterologous proteins is always a goal worthy of elaborate study. In addition to traditional methods including intracellular synthesis process regulation and extracellular environment optimization, some special or extreme conditions can also be employed to create an enhancing effect on heterologous protein production. In this review, we summarize some extreme environmental factors used for the improvement of heterologous protein expression, including low temperature, hypoxia, microgravity and high osmolality. The applications of these strategies are elaborated with examples of well-documented studies. We also demonstrated the confirmed or hypothetical mechanisms of environment stress affecting the host behaviors. In addition, multi-omics techniques driving the stress-responsive research for construction of efficient microbial cell factories are also prospected at the end.

Myxobacteria: Versatile cell factories of novel commercial enzymes for bio-manufacturing.

Microbial cell factories play a pivotal role in the field of biomanufacturing. Enhancing their construction efficiency and product yield is essential for advancing synthetic biology and its industrial implementation. In recent years, the rapid development of large DNA manipulation technologies provided powerful support for cloning, assembling, delivering, and rearranging large DNA. This review hence systematically summarizes the core principles and recent advances in four categories of large DNA manipulation techniques. It highlights their key roles in access to complex biosynthetic gene clusters, constructing multigene biosynthetic pathways, introducing complex genetic modules into microbial chassis, structural rewiring, and modular reconstruction for metabolic network optimization. Furthermore, this review explores the emerging trend of large DNA manipulation technologies to advance microbial cell factories. This review is expected to serve as a technical reference for advancing large DNA manipulation technologies and extending their applications toward the construction of high-performance microbial cell factories.