Lauryl glucoside, a non-ionic surfactant used in various cosmetic products, is valued for its biodegradability and gentleness on the skin. However, its current production through chemical synthesis is considered unsustainable, necessitating the search for alternative methods. In this work, we engineered Escherichia coli BL21(DE3) with a novel lauryl glucoside biosynthetic pathway. Optimisation of 1-dodecanol, a lauryl glucoside precursor, was first implemented. Under optimised conditions, the strain produced 1-dodecanol at a titre of 185.39 ​± ​3.62 ​mg/L and a yield of 11.60 ​± ​0.29 ​mg/g glucose. These conditions were subsequently used to identify UDP-glycosyltransferases capable of converting 1-dodecanol to lauryl glucoside. Among six UDP-glycosyltransferases, MtH2 from Medicago truncatula showed the highest activity, with a titre and a yield of 0.72 ​± ​0.07 ​mg/L and 0.06 ​± ​0.004 ​mg/g glucose, respectively. The lauryl glucoside biosynthesis by MtH2 was confirmed using HPLC and targeted LC-MS. Moreover, the limited availability of 1-dodecanol was primarily identified as the bottleneck in this pathway. Supplementing the cells with twice the amount of 1-dodecanol led to an increase in lauryl glucoside production, achieving a titre of 13.44 ​± ​0.21 ​mg/L and a yield of 1.35 ​± ​0.04 ​mg/g glucose. Fermentation products of all strains were also monitored and suggested the redirection of carbon flux from acetate to the desired products. These findings demonstrate the successful characterisation of a newly designed lauryl glucoside biosynthetic pathway in engineered E. coli and highlight substrate limitation as a bottleneck in the pathway offering a sustainable alternative to traditional production methods.

With the rapid advancements in sustainable development and green chemistry, biotransformation has become increasingly pivotal in the synthesis of bulk chemicals and high-value products. Because natural evolution predominantly favors cellular survival, many valuable compounds, such as 2,4-dihydroxybutanoic acid and 1,2-butanediol, lack corresponding biosynthetic pathways in nature. This limitation calls for the development of fully nonnatural metabolic pathways. By enabling modular design and incorporating novel reactions, such pathways allow efficient de novo synthesis of compounds without known natural biosynthetic pathways. Nonetheless, their implementation may introduce new challenges, such as increased metabolic burden and the accumulation of toxic intermediates. Expanding the scope and efficiency of biotransformation through rational nonnatural pathways has become a key challenge. To address this, researchers have developed various computational methods for nonnatural pathway design, and two major types of methods, template-based and template-free methods, are reviewed here. We evaluate their practical applications in guiding the construction of microbial cell factories and analyze their effectiveness. Additionally, we compiled 55 experimentally validated nonnatural pathways from recent literature to establish a dataset for evaluating the strengths and limitations of these pathway design methods. By simulating a wide range of experimentally verified pathways, we highlight the gaps between computational predictions and empirical feasibility. Finally, we propose potential strategies to bridge these gaps, offering theoretical insights and practical guidance for integrating computational tools with experimental synthetic biology.

Glycerol, a major byproduct of biodiesel production, serves as a promising and versatile feedstock for synthesizing high-value chemicals. In this study, the NZN111, a potential succinic acid (SA) producer, was subjected to adaptive laboratory evolution (ALE) under selective pressure imposed by sodium acetate (NaAC) pressure. By analyzing the differences in the transcript levels of genes related to substrate utilization and product generation in the evolved strains, it was found that NaAC metabolism in Escherichia coli involves a series of enzymatic reactions that are apparently benefit for glycerol metabolism and SA biosynthesis. To further enhance the strain’s performance, a metabolic engineering strategy was implemented, including introduction of exogenous carboxykinases and HCO3− transporter proteins. The engineered strain produced 84.27 ​g/L SA in an anaerobic fermentation medium containing 100 ​g/L glycerol with a yield of 1.25 ​g/g glycerol, which was 1.38 and 1.16 times higher than those of the original strain, respectively. These findings highlight the potential of combining ALE with targeted metabolic engineering to develop robust microbial platforms for the efficient conversion of glycerol into high-value chemicals, contributing to the sustainable utilization of biodiesel byproducts.

Microbial cell factories (MCFs) are extensively used to produce a wide array of bioproducts, such as bioenergy, biochemical, food, nutrients, and pharmaceuticals, and have been regarded as the "chips" of biomanufacturing that will fuel the emerging bioeconomy era. Biotechnology advances have led to the screening, investigation, and engineering of an increasing number of microorganisms as diverse MCFs, which are the workhorses of biomanufacturing and help develop the bioeconomy. This review briefly summarizes the progress and strategies in the development of robust and efficient MCFs for sustainable and economic biomanufacturing. First, a comprehensive understanding of microbial chassis cells, including accurate genome sequences and corresponding annotations; metabolic and regulatory networks governing substances, energy, physiology, and information; and their similarity and uniqueness compared with those of other microorganisms, is needed. Moreover, the development and application of effective and efficient tools is crucial for engineering both model and nonmodel microbial chassis cells into efficient MCFs, including the identification and characterization of biological parts, as well as the design, synthesis, assembly, editing, and regulation of genes, circuits, and pathways. This review also highlights the necessity of integrating automation and artificial intelligence (AI) with biotechnology to facilitate the development of future customized artificial synthetic MCFs to expedite the industrialization process of biomanufacturing and the bioeconomy.

Probiotics are the treasure of the microbiology fields. They have been widely used in the food industry, clinical treatment, and other fields. The equivocal health-promoting effects and the unknown action mechanism were the largest obstacles for further probiotic's developed applications. In recent years, various genome editing techniques have been developed and applied to explore the mechanisms and functional modifications of probiotics. As important genome editing tools, CRISPR-Cas systems that have opened new improvements in genome editing dedicated to probiotics. The high efficiency, flexibility, and specificity are the advantages of using CRISPR-Cas systems. Here, we summarize the classification and distribution of CRISPR-Cas systems in probiotics, as well as the editing tools developed on the basis of them. Then, we discuss the genome editing of probiotics based on CRISPR-Cas systems and the applications of the engineered probiotics through CRISPR-Cas systems. Finally, we proposed a design route for CRISPR systems that related to the genetically engineered probiotics.

Genetic variations such as mutations and recombinations arise spontaneously in all cultured organisms. Although it is possible to identify nonneutral mutations by selection or counterselection, the identification of neutral mutations in a heterogeneous population usually requires expensive and time-consuming methods such as quantitative or droplet polymerase chain reaction and high-throughput sequencing. Neutral mutations could even become dominant under changing environmental conditions enforcing transitory selection or counterselection. We propose a novel method, which we called qSanger, to quantify DNA using amplitude ratios of aligned electropherogram peaks from mixed Sanger sequencing reads. Plasmids expressing enhanced green fluorescent protein and mCherry fluorescent markers were used to validate qSanger both in vitro and in cotransformed Escherichia coli via quantitative polymerase chain reaction and fluorescence quantifications. We show that qSanger allows the quantification of genetic variants, including single-base natural polymorphisms or de novo mutations, from mixed Sanger sequencing reads, with substantial reduction of labor and costs compared to canonical approaches.

The third-generation (3G) biorefinery aims to use microbial cell factories or enzymatic systems to synthesize value-added chemicals from one-carbon (C1) sources, such as CO2, formate, and methanol, fueled by renewable energies like light and electricity. This promising technology represents an important step toward sustainable development, which can help address some of the most pressing environmental challenges faced by modern society. However, to establish processes competitive with the petroleum industry, it is crucial to determine the most viable pathways for C1 utilization and productivity and yield of the target products. In this review, we discuss the progresses that have been made in constructing artificial biological systems for 3G biorefineries in the last 10 years. Specifically, we highlight the representative works on the engineering of artificial autotrophic microorganisms, tandem enzymatic systems, and chemo-bio hybrid systems for C1 utilization. We also prospect the revolutionary impact of these developments on biotechnology. By harnessing the power of 3G biorefinery, scientists are establishing a new frontier that could potentially revolutionize our approach to industrial production and pave the way for a more sustainable future.

The international Genetically Engineered Machine (iGEM) Foundation has continued to promote synthetic biology education throughout its 2021 competition. The 2021 Virtual iGEM Jamboree was the culmination of the competition's growth, with 350 projects from 7314 innovators globally. Collegiate, high school, and community lab teams applied their ideas to the Registry of Standard Biological Parts, designing biological systems that provide solutions to an international scope of issues. The environmental, diagnostics, and therapeutics tracks continue to be the most prevalent focal points for projects, as students devise approaches to detrimental impacts of climate change and the COVID-19 pandemic. The competition exemplifies high standards of human practices, biosafety, and biosecurity through responsible biological engineering. As the iGEM Foundation continues pioneering STEM education into the future, equal developments of the competition's economic accessibility, global diversity, and long-term impact are necessary to allow a larger range of thinkers to access the power of synthetic biology.

A grand challenge facing society is climate change caused mainly by rising CO2 concentration in Earth’s atmosphere. Terrestrial plants are linchpins in global carbon cycling, with a unique capability of capturing CO2 via photosynthesis and translocating captured carbon to stems, roots, and soils for long-term storage. However, many researchers postulate that existing land plants cannot meet the ambitious requirement for CO2 removal to mitigate climate change in the future due to low photosynthetic efficiency, limited carbon allocation for long-term storage, and low suitability for the bioeconomy. To address these limitations, there is an urgent need for genetic improvement of existing plants or construction of novel plant systems through biosystems design (or biodesign). Here, we summarize validated biological parts (e.g., protein-encoding genes and noncoding RNAs) for biological engineering of carbon dioxide removal (CDR) traits in terrestrial plants to accelerate land-based decarbonization in bioenergy plantations and agricultural settings and promote a vibrant bioeconomy. Specifically, we first summarize the framework of plant-based CDR (e.g., CO2 capture, translocation, storage, and conversion to value-added products). Then, we highlight some representative biological parts, with experimental evidence, in this framework. Finally, we discuss challenges and strategies for the identification and curation of biological parts for CDR engineering in plants.