Intracellularly synthesized ssDNA for continuous genome engineering.

Despite the prevalence of genome editing tools, there are still some limitations in dynamic and continuous genome editing. In vivo single-stranded DNA (ssDNA)-mediated genome mutation has emerged as a valuable and promising approach for continuous genome editing. In this review, we summarize the various types of intracellular ssDNA production systems and notable achievements in genome engineering in both prokaryotic and eukaryotic cells.

Engineering Living Material for Controlled Fragrance Release Utilizing CBS6556 and Adaptive Hydrogel.

The demand for controllable fragrance materials is substantial owing to their potential to impart enduring scents in a variety of applications. However, the practical application of such materials has been limited by challenges in tunable morphogenesis, structural variability, and adaptability to diverse conditions. In our study, we introduce a hybrid living material that integrates a genetically engineered strain of CBS6556 with an adaptive hydrogel.

Transportation engineering for enhanced production of plant natural products in microbial cell factories.

Plant natural products (PNPs) exhibit a wide range of biological activities and have essential applications in various fields such as medicine, agriculture, and flavors. Given their natural limitations, the production of high-value PNPs using microbial cell factories has become an effective alternative in recent years. However, host metabolic burden caused by its massive accumulation has become one of the main challenges for efficient PNP production.

Intein-mediated temperature control for complete biosynthesis of sanguinarine and its halogenated derivatives in yeast.

While sanguinarine has gained recognition for antimicrobial and antineoplastic activities, its complex conjugated structure and low abundance in plants impede broad applications. Here, we demonstrate the complete biosynthesis of sanguinarine and halogenated derivatives using highly engineered yeast strains. To overcome sanguinarine cytotoxicity, we establish a splicing intein-mediated temperature-responsive gene expression system (SIMTeGES), a simple strategy that decouples cell growth from product synthesis without sacrificing protein activity.

Metabolic engineering of Pichia pastoris for overproduction of cis-trans nepetalactol.

Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs.

Intricate Metabolic Network for Paclitaxel Biosynthesis.

Paclitaxel is a renowned broad-spectrum anticancer drug. With the establishment of a chromosome-level high-quality reference genome map of , recent research on paclitaxel biosynthesis has flourished. The oxetane ring is a distinctive chemical moiety of paclitaxel, and three recent studies have proposed different enzymes involved in its formation, reflecting divergent opinions on whether the pathway proceeds via acetylation followed by epoxidation or vice versa.

Reconstitution and optimization of complex plant natural product biosynthetic pathways in microbial expression systems.

Plant natural products (PNPs) are a diverse group of chemical compounds synthesized by plants for various biological purposes and play a significant role in the fields of medicine, agriculture, and industry. In recent years, the development of synthetic biology promises the production of PNPs in microbial expression systems in a sustainable, low-cost, and large-scale manner. This review first introduces multiplex genome editing and PNP pathway assembly in microbial expression systems.

Development of a Pichia pastoris cell factory for efficient production of germacrene A: a precursor of β-elemene.

β-Elemene, an active ingredient found in medicinal plants like turmeric and zedoary, is a sesquiterpene compound with antitumor activity against various cancers. However, its current mode of production through plant extraction suffers from low efficiency and limited natural resources. Recently, there has been an increased interest in establishing microbial cell factories to produce germacrene A, which can be converted to β-elemene by a one-step reaction in vitro.

Efficient production of l-glutathione by whole-cell catalysis with ATP regeneration from adenosine.

l-glutathione (GSH) is an important tripeptide compound with extensive applications in medicine, food additives, and cosmetics industries. In this work, an innovative whole-cell catalytic strategy was developed to enhance GSH production by combining metabolic engineering of GSH biosynthetic pathways with an adenosine-based adenosine triphosphate (ATP) regeneration system in Escherichia coli. Concretely, to enhance GSH production in E.

Characterization and engineering of peroxisome targeting sequences for compartmentalization engineering in Pichia pastoris.

Peroxisomal compartmentalization has emerged as a highly promising strategy for reconstituting intricate metabolic pathways. In recent years, significant progress has been made in the peroxisomes through harnessing precursor pools, circumventing metabolic crosstalk, and minimizing the cytotoxicity of exogenous pathways. However, it is important to note that in methylotrophic yeasts (e.

Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.

Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P.

Peroxisome-based metabolic engineering for biomanufacturing and agriculture.

Subcellular compartmentalization of metabolic pathways plays a crucial role in metabolic engineering. The peroxisome has emerged as a highly valuable and promising compartment for organelle engineering, particularly in the fields of biological manufacturing and agriculture. In this review, we summarize the remarkable achievements in peroxisome engineering in yeast, the industrially popular biomanufacturing chassis host, to produce various biocompounds.

XylR Overexpression in Escherichia coli Alleviated Transcriptional Repression by Arabinose and Enhanced Xylitol Bioproduction from Xylose Mother Liquor.

Xylitol is a polyol widely used in food, pharmaceuticals, and light industries. It is currently produced through the chemical catalytic hydrogenation of xylose and generates xylose mother liquor as a substantial byproduct in the procedure of xylose extraction. If xylose mother liquor could also be efficiently bioconverted to xylitol, the greenness and atom economy of xylitol production would be largely improved.

Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in .

H16 is a "Knallgas" bacterium with the ability to utilize various carbon sources and has been employed as a versatile microbial cell factory to produce a wide range of value-added compounds. However, limited genome engineering, especially gene regulation methods, has constrained its full potential as a microbial production platform. The advent of CRISPR/Cas9 technology has shown promise in addressing this limitation.

Nanopore sequencing improves construction of customized CRISPR-based gene activation libraries.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based screening has emerged as a powerful tool for identifying new gene targets for desired cellular phenotypes. The construction of guide RNA (gRNA) pools largely determines library quality and is usually performed using Golden Gate assembly or Gibson assembly. To date, library construction methods have not been systematically compared, and the quality check of each batch has been slow.

De novo biosynthesis of antiarrhythmic alkaloid ajmaline.

The antiarrhythmic drug ajmaline is a monoterpenoid indole alkaloid (MIA) isolated from the Ayurvedic plant Rauvolfia serpentina (Indian Snakeroot). Research into the biosynthesis of ajmaline and another renowned MIA chemotherapeutic drug vinblastine has yielded pivotal advancements in the fields of plant specialized metabolism and engineering over recent decades. While the majority of vinblastine biosynthesis has been recently elucidated, the quest for comprehending ajmaline biosynthesis remains incomplete, marked by the absence of two critical enzymes.

Multisite Mutation of the cAMP Receptor Protein: Enhancing Xylitol Biosynthesis by Activating Xylose Catabolism and Improving Strain Tolerance.

The bioproduction of xylitol from hemicellulose hydrolysate has good potential for industrial development. However, xylitol productivity has always been limited due to corncob hydrolysate toxicity and glucose catabolic repression. To address these challenges, this work selected the S83 and S128 amino acid residues of the cyclic AMP receptor protein (CRP) as the modification target.

De Novo Biosynthesis of Vindoline and Catharanthine in .

Vinblastine has been used clinically as one of the most potent therapeutics for the treatment of several types of cancer. However, the traditional plant extraction method suffers from unreliable supply, low abundance, and extremely high cost. Here, we use synthetic biology approach to engineer for de novo biosynthesis of vindoline and catharanthine, which can be coupled chemically or biologically to vinblastine.

De Novo Design and Synthesis of Polypeptide Immunomodulators for Resetting Macrophage Polarization.

Modulating the extracellular matrix microenvironment is critical for achieving the desired macrophage phenotype in immune investigations or tumor therapy. Combining de novo protein design and biosynthesis techniques, herein, we designed a biomimetic polypeptide self-assembled nano-immunomodulator to trigger the activation of a specific macrophage phenotype. It was intended to be made up of (​GGS​GGP​GGG​PAS​AAA​NSA​SRA​TSN​SP), the RGD motif from collagen, and the IKVAV motif from laminin.

Biomanufacturing of γ-linolenic acid-enriched galactosyldiacylglycerols: Challenges in microalgae and potential in oleaginous yeasts.

γ-Linolenic acid-enriched galactosyldiacylglycerols (GDGs-GLA), as the natural form of γ-linolenic acid in microalgae, have a range of functional activities, including anti-inflammatory, antioxidant, and anti-allergic properties. The low abundance of microalgae and the structural stereoselectivity complexity impede microalgae extraction or chemical synthesis, resulting in a lack of supply of GDGs-GLA with a growing demand. At present, there is a growing interest in engineering oleaginous yeasts for mass production of GDGs-GLA based on their ability to utilize a variety of hydrophobic substrates and a high metabolic flux toward fatty acid and lipid (triacylglycerol, TAG) production.

Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.

While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively.