Transcriptome Analysis Reveals the Role of Sucrose in the Production of Latilactobacillus sakei L3 Exopolysaccharide.

Latilactobacillus (L.) sakei is a species of lactic acid bacteria (LAB) mostly studied according to its application in food fermentation. Previously, L. sakei L3 was isolated by our laboratory and possessed the capability of high exopolysaccharide (EPS) yield during sucrose-added fermentation. However, the understanding of sucrose promoting EPS production is still limited. Here, we analyzed the growth characteristics of L. sakei L3 and alterations of its transcriptional profiles during sucrose-added fermentation. The results showed that L. sakei L3 could survive between pH 4.0 and pH 9.0, tolerant to NaCl (<10%, w/v) and urea (<6%, w/v). Meanwhile, transcriptomic analysis showed that a total of 426 differentially expressed genes and eight non-coding RNAs were identified. Genes associated with sucrose metabolism were significantly induced, so L. sakei L3 increased the utilization of sucrose to produce EPS, while genes related to uridine monophosphate (UMP), fatty acids and folate synthetic pathways were significantly inhibited, indicating that L. sakei L3 decreased self-growth, substance and energy metabolism to satisfy EPS production. Overall, transcriptome analysis provided valuable insights into the mechanisms by which L. sakei L3 utilizes sucrose for EPS biosynthesis. The study provided a theoretical foundation for the further application of functional EPS in the food industry.

Isopropanol production using engineered Corynebacterium glutamicum from waste rice straw biomass.

Isopropanol, a well-known biofuel, is a widely used precursor for chemical products that can replace nonrenewable petroleum energy. Here, engineered Corynebacterium glutamicum that can effectively utilize all xylose and glucose in agricultural waste rice straw to produce isopropanol was described. First, codon mutations were introduced into transporters and glycolytic-related genes to decrease the glucose preference of C. glutamicum. A more energetically favorable xylose oxidative pathway was constructed that replaced traditional xylose isomerization pathways, saving twice the number of enzymatic steps. A succinate auxiliary module was incorporated into the tricarboxylic acid cycle (TCA), connecting the xylose-utilized pathway with the isopropanol pathway to maximize xylose orientation towards the product. The final engineered strain successfully consumed 100 % of the xylose from NaOH-pretreated, enzyme-hydrolyzed rice straw and effectively synthesized 4.91 g/L isopropanol. This study showcases the successful conversion of agricultural waste into renewable energy, unveiling new possibilities for advancing biological fermentation technology.

Microbial host engineering for sustainable isobutanol production from renewable resources.

Due to the limited resources and environmental problems associated with fossil fuels, there is a growing interest in utilizing renewable resources for the production of biofuels through microbial fermentation. Isobutanol is a promising biofuel that could potentially replace gasoline. However, its production efficiency is currently limited by the use of naturally isolated microorganisms. These naturally isolated microorganisms often encounter problems such as a limited range of substrates, low tolerance to solvents or inhibitors, feedback inhibition, and an imbalanced redox state. This makes it difficult to improve their production efficiency through traditional process optimization methods. Fortunately, recent advancements in genetic engineering technologies have made it possible to enhance microbial hosts for the increased production of isobutanol from renewable resources. This review provides a summary of the strategies and synthetic biology approaches that have been employed in the past few years to improve naturally isolated or non-natural microbial hosts for the enhanced production of isobutanol by utilizing different renewable resources. Furthermore, it also discusses the challenges that are faced by engineered microbial hosts and presents future perspectives to enhancing isobutanol production. KEY POINTS: • Promising potential of isobutanol to replace gasoline • Engineering of native and non-native microbial host for isobutanol production • Challenges and opportunities for enhanced isobutanol production.

A Synthetic Pathway for the Production of Benzylsuccinate in Escherichia coli.

(R)-Benzylsuccinate is generated in anaerobic toluene degradation by the radical addition of toluene to fumarate and further degraded to benzoyl-CoA by a ß-oxidation pathway. Using metabolic modules for benzoate transport and activation to benzoyl-CoA and the enzymes of benzylsuccinate ß-oxidation, we established an artificial pathway for benzylsuccinate production in Escherichia coli, which is based on its degradation pathway running in reverse. Benzoate is supplied to the medium but needs to be converted to benzoyl-CoA by an uptake transporter and a benzoate-CoA ligase or CoA-transferase. In contrast, the second substrate succinate is endogenously produced from glucose under anaerobic conditions, and the constructed pathway includes a succinyl-CoA:benzylsuccinate CoA-transferase that activates it to the CoA-thioester. We present first evidence for the feasibility of this pathway and explore product yields under different growth conditions. Compared to aerobic cultures, the product yield increased more than 1000-fold in anaerobic glucose-fermenting cultures and showed further improvement under fumarate-respiring conditions. An important bottleneck to overcome appears to be product excretion, based on much higher recorded intracellular concentrations of benzylsuccinate, compared to those excreted. While no export system is known for benzylsuccinate, we observed an increased product yield after adding an unspecific mechanosensitive channel to the constructed pathway.

Way to efficient microbial paclitaxel mass production.

The microbial synthesis of paclitaxel is attractive for its short-cycle, cost-effectiveness, and sustainability. However, low paclitaxel productivity, depleted capacity during subculture and storage, and unclear biosynthesis mechanisms restrain industrial microbial synthesis. Along with the isolation of various paclitaxel-producing microorganisms and the development of versatile molecular tools, tremendous promises for microbial paclitaxel synthesis have become increasingly prominent. In this review, we summarize the progress of microbial synthesis of paclitaxel in recent years, focusing on paclitaxel-producing endophytes and representative engineering microorganism hosts that were used as chassis for paclitaxel precursor synthesis. Numerous wide-type microbes can manufacture paclitaxel, and fermentation process optimization and strain improvement can greatly enhance the productivity. Engineered microbes can efficiently synthesize precursors of paclitaxel by introducing exogenous synthetic pathway. Mining paclitaxel synthetic pathways and genetic manipulation of endophytes will accelerate the construction of microbial cell factories, indefinitely contributing to paclitaxel mass production by microbes. This review emphasizes the potential and provides solutions for efficient microbial paclitaxel mass production.

Integrated of multi-omics and molecular docking reveal PHGDH, PSAT1 and PSPH in the serine synthetic pathway as potential targets of T-2 toxin exposure in pig intestinal tract.

T-2 toxin (T-2) with a molecular weight of 466.52 g/mol is an inevitable mycotoxin in food products and feeds, posing a significant threat to human and animal health. However, the underlying molecular mechanisms of the cytotoxic effects of T-2 exposure on porcine intestinal epithelial cells (IPEC-J2) remain unclear. Here, we investigated the cytotoxic effects of T-2 exposure on IPEC-J2 through the detection of cell viability, cell morphology, mitochondrial membrane potential, ROS, apoptosis and autophagy. Further transcriptomic and proteomic analyses of IPEC-J2 upon T-2 exposure were performed by using RNA-seq and TMT techniques. A total of 546 differential expressed genes (DEGs) and 269 differentially expressed proteins (DEPs) were detected. Among these, 24 common DEGs/DEPs were involved in IPEC-J2 upon T-2 exposure. Interestingly, molecular docking analysis revealed potential interactions between T-2 and three key enzymes (PHGDP, PSAT1, and PSPH) in the serine biosynthesis pathway. Besides, further experimental showed that PSAT1 knockdown exacerbated T-2-induced oxidative damage. Together, our findings indicated that the serine biosynthesis pathway including PHGDP, PSAT1, PSPH genes probably acts critical roles in the regulation of T-2-induced cell damage. This study provided new insights into the global molecular effects of T-2 exposure and identified the serine biosynthesis pathway as molecular targets and potential treatment strategies against T-2.

Profiling of lipids in Thermus thermophilus HB8 grown under various conditions.

The membrane lipids of Thermus species have unique structures. Only four polar lipid species have so far been identified in Thermus thermophilus HB8; namely, are two phosphoglycolipids and two glycolipids, both of which have three branched fatty acid chains. Other lipid molecules may be present; however, they have not been identified so far. To clarify the whole lipid profile of T. thermophilus HB8, we cultured this organism under four different growth (temperature and/or nutrition) conditions and analyzed the compositions of polar lipids and fatty acids by high-performance thin-layer chromatography (HPTLC) and gas chromatograph-mass spectrometry (GCï½°MS), respectively. Thirty-one lipid spots were detected on HPTLC plates and profiled in terms of the presence or absence of phosphate, amino, and sugar groups. Then, we allocated ID numbers to all the spots. Comparative analyses of these polar lipids showed that the diversity of lipid molecules increased under high temperature and minimal medium conditions. In particular, aminolipid species increased under high temperature conditions. As for the fatty acid comparison by GC-MS, iso-branched even-numbered carbon atoms, which are unusual in this organism, significantly increased under the minimal medium condition, suggesting that kinds of branched amino acids at the fatty acid terminus varies under different nutrition conditions. In this study, several unidentified lipids were detected, and elucidation of the lipid structures will provide important information on the environmental adaptation of bacteria.

[Research progress in strigolactones and application prospect in medicinal plants].

Strigolactones(SLs) are a class of sesquiterpenoids derived from the carotenoid biosynthesis pathway with the core carbon skeleton consisting of tricyclic lactone(ABC tricyclic ring) and α,ß-unsaturated furan ring(D ring). SLs are widely distributed in higher plants and are symbiotic signals between plants and Arbuscular mycorrhiza(AM), which play key roles in the evolution of plant colonizing terrestrial habitats. As a new type of plant hormone, SLs possess such important biological functions as inhibiting shoot branching(tillers), regulating root architecture, promoting secondary growth, and improving plant stress resistance. Therefore, SLs have attracted wide attention. The biological functions of SLs are not only closely related to the formation of "excellent shape and quality" of Chinese medicinal materials but also have important practical significance for the production of high-quality medicinal materials. However, SLs have been currently widely studied in model plants and crops such as Oryza sativa and Arabidopsis thaliana, and few related studies have been reported on SLs in medicinal plants, which need to be strengthened. This review focused on the latest research progress in the isolation and identification, biological and artificial synthesis pathways, biosynthesis sites and transport modes, signal transduction pathways and mechanisms, and biological functions of SLs, and prospected the research on the regulation mechanism of SLs in the growth and development of medicinal plants and their related application on targeted regulation of Chinese herbal medicine production, which is expected to provide some references for the in-depth research on SLs in the field of Chinese medicinal resources.

Reprogramming cellular metabolism to increase the efficiency of microbial cell factories.

Recent studies are increasingly focusing on advanced biotechnological tools, self-adjusting smart microorganisms, and artificial intelligent networks, to engineer microorganisms with various functions. Microbial cell factories are a vital platform for improving the bioproduction of medicines, biofuels, and biomaterials from renewable carbon sources. However, these processes are significantly affected by cellular metabolism, and boosting the efficiency of microbial cell factories remains a challenge. In this review, we present a strategy for reprogramming cellular metabolism to enhance the efficiency of microbial cell factories for chemical biosynthesis, which improves our understanding of microbial physiology and metabolic control. Current methods are mainly focused on synthetic pathways, metabolic resources, and cell performance. This review highlights the potential biotechnological strategy to reprogram cellular metabolism and provide novel guidance for designing more intelligent industrial microbes with broader applications in this growing field.

Engineering Siderophore Biosynthesis and Regulation Pathways to Increase Diversity and Availability.

Siderophores are small metal chelators synthesized by numerous organisms to access iron. These secondary metabolites are ubiquitously present on Earth, and because their production represents the main strategy to assimilate iron, they play an important role in both positive and negative interactions between organisms. In addition, siderophores are used in biotechnology for diverse applications in medicine, agriculture and the environment. The generation of non-natural siderophore analogs provides a new opportunity to create new-to-nature chelating biomolecules that can offer new properties to expand applications. This review summarizes the main strategies of combinatorial biosynthesis that have been used to generate siderophore analogs. We first provide a brief overview of siderophore biosynthesis, followed by a description of the strategies, namely, precursor-directed biosynthesis, the design of synthetic or heterologous pathways and enzyme engineering, used in siderophore biosynthetic pathways to create diversity. In addition, this review highlights the engineering strategies that have been used to improve the production of siderophores by cells to facilitate their downstream utilization.

Enhanced vitamin K2 production by engineered Bacillus subtilis during leakage fermentation.

Menaquinone-7 (MK-7), a valuable member of the vitamin K2 series, is an essential nutrient for humans. It is used for treating coagulation disorders, and osteoporosis, promoting liver function recovery, and preventing cardiovascular diseases. In this study, to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of surfactants on the metabolic synthesis of MK-7 by the mutant strain Bacillus subtilis 168 KO-SinR (BS168 KO-SinR) was analyzed. The scanning electron microscopy and flow cytometry results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. Quantitative real-time PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the electron microscopy results showed that the addition of surfactant changed the permeability of the cell membrane. The research results of this paper can serve as a reference for the industrial development of MK-7 prepared by fermentation.

Machine learning-assisted medium optimization revealed the discriminated strategies for improved production of the foreign and native metabolites.

The composition of medium components is crucial for achieving the best performance of synthetic construction in genetically engineered cells. Which and how medium components determine the performance, e.g., productivity, remain poorly investigated. To address the questions, a comparative survey with two genetically engineered Escherichia coli strains was performed. As a case study, the strains carried the synthetic pathways for producing the aromatic compounds of 4-aminophenylalanine (4APhe) or tyrosine (Tyr), common in the upstream but differentiated in the downstream metabolism. Bacterial growth and compound production were examined in hundreds of medium combinations that comprised 48 pure chemicals. The resultant data sets linking the medium composition to bacterial growth and production were subjected to machine learning for improved production. Intriguingly, the primary medium components determining the production of 4PheA and Tyr were differentiated, which were the initial resource (glucose) of the synthetic pathway and the inducer (IPTG) of the synthetic construction, respectively. Fine-tuning of the primary component significantly increased the yields of 4APhe and Tyr, indicating that a single component could be crucial for the performance of synthetic construction. Transcriptome analysis observed the local and global changes in gene expression for improved production of 4APhe and Tyr, respectively, revealing divergent metabolic strategies for producing the foreign and native metabolites. The study demonstrated that ML-assisted medium optimization could provide a novel point of view on how to make the synthetic construction meet the designed working principle and achieve the expected biological function.

Triterpenoids in Jujube: A Review of Composition, Content Diversity, Pharmacological Effects, Synthetic Pathway, and Variation during Domestication.

Chinese jujube (Ziziphus jujuba Mill.) and its wild ancestor, sour jujube (Z. acidojujuba C.Y. Cheng & M.J. Liu), is a Ziziphus genus in the Rhamnaceae family. ZJ and ZA are rich in a variety of active ingredients, with triterpenoids being a unique active ingredient, which are present in the fruit, leaves, branches, and roots. More than 120 triterpenoids have been identified in ZJ and ZA, and have various biological activities. For example, betulinic and ursolic acids have anticancer, antioxidant, antibacterial and antiviral activities. ceanothic, alphitolic, and zizyberanalic acids possess anti-inflammatory activities. The MVA pathway is a synthetic pathway for triterpenoids in ZJ and ZA, and 23 genes of the MVA pathway are known to regulate triterpene synthesis in ZJ and ZA. In order to better understand the basic situation of triterpenoids in ZJ and ZA, this paper reviews the types, content dynamic changes, activities, pharmacokinetics, triterpenoid synthesis pathways, and the effects of domestication on triterpenoids in ZJ and ZA, and provides some ideas for the future research of triterpenoids in ZJ and ZA. In addition, there are many types of ZJ and ZA triterpenoids, and most of the studies on their activities are on lupane- and ursane-type triterpenes, while the activities of the ceanothane-type and saponin are less studied and need additional research.

Natural carbon fixation and advances in synthetic engineering for redesigning and creating new fixation pathways.

BACKGROUND: Autotrophic carbon fixation is the primary route through which organic carbon enters the biosphere, and it is a key step in the biogeochemical carbon cycle. The Calvin-Benson-Bassham pathway, which is predominantly found in plants, algae, and some bacteria (mainly cyanobacteria), was previously considered to be the sole carbon-fixation pathway. However, the discovery of a new carbon-fixation pathway in sulfurous green bacteria almost two decades ago encouraged further research on previously overlooked ancient carbon-fixation pathways in taxonomically and phylogenetically distinct microorganisms. AIM OF REVIEW: In this review, we summarize the six known natural carbon-fixation pathways and outline the newly proposed additions to this list. We also discuss the recent achievements in synthetic carbon fixation and the importance of the metabolism of thermophilic microorganisms in this field. KEY SCIENTIFIC CONCEPTS OF REVIEW: Currently, at least six carbon-fixation routes have been confirmed in Bacteria and Archaea. Other possible candidate routes have also been suggested on the basis of emerging "omics" data analyses, expanding our knowledge and stimulating discussions on the importance of these pathways in the way organisms acquire carbon. Notably, the currently known natural fixation routes cannot balance the excessive anthropogenic carbon emissions in a highly unbalanced global carbon cycle. Therefore, significant efforts have also been made to improve the existing carbon-fixation pathways and/or design new efficient in vitro and in vivo synthetic pathways.

Research progress in strigolactones and application prospect in medicinal plants / 中国中药杂志

Strigolactones(SLs) are a class of sesquiterpenoids derived from the carotenoid biosynthesis pathway with the core carbon skeleton consisting of tricyclic lactone(ABC tricyclic ring) and α,β-unsaturated furan ring(D ring). SLs are widely distributed in higher plants and are symbiotic signals between plants and Arbuscular mycorrhiza(AM), which play key roles in the evolution of plant colonizing terrestrial habitats. As a new type of plant hormone, SLs possess such important biological functions as inhibiting shoot branching(tillers), regulating root architecture, promoting secondary growth, and improving plant stress resistance. Therefore, SLs have attracted wide attention. The biological functions of SLs are not only closely related to the formation of "excellent shape and quality" of Chinese medicinal materials but also have important practical significance for the production of high-quality medicinal materials. However, SLs have been currently widely studied in model plants and crops such as Oryza sativa and Arabidopsis thaliana, and few related studies have been reported on SLs in medicinal plants, which need to be strengthened. This review focused on the latest research progress in the isolation and identification, biological and artificial synthesis pathways, biosynthesis sites and transport modes, signal transduction pathways and mechanisms, and biological functions of SLs, and prospected the research on the regulation mechanism of SLs in the growth and development of medicinal plants and their related application on targeted regulation of Chinese herbal medicine production, which is expected to provide some references for the in-depth research on SLs in the field of Chinese medicinal resources.

PGLN: A newly identified amino phosphoglycolipid species in Thermus thermophilus HB8.

Thermus thermophilus has several minor lipid molecules with structures that have not been described yet. In this study, we identified a new lipid molecule in T. thermophilus HB8 with an amino group at the polar head, by detecting lipid spots with HPTLC and mass spectrometry. The structure of the lipid resembles an amino sugar phospholipid, except for the glucosamine that lacks an acetyl group. We named this amino phosphoglycolipid PGLN, and proposed its synthetic pathway from a precursor, phosphatidyl-glyceric alkylamine. The primary amine structure of PGLN may contribute to high temperature adaptation through electrostatic interactions between the head groups.

Advances and trends in microbial production of polyhydroxyalkanoates and their building blocks.

With the rapid development of synthetic biology, a variety of biopolymers can be obtained by recombinant microorganisms. Polyhydroxyalkanoates (PHA) is one of the most popular one with promising material properties, such as biodegradability and biocompatibility against the petrol-based plastics. This study reviews the recent studies focusing on the microbial synthesis of PHA, including chassis engineering, pathways engineering for various substrates utilization and PHA monomer synthesis, and PHA synthase modification. In particular, advances in metabolic engineering of dominant workhorses, for example Halomonas, Ralstonia eutropha, Escherichia coli and Pseudomonas, with outstanding PHA accumulation capability, were summarized and discussed, providing a full landscape of diverse PHA biosynthesis. Meanwhile, we also introduced the recent efforts focusing on structural analysis and mutagenesis of PHA synthase, which significantly determines the polymerization activity of varied monomer structures and PHA molecular weight. Besides, perspectives and solutions were thus proposed for achieving scale-up PHA of low cost with customized material property in the coming future.

Production of Gluconic Acid and Its Derivatives by Microbial Fermentation: Process Improvement Based on Integrated Routes.

Gluconic acid (GA) and its derivatives, as multifunctional biological chassis compounds, have been widely used in the food, medicine, textile, beverage and construction industries. For the past few decades, the favored production means of GA and its derivatives are microbial fermentation using various carbon sources containing glucose hydrolysates due to high-yield GA production and mature fermentation processes. Advancements in improving fermentation process are thriving which enable more efficient and economical industrial fermentation to produce GA and its derivatives, such as the replacement of carbon sources with agro-industrial byproducts and integrated routes involving genetically modified strains, cascade hydrolysis or micro- and nanofiltration in a membrane unit. These efforts pave the way for cheaper industrial fermentation process of GA and its derivatives, which would expand the application and widen the market of them. This review summarizes the recent advances, points out the existing challenges and provides an outlook on future development regarding the production of GA and its derivatives by microbial fermentation, aiming to promote the combination of innovative production of GA and its derivatives with industrial fermentation in practice.

Caproic Acid-Producing Bacteria in Chinese Baijiu Brewing.

Caproic acid can be used as spices, preservatives, animal feed additives, and biofuels. At the same time, caproic acid plays an important role in Chinese Baijiu. It is the precursor substance for the synthesis of ethyl caproate, which directly affects the quality of Chinese Baijiu. Caproic acid-producing bacteria are the main microorganisms that synthesize caproic acid in Chinese Baijiu, and the most common strain is Clostridium kluyveri. Caproic acid-producing bacteria synthesize n-caproic acid through reverse ß-oxidation to extend the carboxylic acid chain. This method mainly uses ethanol and lactic acid as substrates. Ethanol and lactic acid are converted into acetyl-CoA, and acetyl-CoA undergoes a series of condensation, dehydrogenation, dehydration, and reduction to extend the carboxylic acid chain. This review addresses the important issues of caproic acid-producing bacteria in the brewing process of Baijiu: the common caproic acid-producing bacteria that have been reported metabolic pathways, factors affecting acid production, biological competition pathways, and the effect of mixed bacteria fermentation on acid production. It is hoped that this will provide new ideas for the study of caproic acid-producing bacteria in Chinese Baijiu.

Design, Analysis, and Implementation of a Novel Biochemical Pathway for Ethylene Glycol Production in Clostridium autoethanogenum.

The platform chemical ethylene glycol (EG) is used to manufacture various commodity chemicals of industrial importance, but largely remains synthesized from fossil fuels. Although several novel metabolic pathways have been reported for its bioproduction in model organisms, none has been reported for gas-fermenting, non-model acetogenic chassis organisms. Here, we describe a novel, synthetic biochemical pathway to convert acetate into EG in the industrially important gas-fermenting acetogen,Clostridium autoethanogenum. We not only developed a computational workflow to design and analyze hundreds of novel biochemical pathways for EG production but also demonstrated a successful pathway construction in the chosen host. The EG production was achieved using a two-plasmid system to bypass unfeasible expression levels and potential toxic enzymatic interactions. Although only a yield of 0.029 g EG/g fructose was achieved and therefore requiring further strain engineering efforts to optimize the designed strain, this work demonstrates an important proof-of-concept approach to computationally design and experimentally implement fully synthetic metabolic pathways in a metabolically highly specific, non-model host organism.