Deciphering and designing microbial communities by genome-scale metabolic modelling.

Microbial communities are shaped by the complex interactions among organisms and the environment. Genome-scale metabolic models (GEMs) can provide deeper insights into the complexity and ecological properties of various microbial communities, revealing their intricate interactions. Many researchers have modified GEMs for the microbial communities based on specific needs. Thus, GEMs need to be comprehensively summarized to better understand the trends in their development. In this review, we summarized the key developments in deciphering and designing microbial communities using different GEMs. A timeline of selected highlights in GEMs indicated that this area is evolving from the single-strain level to the microbial community level. Then, we outlined a framework for constructing GEMs of microbial communities. We also summarized the models and resources of static and dynamic community-level GEMs. We focused on the role of external environmental and intracellular resources in shaping the assembly of microbial communities. Finally, we discussed the key challenges and future directions of GEMs, focusing on the integration of GEMs with quorum sensing mechanisms, microbial ecology interactions, machine learning algorithms, and automatic modeling, all of which contribute to consortia-based applications in different fields.

Enzyme and Metabolic Engineering Strategies for Biosynthesis of α-Farnesene in Saccharomyces cerevisiae.

α-Farnesene, a type of acyclic sesquiterpene, is an important raw material in agriculture, aircraft fuel, and the chemical industry. In this study, we constructed an efficient α-farnesene-producing yeast cell factory by combining enzyme and metabolic engineering strategies. First, we screened different plants for α-farnesene synthase (AFS) with the best activity and found that AFS from Camellia sinensis (CsAFS) exhibited the most efficient α-farnesene production in Saccharomyces cerevisiae 4741. Second, the metabolic flux of the mevalonate pathway was increased to improve the supply of the precursor farnesyl pyrophosphate. Third, inducing site-directed mutagenesis in CsAFS, the CsAFSW281C variant was obtained, which considerably increased α-farnesene production. Fourth, the N-terminal serine-lysine-isoleucine-lysine (SKIK) tag was introduced to construct the SKIK∼CsAFSW281C variant, which further increased α-farnesene production to 2.8 g/L in shake-flask cultures. Finally, the α-farnesene titer of 28.3 g/L in S. cerevisiae was obtained by fed-batch fermentation in a 5 L bioreactor.

Transcriptome Analysis and Identification of Sesquiterpene Synthases in Liverwort Jungermannia exsertifolia.

The liverwort Jungermannia exsertifolia is one of the oldest terrestrial plants and rich in structurally specific sesquiterpenes. There are several sesquiterpene synthases (STSs) with non-classical conserved motifs that have been discovered in recent studies on liverworts; these motifs are rich in aspartate and bind with cofactors. However, more detailed sequence information is needed to clarify the biochemical diversity of these atypical STSs. This study mined J. exsertifolia sesquiterpene synthases (JeSTSs) through transcriptome analysis using BGISEQ-500 sequencing technology. A total of 257,133 unigenes was obtained, and the average length was 933 bp. Among them, a total of 36 unigenes participated in the biosynthesis of sesquiterpenes. In addition, the in vitro enzymatic characterization and heterologous expression in Saccharomyces cerevisiae showed that JeSTS1 and JeSTS2 produced nerolidol as the major product, while JeSTS4 could produce bicyclogermacrene and viridiflorol, suggesting a specificity of J. exsertifolia sesquiterpene profiles. Furthermore, the identified JeSTSs had a phylogenetic relationship with a new branch of plant terpene synthases, the microbial terpene synthase-like (MTPSL) STSs. This work contributes to the understanding of the metabolic mechanism for MTPSL-STSs in J. exsertifolia and could provide an efficient alternative to microbial synthesis of these bioactive sesquiterpenes.

Association of lung-intestinal microecology and lung cancer therapy.

In recent years, the incidence of lung cancer is increasing. Lung cancer has become one of the most malignant tumors with the highest incidence in the world, which seriously affects people's health. The most important cause of death of lung cancer is metastasis. Therefore, it is crucial to understand the mechanism of lung cancer progression and metastasis. This review article discusses the physiological functions, pathological states and disorders of the lung and intestine based on the concepts of traditional Chinese medicine (TCM), and analyzes the etiology and mechanisms of lung cancer formation from the perspective of TCM. From the theory of "the exterior and interior of the lung and gastrointestinal tract", the theory of "the lung-intestinal axis" and the progression and metastasis of lung cancer, we proposed e "lung-gut co-treatment" therapy for lung cancer. This study provides ideas for studying the mechanism of lung cancer and the comprehensive alternative treatment for lung cancer patients.

Development of Base Editors for Simultaneously Editing Multiple Loci in Lactococcus lactis.

Lactococcus lactis serves as the most extensively studied model organism and an important dairy species. Though CRISPR-Cas9 systems have been developed for robust genetic manipulations, simultaneously editing multiple endogenous loci in L. lactis is still challenging. Herein, we first report the development of a double-strand break-free, robust, multiloci editing system CRISPR-deaminase-assisted base editor (CRISPR-DBE), which comprises a cytidine (CRISPR-cDBE) and an adenosine deaminase-assisted base editor (CRISPR-aDBE). Specifically targeted by a sgRNA, CRISPR-cDBE can efficiently introduce a cytidine-to-thymidine mutation and CRISPR-aDBE can high-efficiently convert adenosine to guanosine within a 5 nt editing window. CRISPR-cDBE was validated and successfully applied to simultaneously inactivate multiple genes using a single plasmid in L. lactis strain NZ9000. Meanwhile, the temperature-sensitive plasmid of CRISPR-DBE can be cured quickly, and the continuous gene editing of L. lactis has been achieved. Furthermore, CRISPR-cDBE can also efficiently convert the targeted C to T in a nisin-producing, industrial L. lactis strain F44. Finally, we applied genome-wide bioinformatics analysis to determine the scope of gene inactivation for these base editors using different Cas9 variants and evaluated the preference of SpGn and SpRYn variants for the protospacer adjacent motif in L. lactis NZ9000. Taken together, our study provides a powerful tool for simultaneously editing multiple loci in L. lactis, which may have a wide range of industrial applications in the future.

Nisin Variants Generated by Protein Engineering and Their Properties.

Nisin, a typical lantibiotic, has robust antimicrobial activity combined with limited cytotoxicity, and the development of resistance to it is slow. These properties make nisin a promising antimicrobial agent to control pathogenic microorganisms in dairy foods. However, its low solubility, poor stability and short half-life at neutral pH limit its application within the dairy industry. Protein engineering technology has revealed the potential of modifying nisin to improve its properties, and many valuable variants have emerged. This review summarizes progress in the generation of nisin variants for the dairy industry and for other purposes. These nisin variants with additional modification have improved properties and can even expand the inhibition spectrum range of nisin. Nisin, as the most thoroughly studied lantibiotic, and its variants can also guide the modification of other lantibiotics.

Research Progress on the Synthetic Biology of Botanical Biopesticides.

The production and large-scale application of traditional chemical pesticides will bring environmental pollution and food safety problems. With the advantages of high safety and environmental friendliness, botanical biopesticides are in line with the development trend of modern agriculture and have gradually become the mainstream of modern pesticide development. However, the traditional production of botanical biopesticides has long been faced with prominent problems, such as limited source and supply, complicated production processes, and excessive consumption of resources. In recent years, the rapid development of synthetic biology will break through these bottlenecks, and many botanical biopesticides are produced using synthetic biology, such as emodin, celangulin, etc. This paper reviews the latest progress and application prospect of synthetic biology in the development of botanical pesticides so as to provide new ideas for the analysis of synthetic pathways and heterologous and efficient production of botanical biopesticides and accelerate the research process of synthetic biology of natural products.

Molecular and Functional Evolution of the Spermatophyte Sesquiterpene Synthases.

Sesquiterpenes are important defense and signal molecules for plants to adapt to the environment, cope with stress, and communicate with the outside world, and their evolutionary history is closely related to physiological functions. In this study, the information of plant sesquiterpene synthases (STSs) with identified functions were collected and sorted to form a dataset containing about 500 members. The phylogeny of spermatophyte functional STSs was constructed based on the structural comparative analysis to reveal the sequence-structure-function relationships. We propose the evolutionary history of plant sesquiterpene skeletons, from chain structure to small rings, followed by large rings for the first time and put forward a more detailed function-driven hypothesis. Then, the evolutionary origins and history of spermatophyte STSs are also discussed. In addition, three newly identified STSs CaSTS2, CaSTS3, and CaSTS4 were analyzed in this functional evolutionary system, and their germacrene D products were consistent with the functional prediction. This demonstrates an application of the structure-based phylogeny in predicting STS function. This work will help us to understand evolutionary patterns and dynamics of plant sesquiterpenes and STSs and screen or design STSs with specific product profiles as functional elements for synthetic biology application.

ComX improves acid tolerance by regulating the expression of late competence proteins in Lactococcus lactis F44.

ComX can improve bacterial competence by modulating global gene expression. Although competence induction may also be a protective mechanism under stress, this has not been investigated in detail. Here, we demonstrated that ComX improved the acid tolerance and nisin yield of Lactococcus lactis, which is an important gram-positive bacterium increasingly used in modern biotechnological applications. We found that overexpression of comX could improve the survival rate up to 36.5% at pH 4.0, compared with only 5.4% and 1.1% with the wild-type and comX knockout strains, respectively. Moreover, quantitative real-time PCR results indicated that comX overexpression stimulated the expression of late competence genes synergistically with exposure to acid stress. Finally, electrophoretic mobility shift assay demonstrated the binding of purified ComX to the cin-box in the promoters of these genes. Taken together, our results reveal a regulation mechanism by which ComX and acid stress can synergistically modulate the expression of late competence genes to enhance cells' acid tolerance and nisin yield.

Comparative transcriptome analysis of sesquiterpene biosynthesis and functional characterization of sesquiterpene synthases in Leonurus sibiricus L.

MAIN CONCLUSION: Two sesquiterpene synthases were identified through comparative transcriptome analysis of Leonurus sibiricus. LsSqTPS2 could produce high titer of δ-cadinene in vivo which suggests the terpene specificity of L. sibiricus. Leonurus sibiricus L., a medicinal herb, is widely used in China due to its pharmacological activities. Cadinene type sesquiterpenes, one of major bioactive components mainly present in aerial parts of L. sibiricus, showed antibacterial, anti-inflammatory, antioxidant and antiproliferative properties. However, there is no report about the sesquiterpene biosynthesis in L. sibiricus. This study identified L. sibiricus sesquiterpene synthases (LsSqTPSs) through comparative transcriptome analysis of L. sibiricus leaf and root samples using the BGISEQ-500 sequencing technique. A total of 83,244 unigenes were obtained with an average length of 1025 bp. Among them, 50,356 unigenes (60.49%) acquired annotations according to the BLAST searching results. A total of 68 differentially expressed genes (DEGs) were potentially involved in the sesquiterpene biosynthesis. Furthermore, four candidate DEGs encoding LsSqTPSs were characterized. The enzymatic characterization in engineered yeast showed that LsSqTPS1 produced α-farnesene as the single product and LsSqTPS2 mainly produced 76.23 mg/L of δ-cadinene, which constituted the major component of L. sibiricus leaf essential oil. This work contributes to the investigation of sesquiterpene biosynthesis in L. sibiricus.

Characterization of trans-Nerolidol Synthase from Celastrus angulatus Maxim and Production of trans-Nerolidol in Engineered Saccharomyces cerevisiae.

Volatile terpenoids are a large group of important secondary metabolites and possess many biological activities. The acyclic sesquiterpene trans-nerolidol is one of the typical representatives and widely used in cosmetics and agriculture. Here, the accumulation of volatile terpenes in different tissues of Celastrus angulatus was investigated, and two trans-nerolidol synthases, CaNES1 and CaNES2, were identified and characterized by in vitro enzymatic assays. Both genes are differentially transcribed in different tissues of C. angulatus. Next, we constructed a Saccharomyces cerevisiae cell factory to enable high-level production of trans-nerolidol. Glucose was the sole carbon source to sequentially control gene expression between the competitive squalene and trans-nerolidol pathways. Finally, the trans-nerolidol production of recombinant strain LWG003-CaNES2 was 7.01 g/L by fed-batch fermentation in a 5 L bioreactor. The results clarify volatile terpenoid biosynthesis in C. angulatus and provide a promising potential for industrial production of trans-nerolidol in S. cerevisiae.

De novo assembly of the Mylia taylorii transcriptome and identification of sesquiterpene synthases.

Mylia taylorii is an ancient nonseed land plant that accumulates various sesquiterpenes with insecticidal and antibacterial activities. Recently, microbial-type sesquiterpene synthases (STSs) with atypical aspartate-rich metal ion binding motifs have been identified in some liverworts. Here, transcriptome analysis of M. taylorii was performed to identify M. taylorii sesquiterpene synthases (MtSTSs) that are potentially involved in sesquiterpene biosynthesis and diversity. A total of 255,669 unigenes were obtained with an average length of 963 bp in the transcriptome data of M. taylorii, among which 148,093 (57.92%) unigenes had BLAST results. Forty-eight unigenes were related to the sesquiterpene backbone biosynthesis according to KEGG annotation. In addition, MtSTS1, MtSTS2 and MtSTS3 identified from putative MtSTSs display sesquiterpene catalytic activities on the basis of functional characterizations in yeast. Interestingly, MtSTSs exhibit a noncanonical metal ion binding motif and the structural composition of a single α-domain, which are features of microbial STSs instead of archetypical plant STSs. This study revealed new microbial-type STS members of nonseed plants, and functionally identified that MtSTSs may contribute to the investigation of the biosynthesis and biological role of sesquiterpenes in M. taylorii.

NisI Maturation and Its Influence on Nisin Resistance in Lactococcus lactis.

NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant (KD ) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.IMPORTANCE Nisin, a safe and natural antimicrobial peptide, has a long and impressive history as a food preservative and is also considered a novel candidate to alleviate the increasingly serious threat of antibiotic resistance. Nisin is produced by certain L. lactis strains. The nisin immunity protein NisI, a membrane-bound lipoprotein, is expressed by nisin producers to avoid suicidal action. Here, we report the roles of Lsp and Lgt in NisI maturation and nisin resistance of L. lactis F44. The results verified the importance of Lsp to NisI-conferred immunity and Lgt to localization. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.

[Construction of five-level practical teaching system for bioengineering under Emerging Engineering Education background].

Strengthening practical teaching, together with improving innovation ability is one of the key tasks of Emerging Engineering Education. This paper is based on the revision of the training program of bioengineering in School of Chemical Engineering and Technology, Tianjin University, improved the practical teaching system and curriculum content, built a five-level teaching system for basic experiment, comprehensive experiment, course design, scientific research and practical training. In order to cultivate outstanding innovative talents with practical ability and innovative spirit, innovative teaching reform mode is proposed. Furthermore the new thought and new schemes for Emerging Engineering Education are put forward.

d-Methionine and d-Phenylalanine Improve Lactococcus lactis F44 Acid Resistance and Nisin Yield by Governing Cell Wall Remodeling.

Lactococcus lactis encounters various environmental challenges, especially acid stress, during its growth. The cell wall can maintain the integrity and shape of the cell under environmental stress, and d-amino acids play an important role in cell wall synthesis. Here, by analyzing the effects of 19 different d-amino acids on the physiology of L. lactis F44, we found that exogenously supplied d-methionine and d-phenylalanine increased the nisin yield by 93.22% and 101.29%, respectively, as well as significantly increasing the acid resistance of L. lactis F44. The composition of the cell wall in L. lactis F44 with exogenously supplied d-Met or d-Phe was further investigated via a vancomycin fluorescence experiment and a liquid chromatography-mass spectrometry assay, which demonstrated that d-Met could be incorporated into the fifth position of peptidoglycan (PG) muropeptides and d-Phe could be added to the fourth and fifth positions. Moreover, overexpression of the PG synthesis gene murF further enhanced the levels of d-Met and d-Phe involved in PG and increased the survival rate under acid stress and the nisin yield of the strain. This study reveals that the exogenous supply of d-Met or d-Phe can change the composition of the cell wall and influence acid tolerance as well as nisin yield in L. lactisIMPORTANCE As d-amino acids play an important role in cell wall synthesis, we analyzed the effects of 19 different d-amino acids on L. lactis F44, demonstrating that d-Met and d-Phe can participate in peptidoglycan (PG) synthesis and improve the acid resistance and nisin yield of this strain. murF overexpression further increased the levels of d-Met and d-Phe incorporated into PG and contributed to the acid resistance of the strain. These findings suggest that d-Met and d-Phe can be incorporated into PG to improve the acid resistance and nisin yield of L. lactis, and this study provides new ideas for the enhancement of nisin production.

Co-production of Nisin and γ-Aminobutyric Acid by Engineered Lactococcus lactis for Potential Application in Food Preservation.

Microbiological contamination and oxidative damage are the two main challenges in maintaining quality and improving shelf-life of foods. Here, we developed a Lactococcus lactis fermentation system that could simultaneously produce nisin, an antimicrobial peptide, and γ-aminobutyric acid (GABA), an antioxidant agent. In this system, we metabolically engineered a nisin producing strain L. lactis F44 for GABA production by expression of glutamate decarboxylase and glutamate/GABA antiporter. GABA biosynthesis could facilitate nisin production through enhancing acid resistance of the strain. By applying a two-stage pH-control fermentation strategy, the engineered strain yielded up to 9.12 g/L GABA, which was 2.2 times higher than that of pH-constant fermentation. Furthermore, we demonstrated the potential application of the freeze-dried fermentation product as a preservative to improve the storage performance of meat and fruit. These results suggested that the fermentation product of nisin-GABA co-producing strain could serve as a cost-effective, easily prepared, and high-performance food preservative.

De novo leaf and root transcriptome analysis to explore biosynthetic pathway of Celangulin V in Celastrus angulatus maxim.

BACKGROUND: Celastrus angulatus Maxim is a kind of crucial and traditional insecticidal plant widely distributed in the mountains of southwest China. Celangulin V is the efficient insecticidal sesquiterpenoid of C. angulatus and widely used in pest control in China, but the low yield and discontinuous supply impeded its further popularization and application. Fortunately, the development of synthetic biology provided an opportunity for sustainable supply of Celangulin V, for which understanding its biosynthetic pathway is indispensable. RESULTS: In this study, six cDNA libraries were prepared from leaf and root of C. angulatus before global transcriptome analyses using the BGISEQ-500 platform. A total of 104,950 unigenes were finally obtained with an average length of 1200 bp in six transcriptome databases of C. angulatus, in which 51,817 unigenes classified into 25 KOG classifications, 39,866 unigenes categorized into 55 GO functional groups, and 48,810 unigenes assigned to 135 KEGG pathways, 145 of which were putative biosynthetic genes of sesquiterpenoid and triterpenoid. 16 unigenes were speculated to be related to Celangulin V biosynthesis. De novo assembled sequences were verified by Quantitative Real-Time PCR (qRT-PCR) analysis. CONCLUSIONS: This study is the first report on transcriptome analysis of C. angulatus, and 16 unigenes probably involved in the biosynthesis of Celangulin V were finally collected. The transcriptome data will make great contributions to research for this specific insecticidal plant and the further gene mining for biosynthesis of Celangulin V and other sesquiterpene polyol esters.

Two-stage carbon distribution and cofactor generation for improving l-threonine production of Escherichia coli.

L-Threonine, a kind of essential amino acid, has numerous applications in food, pharmaceutical, and aquaculture industries. Fermentative l-threonine production from glucose has been achieved in Escherichia coli. However, there are still several limiting factors hindering further improvement of l-threonine productivity, such as the conflict between cell growth and production, byproduct accumulation, and insufficient availability of cofactors (adenosine triphosphate, NADH, and NADPH). Here, a metabolic modification strategy of two-stage carbon distribution and cofactor generation was proposed to address the above challenges in E. coli THRD, an l-threonine producing strain. The glycolytic fluxes towards tricarboxylic acid cycle were increased in growth stage through heterologous expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and citrate synthase, leading to improved glucose utilization and growth performance. In the production stage, the carbon flux was redirected into l-threonine synthetic pathway via a synthetic genetic circuit. Meanwhile, to sustain the transaminase reaction for l-threonine production, we developed an l-glutamate and NADPH generation system through overexpression of glutamate dehydrogenase, formate dehydrogenase, and pyridine nucleotide transhydrogenase. This strategy not only exhibited 2.02- and 1.21-fold increase in l-threonine production in shake flask and bioreactor fermentation, respectively, but had potential to be applied in the production of many other desired oxaloacetate derivatives, especially those involving cofactor reactions.

The genome and transcriptome of Lactococcus lactis ssp. lactis F44 and G423: Insights into adaptation to the acidic environment.

Nisin, as a common green (environmentally friendly), nontoxic antibacterial peptide secreted by Lactococcus lactis, is widely used to prevent the decomposition of meat and dairy products and maintains relatively high stability at low pH. However, the growth of Lc. lactis is frequently inhibited by high lactic acid concentrations produced during fermentation. This phenomenon has become a great challenge in enhancing the nisin yield for this strain. Here, the shuffled strain G423 that could survive on a solid plate at pH 3.7 was generated through protoplast fusion-mediated genome shuffling. The nisin titer of G423 peaked at 4,543 IU/mL, which was 59.9% higher than that of the same batch of the initial strain Lc. lactis F44. The whole genome comparisons between G423 and F44 indicated that 6 large fragments (86,725 bp) were inserted in G423 compared with that of Lc. lactis F44. Transcriptome data revealed that 4 novel noncoding transcripts, and the significantly upregulated genes were involved in multiple processes in G423. In particular, the expression of genes involved in cell wall and membrane biosynthesis was obviously perturbed under acidic stress. Quantitative real-time PCR analysis showed that the transcription of noncoding small RNA NC-1 increased by 2.35-fold at pH 3.0 compared with that of the control (pH 7.0). Overexpression assays indicated that small RNA NC-1 could significantly enhance the acid tolerance and nisin production of G423 and F44. Our work provided new insights into the sophisticated genetic mechanisms involved in Lc. lactis in an acidic environment, which might elucidate its potential application in food and dairy industries.

Engineering Saccharomyces cerevisiae for Enhanced Production of Protopanaxadiol with Cofermentation of Glucose and Xylose.

Protopanaxadiol (PPD), an active triterpene compound, is the precursor of high-value ginsenosides. In this study, we report a strategy for the enhancement of PPD production in Saccharomyces cerevisiae by cofermentation of glucose and xylose. In mixed sugar fermentation, strain GW6 showed higher PPD titer and yield than that obtained from glucose cultivation. Then, engineering strategies were implemented on GW6 to enhance the PPD yields, such as adjustment of the central carbon metabolism, optimization of the mevalonate pathway, reinforcement of the xylose assimilation pathway, and regulation of cofactor balance, namely, overexpression of xPK/PTA, ERG10/ERG12/ERG13, XYL1/XYL2/TAL1, and POS5, respectively. In particular, the final obtained strain GW10, harboring overexpressed POS5, exhibited the highest PPD yield, which was 2.06 mg of PPD/g of mixed sugar. In a 5-L fermenter, PPD titer reached 152.37 mg/L. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of PPD.