Sclareol, a diterpene alcohol, is widely utilized in the perfume industry, particularly as a substitute for natural ambergris. Microbial synthesis of sclareol serves as an alternative way to produce this valuable and marketable compound; however, it was still challenged by ineffective expression of heterogeneous enzymes, insufficient activity of key enzymes, byproduct accumulation, scale-up, especially downstream separation, etc. In this study, sclareol biosynthesis was established in Yarrowia lipolytica by coexpressing the truncated exogenous sclareol synthase genes tSsTPS and tSsLPPS. Through semirational mutagenesis of the exogenous enzyme tSsTPS, the mutant tSsTPS(V325I) was screened, exhibiting a 70% increase in catalytic activity. Multicopy optimization of the genes tSsTPS and tSsLPPS enabled the strain to produce 348.6 mg/L sclareol. Regulating the supply and consumption of GGPP further improved the titer of sclareol to 1565.4 mg/L. Furthermore, the efflux capacity of various transporters for sclareol was evaluated. Among them, the ABC transporter YALI1_C28310p increased extracellular sclareol production by 8.6-fold compared to the control without the solvent extractant. Additionally, citrate overflow metabolism in Y. lipolytica was eliminated by knocking out CEX1, achieving a final sclareol titer of 3307.3 mg/L in shake flasks and 13.9 g/L in a 5 L bioreactor without solvent overlay by fed-batch fermentation, both representing the highest reported sclareol titer to date. This achievement establishes a robust de novo biosynthesis platform for sclareol in Y. lipolytica, which can be broadly adapted for synthesizing other natural terpenoids.

Sabinene is a type of monoterpene that is widely used in flavors, fragrances and pharmaceuticals. Though sabinene biosynthesis has been investigated in a variety of microorganisms, application of sabinene is still limited due to its high production cost and lesser yielding strains. The baker’s yeast Saccharomyces cerevisiae, which is generally recognized as safe (GRAS), is a suitable cell factory for the food and beverage industries. In this study, we aimed to enhance the production of sabinene from corn hydrolysates by employing genetic engineering techniques on S. cerevisiae. Here, we engineered S. cerevisiae for the production of sabinene by overexpressing sabinene synthase (SabS) and geranyl diphosphate synthase (GPPS) via CRISPR-Cas9, which is a simple and efficient tool for targeted and marker-free genome engineering. Subsequently, the culture medium and process conditions were optimized to enhance sabinene production and achieve 23.6 mg/L under flask fermentation conditions. Based on the optimized culture conditions, we further investigated the production of sabinene from corn hydrolysates, which is a major source of dietary nutrients worldwide and an inexpensive source of sugars, and a high-level production of 60.0 mg/L was achieved in shake-flask fermentation. Our results implied that corn hydrolysates was a suitable medium for sabinene production and that CRISPR-Cas9 could boost the marker-free engineered yeast strain, which was more suitable for the food and beverage industry. Altogether, our work represents the progress in the bioproduction of food-grade sabinene from an inexpensive raw material.

The sesquiterpene (+)-valencene, with its flavor and diverse biological functions, holds promise for applications in the food, fragrance, and pharmaceutical industries. However, the low concentration in nature and high cost of extraction limit its application. This study aimed to construct a microbial cell factory to efficiently produce (+)-valencene. The strain Yarrowia lipolytica YL238, possessing a stronger capacity for (+)-valencene synthesis, was selected and utilized as the chassis for further modifications. By fine-tuning the mevalonate and squalene synthesis pathways we achieved a remarkable 13.2-fold increase in (+)-valencene titer compared to the original strain. Following directed evolution was employed to screen for efficient (+)-valencene synthase, which further enhanced (+)-valencene production by 138%. Consequently, the engineered strain overproduced 813 mg/L of (+)-valencene in shake flasks, marking the highest titer reported in microbials to date. Furthermore, in fed-batch fermentation, this engineered strain showed the capacity to produce 3.3 g/L of (+)-valencene. This study offers a successful model for the application of the "strain-pathway-enzyme" triune strategy in the metabolic engineering of Y. lipolytica, and these methodologies could be broadly utilized for the synthesis of other natural terpenes.

Energy crops play a vital role in meeting future energy and chemical demands while addressing climate change. However, the idealization of low-carbon workflows and careful consideration of cost-benefit equations are crucial for their more sustainable implementation. Here, we propose tobacco as a promising energy crop because of its exceptional water solubility, mainly attributed to a high proportion of water-soluble carbohydrates and nitrogen, less lignocellulose, and the presence of acids. We then designed a strategy that maximizes biomass conversion into bio-based products while minimizing energy and material inputs. By autoclaving tobacco leaves in water, we obtained a nutrient-rich medium capable of supporting the growth of microorganisms and the production of bioproducts without the need for extensive pretreatment, hydrolysis, or additional supplements. Additionally, cultivating tobacco on barren lands can generate sufficient biomass to produce approximately 573 billion gallons of ethanol per year. This approach also leads to a reduction of greenhouse gas emissions by approximately 76% compared to traditional corn stover during biorefinery processes. Therefore, our study presents a novel and direct strategy that could significantly contribute to the goal of reducing carbon emissions and global sustainable development compared to traditional methods.

Utilization of microbial hosts to produce natural plant products is regarded as a promising and sustainable approach. However, achieving highly efficient production of terpenoids using microorganisms remains a significant challenge. Here, mevalonate, a building block of terpenoids, was used as a demo product to explore the potential metabolic constraints for terpenoid biosynthesis in Yarrowia lipolytica. First, by regulation of the expression of ERG12 and HMGR, the mevalonate titer was improved by 7660%. Subsequently, the native mevalonate pathway (MVA pathway) was enhanced, and the production of mevalonate increased to 4.16 g/L. To ensure a sufficient supply of acetyl-CoA, the citrate route and TCA cycle were simultaneously engineered, and the mevalonate titer was further improved to 5.25 g/L in shake flasks. Ultimately, the citrate overflow metabolism of Y. lipolytica was eliminated by deleting CEX1, resulting in the highest mevalonate titer of 101 g/L with a yield of 0.255 g/g of glucose in eukaryotes. These insights could be applied to the effective production of terpenoids and biochemicals derived from central carbon metabolic pathways.

Xylanase is widely used in various industries such as food processing, paper, textiles, and leather tanning. In this study, Bacillus cereus L-1 strain was isolated and identified as capable of producing low molecular weight xylanase through 16 s rRNA sequencing. Maximum xylanase yield of 15.51 ± 2.08 U/mL was achieved under optimal fermentation conditions (5

Many bacteria secrete secondary metabolites to compete or cooperate with other microbes or hosts in diverse and dynamic ecological niches. 2-Phenylethanol (2-PE) and indole-3-acetic acid (IAA) are small metabolites that play important roles in biological and ecological functions, produced by microorganisms. They are synthesized via expanded shikimate pathways, and required the key enzyme ��-ketoacid decarboxylase. Here we show an adaptive strategy driven by secondary metabolites in accordance with bacteria survival state. A soil derived Enterobacter strain CGMCC 5087 produces 2-PE in exponential growth phase whenever in nutrient rich or limited environments that suppresses microbial competitors, but produces IAA at the onset of stationary phase only in a tryptophann rich environment enabling plant growth promotion, which is in an ��-ketoacid decarboxylase KDC4427 dependent manner. The metabolic fluxes of 2-PE and IAA are mediated by the ratio of KDC4427 and an L-glyceraldehyde 3-phosphate reductase gene ADH4428, which are transcribed divergently by a bidirectional promoter in one operon, and by the enzyme activity characteristics of KDC4427. The expression of KDC4427 is up-regulated with bacteria growth, while ADH4428 is down-regulated; simultaneously, KDC4427 shows a higher kcat value for phenylpyruvate, and has a higher affinity for indolepyruvate, thus making the reaction flow towards the production of 2-PE in exponential growth phase, however as the growth of bacteria enters the stationary phase, the production of IAA is increased. Additionally, we demonstrated that TyrR and RpoS activate and repress the expression of KDC4427 and ADH4428 through direct binding to the bidirectional promoter. These results reveal an ingenious control of competition and cooperation behaviours through fine-tuning the sequential synthesis of 2-PE and IAA in response to growth and environmental conditions.

The highest titer of the anticancer precursor sesquiterpene germacrene A was observed in oleaginous yeast using multi-layered systematic metabolic engineering strategies.

Background In the recombinant protein market with broad economic value, the rapid development of synthetic biology has made it necessary to construct an efficient exocrine expression system for the different heterologous proteins. Yarrowia lipolytica possesses unique advantages in nascent protein transport and glycosylation modification, so it can serve as a potential protein expression platform. Although the Po1 series derived from W29 is often used for the expression of the various heterologous proteins, the ability of W29 to secrete proteins has not been verified and the Po1 series has been found to be not convenient for further gene editing. Results A total of 246 Y. lipolytica strains were evaluated for their secretory capacity through performing high-throughput screening in 48-well plate. Thereafter, following two rounds of shake flask re-screening, a high-secreting protein starting strain DBVPG 5851 was obtained. Subsequently, combined with the extracellular protein types and relative abundance information provided by the secretome of the starting strain, available chassis cell for heterologous protein expression were preliminarily constructed, and it was observed that the most potential signal peptide was derived from YALI0D20680g. Conclusions This study offers a novel perspective on the diversification of Y. lipolytica host cells for the heterologous protein expression and provides significant basis for expanding the selection space of signal peptide tools in the future research.

Abstract Background: Microbial communities that inhabit aging tobacco leaves play a key role in improving quality by interactions with themselves and tobacco leaves to release tannins, increase sugar levels, promote aromatic taste and degrade harmful compounds. A better understanding of microbial communities on the aging of tobacco leaves could provide an important microbial repository for the industrial applications. Results: Here, we examined the structural and compositional changes of microbial communities and identified the potential metabolic pathways of bacteria and fungi throughout the aging process. The results showed that the diversity and structure of the microbial communities keep changing along with the aging process went on. The richness and diversity of bacterial community decreased, while the richness of fungal community was in an inverse trend. At the phylum level, the bacterial community was dominated by Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidete, while Ascomycota and Basidiomycota were the dominant species in the fungal community. In the bacterial community, metabolic functions related to the carbon and nitrogen cycles which response to the degradation of harmful components, and the metabolism of aromatic hydrocarbons showed extremely dynamic at different aging periods. The change of the main nutritional mode of the fungal community also led to an increase in the abundance of saprophytic fungi. Conclusion: These results provide information on the succession of microbial community structure and function in the whole process of tobacco aging and suggest that the aging process of tobacco leaves can be a natural microbial collection for target microorganism and their metabolites. It also enables the further investigation of coordination mechanisms between beneficial microbial regulation and pathogenicity during aging process.

Methyl ketones (MK) are highly valuable fatty acid derivatives with broad applications. Microbes based biosynthesis represents an alternative route for production of these usually fossil based chemicals. In this study, we reported metabolic engineering of Saccharomyces cerevisiae to produce MK, including 2-nonanone, 2-undecanone, 2-tridecanone and 2-pentadecanone. Besides enhancing inherent peroxisomal fatty acids β-oxidation cycle, a novel heterologous cytosolic fatty acids β-oxidation pathway was constructed, and this resulted in an increased production of MK by 2-fold. To increase carbon fluxes to methyl ketones, the supply of precursors was enhanced by engineering lipid metabolism, including improving the intracellular biosynthesis of acyl-CoAs, weakening the consumption of acyl-CoAs for lipids storage, and reinforcing activation of free fatty acids to acyl-CoAs. Hereby the titer of MK was improved by 7-fold, reaching 143.72 mg/L. Finally, transcription factor engineering was employed to increase the biosynthesis of methyl ketones and it was found that overexpression of ADR1 can mimic the oleate activated biogenesis and proliferation of peroxisomes, which resulted in a further increased production of MK by 28%. With these modifications and optimization, up to 845 mg/L total MK were produced from glucose in fed-batch fermentation, which is the highest titer of methyl ketones reported produced by fungi.

[背景]烟草在生产和加工中会产生高浓度的尼古丁废弃物,对环境造成较大的污染.[目的]筛选降解尼古丁的微生物菌种并解析其降解尼古丁的代谢途径,理解微生物如何降解尼古丁.[方法]用常规分离筛选方法、结合形态学观察和分子鉴定手段分离和鉴定菌株类别,进而利用单因素试验方法,通过设置不同的尼古丁浓度、温度和pH确定菌株降解尼古丁的最适发酵条件和降解率,利用气相色谱-质谱联用技术检测菌株在尼古丁降解过程中的主要代谢产物.[结果]获得一株以尼古丁为唯一碳源和氮源的节杆菌属(Arthrobacter)菌株,编号为D4;该菌株降解尼古丁的最适温度和pH分别为30.0℃和7.0;在1 g/L的尼古丁浓度下具备较快的尼古丁降解速率,培养18 h时尼古丁降解率可达到90％以上;尼古丁浓度＞4 g/L时菌株生长受到明显抑制;与目前报道的节杆菌属降解途径不同,该菌株降解尼古丁过程中产生了新的终产物N-甲基吡咯烷酮、可替宁及中间产物麦斯明.[结论]本研究分离鉴定到一株具有较快尼古丁降解速率的节杆菌,该菌株很可能存在新的尼古丁降解途径.

Microbial communities that inhabit aging tobacco leaves play a key role in improving products quality. A better understanding of microbial communities on the aging of tobacco leaves could provide an important microbial repository for the industrial applications. Here, we examined the structural and compositional changes of microbial communities throughout the aging process of by tobacco leaves 16 S and ITS rRNA amplicon sequencing techniques and identified the potential metabolic pathways of bacteria and fungi using Functional Annotation of Prokaryotic Taxa (FAPROTAX) and Fungi Functional Guild (FUNGuild), respectively. The results showed that the diversity and structure of the microbial communities keep changing along with the aging process went on. The richness and diversity of bacterial community decreased, while the richness of fungal community was in an inverse trend. At the phylum level, the bacterial community was dominated by Proteobacteria, Actinobacteria, and Firmicutes, while Ascomycota and Basidiomycota were the dominant species in the fungal community. In the bacterial community, metabolic functions related to the carbon and nitrogen cycles which response to the degradation of harmful components, and the metabolism of aromatic hydrocarbons showed extremely dynamic at different aging periods. The change of the main nutritional mode of the fungal community also led to an increase in the abundance of saprophytic fungi. These results provide information on the succession of microbial community structure and function in the whole process of tobacco aging and suggest that the aging process of tobacco leaves can be a natural microbial collection for target microorganism and their metabolites. It also enables the further investigation of coordination mechanisms between beneficial microbial regulation and pathogenicity during aging process.

Green tea is popularly known for its pleasant flavor and health-care functions. Bitterness and astringency are the two important quality attributes of green tea that enrich tea flavor. Although many research works have focused on the flavor formation of green tea, the review articles about bitterness and astringency is limited. This review article summarizes the major components of bitter and astringent substances in green tea, their sensory perception mechanism, factors influencing the formation of these substances, and the evaluation methods of bitterness and astringency. This review will shed light on the subsequent studies in tea flavor, and provide deeper insight for the research of bitterness and astringency in other foods.

Microbial biomass and waste materials conversion for biochemicals production has been an alternative for energy conservation and emission reduction. While toxic substances in biomass materials and high osmotic pressure formed in fermentation-based systems block the bioconversion processes of microorganisms. In the present study, strain T4 that isolated from tobacco waste could resist toxic inhibitors such as nicotine and was suitable for generation of 2, 3-butanediol (2, 3-BD) with a high concentration of glucose (up to 20%). 30.06 and 1.54 g/L of 2, 3-BD was generated respectively from 50 g/L of tobacco waste with and without 200 g/L glucose after fermentation for 48 h. Besides, the results of biochemical tests showed that it was gram-positive and able to liquefy gelatin, hydrolyze starch and produce catalases. It could utilize glucose but not lactose as carbohydrates during fermentation. The 16S rRNA sequence and systematic analysis revealed that T4 was identified to be a Bacillus amyloliquefaciens (B. amyloliquefaciens). This work presents a promising model microorganism chassis to use the biomass waste for high value-added biochemicals production.

Terpenoids are a large family of natural products with diversified structures and functions that are widely used in the food, pharmaceutical, cosmetic, and agricultural fields. However, the traditional methods of terpenoids production such as plant extraction and chemical synthesis are inefficient due to the complex processes, high energy consumption, and low yields. With progress in metabolic engineering and synthetic biology, microbial cell factories provide an interesting alternative for the sustainable production of terpenoids. The non-conventional yeast, Yarrowia lipolytica, is a promising host for terpenoid biosynthesis due to its inherent mevalonate pathway, high fluxes of acetyl-CoA and NADPH, and the naturally hydrophobic microenvironment. In this review, we highlight progress in the engineering of Y. lipolytica as terpenoid biomanufacturing factories, describing the different terpenoid biosynthetic pathways and summarizing various metabolic engineering strategies, including progress in genetic manipulation, dynamic regulation, organelle engineering, and terpene synthase variants.

Glucose oxidase, which uses molecular oxygen as an electron acceptor to specifically catalyze the conversion of β-d-glucose to gluconic acid and hydrogen peroxide (H2O2), has been considered an important enzyme in increasing environmental sustainability and food security. However, achieving the high yield, low price and high activity required for commercial viability remains challenging. In this review, we first present a brief introduction, looking at the sources, characteristics, catalytic process, and applications of glucose oxidase. Then, the predictive structures of glucose oxidase from two different sources are comparatively discussed. We summarize the inhibitors of glucose oxidase. Finally, we highlight how the production of glucose oxidase can be improved by optimizing the culture conditions and microbial metabolic engineering.

[目的]探索海南H382雪茄烟叶发酵中细菌群落组成和演替规律,为揭示发酵机制提供依据,为筛选功能细菌提供参考.[方法]通过16SrRNA测序技术对海南H382雪茄烟叶不同发酵时期的细菌群落多样性及群落演替规律进行表征,并利用PICRUSt预测了不同发酵时期细菌群落的功能.[结果](1)雪茄烟叶发酵中细菌群落多样性较丰富,7个样本共聚类299个OTU,属于27个属;不同发酵时期样本中OTU数目不同,发酵前期呈增加趋势,随后呈现下降趋势,以YB 3(样本-3)中OTU最多,共222个;(2)以细菌占比多少为标准,H382雪茄烟叶发酵过程中细菌群落演替规律为:含量最高由YB_2中的葡萄球菌属(Staphylococcus)到YB_3中海洋芽孢杆菌属(Oceanobacillus),随后为YB_4(样本-4)中假单胞菌属(Pseudomonas),最后为YB_5、YB_6和YB_7中的葡萄球菌属(Staphylococcus);(3)PICRUSt功能预测结果显示,样本间预测功能种类差异小,而样本间功能种类的丰度差异较大.[结论]H382雪茄烟叶发酵过程中细菌群落组成多样性丰富,优势菌群在本实验条件下是演替的.发酵过程中主要的细菌群落由发酵初期的Staphylococcus、Enterococcus和Pantoea,演变为中期的Oceanobacillus、Paracoccus、Staphylococcus 和 Bacillus,随后演变为Pseudomonas和 Enterobacter,最后稳定为Staphylococcus和Terribacillus.根据细菌的功能预测,它们主要参与氨基酸运输和代谢、碳水化合物的运输和代谢等.

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.

The invention relates to an analysis method for succession of microbial communities during tobacco mellowing, and belongs to the technical field of environmental microbes. In order to define microbialpopulation structures and dynamic changes thereof and predict microbial gene functions during tobacco mellowing, the invention provides the analysis method for the succession of the microbial communities during tobacco mellowing, and the analysis method comprises the following steps of (1) performing sampling: taking naturally mellowed tobacco samples at the beginning of mellowing at intervals ofthe same time; (2) constructing gene libraries: extracting genomic DNA of the samples collected in each time period, and constructing a 16S ribosomal RNA (16S rRNA) gene library and an internal transcribed spacer region gene library; and (3) analyzing data of each gene library constructed in the step (2) to obtain the rule of the succession of the microbial communities during tobacco mellowing. The analysis method can be used for giving guidance on artificially mellowing tobacco and establishing the microbial communities.