Efficient biosynthesis of transglutaminase in Streptomyces mobaraensis via systematic engineering strategies.

Transglutaminases (TGases) have been widely used in food, pharmaceutical, biotechnology, and other industries because of their ability to catalyze deamidation, acyl transfer, and crosslinking reactions between Ƴ-carboxamide groups of peptides or protein-bound glutamine and the Ɛ-amino group of lysine. In this study, we demonstrated an efficient systematic engineering strategy to enhance the synthesis of TGase in a recombinant Streptomyces mobaraensis smL2020 strain in a 1000-L fermentor. Briefly, the enzymatic properties of the TGase TGL2020 from S. mobaraensis smL2020 and TGase TGLD from S. mobaraensis smLD were compared to obtain the TGase TGLD with perfected characteristics for heterologous expression in a recombinant S. mobaraensis smL2020ΔTG without the gene tgL 2020. Through multiple engineering strategies, including promoter engineering, optimizing the signal peptides and recombination sites, and increasing copies of the expression cassettes, the final TGLD activity in the recombinant S. mobaraensis smL2020ΔTG: (PL2020-spL2020-protgLD-tgLD)2 (tgL2020and BT1) reached 56.43 U/mL and 63.18 U/mL in shake flask and 1000-L fermentor, respectively, which was the highest reported to date. With the improvement of expression level, the application scope of TGLD in the food industry will continue to expand. Moreover, the genetic stability of the recombinant strain maintained at more than 20 generations. These findings proved the feasibility of multiple systematic engineering strategies in synthetic biology and provided an emerging solution to improve biosynthesis of industrial enzymes.

Metabolic engineering of Candida tropicalis for efficient 1,2,4-butanetriol production.

1,2,4-Butanetriol serves as a precursor in the manufacture of diverse pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. The study involved further modifications to an engineered Candida tropicalis strain, aimed at improving the production efficiency of 1,2,4-butanetriol. Faced with the issue of xylonate accumulation due to the low activity of heterologous xylonate dehydratase, we modulated iron metabolism at the transcriptional level to boost intracellular iron ion availability, thus enhancing the enzyme activity by 2.2-fold. Addressing the NADPH shortfall encountered during 1,2,4-butanetriol biosynthesis, we overexpressed pivotal genes in the NADPH regeneration pathway, achieving a 1,2,4-butanetriol yield of 3.2 g/L. The introduction of calcium carbonate to maintain pH balance led to an increased yield of 4 g/L, marking a 111% improvement over the baseline strain. Finally, the use of corncob hydrolysate as a substrate culminated in 1,2,4-butanetriol production of 3.42 g/L, thereby identifying a novel host for the conversion of corncob hydrolysate to 1,2,4-butanetriol.

Development of a gene-coded biosensor to establish a high-throughput screening platform for salidroside production.

Metabolic engineering reconfigures cellular networks to produce value-added compounds from renewable substrates efficiently. However, identifying strains with desired phenotypes from large libraries through rational or random mutagenesis remains challenging. To overcome this bottleneck, an effective high-throughput screening (HTS) method must be developed to detect and analyze target candidates rapidly. Salidroside is an aromatic compound with broad applications in food, healthcare, medicine, and daily chemicals. However, there currently needs to be HTS methods available to monitor salidroside levels or to screen enzyme variants and strains for high-yield salidroside biosynthesis, which severely limits the development of microbial cell factories capable of efficiently producing salidroside on an industrial scale. This study developed a gene-encoded whole-cell biosensor that is specifically responsive to salidroside. The biosensor was created by screening a site-saturated mutagenic library of uric acid response regulatory protein binding bags. This work demonstrates the feasibility of monitoring metabolic flux with whole-cell biosensors for critical metabolites. It provides a promising tool for building salidroside high-yielding strains for high-throughput screening and metabolic regulation to meet industrial needs.

Biosynthetic pathway redesign in non-conventional yeast for enhanced production of cembratriene-ol.

Cembratriene-ol (CBT-ol), a plant-derived macrocyclic diterpene with notable insecticidal activity, has attracted considerable attention with respect to the development of sustainable and green biopesticides. Currently, CBT-ol production is limited by an inefficient and costly plant extraction strategy. Herein, CBT-ol production was enhanced by redesigning the CBT-ol biosynthetic pathway in Candida tropicalis, with subsequent truncation of CBT-ol synthase further increasing CBT-ol production. Moreover, bottlenecks in the CBT-ol biosynthetic pathway were eliminated by adjusting the gene dosage of the rate-limiting enzymes. Ultimately, the resulting strain C. tropicalis CPPt-03D produced 129.17 mg/L CBT-ol in shaking flasks (a 144-fold increase relative to that of the initial strain C01-CD) with CBT-ol production reaching 1,425.76 mg/L in a 5-L bioreactor, representing the highest CBT-ol titer reported to date. These findings provide a green process and promising platform for the industrial production of CBT-ol and lays the foundation for organic farming.

Molecular insights into the microbial degradation of sediment-derived DOM in a macrophyte-dominated lake under aerobic and hypoxic conditions.

The mineralization of dissolved organic matter (DOM) in sediments is an important factor leading to the eutrophication of macrophyte-dominated lakes. However, the changes in the molecular characteristics of sediment-derived DOM during microbial degradation in macrophyte-dominated lakes are not well understood. In this study, the microbial degradation process of sediment-derived DOM in Lake Caohai under aerobic and hypoxic conditions was investigated using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and metagenomics. The results revealed that the microbial degradation of sediment-derived DOM in macrophyte-dominated lakes was more intense under aerobic conditions. The microorganisms mainly metabolized the protein-like substances in the macrophyte-dominated lakes, and the carbohydrate-active enzyme genes and protein/lipid-like degradation genes played key roles in sediment-derived DOM degradation. Organic compounds with high H/C ratios such as lipids, carbohydrates, and protein/lipid-like compounds were preferentially removed by microorganisms during microbial degradation. Meanwhile, there was an increase in the abundance of organic molecular formula with a high aromaticity such as tannins and unsaturated hydrocarbons with low molecular weight and low double bond equivalent. In addition, aerobic/hypoxic environments can alter microbial metabolic pathways of sediment-derived DOM by affecting the relative abundance of microbial communities (e.g., Gemmatimonadetes and Acidobacteria) and functional genes (e.g., ABC.PE.P1 and ABC.PE.P) in macrophyte-dominated lakes. The abundances of lipids, unsaturated hydrocarbons, and protein compounds in aerobic environments decreased by 58 %, 50 %, and 44 %, respectively, compared to in hypoxic environments under microbial degradation. The results of this study deepen our understanding of DOM biodegradation in macrophyte-dominated lakes under different redox environments and provide new insights into nutrients releases from sediment and continuing eutrophication in macrophyte-dominated lakes.

Boosting production of cembratriene-ol in Saccharomyces cerevisiae via systematic optimization.

Cembratriene-ol is a good biodegradable biopesticide ingredient with future potential applications in the field of sustainable agriculture. Cembratriene-ol is a monocyclic diterpenoid compound that is synthesized only in the trichome gland of Nicotiana plants. In this study, geranylgeranyl diphosphate synthase gene ggpps from Taxus canadensis and cbts*Δp were heterologously expressed in Saccharomyces cerevisiae W303-1A to successfully synthesize cembratriene-ol. The titer of cembratriene-ol was increased by 1.84-fold compared to the control by overexpressing the S. cerevisiae bifunctional (2E,6E)-farnesyl diphosphate synthase genes ERG20 and cbts*Δp under one promoter PGAP . The titer of cembratriene-ol in the engineered S. cerevisiae BY4741 was increased by 1.39-fold compared to the engineered S. cerevisiae W303-1A. The titer of cembratriene-ol in the engineered S. cerevisiae BY4741 was increased by 2.22-fold compared to the control by overexpressing ERG20 and cbts*Δp, respectively, using two promoters PGAP . Cembratriene-ol was found to be successfully synthesized via the integrated expression of cbts*Δp, ggpps and ERG20 on the genome of S. cerevisiae BY4741. The titer of cembratriene-ol in S. cerevisiae S25 was further increased by 1.80-fold compared to the control via dynamic control of the squalene synthase gene ERG9. Overexpression of the genes cbts*Δp and ggpps using pY26-GPD-TEF in S. cerevisiae S25 with their integration expression increased the titer of cembratriene-ol by 26.1-fold compared to S. cerevisiae S25. The titer of cembratriene-ol was significantly enhanced by mitochondrial compartmentalized expression of cbts*Δp and ggpps, which was 76.3-fold higher than that of the initial strain constructed. It was indicated that the systematic optimization has great potential in facilitating high-level production of cembratriene-ol production in S. cerevisiae.

[Production of limonene and its derivative in Saccharomyces cerevisiae via metabolic engineering].

Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.

CRISPR-Cas Technology for Bioengineering Conventional and Non-Conventional Yeasts: Progress and New Challenges.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system has undergone substantial and transformative progress. Simultaneously, a spectrum of derivative technologies has emerged, spanning both conventional and non-conventional yeast strains. Non-conventional yeasts, distinguished by their robust metabolic pathways, formidable resilience against diverse stressors, and distinctive regulatory mechanisms, have emerged as a highly promising alternative for diverse industrial applications. This comprehensive review serves to encapsulate the prevailing gene editing methodologies and their associated applications within the traditional industrial microorganism, Saccharomyces cerevisiae. Additionally, it delineates the current panorama of non-conventional yeast strains, accentuating their latent potential in the realm of industrial and biotechnological utilization. Within this discourse, we also contemplate the potential value these tools offer alongside the attendant challenges they pose.

[Optimization of retinin expression and the application with wax emulsion in nanocoatings].

Anti-reflective nanocoatings that mimic the eyes of fruit flies are biodegradable materials with great market potential for a variety of optical devices that require anti-reflective properties. Microbial expression of retinin provides a new idea for the preparation of nanocoatings under mild conditions compared to physicochemical methods. However, the current expression level of retinin, the key to anti-reflective coating, is low and difficult to meet mass production. In this study, we analyzed and screened the best expression hosts for Drosophila-derived retinin protein, and optimized its expression. Chinese hamster ovary (CHO) cells were identified as the efficient expression host of retinin, and purified retinin protein was obtained. At the same time, the preparation method of lanolin nanoemulsion was explored, and the best anti-reflective ability of the nano-coating was determined when the ratio of specific concentration of retinin protein and wax emulsion was 16:4, the pH of the nano-coating formation system was 7.0, and the temperature was 30 ℃. The enhanced antireflective ability and reduced production cost of artificial antireflective nanocoatings by determining the composition of nanocoatings and optimizing the concentration, pH and temperature of system components may facilitate future application of artificial green degradable antireflective coatings.

Efficient production of hydroxytyrosol by directed evolution of HpaB in Escherichia coli.

Hydroxytyrosol (HT) is an olive-derived phenolic phytochemical that has gained increasing commercial interest due to its natural antioxidant properties. It is widely used in the field of food supplement and medicine. It is reported that 4-hydroxyphenylacetate 3-hydroxylase (EcHpaB) and flavin reductase (EcHpaC) from E. coli BL21(DE3) can successfully express and catalyze the production of HT from tyrosol. In this study, the tyrosol production strain YMG5∗R as chassis cells, and a random mutant library of EcHpaB was established using error-prone PCR to improve the ability of EcHpaB to convert tyrosol to HT. Finally, a highly efficient HT synthetic mutant strainYMG5∗R-HpaBTLEHC with high transformation efficiency was screened by directed evolution. The YMG5∗R-HpaBTLEHC strain efficiently converted 50 mM tyrosol, with a yield of hydroxytyrosol reaching 48.2 mM (7.43 g/L) and a space-time yield reached 0.62 g/L·h. Overall, our study demonstrates the successful development of a highly efficient synthetic enzyme mutant for the production of HT, which has the potential to significantly improve the commercial viability of this natural antioxidant.

Efficient production of cembratriene-ol in Escherichia coli via systematic optimization.

BACKGROUND: The tobacco leaf-derived cembratriene-ol exhibits anti-insect effects, but its content in plants is scarce. Cembratriene-ol is difficult and inefficiently chemically synthesised due to its complex structure. Moreover, the titer of reported recombinant hosts producing cembratriene-ol was low and cannot be applied to industrial production. RESULTS: In this study, Pantoea ananatis geranylgeranyl diphosphate synthase (CrtE) and Nicotiana tabacum cembratriene-ol synthase (CBTS) were heterologously expressed to synthsize the cembratriene-ol in Escherichia coli. Overexpression of cbts*, the 1-deoxy-D-xylulose 5-phosphate synthase gene dxs, and isopentenyl diphosphate isomerase gene idi promoted the production of cembratriene-ol. The cembratriene-ol titer was 1.53-folds higher than that of E. coli Z17 due to the systematic regulation of ggpps, cbts*, dxs, and idi expression. The production of cembratriene-ol was boosted via the overexpression of genes ispA, ispD, and ispF. The production level of cembratriene-ol in the optimal medium at 72 h was 8.55-folds higher than that before fermentation optimisation. The cembratriene-ol titer in the 15-L fermenter reached 371.2 mg L- 1, which was the highest titer reported. CONCLUSION: In this study, the production of cembratriene-ol in E. coli was significantly enhanced via systematic optimization. It was suggested that the recombinant E. coli producing cembratriene-ol constructed in this study has potential for industrial production and applications.

Control over Selectivity in Alkene Hydrosilylation Catalyzed by Cobalt(III) Hydride Complexes.

Two new bisphosphine [PCP] pincer cobalt(III) hydrides, [(L1)Co(PMe3)(H)(Cl)] (L11, L1 = 2,6-((Ph2P)(Et)N)2C6H3) and [(L2)Co(PMe3)(H)(Cl)] (L21, L2 = 2,6-((iPr2P)(Et)N)2C6H3), as well as one new bissilylene [SiCSi] pincer cobalt(III) hydride, [(L3)Co(PMe3)(H)(Cl)] (L31, L3 = 1,3-((PhC(tBuN)2Si)(Et)N)2C6H3), were synthesized by reaction of the corresponding protic [PCP] or [SiCSi] pincer ligands L1H, L2H, and L3H with CoCl(PMe3)3. Despite the similarities in the ligand scaffolds, the three cobalt(III) hydrides show remarkably different performance as catalysts in alkene hydrosilylation. Among the PCP pincer complexes, L11 has higher catalytic activity than complex L21, and both catalysts afford anti-Markovnikov selectivity for both aliphatic and aromatic alkenes. In contrast, the catalytic activity for alkene hydrosilylation of silylene complex L31 is comparable to phosphine complex L11, but a dependence of regioselectivity on the substrates was observed: While aliphatic alkenes are converted in an anti-Markovnikov fashion, the hydrosilylation of aromatic alkenes affords Markovnikov products. The substrate scope was explored with 28 examples. Additional experiments were conducted to elucidate these mechanisms of hydrosilylation. The synthesis of cobalt(I) complex (L1)Co(PMe3)2 (L17) and its catalytic properties for alkene hydrosilylation allowed for the proposal of the mechanistic variations that occur in dependence of reaction conditions and substrates.

Systematic Review of Actinomycetes in the Baijiu Fermentation Microbiome.

Actinomycetes (a group of filamentous bacteria) are the dominant microbial order in the Daqu (DQ) fermentation starter and in the pit mud (PM) of the Baijiu fermentation microbiome. Actinomycetes produce many of the key enzymes and flavor components, and supply important precursors, which have a major influence on its characteristic aroma components, to other microorganisms during fermentation. This paper reviews the current progress on actinomycete research related to Baijiu fermentation, including the isolation and identification, distribution, interspecies interactions, systems biology, and main metabolites. The main metabolites and applications of the actinomycetes during Baijiu fermentation are also discussed.

Simple phase unwrapping method with continuous convex minimization.

Phase unwrapping is a problem to reconstruct true phase values from modulo 2π phase values measured using various phase imaging techniques. This procedure is essentially formulated as a discrete optimization problem. However, most energy minimization methods using continuous optimization techniques have ignored the discrete nature and solved it as a continuous minimization problem directly, leading to losing exactness of the algorithms. We propose a new minimum norm method that can yield the optimal solution of the discrete problem by minimizing a continuous energy function. In contrast to the graph-cuts method, which is state of the art in this field, the proposed method requires much less memory space and a very simple implementation. Therefore, it can be simply extended to 3D or 4D phase unwrapping problems.

Abundance and characteristics of sediment-bound magnetic minerals and trace elements in karst ditch wetland: A case study from Guizhou Province, China.

This study investigated sediment-bound magnetic properties and selected trace elements level in the karst ditch wetland, Caohai National Nature Reserve, Guizhou Province, China. Sediment-bound magnetic signals were quantified using low-frequency mass magnetic susceptibility (χLF), anhysteretic remanent magnetization susceptibility (χARM), saturation isothermal remanent magnetization (SIRM), and percentage frequency-dependent susceptibility (χfd%). Concentrations of Cd, Cr, Sb and Zn were determined using inductively coupled plasma mass spectrometry. Sediment χLF, χARM, SIRM, and χfd% were higher than those of bedrocks and mainly altered by the pedogenic processes. The estimated χfd% ranged from 6.15 % to 14.62 % and reflected the magnetic grain sizes were largely concentrated in the range of superparamagnetic particles. The elevated concentrations of sediment-bound Cd, Cr, Sb, and Zn supported the significant contribution of the anthropogenic sources in the karst ditch wetlands. The weak relationship between magnetic signals and selected trace elements (p < 0.05) suggested the occurrence of few sediment-bound iron-containing minerals associated with selected trace elements. These results indicated that a minor contribution of anthropogenic sources of selected trace elements to the elevated sediment magnetic signals in the karst ditch wetlands.

Efficient extracellular production of recombinant proteins in E. coli via enhancing expression of dacA on the genome.

D, D-carboxypeptidase DacA plays an important role in the synthesis and stabilization of Escherichia coli cell wall peptidoglycan. The production level of extracellular recombinant proteins in E. coli can be enhanced by high D, D-carboxypeptidase activity. Construction of expression systems under optimal promoters is one of the main strategies to realize high protein production in E. coli. In this study, the promoter PdacA-3 from DacA on the genome of E. coli BL21 (DE3) was verified to be efficient for recombinant green fluorescent protein using the plasmid mutant pET28a-PdacA with PdacA-3. Meanwhile, the promoter PdacA-3 was engineered to increase the production level of proteins via inserting one or two Shine-Dalgarno (SD) sequences between the promoter PdacA-3 and the target genes. The expression level of dacA on the genome was increased by the improved transcription of the engineered promoters (especially after inserting one additional SD sequence). The engineered promoters increased cell membrane permeabilities to significantly enhance the secretion production of extracellular recombinant proteins in E. coli. Among them, the extracellular recombinant amylase activities in E. coli BL21::1SD-pET28a-amyK and E. coli BL21::2SD-pET28a-amyK were increased by 2.0- and 1.6-fold that of the control (E. coli BL21-pET28a-amyK), respectively. Promoter engineering also affected the morphology and growth of the E. coli mutants. It was indicated that the engineered promoters enhanced the expression of dacA on the genome to disturb the synthesis and structural stability of cell wall peptidoglycans.

Significant Improvement of Both Catalytic Efficiency and Stability of Fructosyltransferase from Aspergillus niger by Structure-Guided Engineering of Key Residues in the Conserved Sequence of the Catalytic Domain.

Fructosyltransferase is a key enzyme in fructo-oligosaccharide production, while the highly demanding conditions of industrial processes may reduce its stability and activity. This study employs sequence alignment and structural analysis to target three potential residues (Gln38, Ile39, and Cys43) around the active center of FruSG from Aspergillus niger, and mutants with greatly improved activity and stability were obtained through site-directed mutagenesis. The Km values of C43N and Q38Y were, respectively, reduced to 60.8 and 93.1% compared to those of WT. Meanwhile, the kcat of C43N was increased by 21.2-fold compared to that of WT. These imply that both the affinity and catalytic efficiency of C43N were significantly enhanced compared to WT. The Glide docking score of sucrose inside C43N was calculated to be -5.980, which was lower than that of WT (-4.887). What is more, the proposed general acid/base catalyst Glu273 with a lower pKa value of C43N calculated by PROPKA might contribute to an easier catalytic reaction compared to that of WT. The thermal stability and pH stability of the mutant C43N were significantly enhanced compared to those of WT, and more hydrogen bonds formed during molecular dynamics simulations might contribute to the improved stability of C43N.

Engineering the oleaginous yeast Candida tropicalis for α-humulene overproduction.

BACKGROUND: α-Humulene is a plant-derived monocyclic sesquiterpenoid with multiple pharmacological activities, and far-reaching potential for the development of new drugs. Currently, the production of α-humulene is typically achieved via plant extraction, which is not sustainable and limited by low yields. The oleaginous yeast Candida tropicalis has recently emerged as a valuable host for producing high-value-added chemicals. However, the potential of C. tropicalis for terpenoid production has not been exploited. RESULTS: In this study, C. tropicalis was engineered for de novo synthesis of α-humulene from glucose. To improve α-humulene production, the codon-optimised α-humulene synthase gene and the entire endogenous farnesyl diphosphate synthesis pathway were co-overexpressed. Furthermore, bottlenecks in the α-humulene synthase pathway were identified and relieved by overexpressing α-humulene synthase, acetoacetyl-CoA thiolase and NADH-dependent HMG-CoA reductase. Combined with fermentation medium optimisation, the engineered strain produced 195.31 mg/L of α-humulene in shake flasks and 4115.42 mg/L in a bioreactor through fed-batch fermentation, a 253- and 5345-fold increase over the initial production, respectively. CONCLUSIONS: This study demonstrates the potential of C. tropicalis for α-humulene production, and presents a platform for the biosynthesis of other terpenoids.

Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.

Candida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNAGly sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene GFP3 (green fluorescent protein) and the endogenous gene ADE2 (phosphoribosylaminoimidazole carboxylase). Different regions of GFP3 and ADE2 were targeted with the gRNAs processed by the tRNAGly, and the transcription levels of GFP3 and ADE2 were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence ERG9 (squalene synthase) to enhance ß-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNAGly and the CRISPRi system have great potential for metabolic engineering of C. tropicalis. IMPORTANCE In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating ß-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.

Organic phosphorus regeneration enhanced since eutrophication occurred in the sub-deep reservoir.

Lake eutrophication remains a serious environmental problem of global significance, and phosphorus (P) plays a key role in lake eutrophication. Internal P loading, as a result of P release from sediments, is gathering more and more recognition as an important source governing the P availability in these ecosystems. Anoxic condition can promote the release of P associated with Fe oxides, which has already been a consensus. However, it is still unknown whether the anoxic conditions induced by eutrophication act to intensify or weaken the regeneration of organic P (Porg) in sediments. We selected the Hongfeng Reservoir, a typical sub-deep lake, to study the regeneration behaviours of C and P in the sediments buried before and after eutrophication. The results showed that Porg did not significantly increase with the rapid increase in organic C (Corg) since eutrophication occurred. Furthermore, the organic C/P ratio was much higher in sediments buried after eutrophication than in those buried before, which indicated that Porg regeneration had been significantly enhanced since eutrophication occurred. Based on C/P ratios, our estimation suggested that the Porg regeneration and P release from sediment to water approximately enhanced 45.2% ± 8.7% and 34.5% ± 9.8%, respectively. Elevated primary productivity (algae) and the corresponding hypoxic/anoxic condition, both caused by eutrophication, promoted P biogeochemical cycle in the sub-deep reservoir. This study further verifies the significant contribution of regenerated Porg to the internal P load, and highlights the importance of controlling P release from sediments in order to restore clear water ecosystems in sub-deep lakes or reservoirs.