Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis.

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production.

[Advances in efficient biosynthesis of erythritol by metabolic engineering of Yarrowia lipolytica].

Erythritol is a novel 4-carbon sugar alcohol produced by microbes in the presence of hyper-osmotic stress. It has excellent potential to serve as an alternative sugar for people with diabetes and also a platform compound for synthesizing various C4 compounds, such as 1, 3-butadiene, 1, 4-butanediol, 2, 5-dihydrofuran and so on. Compared with other polyols, the fermentative production of erythritol is more challenging. Yarrowia lipolytica is the preferred chassis of erythritol biosynthesis for its high-titer and high-productivity. At present, there are still some bottlenecks in the production of erythritol by Y. lipolytica, such as weak metabolic activity, abundant by-products, and low industrial attributes. Progress has been made in tailoring high version strains according to industrial needs. For example, the highest titer of erythritol produced by the metabolically engineered Y. lipolytica reached 196 g/L and 150 g/L, respectively, by using glucose or glycerol as the carbon sources. However, further improving its production performance becomes challenging. This review summarizes the research progress in the synthesis of erythritol by Y. lipolytica from the perspectives of erythritol producing strains, metabolic pathways, modular modifications, and auxiliary strategies to enhance the industrial properties of the engineered strain. Key nodes in the metabolic pathway and their combination strategies are discussed to guide the research on promoting the production of erythritol by Y. lipolytica.

Highly efficient production of rhamnolipid in P. putida using a novel sacB-based system and mixed carbon source.

Pseudomonas putida KT2440, a GRAS strain, has been used for synthesizing bulk and fine chemicals. However, the gene editing tool to metabolically engineer KT2440 showed low efficiency. In this study, a novel sacB-based system pK51mobsacB was established to improve the efficiency for marker-free gene disruption. Then the rhamnolipid synthetic pathway was introduced in KT2440 and genes of the competitive pathways were deleted to lower the metabolic burden based on pK51mobsacB. A series of endogenous and synthetic promoters were used for fine tuning rhlAB expression. The limited supply of dTDP-L-rhamnose was enhanced by heterologous rmlBDAC expression. Cell growth and rhamnolipid production were well balanced by using glucose/glycerol as mixed carbon sources. The final strain produced 3.64 g/L at shake-flask and 19.77 g/L rhamnolipid in a 5 L fermenter, the highest obtained among metabolically engineered KT2440, which implied the potential of KT2440 as a promising microbial cell factory for industrial rhamnolipid production.

Designing a novel (R)-ω-transaminase for asymmetric synthesis of sitagliptin intermediate via motif swapping and semi-rational design.

The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a "best" engineered variant MwTAM8, which exhibited 79.2-fold higher activity than the chimeric scaffold MwTAMc. Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates.

Multiplex modification of Yarrowia lipolytica for enhanced erythritol biosynthesis from glycerol through modularized metabolic engineering.

Erythritol is a novelty 4-carbon sugar polyol and has great potential to be used as the precursor of some platform chemicals. The increasing cost of glucose poses researchers shifting insights to the cheaper biodiesel raw materials. Herein, we engineered a non-degradation, non-byproducts Yarrowia lipolytica for the erythritol production with high-titer from glycerol. Initially, the degradation and competition modules were blocked by URA3 counter-selection marker. Subsequently, a shortened biosynthetic pathway was explored to elevate its synthetic flux by multi-modules combination expression of functional genes. Furthermore, a screened glycerol transporter ScFPS1 was integrated into ERY6 genome to promote the glycerol uptake. The constructed strain ERY8 produced 176.66 g/L erythritol in the 5-L bioreactor with a yield and productivity of 0.631 g/g and 1.23 g/L/h, respectively, which achieved the highest fermentation production efficiency till date. This study proposed a novel multi-modules combination strategy for effectively engineering Y. lipolytica to produce erythritol using glycerol.

Metabolic engineering and pathway construction for O-acetyl-L-homoserine production in Escherichia coli.

O-Acetyl-L-homoserine (OAH) is a potentially important platform metabolic intermediate for the production of homoserine lactone, methionine, 1,4-butanediol and 1,3-propanediol which have giant market value. Currently, multiple strategies have been adopted to explore sustainable production of OAH. However, the production of OAH by consuming cheap bio-based feedstocks with Escherichia coli as the chassis is still in its infancy. Construction of high yield OAH-producing strains is of great significance in industry. In this study, we introduced an exogenous metA from Bacillus cereus (metXbc) and engineered an OAH-producing strain by combinatorial metabolic engineering. Initially, exogenous metXs/metA were screened and used to reconstruct an initial biosynthesis pathway of OAH in E. coli. Subsequently, the disruption of degradation and competitive pathways combined with optimal expression of metXbc were carried out, accumulating 5.47 g/L OAH. Meanwhile, the homoserine pool was enriched by overexpressing metL with producing 7.42 g/L OAH. Lastly, the carbon flux of central carbon metabolism was redistributed to balance the metabolic flux of homoserine and acetyl coenzyme A (acetyl-CoA) in OAH biosynthesis with accumulating 8.29 g/L OAH. The engineered strain produced 24.33 g/L OAH with a yield of 0.23 g/g glucose in fed-batch fermentation. By these strategies, the key nodes for OAH synthesis were clarified and corresponding strategies were proposed. This study would lay a foundation for OAH bioproduction. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03564-5.

Gibberellic acid overproduction in Fusarium fujikuroi using regulatory modification and transcription analysis.

Gibberellic acid (GA3), one of the natural diterpenoids produced by Fusarium fujikuroi, serves as an important phytohormone in agriculture for promoting plant growth. Presently, the metabolic engineering strategies for increasing the production of GA3 are progressing slowly, which seriously restricted the advancing of the cost-effective industrial production of GA3. In this study, an industrial strain with high-yield GA3 of F. fujikuroi was constructed by metabolic modification, coupling with transcriptome analysis and promoter engineering. The over-expression of AreA and Lae1, two positive factors in the regulatory network, generated an initial producing strain with GA3 production of 2.78 g L-1. Compared with a large abundance of transcript enrichments in the GA3 synthetic gene cluster discovered by the comparative transcriptome analysis, geranylgeranyl pyrophosphate synthase 2 (Ggs2), and cytochrome P450-3 genes, two key genes that respectively participated in the initial and final step of biosynthesis, were identified to be downregulated when the highest GA3 productivity was obtained. Employing with a nitrogen-responsive bidirectional promoter, the two rate-limiting genes were dynamically upregulated, and therefore, the production of GA3 was increased to 3.02 g L-1. Furthermore, the top 20 upregulated genes were characterized in GA3 over-production, and their distributions in chromosomes suggested potential genomic regions with a high transcriptional level for further strain development. The construction of a GA3 high-yield-producing strain was successfully achieved, and insights into the enriched functional transcripts provided novel strain development targets of F. fujikuroi, offering an efficient microbial development platform for industrial GA3 production. KEY POINTS: • Global regulatory modification was achieved in F. fujikuroi for GA3 overproduction. • Comparative transcriptome analysis revealed bottlenecks in GA specific-pathway. • A dynamically nitrogen-regulated bidirectional promoter was cloned and employed.

Multivariate modular metabolic engineering for enhanced gibberellic acid biosynthesis in Fusarium fujikuroi.

Gibberellic acid (GA3) is one of natural phytohormones, widely used in agriculture and downstream fields. Qualified for the nature productivity, Fusarium fujikuroi was currently employed for the industrial biotransformation from agriculture residues into GA3. Herein, Multivariate modular metabolic engineering (MMME) was assigned to reconstitute the metabolic balance in F. fujikuroi for enhancing GA3 production. Three modules including precursor pool, cluster-specific channel and P450-mediated oxidation in GA3 biosynthetic pathway were defined and optimized separately. The enhancement of both precursor pool and cluster-specific channel pushed metabolic flux transfer into the GA3-specific pathway. Moreover, both introduction of Vitreoscilla hemoglobin and reinforcement of NADPH-dependent cytochrome P450 reductase facilitated oxidation cofactor transfer and subsequently boosted mycelium growth and GA3 biosynthesis. Integration of three modules in the engineered strain accumulated 2.89 g/L GA3 in shake flask via submerged fermentation, presenting a promising modular metabolic engineering model for efficient microbial transformation in agro-industrial application.

Profiling of chromatin accessibility identifies transcription factor binding sites across the genome of Aspergillus species.

BACKGROUND: The identification of open chromatin regions and transcription factor binding sites (TFBs) is an important step in understanding the regulation of gene expression in diverse species. ATAC-seq is a technique used for such purpose by providing high-resolution measurements of chromatin accessibility revealed through integration of Tn5 transposase. However, the existence of cell walls in filamentous fungi and associated difficulty in purifying nuclei have precluded the routine application of this technique, leading to a lack of experimentally determined and computationally inferred data on the identity of genome-wide cis-regulatory elements (CREs) and TFBs. In this study, we constructed an ATAC-seq platform suitable for filamentous fungi and generated ATAC-seq libraries of Aspergillus niger and Aspergillus oryzae grown under a variety of conditions. RESULTS: We applied the ATAC-seq assay for filamentous fungi to delineate the syntenic orthologue and differentially changed chromatin accessibility regions among different Aspergillus species, during different culture conditions, and among specific TF-deleted strains. The syntenic orthologues of accessible regions were responsible for the conservative functions across Aspergillus species, while regions differentially changed between culture conditions and TFs mutants drove differential gene expression programs. Importantly, we suggest criteria to determine TFBs through the analysis of unbalanced cleavage of distinct TF-bound DNA strands by Tn5 transposase. Based on this criterion, we constructed data libraries of the in vivo genomic footprint of A. niger under distinct conditions, and generated a database of novel transcription factor binding motifs through comparison of footprints in TF-deleted strains. Furthermore, we validated the novel TFBs in vivo through an artificial synthetic minimal promoter system. CONCLUSIONS: We characterized the chromatin accessibility regions of filamentous fungi species, and identified a complete TFBs map by ATAC-seq, which provides valuable data for future analyses of transcriptional regulation in filamentous fungi.

CRISPR/dCas9-mediated epigenetic modification reveals differential regulation of histone acetylation on Aspergillus niger secondary metabolite.

Epigenetic studies on secondary metabolites (SMs) mainly relied so far on non-selective epigenetic factors deletion or feeding epigenetic inhibitors in Aspergillus niger. Although technologies developed for epigenome editing at specific loci now enable the direct study of the functional relevance of precise gene regulation and epigenetic modification, relevant assays are limited in filamentous fungi. Herein, we show that CRISPR/dCas9-mediated histone epigenetic modification systems efficiently reprogramed the expression of target genes in A. niger. First, we constructed a p300-dCas9 system and demonstrated the activation of a EGFP fluorescent reporter. Second, by precisely locating histone acetylase p300 on ATG adjacent region of secondary metabolic gene breF, the transcription of breF was activated. Third, p300-dCas9 was guided to the native polyketide synthase (PKS) gene fuml, which increased production of the compound fumonisin B2 detected by HPLC and LC-MS. Then, endogenous histone acetylase GcnE-dCas9 and histone deacetylases HosA-dCas9 and RpdA-dCas9 repressed the transcription of breF. Finally, by targeting HosA-dCa9 fusion to pigment gene fwnA, we confirmed that histone deacetylase HosA activated the expression of fwnA, accelerated the synthesis of melanin. Targeted epigenome editing is a promising technology and this study is the first time to apply the epigenetic CRISPR/dCas9 system on regulating the expression of the secondary metabolic genes in A. niger.

The transcription factor PrtT and its target protease profiles in Aspergillus niger are negatively regulated by carbon sources.

OBJECTIVE: To survey genome-scale protease profiles regulated by the Aspergillus niger transcription factor PrtT and further controlled by carbon sources. RESULTS: The PrtT disruption mutant (delprtT) and overexpression (OEprtT) strains were successfully generated and further confirmed by phenotypic and protease activity analysis. RNA-seq analysis of WT and mutants identified 32 differentially expressed protease genes, which mostly belonged to serine-type peptidases, aspartic-type endopeptidases, aminopeptidases and carboxypeptidases. Furthermore, based on the MEME predicted motif analysis of the PrtT promoter, EMSA and phenotypic and qRT-PCR analyses confirmed that the carbon metabolism regulator AmyR directly regulated the protease genes and their regulatory factor PrtT. CONCLUSION: Thirty-two PrtT-regulated protease genes were identified by RNA-seq, and the secondary carbon source regulator AmyR was found to have a negative regulatory effect on the expression of PrtT and its target protease genes.

High-level expression of highly active and thermostable trehalase from Myceliophthora thermophila in Aspergillus niger by using the CRISPR/Cas9 tool and its application in ethanol fermentation.

Trehalase catalyzes the hydrolysis of the non-reducing disaccharide trehalose. The highly active trehalase MthT from Myceliophthora thermophila was screened from the trehalase genes of six species of filamentous fungi. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool and medium optimization were used to improve MthT production in Aspergillus niger, up to 1698.83 U/mL. The protein background was dramatically abated due to insertion. The recombinant MthT showed optimal activity at pH 5.5 and 60 °C, and exhibited prominent thermal stability between 50 and 60 °C under acid conditions (pH 4.5-6.5). The ethanol conversion rate (ethanol yield/total glucose) was significantly improved by addition of MthT (51.88%) compared with MthT absence (34.38%), using 30% starch saccharification liquid. The results of this study provided an effective strategy, established a convenient platform for heterologous expression in A. niger and showed a potential strategy to decrease production costs in industrial ethanol production.

Highly efficient single base editing in Aspergillus niger with CRISPR/Cas9 cytidine deaminase fusion.

Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.

The global transcriptional landscape of Bacillus amyloliquefaciens XH7 and high-throughput screening of strong promoters based on RNA-seq data.

Bacillus amyloliquefaciens is an important industrial microbe for the production of many industrial enzymes and primary metabolites. Although the complete genome sequence of B. amyloliquefaciens has been now published, transcript structures of B. amyloliquefaciens remain poorly defined. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of B. amyloliquefaciens strain XH7. In total, 3936 out of a total of 4204 B. amyloliquefaciens genes (93.6%) were transcribed under the selected growth condition. Transcriptional start sites (TSS) of 1064 annotated genes and 749 operons were identified. To screen for strong promoters, a beta-galactoside reporter was fused to eight candidate promoters from 288 genes with higher expression levels (RPKM values) than the control gene P43-bgaB. The results illustrated that the candidate promoter Pr2 (promoter for the sigW gene) displayed the strongest beta-galactosidase specific activity during the post-log phase, suggesting that it could be used effectively for heterologous gene expression. The presented data will contribute to the further study of the B. amyloliquefaciens transcriptome by identifying useful promoters for industrial uses.