Engineering Microbiology最新文献

Myxobacteria are famous for their capacity for social behavior and natural product biosynthesis. The unique sociality of myxobacteria is not only an intriguing scientific topic but also the main limiting factor for their manipulation. After more than half a century of research, a series of genetic techniques for myxobacteria have been developed, rendering these mysterious bacteria manipulable. Here, we review the advances in genetic manipulation of myxobacteria, with a particular focus on the exploitation of secondary metabolism. We emphasize the necessity and urgency of constructing the myxobacterial chassis for synthetic biology research and the exploitation of untapped secondary metabolism.

The decarbonization of the chemical industry and a shift toward circular economies because of high global CO₂ emissions make CO₂ an attractive feedstock for manufacturing chemicals. Moreover, H₂ is a low-cost and carbon-free reductant because technologies such as solar-driven electrolysis and supercritical water (scH₂O) gasification enable sustainable production of molecular hydrogen (H₂). We review the recent advances in engineering Ralstonia eutropha, the representative species of “Knallgas” bacteria, for utilizing CO₂ and H₂ to autotrophically produce 2,3-butanediol (2,3-BDO). This assessment is focused on state-of-the-art approaches for splitting H₂ to supply energy in the form of ATP and NADH to power cellular reactions and employing the Calvin-Benson-Bassham cycle for CO₂ fixation. Major challenges and opportunities for application and future perspectives are discussed in the context of developing other promising CO₂ and H₂-utilizing microorganisms, exemplified by Zymomonas mobilis.

Trichoderma reesei Rut-C-30 is a well-known robust producer of cellulolytic enzymes, which are used to degrade lignocellulosic biomass for the sustainable production of biofuels and biochemicals. However, studies of its secondary metabolism and regulation remain scarce. Ypr1 was previously described as a regulator of the biosynthesis of the yellow pigment sorbicillin (a bioactive agent with great pharmaceutical interest) in T. reesei and several other fungi. However, the manner in which this regulator affects global gene transcription has not been explored. In this study, we report the effect of Ypr1 on the regulation of both the secondary and primary metabolism of T. reesei Rut-C30. A global gene transcription profile was obtained using a comparative transcriptomic analysis of the wild-type strain T. reesei Rut-C-30 and its ypr1 deletion mutant. The results of this analysis suggest that, in addition to its role in regulating sorbicillin and the major extracellular (hemi)cellulases, Ypr1 also affects the transcription of genes encoding several other secondary metabolites. Although the primary metabolism of T. reesei ∆ypr1 became less active compared with that of T. reesei Rut-C-30, several gene clusters involved in its secondary metabolism were activated, such as the gene clusters for the biosynthesis of specific polyketides and non-ribosomal peptides, together with the “sorbicillinoid–cellulase” super cluster, indicating that specific secondary metabolites and cellulases may be co-regulated in T. reesei Rut-C-30. The results presented in this study may benefit the development of genetic engineering strategies for the production of sorbicillin by T. reesei Rut-C-30, and provide insights for enhancing sorbicillin production in other filamentous fungal producers.

Numerous studies have investigated the biosynthesis of pyridine heterocycles derived from nicotinic acid. However, metabolic pathways generating pyridine heterocycles in nature remain uninvestigated. Here, we summarize recent contributions conducted in the last decade on the biosynthetic pathways of non-derivate from nicotinic acid pyridine rings and discuss their implication on the study of natural products with pyridine structures.

Genistein, an isoflavone found mainly in legumes, has been shown to have numerous health benefits for humans. Therefore, there is substantial interest in producing it using microbial cell factories. To aid in screening for high genistein producing microbial strains, a cell-based biosensor for genistein was developed by repurposing the Gal4DBD-ERα-VP16 (GEV) transcriptional activator in Saccharomyces cerevisiae. In the presence of genistein, the GEV sensor protein binds to the GAL1 promoter and activates transcription of a downstream GFP reporter. The performance of the biosensor, as measured by fold difference in GFP signal intensity after external genistein induction, was improved by engineering the sensor protein, its promoter and the reporter promoter. Biosensor performance increased when the weak promoter REV1p was used to drive GEV sensor gene expression and the VP16 transactivating domain on GEV was replaced with the tripartite VPR transactivator that had its NLS removed. The biosensor performance further improved when the binding sites for the inhibitor Mig1 were removed from and two additional Gal4p binding sites were added to the reporter promoter. After genistein induction, our improved biosensor output a GFP signal that was 20 times higher compared to the uninduced state. Out of the 8 flavonoids tested, the improved biosensor responded only to genistein and in a somewhat linear manner. The improved biosensor also responded to genistein produced in vivo, with the GFP reporter intensity directly proportional to intracellular genistein concentration. When combined with fluorescence-based cell sorting technology, this biosensor could facilitate high-throughput screening of a genistein-producing yeast cell factory.

Biosynthetic pathways without any identifiable core enzymes may encode unknown (biosynthetic route)–unknown (molecular structure) natural products. However, bioinformatics-guided mining for such unknown-unknown metabolites is challenging. Recently, an unknown-unknown biosynthetic route has been deciphered in fungi. It was found that a class of enzymes previously annotated as hypothetical proteins catalyze the biosynthesis of arginine-containing cyclodipeptides (CDPs). This advances the understanding of the biosynthesis of CDPs and highlights the vast potential of unknown-unknown natural products encoded by microbial genomes.

Leech hyaluronidase (LHyal) is a hyperactive hyaluronic acid (HA) hydrolase that belongs to the hyaluronoglucuronidase family. Traditionally, LHyal is extracted from the heads of leeches, but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides. However, at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps. Moreover, the enzyme is deactivated and removed after single use. To solve this problem, we developed a recyclable LHyal biocatalyst using a yeast surface display (YSD) system. After screening and characterization, we found that the cell wall protein Sed1p displayed stronger anchoring to the P. pastoris cell wall than other cell wall proteins. By optimizing the type and length of the linkers between LHyal and Sed1p, we increased the activity of enzymes displayed on the P. pastoris cell wall by 50.34% in flask cultures. LHyal-(GGGS)₆-Sed1p activity further increased to 3.58 × 10⁵ U mL⁻¹ in fed-batch cultivation in a 5 L bioreactor. Enzymatic property analysis results revealed that the displayed LHyal-(GGGS)₆-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme. Moreover, displayed LHyal-(GGGS)₆-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times. YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification process for HA oligosaccharides. Thus, this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Microbially derived, protein-based biopesticides have become a vital element in pest management strategies. Vip3 family proteins from Bacillus thuringiensis have distinct characteristics from known insecticidal Cry toxins and show efficient insecticidal activity against several detrimental lepidopteran pests. They are considered to be a promising toxic candidate for the management of various detrimental pests. In this study, we found that in addition to the preliminary digestion sites lysine, there are multiple cleavage activation sites in the linker region between domain I (DI) and DII of Vip3Aa. We further demonstrated that by adding more cleavage sites between DI and DII of Vip3Aa, its proteolysis efficiency by midgut proteases can be significantly increased, and correspondingly enhance its insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera larvae. Our study promotes the understanding of the insecticidal mechanism of Vip3 proteins and illustrates an easily implementable strategy to increase the insecticidal potency of Vip3Aa. This facilitates their potential future development and efficient application for sustainable agriculture.

Fe(II)/α-ketoglutarate (αKG)-dependent oxygenases catalyze the oxidative modification of various molecules, from DNA, RNA, and proteins to primary and secondary metabolites. They also catalyze a variety of biochemical reactions, including hydroxylation, halogenation, desaturation, epoxidation, cyclization, peroxidation, epimerization, and rearrangement. Given the versatile catalytic capability of such oxygenases, numerous studies have been conducted to characterize their functions and elucidate their structure–function relationships over the past few decades. Amino acids, particularly nonproteinogenic amino acids, are considered as important building blocks for chemical synthesis and components for natural product biosynthesis. In addition, the Fe(II)/αKG-dependent oxygenase superfamily includes important enzymes for generating amino acid derivatives, as they efficiently modify various free-standing amino acids. The recent discovery of new Fe(II)/αKG-dependent oxygenases and the repurposing of known enzymes in this superfamily have promoted the generation of useful amino acid derivatives. Therefore, this study will focus on the recent progress achieved from 2019 to 2022 to provide a clear view of the mechanism by which these enzymes have expanded the repertoire of free amino acid oxidative modifications.