The small subunit, ssPOXA3a/b, and the large subunit, POXA3, are indispensable components of typical heterodimeric laccase (Lacc2) in white rot fungi. However, the enzymatic and biological functions of ssPOXA3a/b remain unclear. The present study revealed that neither ssPOXA3a nor ssPOXA3b per se has a catalytic ability, whereas their combination with POXA3 (and especially ssPOXA3b) enhances the activity, thermostability, and pH stability of POXA3. In Pleurotus eryngii var. ferulae, there was no regulatory relationship between ssPOXA3a/b and POXA3 at the transcriptional level. However, sspoxa3a/b overexpression had a negative feedback effect on lacc6 transcription. By contrast, poxa3 transcripts had no effect on any other laccase isoenzyme. Overexpression of sspoxa3a/b resulted in small fungal pellets, thin mycelial walls, and facilitated laccase secretion. However, poxa3 overexpression had no influence on pellet morphology. Collectively, this work elucidated the functions of ssPOXA3a/b and laid an empirical foundation for the development of high-yield laccase.

3-Hydroxy-L-tyrosine (L-DOPA) is a promising drug for treating Parkinson's disease. Tyrosine hydroxylase catalyzes the microbial synthesis of L-DOPA, which is hindered by the efficiency of catalysis, the supply of cofactor tetrahydrobiopterin, and the regulation of the pathway. In this study, the modular engineering strategy in Bacillus licheniformis was identified to effectively enhance L-DOPA production. First, the catalytic efficiency of biocatalyst tyrosine hydroxylase from Streptosporangium roseum DSM 43021 (SrTH) was improved by 20.3% by strengthening its affinity toward tetrahydrobiopterin. Second, the tetrahydrobiopterin supply pool was increased by bottleneck gene expression, oxygen transport facilitation, budC (encoding meso-2,3butanediol dehydrogenase) deletion, and tetrahydrobiopterin regeneration using a native YfkO nitroreductase. The strain 45AB v C successfully produced tetrahydrobiopterin, which was detected as pterin (112.48 mg/L), the oxidation product of tetrahydrobiopterin. Finally, the yield of precursor L-tyrosine reached 148 mg/g DCW , with an increase of 71%, with the deletion of a novel spliced transcript 41sRNA associated with the regulation of the shikimate pathway. The engineered strain 45AB v CS::PD produced 167.14 mg/L (2.41 times of wild-type strain) and 1290 mg/L L-DOPA in a shake flask and a 15 L bioreactor, respectively, using a fermentation strategy on a mixture of carbon sources. This study holds great potential for constructing a microbial source of L-DOPA and its high-value downstream pharmaceuticals.

This study aims to find the targets that may influence the production of bacitracin based on RNA sequencing in Bacillus licheniformis. Transcriptional profiling revealed that (i) the expression of the bacT gene, encoding a type II thioesterase (TEII bac ), was positively correlated with bacitracin production and (ii) the oxygen uptake exhibited significant influence on precursor synthesis. The verified experiments showed that the overexpression of TEII bac with an endogenous promoter increased the bacitracin A titer by 37.50%. Furthermore, the increase of oxygen availability through Vitreoscilla hemoglobin (VHb) expression increased the bacitracin A titer by 126.67% under oxygen-restricted conditions. From the transcriptome perspective, the results of this paper demonstrate that TEII bac and oxygen supply are crucial to bacitracin production. This study also provides insights into the construction of chassis cells for the industrial production of secondary metabolites with a preference for aerobic conditions.

The aging process is the final step in cigar production and plays a vital role in the quality. There are complex microbial growth and metabolism, enzymatic catalysis, and chemical reactions in this process. Among them, microorganisms function as a bridge between organic and inorganic contents. At present, we do not yet have sufficient knowledge of the evolution of microorganisms in the aging process of cigars. Neither are we clear about the mechanisms by which environmental conditions may impact the quality of cigars through the interaction of microorganisms and chemical substances. In this study, we used a metagenomics sequence-guided strategy to identify the microbes and enzymes involved in the degradation of key compounds, biomass and alkaloids, under different environmental conditions. The results showed that different environmental factors including temperature, humidity, aging cycle, and turning frequency resulted varied contents of total sugar, and alkaloids in cigars. Microbial communities showed significant correlations with starch, reducing sugars, total sugars and alkaloids. Vital species in the catabolism of starch (Bacillus pumilus, Pseudomonas sp. 286 and Aspergillus cristatus), reducing sugars and total sugars (Aspergillus cristatus and Nitrolancea hollandica) were identified. Corynespora cassiicola and Pseudomonas fulva were proposed to contribute to the degradation of nornicotine and neonicotinoid. Our work should deepen the understanding of microbial roles in cigar aging and provide a new viewpoint for applying specific microbial consortia to modulate the degradation of biomass and alkaloids in cigar leaves.

A significant distinction between cigar production and tobacco lies in the necessary aging process, where intricate microbial growth, metabolic activities, enzymatic catalysis, and chemical reactions interact. Despite its crucial role in determining the final quality of cigars, our comprehension of the underlying chemical and biological mechanisms within this process remains insufficient. Biomass and alkaloids are the primary constituents that influence the flavor of cigars. Consequently, investigating the entire aging process could begin by exploring the involvement of microbes and enzymes in their biodegradation. In this study, handmade cigars were aged under different conditions. Metagenomic sequencing was employed to identify the microbes and enzymes responsible for the degradation of biomass and alkaloids derived from tobacco leaves. The results revealed that various environmental factors, including temperature, humidity, duration time, and turning frequency, yielded varying contents of total sugar and alkaloids in the cigars. Significant correlations were observed between microbial communities and starch, reducing sugars, total sugars, and alkaloids. Key species involved in the breakdown of biomass constituents, such as starch (Bacillus pumilus, Pseudomonas sp. 286, and Aspergillus cristatus), reducing sugars and total sugars (Aspergillus cristatus and Nitrolancea hollandica), were identified. Furthermore, Corynespora cassiicola and Pseudomonas fulva were found to potentially contribute to the degradation of alkaloid compounds, specifically nornicotine and neonicotinoid. Our work contributes to a deeper understanding of the microbial roles in the aging of cigars. Moreover, the selection of specific microbial strains or starter cultures can be employed to control and manipulate the aging process, thereby further refining the flavor development in cigar products. Graphical Abstract

Synthetic biology has rapidly advanced from the setup of native genetic devices to the design of artificial elements able to provide organisms with highly controllable functions. In particular, genetic switches are crucial for deploying new layers of regulation into the engineered organisms. While the assembly and mutagenesis of native elements have been extensively studied, limited progress has been made in rational design of genetic switches due to a lack of understanding of the molecular mechanism by which a specific transcription factor interacts with its target gene. Here, a reliable workflow is presented for designing two categories of genetic elements, one is the switch element-MtlR box and the other is the transcriptional regulatory element- catabolite control protein A (CcpA) box. The MtlR box was designed for ON/OFF-state selection and is controlled by mannitol. The rational design of MtlR box-based molecular structures can flexibly tuned the selection of both ON and OFF states with different output switchability in response to varied kind effectors. Different types of CcpA boxes made the switches with more markedly inducer sensitivities. Ultimately, the OFF-state value was reduced by 90.69%, and the maximum change range in the presence of two boxes was 15.31-fold. This study presents a specific design of the switch, in a plug-and-play manner, which has great potential for controlling the flow of the metabolic pathway in synthetic biology. Graphical Abstract