l-Tyrosine serves as a common precursor for multiple valuable secondary metabolites. Synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway, but the underlying mechanisms involving metabolic regulation remain unclear. In this work, improved l-tyrosine accumulation was achieved in B. licheniformis via co-overexpression of aroGfbr and tyrAfbr from Escherichia coli to yield strain 45A12, and the l-tyrosine titer increased to 1005 mg/L with controlled glucose feeding. Quantitative RT-PCR results indicated that aroA, encoding DAHP synthase, and aroK, encoding shikimate kinase, were feedback-repressed by the end product l-tyrosine in the modified strain. Therefore, the native aroK was first expressed with multiple copies to yield strain 45A13, which could accumulate 1201 mg/L l-tyrosine. Compared with strain 45A12, the expression of aroB and aroF in strain 45A13 was upregulated by 21% and 27%, respectively, which may also have resulted in the improvement of l-tyrosine production. Furthermore, supplementation with 5 g/L shikimate enhanced the l-tyrosine titers of 45A12 and 45A13 by 29.1% and 24.0%, respectively. However, the yield of l-tyrosine per unit of shikimate decreased from 0.365 to 0.198 mol/mol after aroK overexpression in strain 45A12, which suggested that the gene product was also involved in uncharacterized pathways. This study provides a good starting point for further modification to achieve industrial-scale production of l-tyrosine using B. licheniformis, a generally recognized as safe workhorse.

l-Tyrosine hydroxylation by tyrosine hydroxylase is a significant reaction for preparing many nutraceutical and pharmaceutical chemicals.

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.