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The natural compound ( R )-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 ( pks8 ) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous ( R )-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in β-oxidation ( fadD , fadE , fadB , and fadA ). Cell growth inhibition by ( R )-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced ( R )-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous ( R )-(-)-mellein synthesis in microorganism with a high titer. ( R )-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.

Bacitracin, a type of cyclic dodecapeptide antibiotic mainly produced by Bacillus , is widely used in fields of veterinary drug and feed additive. Modularization of metabolic pathways based on the concept of synthetic biology has been widely used in the efficient synthesis of target products. Here, we want to improve bacitracin production through strengthening aspartic acid (Asp) supply in B. licheniformis DW2. First, exogenous Asp addition assays implied that strengthening Asp supply benefited bacitracin production. Second, Asp synthetic pathways were strengthened via overexpressing aspartate dehydrogenase AspD and asparaginase AnsB, attaining recombinant strain DW2-ASP2, and bacitracin yield produced by DW2-ASP2 was 862.81 U/mL, increased by 14.05% compared with that of DW2 (756.49 U/mL). Then, to improve precursor oxaloacetate (OAA) accumulation for Asp synthesis, pyruvate carboxylase PycA and carbonic anhydrase EcaA were co-overexpressed in DW2-ASP2, and malic enzyme gene malS was deleted to weak overflow metabolism of tricarboxylic acid, and the attained strain DW2-ASP7 showed further increased bacitracin production from 862.81 to 989.23 U/mL. Subsequently, transporter YveA was identified as an Asp exporter, and bacitracin yield was increased to 1025.26 U/mL via deleting yveA , attaining strain DW2-ASP9. Finally, Asp ammonia-lyase gene aspA was disrupted to weaken Asp degradation, and bacitracin yield of attained strain DW2-ASP10 reached 1059.86 U/mL, increased by 40.10% compared to DW2. Taken together, this research demonstrated that metabolic engineering of Asp metabolic modules is an efficient strategy for enhancing bacitracin production, and these strategies could also be applied in the production of other peptide-related metabolites.

Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R. capsulatus . The above strategies enabled a 35-fold higher titer of bisabolene than that of the starting strain, reaching 1089.7 mg/L from glucose in a shake flask. The engineered strain produced 9.8 g/L bisabolene with a yield of >0.196 g/g-glucose under the two-phase fed-batch fermentation, which corresponds to >78% of theoretical maximum. Taken together, our work represents one of the pioneering studies to demonstrate PNSB as a promising platform for terpene-based advanced biofuel production.

Erythromycin is necessary in medical treatment and known to be biosynthesized with propionyl-CoA as direct precursor. Oversupply of propionyl-CoA induced hyperpropionylation, which was demonstrated as harmful for erythromycin synthesis in Saccharopolyspora erythraea. Herein, we identified three propionyl-CoA synthetases regulated by propionylation, and one propionyl-CoA synthetase SACE_1780 revealed resistance to propionylation. A practical strategy for raising the precursor (propionyl-CoA) supply bypassing the feedback inhibition caused by propionylation was developed through two approaches: deletion of the propionyltransferase AcuA, and SACE_1780 overexpression. The constructed Δ acuA strain presented a 10% increase in erythromycin yield; SACE_1780 overexpression strain produced 33% higher erythromycin yield than the wildtype strain NRRL2338 and 22% higher erythromycin yield than the industrial high yield Ab strain. These findings uncover the role of protein acylation in precursor supply for antibiotics biosynthesis and provide efficient post-translational modification-metabolic engineering strategy (named as PTM-ME) in synthetic biology for improvement of secondary metabolites.