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Background: Oxytetracycline which is derived from Streptomyces rimosus, inhibits a wide range of bacteria and is industrially important. The underlying biosynthetic processes are complex and hinder rational engineering, so industrial manufacturing currently relies on classical mutants for production. While the biochemistry underlying oxytetracycline synthesis is known to involve polyketide synthase, hyperproducing strains of S. rimosus have not been extensively studied, limiting our knowledge on fundamental mechanisms that drive production. Results: In this study, a multiomics analysis of S. rimosus is performed and wild-type and hyperproducing strains are compared. Insights into the metabolic and regulatory networks driving oxytetracycline formation were obtained. The overproducer exhibited increased acetyl-CoA and malonyl CoA supply, upregulated oxytetracycline biosynthesis, reduced competing byproduct formation, and streamlined morphology. These features were used to synthesize bhimamycin, an antibiotic, and a novel microbial chassis strain was created. A cluster deletion derivative showed enhanced bhimamycin production. Conclusions: This study suggests that the precursor supply should be globally increased to further increase the expression of the oxytetracycline cluster while maintaining the natural cluster sequence. The mutagenized hyperproducer S. rimosus HP126 exhibited numerous mutations, including large genomic rearrangements, due to natural genetic instability, and single nucleotide changes. More complex mutations were found than those typically observed in mutagenized bacteria, impacting gene expression, and complicating rational engineering. Overall, the approach revealed key traits influencing oxytetracycline production in S. rimosus, suggesting that similar studies for other antibiotics could uncover general mechanisms to improve production. [ABSTRACT FROM AUTHOR]

Background: Transforming waste and nonfood materials into bulk biofuels and chemicals represents a major stride in creating a sustainable bioindustry to optimize the use of resources while reducing environmental footprint. However, despite these advancements, the production of high-value natural products often continues to depend on the use of first-generation substrates, underscoring the intricate processes and specific requirements of their biosyntheses. This is also true for Streptomyces lividans, a renowned host organism celebrated for its capacity to produce a wide array of natural products, which is attributed to its genetic versatility and potent secondary metabolic activity. Given this context, it becomes imperative to assess and optimize this microorganism for the synthesis of natural products specifically from waste and nonfood substrates. Results: We metabolically engineered S. lividans to heterologously produce the ribosomally synthesized and posttranslationally modified peptide bottromycin, as well as the polyketide pamamycin. The modified strains successfully produced these compounds using waste and nonfood model substrates such as protocatechuate (derived from lignin), 4-hydroxybenzoate (sourced from plastic waste), and mannitol (from seaweed). Comprehensive transcriptomic and metabolomic analyses offered insights into how these substrates influenced the cellular metabolism of S. lividans. In terms of production efficiency, S. lividans showed remarkable tolerance, especially in a fed-batch process using a mineral medium containing the toxic aromatic 4-hydroxybenzoate, which led to enhanced and highly selective bottromycin production. Additionally, the strain generated a unique spectrum of pamamycins when cultured in mannitol-rich seaweed extract with no additional nutrients. Conclusion: Our study showcases the successful production of high-value natural products based on the use of varied waste and nonfood raw materials, circumventing the reliance on costly, food-competing resources. S. lividans exhibited remarkable adaptability and resilience when grown on these diverse substrates. When cultured on aromatic compounds, it displayed a distinct array of intracellular CoA esters, presenting promising avenues for polyketide production. Future research could be focused on enhancing S. lividans substrate utilization pathways to process the intricate mixtures commonly found in waste and nonfood sources more efficiently. [ABSTRACT FROM AUTHOR]

The Great Pacific Garbage Patch, a significant collection of plastic introduced by human activities, provides an ideal environment to study bacterial lifestyles on plastic substrates. We proposed that bacteria colonizing the floating plastic debris would develop strategies to deal with the ultraviolet‐exposed substrate, such as the production of antioxidant pigments. We observed a variety of pigmentation in 67 strains that were directly cultivated from plastic pieces sampled from the Garbage Patch. The genomic analysis of four representative strains, each distinct in taxonomy, revealed multiple pathways for carotenoid production. These pathways include those that produce less common carotenoids and a cluster of photosynthetic genes. This cluster appears to originate from a potentially new species of the Rhodobacteraceae family. This represents the first report of an aerobic anoxygenic photoheterotrophic bacterium from plastic biofilms. Spectral analysis showed that the bacteria actively produce carotenoids, such as beta‐carotene and beta‐cryptoxanthin, and bacteriochlorophyll a. Furthermore, we discovered that the genetic ability to synthesize carotenoids is more common in plastic biofilms than in the surrounding water communities. Our findings suggest that plastic biofilms could be an overlooked source of bacteria‐produced carotenoids, including rare forms. It also suggests that photoreactive molecules might play a crucial role in bacterial biofilm communities in surface water. [ABSTRACT FROM AUTHOR]

The Gram‐positive bacteria Streptomyces davaonensis and Streptomyces cinnabarinus have been the only organisms known to produce roseoflavin, a riboflavin (vitamin B2) derived red antibiotic. Using a selective growth medium and a phenotypic screening, we were able to isolate a novel roseoflavin producer from a German soil sample. The isolation procedure was repeated twice, that is, the same strain could be isolated from the same location in Berlin 6 months and 12 months after its first isolation. Whole genome sequencing of the novel roseoflavin producer revealed an unusual chromosomal arrangement and the deposited genome sequence of the new isolate (G + C content of 71.47%) contains 897 genes per inverted terminal repeat, 6190 genes in the core and 107 genes located on an illegitimate terminal end. We identified the roseoflavin biosynthetic genes rosA, rosB and rosC and an unusually high number of riboflavin biosynthetic genes. Overexpression of rosA, rosB and rosC in Escherichia coli and enzyme assays confirmed their predicted functions in roseoflavin biosynthesis. A full taxonomic analysis revealed that the isolate represents a previously unknown Streptomyces species and we propose the name Streptomyces berlinensis sp. nov. for this roseoflavin producer. [ABSTRACT FROM AUTHOR]

Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus , making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 μg L−1 of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common P erm* promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that P erm* failed to drive expression during the product synthesis phase but that use of two synthetic promoters (P kasOP* and P 41) enabled strong constitutive expression during the entire process. Using P kasOP* , several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L−1 in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3′-end nyb of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L−1 nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin. • Nybomycin is a promising antibiotic against multi-resistant S. aureus. • Genomic studies identified the minimal nybomycin gene cluster. • Streamlined chassis Streptomyces albidoflavus Del14 used for production. • Systematic engineering of primary and secondary pathways. • Engineered S. albidoflavus NYB-11 forms nybomycin up to 12 mg L−1 [ABSTRACT FROM AUTHOR]

Background: Fatty acid-derived products such as fatty alcohols (FAL) find growing application in cosmetic products, lubricants, or biofuels. So far, FAL are primarily produced petrochemically or through chemical conversion of bio-based feedstock. Besides the well-known negative environmental impact of using fossil resources, utilization of bio-based first-generation feedstock such as palm oil is known to contribute to the loss of habitat and biodiversity. Thus, the microbial production of industrially relevant chemicals such as FAL from second-generation feedstock is desirable. Results: To engineer Corynebacterium glutamicum for FAL production, we deregulated fatty acid biosynthesis by deleting the transcriptional regulator gene fasR, overexpressing a fatty acyl-CoA reductase (FAR) gene of Marinobacter hydrocarbonoclasticus VT8 and attenuating the native thioesterase expression by exchange of the ATG to a weaker TTG start codon. C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) produced in shaking flasks 0.54 ± 0.02 gFAL L−1 from 20 g glucose L−1 with a product yield of 0.054 ± 0.001 Cmol Cmol−1. To enable xylose utilization, we integrated xylA encoding the xylose isomerase from Xanthomonas campestris and xylB encoding the native xylulose kinase into the locus of actA. This approach enabled growth on xylose. However, adaptive laboratory evolution (ALE) was required to improve the growth rate threefold to 0.11 ± 0.00 h−1. The genome of the evolved strain C. glutamicum gX was re-sequenced, and the evolved genetic module was introduced into C. glutamicum ∆fasR cg2692TTG (pEKEx2-maqu2220) which allowed efficient growth and FAL production on wheat straw hydrolysate. FAL biosynthesis was further optimized by overexpression of the pntAB genes encoding the membrane-bound transhydrogenase of E. coli. The best-performing strain C. glutamicum ∆fasR cg2692TTG CgLP12::(Ptac-pntAB-TrrnB) gX (pEKEx2-maqu2220) produced 2.45 ± 0.09 gFAL L−1 with a product yield of 0.054 ± 0.005 Cmol Cmol−1 and a volumetric productivity of 0.109 ± 0.005 gFAL L−1 h−1 in a pulsed fed-batch cultivation using wheat straw hydrolysate. Conclusion: The combination of targeted metabolic engineering and ALE enabled efficient FAL production in C. glutamicum from wheat straw hydrolysate for the first time. Therefore, this study provides useful metabolic engineering principles to tailor this bacterium for other products from this second-generation feedstock. [ABSTRACT FROM AUTHOR]