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The platensimycin (PTM), platencin (PTN), and platensilin (PTL) family of natural products continues to inspire the discovery of new chemistry, enzymology, and medicine. Engineered production of this emerging family of natural products, however, remains laborious due to the lack of practical systems to manipulate their biosynthesis in the native-producing Streptomyces platensis species. Here we report solving this technology gap by implementing a CRISPR-Cas9 system in S. platensis CB00739 to develop an expedient method to manipulate the PTM, PTN, and PTL biosynthetic machinery in vivo. We showcase the utility of this technology by constructing designer recombinant strains S. platensis SB12051, SB12052, and SB12053, which, upon fermentation in the optimized PTM-MS medium, produced PTM, PTN, and PTL with the highest titers at 836 mg L-1, 791 mg L-1, and 40 mg L-1, respectively. Comparative analysis of these resultant recombinant strains also revealed distinct chemistries, catalyzed by PtmT1 and PtmT3, two diterpene synthases that nature has evolved for PTM, PTN, and PTL biosynthesis. The ΔptmR1/ΔptmT1/ΔptmT3 triple mutant strain S. platensis SB12054 could be envisaged as a platform strain to engineer diterpenoid biosynthesis by introducing varying ent-copalyl diphosphate-acting diterpene synthases, taking advantage of its clean metabolite background, ability to support diterpene biosynthesis in high titers, and the promiscuous tailoring biosynthetic machinery., One-Sentence Summary: Implementation of a CRISPR-Cas9 system in Streptomyces platensis CB00739 enabled the construction of a suite of designer recombinant strains for the overproduction of platensimycin, platencin, and platensilin, discovery of new diterpene synthase chemistries, and development of platform strains for future diterpenoid biosynthesis engineering., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Carotenoids are naturally occurring pigments that are abundant in the natural world. Due to their excellent antioxidant attributes, carotenoids are widely utilized in various industries, including the food, pharmaceutical, cosmetic industries, and others. Plants, algae, and microorganisms are presently the main sources for acquiring natural carotenoids. However, due to the swift progress in metabolic engineering and synthetic biology, along with the continuous and thorough investigation of carotenoid biosynthetic pathways, recombinant strains have emerged as promising candidates to produce carotenoids. The identification and manipulation of gene targets that influence the accumulation of the desired products is a crucial challenge in the construction and metabolic regulation of recombinant strains. In this review, we provide an overview of the carotenoid biosynthetic pathway, followed by a summary of the methodologies employed in the discovery of gene targets associated with carotenoid production. Furthermore, we focus on discussing the gene targets that have shown potential to enhance carotenoid production. To facilitate future research, we categorize these gene targets based on their capacity to attain elevated levels of carotenoid production.

Bacterial aromatic polyketides are mainly biosynthesized by type II polyketide synthases (PKSs). The PKSs cannot be functional unless their acyl carrier proteins (ACPs) are phosphopantetheinylated by phosphopantetheinyl transferases (PPTases). Gra-ORF32 was identified as an in-cluster PPTase dedicated for granaticin biosynthesis in Streptomyces vietnamensis and the Arg- and Pro-rich N terminus was found to be crucial for catalytic activity. Overexpression of the encoding genes of the holo -ACP synthases of fatty acid synthases (FAS ACPSs) of both E. coli and S. vietnamensis could efficiently activate the production of granaticins in the Δ gra-orf32 mutant, suggesting the ACP of granaticin (graACP) is an efficient substrate for FAS ACPSs. However, Gra-ORF32, the cognate PPTase of the graACP, could not compensate the conditional deficiency of ACPS in E. coli HT253, indicating that it has evolved to be functionally segregated from fatty acid biosynthesis. Nine out of eleven endogenous and all the tested exogenous non-cognate PPTases could activate the production of granaticins to varied extents when overexpressed in the Δ gra-orf32 mutant, indicating that ACPs of type II PKSs could also be widely recognized as effective substrates by the Sfp-type PPTases. The exogenous PPTases of type II PKSs activated the production of granaticins with much higher efficiency, suggesting that the phylogenetically distant in-cluster PPTases of type II PKSs could share substrate preferences for the ACPs of type II PKSs. A significantly elevated production of granaticins was observed when the mutant Δ gra-orf32 was cultivated on ISP2 plates, which was a consequence of crosstalk between the granaticin pathway and a kinamycin-like pathway as revealed by transcriptome analysis and pathway inactivations. Although the host FAS ACPS could efficiently activate the production of granaticins when overexpressed, only Gra-ORF32 activated the efficient production of granaticins under natural physiological conditions, indicating that the activity of the host FAS ACPS was strictly regulated, possibly by binding the FAS holo -ACP product with high affinity. Our findings would contribute to a more comprehensive understanding of how the ACPs of type II PKSs are activated and facilitate the future functional reconstitutions of type II PKSs in E. coli ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Deng, Chik, Li and Zhu.)

Members of the genus Qipengyuania are heterotrophic bacteria frequently isolated from marine environments with great application potential in areas such as carotenoid production. However, the genomic diversity, metabolic function, and adaption of this genus remain largely unclear. Here, 16 isolates related to the genus Qipengyuania were recovered from coastal samples and their genomes were sequenced. The phylogenetic inference of these isolates and reference type strains of this genus indicated that the 16S rRNA gene was insufficient to distinguish them at the species level; instead, the phylogenomic reconstruction could provide the reliable phylogenetic relationships and confirm 15 new well-supported branches, representing 15 putative novel genospecies corroborated by the digital DNA-DNA hybridization and average nucleotide identity analyses. Comparative genomics revealed that the genus Qipengyuania had an open pangenome and possessed multiple conserved genes and pathways related to metabolic functions and environmental adaptation, despite the presence of divergent genomic features and specific metabolic potential. Genetic analysis and pigment detection showed that the members of this genus were identified as carotenoid producers, while some proved to be potentially aerobic anoxygenic photoheterotrophs. Collectively, the first insight into the genetic diversity and metabolic potentials of the genus Qipengyuania will contribute to better understanding of the speciation and adaptive evolution in natural environments. IMPORTANCE The deciphering of the phylogenetic diversity and metabolic features of the abundant bacterial taxa is critical for exploring their ecological importance and application potential. Qipengyuania is a genus of frequently isolated heterotrophic microorganisms with great industrial application potential. Numerous strains related to the genus Qipengyuania have been isolated from diverse environments, but their genomic diversity and metabolic functions remain unclear. Our study revealed a high degree of genetic diversity, metabolic versatility, and environmental adaptation of the genus Qipengyuania using comparative genomics. Fifteen novel species of this genus have been established using a polyphasic taxonomic approach, expanding the number of described species to almost double. This study provided an overall view of the genus Qipengyuania at the genomic level and will enable us to better uncover its ecological roles and evolutionary history.

Granaticins are benzoisochromanequinone polyketides with remarkable antibacterial and anticancer activities. Three sulfur-containing granaticin congeners, mycothiogranaticins A ( 1 ), B ( 2 ) and granaticin MA ( 3 ) were discovered from a granaticin-producing strain of Streptomyces vietnamensis GIMV4.0001. Two of them were structurally determined with mycothiol or N-acetylcysteine moieties and found to be bio-actively reluctant. Disruption of the mshA gene ( SVTN_RS20640 ) that encodes the D-inositol-3-phosphate glycosyltransferase crucial for mycothiol biosynthesis, fully abolished the production of mycothiogranaticins. The result substantiated that the newly discovered mycothiogranaticins are consequences of the combination of the granaticin and mycothiol biosynthetic pathways. The overall granaticin production of the Δ mshA mutant strain was unexpectedly decreased by at least more than 50%, while similar production level of granaticins to that of the wild type strain was observed in an mycothiol-S transferase gene ( SVTN_RS22215 ) disruptant Δ mst . These results indicated that the mycothiol deficiency was responsible for the decreased production of granaticins. Mycothiol may positively regulate the biosynthesis of granaticin possibly by maintaining the cellular redox balance. To the best of our knowledge, this is the first report that mycothiol can not only be a direct building block of polyketides but also play a regulatory role in the polyketide biosynthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Deng, Li, Luo, Zheng, Chen, Zhang, Zhang, Zhou and Zhu.)

Rapid and accurate strain identification of the most closely related genera Myxococcus , Corallococcus , and Pyxidicoccus can enhance the efficiency of the mining of novel secondary metabolites through dereplication. However, the commonly used 16S rRNA gene sequencing cannot accurately differentiate members of the three genera above, and the whole-genome sequencing is unable to rapidly and inexpensively provide species assignation toward a large number of isolates. To overcome the limitations, the gyrB gene was investigated as a candidate genetic marker for exploring the phylogenetic relationships of bacteria within the three genera and for developing the gyrB -based typing method. Here, the bacterial phylogeny and species affiliations of the three genera were determined based on the phylogenomic reconstruction and the analysis of digital DNA-DNA hybridization values among 90 genomes, further confirming nine novel taxa and assigning over one-third of genomes to defined species. The phylogenetic relationships of these strains based on the gyrB gene sequences were congruent with those based on their genome sequences, allowing the use of the gyrB gene as a molecular marker. The gyrB gene-specific primers for the PCR-amplification and sequencing of bacteria within the three genera were designed and validated for 31 isolates from our group collection. The gyrB -based taxonomic tool proved to be able to differentiate closely related isolates at the species level. Based on the newly proposed 98.6% identity threshold for the 966-bp gyrB gene and the phylogenetic inference, these isolates were assigned into two known species and eight additional putative new species. In summary, this report demonstrated that the gyrB gene is a powerful phylogenetic marker for taxonomy and phylogeny of bacteria within the closely related genera Myxococcus , Corallococcus , and Pyxidicoccus , particularly in the case of hundreds or thousands of isolates in environmental studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Pei, Yi, Du, Zhang, Deng, Yao, Deng and Zhu.)

Carotenoids are a large family of health-beneficial compounds that have been widely used in the food and nutraceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol-induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03-D-4, subsequently, an evolved strain M3 was obtained with a 5.1-fold increase in carotenoid yield. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcriptome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain., (© 2021. The Author(s).)

We previously constructed a Saccharomyces cerevisiae carotenoid producer BL03-D-4 which produced much more carotenoid in YPM (modified YPD) media than YPD media. In this study, the impacts of nutritional components on carotenoid accumulation of BL03-D-4 were investigated. When using YPM media, the carotenoid yield was increased 10-fold compared to using the YPD media. To elucidate the hidden mechanism, a transcriptome analysis was performed and showed that 464 genes changed significantly in YPM media. Furthermore, inspired by the differential gene expression analysis which indicated that ADY2 , HES1 , and CUP1 showed the most remarkable changes, we found that the improvement of carotenoid accumulation in YPM media was mainly due to the copper ions, since supplementation of 0.08 mM CuSO 4 in YPD media could increase carotenoid yield 9.2-fold. Reverse engineering of target genes was performed and carotenoid yield could be increased 6.4-fold in YPD media through overexpression of ACE1 . The present study revealed for the first time the prominent promotion of carotenoid yield by copper ions in engineered S. cerevisiae and provided a new target ACE1 for genetic engineering of S. cerevisiae for the bioproduction of carotenoids.

We report the complete genome sequence of Streptomyces vietnamensis GIMV4.0001(T), a new and genetically manipulable producer of the benzoisochromanequinone antibiotic granaticin, whose unique sugar attachment pattern in structure has drawn much attention among chemical and biochemical researchers. The genome of S. vietnamensis GIMV4.0001(T) consists of one linear chromosome (8,867,142 bp, 72.09% G+C content) and one linear megaplasmid named pSVL1 (286,635 bp, 69.04% G+C content), encoding a total of 7356 protein coding genes. Twenty-nine gene clusters for secondary metabolites were predicted on the chromosome., (Copyright © 2015 Elsevier B.V. All rights reserved.)