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The transcription factor complex INO2 and INO4 in Saccharomyces cerevisiae plays a vital role in lipid biosynthesis by activating multiple genes in the biosynthetic pathways of phospholipid, fatty acid, and sterol. Previous studies have reported conflicting results regarding the effects of ino2 and ino4 gene expression levels on target chemicals. Therefore, this study aimed to examine the influence of different ino2 and ino4 expression levels on carotenoid production (e.g., lycopene), which shares a common precursor, acetyl-CoA, with lipid metabolism. Surprisingly, 2.6- and 1.8-fold increase in lycopene yield in the ino2 and ino4 deletion strains were found, respectively. In contrast, ino2 overexpression did not promote lycopene accumulation. Additionally, there was a decrease in intracellular free fatty acids in the ino2 deletion strain. Comparative transcriptome analysis revealed a significant downregulation of genes related to lipid biosynthesis in the ino2 deletion strain. To our knowledge, this is the first report showing that deletion of transcription factor genes ino2 and ino4 can facilitate lycopene accumulation. These findings hold significant implications for the development of metabolically engineered S. cerevisiae with enhanced carotenoid production., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

L-threonine is a crucial amino acid that is extensively employed in the realms of food, animal feed and pharmaceuticals. Unfortunately, the lack of an appropriate biosensor has hindered the establishment of a robust high-throughput screening (HTS) system for the identification of the desired strains from random mutants. In this study, a dual-responding genetic circuit that capitalizes on the L-threonine inducer-like effect, the L-threonine riboswitch, and a signal amplification system was designed for the purpose of screening L-threonine overproducers. This platform effectively enhanced the performance of the enzyme and facilitated the identification of high L-threonine-producing strains from a random mutant library. Consequently, pathway optimization and directed evolution of the key enzyme enhanced L-threonine production by 4 and 7-fold, respectively. These results demonstrate the potential of biosensor design for dynamic metabolite detection and offer a promising tool for HTS and metabolic regulation for the development of L-threonine-hyperproducing strains., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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.

Saccharomyces cerevisiae is increasingly being used for the production of chemicals derived from acetyl coenzyme A (acetyl-CoA). However, the inadequate supply of cytosolic acetyl-CoA often leads to low yields. Here, we developed a novel strategy for balancing acetyl-CoA metabolism and increasing the amount of the downstream product. First, the combination of acetaldehyde dehydrogenase (eutE) and acetoacetyl-CoA thiolase (AtoB) was optimized to redirect the acetyl-CoA flux toward the target pathway, with a 21-fold improvement in mevalonic acid production. Second, pathway engineering and evolutionary engineering were conducted to attenuate the growth deficiency, and a 10-fold improvement of the maximum productivity was achieved. Third, acetyl-CoA carboxylase (ACC 1 ) was dynamically downregulated as the complementary acetyl-CoA pathway, and the yield was improved more than twofold. Fourth, the most efficient and complementary acetyl-CoA pathways were combined, and the final strain produced 68 mg/g CDW lycopene, which was among the highest yields reported in S. cerevisiae . This study demonstrates a new method of producing lycopene products by regulating acetyl-CoA metabolism.

Metabolic engineering of microbial cell factories through integrating the heterologous synthetic pathway into the chromosome is most commonly used for industrial applications. However, the position of the foreign gene in the chromosome can affect its transcriptional level. As a microorganism that is generally regarded as safe (GRAS) and commonly applied in industrial manufacture with large-scale operations, Saccharomyces cerevisiae is also confronted with this position effect. In this study, we characterized 12 different chromosome sites by inserting the lycopene biosynthetic pathway as a reporter cassette. Due to the different integration loci, the gene transcription and lycopene yield exhibited more than 58-fold and 3.8-fold differences, respectively. Furthermore, changing the gene order also revealed a remarkable influence (30-fold and 14-fold) on gene transcription and lycopene yield. Besides, the upstream activation sequence of a strong promoter (defined as an insulator) in S. cerevisiae could reduce the impact by gene order, and increased the gene transcription (tenfold) and lycopene yield (sevenfold). Taken together, our results demonstrated that gene order and insulator affected gene transcription and heterogeneous biosynthesis, opening the opportunity to regulate gene transcription by insulator against position effect in S. cerevisiae., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A novel aerobic, yellow, and rod-shaped bacterial isolate, designated as 1Y8A T , was isolated from aquaculture water sampled in Jiangmen, Guangdong province, P. R. China. Here, the taxonomic position of strain 1Y8A T was conducted based on phenotypic, genomic, and chemotaxonomic characteristics. Strain 1Y8A T was observed to grow at 10-37 °C (optimum 28 °C), at pH 6.0-9.0 (optimum 7.0) and in 0-2% NaCl (optimum 1%, w/v). The 16S rRNA gene-based analysis showed that strain 1Y8A T was closely related to "Flavobacterium sasangense" YC6274 T (99.3%), Flavobacterium aquaticum JC164 T (98.4%), Flavobacterium cucumis R2A45-3 T (98.0%), Flavobacterium celericrescens TWA-26 T (98.0%), and Flavobacterium cheniae NJ-26 T (97.2%). The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain 1Y8A T and reference strains above were far below the recognized thresholds of 70% dDDH and 95-96% ANI for species definition, implying that the strain represents a novel genospecies. The phylogenomic analysis indicated that strain 1Y8A T formed an independent branch distinct from known species. The major cellular fatty acids of strain 1Y8A T were iso-C 15:0 , iso-C 15:1 G and C 15:0 ; the polar lipids comprised phosphatidylethanolamine, glycolipid, and two lipids; the respiratory quinone was MK-6. The G + C content of genomic DNA was 32.5%. Based on the genotypic and phenotypic characteristics such as the utilization of D-glucose and casein hydrolysis, strain 1Y8A T is concluded to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium proteolyticum sp. nov. is proposed. The type strain of the species is 1Y8A T (= GDMCC 1.1933 T  = KACC 22081 T )., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel Gram-staining-negative and short-rod-shaped bacterial strain designated as 1NDH52 T was isolated from a tidal flat sediment and characterized using a polyphasic taxonomic approach. The predominant cellular fatty acids of strain 1NDH52 T were summed feature 8 (C 18:1 ω7c and/or C 18:1 ω6c) and C 14:0 2-OH; the major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipid; the major respiratory quinone was Q-10. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 1NDH52 T was closely related to type strains Croceicoccus sediminis S2-4-2  T (98.9%), Croceicoccus bisphenolivorans H4 T (98.9%) and Croceicoccus pelagius Ery9 T (98.7%). Phylogenomic analysis indicated that strain 1NDH52 T formed an independent branch distinct from all type strains of this genus. The overall genome related indices including the digital DNA-DNA hybridization values, average nucleotide identities and average amino acid identities between strain 1NDH52 T and the three close relatives above indicated that strain 1NDH52 T should represent a novel genospecies. The genomic DNA G + C content was 62.6%. Strain 1NDH52 T could produce carotenoids and its genome contained the complete carotenoids biosynthetic gene cluster. Based on the phenotypic and genotypic characteristics, strain 1NDH52 T is concluded to represent a novel species of the genus Croceicoccus, for which the name Croceicoccus gelatinilyticus sp. nov., is proposed. The type strain of the species is 1NDH52 T (= GDMCC 1.2381  T  = KCTC 82668  T )., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

The two novel bacterial strains designated 1Y17 T and 4Y10 T from aquaculture water were characterized using a polyphasic taxonomic approach. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains 1Y17 T and 4Y10 T belonged to the genus Inhella and were close to Inhella crocodyli CCP-18 T , Inhella inkyongensis IMCC1713 T and Inhella fonticola TNR-25 T . Strains 1Y17 T and 4Y10 T shared 98.6% identity with each other and less than 99.0% identity with their relatives above. The phylogenomic analysis indicated that the two strains formed two independent branches distinct from their relatives. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the two strains were 21.3 and 80.9% below the two thresholds of 70% dDDH and 95-96% ANI for species definition; those between the two novel strains and their relatives were far below thresholds for species definition, implying that they represent two novel genospecies. The predominant fatty acids of the two strains were summed feature 3 (C 16:1 ω7c and/or C 16:1 ω6c) and C 16:0 ; the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol; the major quinone and polyamine were Q-8 and putrescine. Their genomic DNA G + C contents were 69.3 and 65.0%. The two novel strains can produce poly-β-hydroxybutyrate, matching with the presence of the three synthetic related genes of the phaC-phaA-phaB in their genomes. Based on the genotypic and phenotypic characteristics such as aesculin and gelatin hydrolysis, strains 1Y17 T and 4Y10 T are concluded to represent two novel species of the genus Inhella, for which the names Inhella proteolytica sp. nov. (type strain 1Y17 T  = GDMCC 1.1830 T  = KACC 21948 T ) and Inhella gelatinilytica sp. (type strain 4Y10 T  = GDMCC 1.1829 T  = KCTC 82338 T ) are proposed., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel Gram-stain-negative and rod-shaped bacterial strain, designated as 4Y14 T , was isolated from aquaculture water and characterized by using a polyphasic taxonomic approach. Strain 4Y14 T was found to grow at 10-40 °C (optimum, 28 °C), at pH 7.0-9.0 (optimum, 7.0-8.0) and with 0-2 % NaCl (optimum, 1 %, w/v). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 4Y14 T belonged to the genus Chitinilyticum with high levels of similarity to Chitinilyticum litopenaei c1 T (97.8 %) and Chitinilyticum aquatile c14 T (97.2 %). Phylogenomic analysis indicated that strain 4Y14 T formed an independent branch distinct from the two type strains above. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain 4Y14 T and the two type strains were, respectively, 25.3 and 25.0 %, and 81.2 and 80.3 %, which were well below the thresholds of 70 % DDH and 95-96 % ANI for species definition, implying that strain 4Y14 T should represent a novel genospecies. The predominant cellular fatty acids of strain 4Y14 T were summed feature 3 (C 16 : 1 ω7 c and/or C 16 : 1 ω6 c) and iso-C 16 : 0 ; the major polar lipids were diphosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine; and the sole respiratory quinone was Q-8. The genomic DNA G+C content was 60.1 mol%. Based on the phenotypic and genotypic analyses, strain 4Y14 T is concluded to represent a novel species of the genus Chitinilyticum , for which the name Chitinilyticum piscinae sp. nov. is proposed. The type strain of the species is 4Y14 T (=GDMCC 1.1934 T =KACC 22080 T ).

A novel Gram-stain-negative, aerobic and rod-shaped bacterial strain designated as 6D45A T was isolated from mangrove soil and characterized using a polyphasic taxonomic approach. Strain 6D45A T was found to grow at 10-37 °C (optimum, 28 °C), at pH 6.0-9.0 (optimum, 7.0) and in 0-5 % (w/v) NaCl (optimum, 2%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 6D45A T fell into the genus Salipiger and shared 99.1 % identity with the closest type strain Salipiger pacificus CGMCC 1.3455 T and less than 97.2 % identity with other type strains of this genus. The 34.8 % digital DNA-DNA hybridization (dDDH) and 88.3 % average nucleotide identity (ANI) values between strain 6D45A T and the closest relative above were well below recognized thresholds of 70 % DDH and 95-96 % ANI for species definition, implying that strain 6D45A T should represent a novel genospecies. The phylogenomic analysis indicated that strain 6D45A T formed an independent branch distinct from reference strains. The predominant cellular fatty acid of strain 6D45A T was summed feature 8 (C 18 : 1  ω6 c and/or C 18 : 1  ω7 c, 66.9 %); the polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids, two unidentified glycolipids and an unknown lipid; the respiratory quinone was Q-10. The genomic DNA G+C content was 66.5 mol %. Based on the phenotypic and genotypic characteristics, strain 6D45A T is concluded to represent a novel species of the genus Salipiger , for which the name Salipiger mangrovisoli sp. nov., is proposed. The type strain of the species is 6D45A T (=GDMCC 1.1960 T =KCTC 82334 T ). We also propose the reclassification of Paraphaeobacter pallidus as Salipiger pallidus comb. nov. and ' Pelagibaca abyssi ' as a species of the genus Salipiger .

A novel Gram-stain-negative, non-motile, and rod-shaped bacterial strain, designated as 6D36 T , was isolated from mangrove soil and characterized by using a polyphasic taxonomic approach. Strain 6D36 T was found to grow at 10-37 °C (optimum, 28 °C), at pH 6.0-9.0 (optimum, 7.0) and in 0-8% (w/v) NaCl (optimum, 3%). The predominant cellular fatty acids of strain 6D36 T were summed feature 8 (C 19:1 ω7c and/or C 18:1 ω6c) and C 17:1 ω6c; the major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and sphingoglycolipid; the sole respiratory quinone was Q-10. The phylogenetic analysis based on 16S rRNA gene sequences showed that strain 6D36 T fell into the genus Qipengyuania and was closely related to "Erythrobacter mangrovi" MCCC 1K03690 T (98.5%), Qipengyuania citrea CGMCC 1.8703 T (97.6%), and Qipengyuania pelagi JCM 17468 T (97.4%). The phylogenomic analysis indicated that strain 6D36 T formed an independent branch distinct from reference-type strains of species within this genus. The digital DNA-DNA hybridization and average nucleotide identity values between strain 6D36 T and the three type strains above were, respectively, 20.2-21.3% and 79.5-81.5%, of which were far below their respective threshold for species definition, implying that the strain represents a novel genospecies. The genomic DNA G + C content was 63.3%. Based on phenotypic and genotypic characteristics, strain 6D36 T is concluded to represent a novel species of the genus Qipengyuania, for which the name Qipengyuania soli sp. nov., is proposed. The type strain of the species is 6D36 T (= GDMCC 1.1977 T  = KCTC 82333 T ).

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).)

Members of the genus Novosphingobium are well known for their metabolically versatile and great application potential in pollution elimination. The three novel bacterial strains, designated 4Y4 T , 4Y9, and 1Y9A T , were isolated from aquaculture water and characterized by using a polyphasic taxonomic approach. The 16S rRNA gene sequences phylogenetic analysis revealed that the three strains belonged to the genus Novosphingobium. The phylogenomic analysis indicated that the three strains formed two independent and robust branches distinct from all reference strains. The analyses of dDDH values and ANIs between the three strains and their relatives further demonstrated that the three strains represented two different novel genospecies. Comparative genomic analysis of the three isolates and 32 type strains of the genus Novosphingobium showed that the most important central metabolic pathways of these strains appeared to be similar, while specific and specialized metabolic pathways were flexible and variable among these strains. Chemotaxonomic characterization exhibited that the predominant cellular fatty acids were summed feature 8, summed feature 3, and C 14:0 2OH; the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidyldimethylethanolamine, phosphatidylglycerol, and sphingoglycolipid; the major respiratory quinone and polyamine were Q-10 and spermidine. The DNA G + C contents were 67.6 and 64.7 %. Based on the genotypic and phenotypic characteristics, strains 4Y4 T and 1Y9A T are concluded to represent two novel species of the genus Novosphingobium, for which the names Novosphingobium aerophilum sp. nov. (type strain 4Y4 T  = GDMCC 1.1828  T  = KACC 21946  T ) and Novosphingobium jiangmenense sp. nov. (type strain 1Y9A T  = GDMCC 1.1936  T  = KACC 22085  T ) are proposed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier GmbH.)

A novel Gram-stain-negative, facultatively anaerobic, rod-shaped and non-motile bacterial strain, designated as 4C16A T , was isolated from a tidal flat sediment and characterized by using a polyphasic taxonomic approach. Strain 4C16A T was found to grow at 10-40 °C (optimum, 28 °C), at pH 5.0-10.0 (optimum, pH 6.0-7.0) and in 0-6 % (w/v) NaCl (optimum, 1 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 4C16A T fell into the genus Roseibium , and shared the highest identity of 98.9 % with the closest type strain Roseibium suaedae KACC 13772 T and less than 98.0 % identity with other type strains of recognized species within this genus. The phylogenomic analysis indicated that strain 4C16A T formed an independent branch within this genus. The 28.6 % digital DNA-DNA hybridization estimate and 85.0 % average nucleotide identity between strains 4C16A T and R. suaedae KACC 13772 T were the highest, but still far below their respective threshold for species definition, implying that strain 4C16A T should represent a novel genospecies. The predominant cellular fatty acid was summed feature 8; the polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylmonomethylethanolamine; the respiratory quinones were Q-9 and Q-10. The genomic DNA G+C content was 59.8mol %. Based on phylogenetic analyses and phenotypic and chemotaxonomic characteristics, strain 4C16A T is concluded to represent a novel species of the genus Roseibium , for which the name Roseibium litorale sp. nov. is proposed. The type strain of the species is 4C16A T (=GDMCC 1.1932 T =KACC 22078 T ). We also propose the reclassification of Labrenzia polysiphoniae as Roseibium polysiphoniae comb. nov. and ' Labrenzia callyspongiae ' as Roseibium callyspongiae sp. nov.

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.

Myxobacteria are soil microorganisms with the ability to break down biological macromolecules due to the secretion of a large number of extracellular enzymes, but there has been no research report on myxobacterial lytic polysaccharide monooxygenases (LPMOs). In this study, two LPMO10s, ViLPMO10A and ViLPMO10B, from myxobacterium Vitiosangium sp. GDMCC 1.1324 were characterized. Of which, ViLPMO10B is a C1-oxidizing cellulose-active LPMO. Moreover, ViLPMO10B could decrease the degree of polymerization of crop straws cellulose and synergize with commercial cellulase to promote the saccharification. When the weight ratio of commercial cellulase to ViLPMO10B was 9:1, the conversion efficiency of corn stalk, sugarcane bagasse, and rice straw into reducing sugar was improved by 17%, 16%, and 22%, respectively, compared with commercial cellulase without ViLPMO10B. These results indicate that ViLPMO10B has the potential to be a component of a high-efficient cellulase cocktail and has application value in the saccharification of agricultural residual biomasses., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A systematic study of the secondary metabolites of the wild granaticin-producing strain Streptomyces vietnamensis GIMV4.0001 led to the isolation of six known early shunt products related to actinorhodin, SEK34 (3), SEK34b (4), mutactin (5), dehydromutactin (7), EM18 (8) and GTRI-02 (9). While the other shunt products were minor or trace products, the production ratio of SEK34 (3) and SEK34b (4) to granaticins was strikingly high. Nearly 64% of the intermediate with the first ring closed went to the SEK34/SEK34b aberrant pathway. The high level of the aberrant metabolic flow toward the early shunt products SEK34 and SEK34b indicated that the second ring closure of the granaticin (1) biosynthesis is a key limiting step in the granaticin biosynthetic machinery of S. vietnamensis GIMV4.0001.

Nonheme diiron monooxygenases make up a rapidly growing family of oxygenases that are rarely identified in secondary metabolism. Herein, we report the in vivo, in vitro, and structural characterizations of a nonheme diiron monooxygenase, PtmU3, that installs a C-5 β-hydroxyl group in the unified biosynthesis of platensimycin and platencin, two highly functionalized diterpenoids that act as potent and selective inhibitors of bacterial and mammalian fatty acid synthases. This hydroxylation sets the stage for the subsequent A-ring cleavage step key to the unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 adopts an unprecedented triosephosphate isomerase (TIM) barrel structural fold for this class of enzymes and possesses a noncanonical diiron active site architecture with a saturated six-coordinate iron center lacking a μ-oxo bridge. This study reveals the first member of a previously unidentified superfamily of TIM-barrel-fold enzymes for metal-dependent dioxygen activation, with the majority predicted to act on CoA-linked substrates, thus expanding our knowledge of nature's repertoire of nonheme diiron monooxygenases and TIM-barrel-fold enzymes.

Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoids of ent-kauranol and ent-atiserene biosynthetic origin. C7 oxidation in the B-ring plays a key biosynthetic role in generating structural complexity known for ent-kaurane and ent-atisane derived diterpenoids. While all three oxidation patterns, α-hydroxyl, β-hydroxyl, and ketone, at C7 are seen in both the ent-kaurane and ent-atisane derived diterpenoids, their biosynthetic origins remain largely unknown. We previously established that PTM and PTN are produced by a single biosynthetic machinery, featuring cryptic C7 oxidations at the B-rings that transform the ent-kauranol and ent-atiserene derived precursors into the characteristic PTM and PTN scaffolds. Here, we report a three-enzyme cascade affording C7 α-hydroxylation in PTM and PTN biosynthesis. Combining in vitro and in vivo studies, we show that PtmO3 and PtmO6 are two functionally redundant α-ketoglutarate-dependent dioxygenases that generate a cryptic C7 β-hydroxyl on each of the ent-kauranol and ent-atiserene scaffolds, and PtmO8 and PtmO1, a pair of NAD + /NADPH-dependent dehydrogenases, subsequently work in concert to invert the C7 β-hydroxyl to α-hydroxyl via a C7 ketone intermediate. PtmO3 and PtmO6 represent the first dedicated C7 β-hydroxylases characterized to date and, together with PtmO8 and PtmO1, provide an account for the biosynthetic origins of all three C7 oxidation patterns that may shed light on other B-ring modifications in bacterial, plant, and fungal diterpenoid biosynthesis. Given their unprecedented activities in C7 oxidations, PtmO3, PtmO6, PtmO8, and PtmO1 enrich the growing toolbox of novel enzymes that could be exploited as biocatalysts to rapidly access complex diterpenoid natural products.

Although genome mining has advanced the identification, discovery, and study of microbial natural products, the discovery of bacterial diterpenoids continues to lag behind. Herein, we report the identification of 66 putative producers of novel bacterial diterpenoids, and the discovery of the tiancilactone (TNL) family of antibiotics, by genome mining of type II diterpene synthases that do not possess the canonical DXDD motif. The TNLs, which are broad-spectrum antibiotics with moderate activities, are produced by both Streptomyces sp. CB03234 and Streptomyces sp. CB03238 and feature a highly functionalized diterpenoid skeleton that is further decorated with chloroanthranilate and γ-butyrolactone moieties. Genetic manipulation of the tnl gene cluster resulted in TNL congeners, which provided insights into their biosynthesis and structure-activity relationships. This work highlights the biosynthetic potential that bacteria possess to produce diterpenoids and should inspire continued efforts to discover terpenoid natural products from bacteria., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.)

A Gram-stain-negative, non-motile bacterial strain designated 1NM-4(T) was isolated from an abandoned lead-zinc ore mine site in Mei County, Meizhou, Guangdong Province, southern China. The isolate was light yellow, strictly aerobic, oxidase-negative and catalase-positive. Phylogenetic analyses based on 16S rRNA, rpoB and gyrB gene sequences, together with DNA-DNA hybridization values less than 70%, revealed that strain 1NM-4(T) belongs to the genus Acinetobacter and may represent a novel species. The major respiratory quinone was ubiquinone 9 (Q-9) and the major cellular fatty acids consisted of C18:1ω9c, summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16:0 and C12:0. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid and two unidentified phospholipids. The genomic DNA G+C content of strain 1NM-4(T) was 47.17 ± 0.02 mol%. Based on phenotypic, phylogenetic and chemotaxonomic characteristics, strain 1NM-4(T) should be assigned to a novel species of the genus Acinetobacter, for which the name Acinetobacter guangdongensis sp. nov. is proposed. The type strain is 1NM-4(T) ( = GIMCC 1.656(T) = CCTCC AB 2014199(T) = KCTC 42012(T))., (© 2014 IUMS.)

A novel bacterial strain designated 9PNM-6(T) was isolated from an abandoned lead-zinc ore mine site in Meizhou, Guangdong Province, China. The isolate was found to be Gram-negative, rod-shaped, orange-pigmented, strictly aerobic, oxidase- and catalase-positive. Growth occurred at 0-4 % NaCl (w/v, optimum, 0 %), at pH 6.0-8.0 (optimum, pH 7.0) and at 15-32 °C (optimum, 28-30 °C). Phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strain 9PNM-6(T) belongs to the genus Sphingomonas, with the highest sequence similarities with Sphingomonas jejuensis NBRC 107775(T) (99.7 %), Sphingomonas koreensis KCTC 2882(T) (95.1 %) and Sphingomonas dokdonesis KCTC 12541(T) (95.1 %). The chemotaxonomic characteristics of strain 9PNM-6(T) were consistent with those of the genus Sphingomonas. The predominant respiratory quinone was identified as ubiquinone Q-10, the major polyamine as sym-homospermidine, and the major cellular fatty acids as C18:1 ω7c, C16:0, C16:1 ω7c and/or C16:1 ω6c and C14:0 2-OH. The major polar lipids are sphingoglycolipid, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatideylcholine, an unidentified phospholipid and four unidentified aminolipids. The genomic DNA G+C content of strain 9PNM-6(T) was determined to be 69.2 ± 0.6 mol%. Based on comparative analyses of morphological, physiological and chemotaxonomic data, and levels of DNA-DNA relatedness values, strain 9PNM-6(T) is considered to represent a novel species of the genus Sphingomonas, for which the name Sphingomonas gimensis sp. nov. (Type strain 9PNM-6(T) = GIMCC 1.655(T) = CGMCC 1.12671(T) = DSM 27569(T)) is proposed.

A Gram-stain-negative bacterial strain, designated 9NM-8T, was isolated from an abandoned lead-zinc ore in Mei county, Meizhou, Guangdong province, PR China. The isolate was orange-pigmented, aerobic, oxidase- and catalase-positive, motile with lophotrichous flagella and rod-shaped. Strain 9NM-8T grew optimally at pH 7.0 and 30 °C and in the absence of NaCl on R2A agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 9NM-8T belongs to the genus Sphingomonas, with highest sequence similarities to Sphingomonas azotifigens KACC 14484T (96.1%), Sphingomonas trueperi DSM 7225T (96.0%) and Sphingomonas pituitosa DSM 13101T (95.6 %). Strain 9NM-8T contained Q-10 as the predominant ubiquinone. The major fatty acids included C18:1ω7c, C16:0, C16:1ω7c and/or C16 : 1ω6c (summed feature 3) and 11-methyl C18:1ω7c. The DNA G+C content was 69.6±1.3 mol%. The major component in the polyamine pattern was sym-homospermidine and the polar lipid profile contained sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified glycolipid and two unidentified phospholipids. Based on comparative analysis of physiological, chemotaxonomic and phylogenetic characteristics, strain 9NM-8T should be considered to represent a novel species of the genus Sphingomonas, for which the name Sphingomonas guangdongensis sp. nov. is proposed. The type strain is 9NM-8T (=GIMCC 1.653T=CGMCC 1.12672T=DSM 27570T).

Streptomyces vietnamensis, a recently designated species isolated from tropical forest soil, was found to be a new granaticin producer. The granaticin biosynthetic gene cluster (gra) and flanking genes from S. vietnamensis were cloned and sequenced by a sequential cloning strategy. All biosynthetic genes were found as expected. The high overall homology of the gra cluster from S. vietnamensis to that of Streptomyces violaceoruber Tü22 indicated a recent common ancestor of the two clusters. However, a flanking gene orf35 was missing from the gra cluster of S. vietnamensis, and high frequency of insertions and deletions of short fragment (shorter than 63 bp) were observed throughout the sequenced region compared to that of S. violaceoruber Tü22. These revealed a rapid evolution of the gra cluster and suggested that small insertions and deletions might be one of the basic evolution mechanisms for streptomycete genomes. The phylogenetic incongruence between 16S rDNA and the gra cluster and the scattered distribution of the granaticin producers within Streptomyces implicated horizontal gene transfer (HGT) being involved in the gra cluster dispersion. The remnants of orf35 found in S. vietnamensis present a scenario on how the antibiotic gene clusters evolved after HGT. The contemporary gra cluster residing in S. vietnamensis could be interpreted as a combination of HGT and highly variable vertical transmission.

An actinomycete, designated strain GIMN4.002(T), was isolated from a tomato rhizosphere soil sample in Guangzhou, China. The strain produces white aerial mycelium and dark blue diffusible pigment on Gause's synthetic agar, and microscopic observation revealed that it produces looped chains of spiny spores. Morphological and chemotaxonomic characteristics of the strain are typical of the genus Streptomyces. Melanin was produced and antibacterial activity was detected against Gram-positive micro-organisms, such as Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus. The 16S rRNA gene sequence of strain GIMN4.002(T) had highest similarity (99.4  %) to Streptomyces lincolnensis B91; however, DNA-DNA relatedness between strain GIMN4.002(T) and S. lincolnensis NBRC 13054(T) was only 32.17  %. Further, the morphological, physiological and biochemical characteristics of strain GIMN4.002(T) are distinct from S. lincolnensis and other species of the genus Streptomyces with which this strain has high 16S rRNA gene sequence similarity (98-99  %). On the basis of the physiological and molecular properties observed, it is proposed that strain GIMN4.002(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces caeruleatus sp. nov. is proposed, with GIMN4.002(T) (=CCTCC M 208213(T) =NRRL B-24802(T)) as the type strain.

Livestock feces and domestic sewage are the one of the main factors inducing water pollution, while the identification of the pollution source is particularly important in pollution control and management. Because of this, microbial source tracking (MST) has recently been paid more and more attention by the related researchers around the world. In this paper, the research progress of two types of MST methods, their advantages and disadvantages, and existing problems in application were reviewed and discussed. It was considered that in the library- and culture-dependent MST methods, PCR genotyping based on repetitive sequences was most practicable, while in the library- and culture-independent MST methods, PCR-DGGE based on the gene of specificity in Escherichia coli had a very glaring sight. Future researches should be more focused on the library- and culture-independent MST, and the combination of library- and culture-dependent MST with library- and culture-independent MST could make the tracking results more credible.

An actinomycete, designated strain GIMV4.0001(T), was isolated from a forest soil sample in Vietnam. It produced white aerial mycelium and violet-blue diffusible pigment on Gause's synthetic agar. The substrate mycelium colour was not sensitive to pH. Micoscopic observations revealed that strain GIMV4.0001(T) produced long, straight chains of cylindrical spores, and chemotaxonomic data confirmed that it belongs to the genus Streptomyces. Melanin was produced, but no antibacterial activity was evident against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans or Penicillium citrinum. Analysis of the 16S rRNA gene sequence of strain GIMV4.0001(T) revealed that the highest similarity (99.4 %) was to Streptomyces bikiniensis ATCC 11062(T). However, the DNA-DNA relatedness between strain GIMV4.0001(T) and S. bikiniensis ATCC 11062(T) was found to be 50.3 %. Strain GIMV4.0001(T) could also be differentiated from S. bikiniensis ATCC 11062(T) and other Streptomyces species showing high 16S rRNA gene sequence similarity (98-99 %) based on morphological, physiological and biochemical characteristics. On the basis of its physiological and molecular properties, it is evident that strain GIMV4.0001(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces vietnamensis sp. nov. is proposed. The type strain is GIMV4.0001(T) (=CCTCC M 205143(T)=IAM 15340(T)).