Corrigendum: An in-cluster Sfp-type phosphopantetheinyl transferase instead of the holo-ACP synthase activates the granaticin biosynthesis under natural physiological conditions.

[This corrects the article DOI: 10.3389/fchem.2022.1112362.].

Global rewiring of lipid metabolism to produce carotenoid by deleting the transcription factor genes ino2/ino4 in Saccharomyces cerevisiae.

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.

Development of platensimycin, platencin, and platensilin overproducers by biosynthetic pathway engineering and fermentation medium optimization.

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.

Design of a dual-responding genetic circuit for high-throughput identification of L-threonine-overproducing Escherichia coli.

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.

Advances in the Discovery and Engineering of Gene Targets for Carotenoid Biosynthesis in Recombinant Strains.

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.

Comparative Genomics Reveals Genetic Diversity and Metabolic Potentials of the Genus Qipengyuania and Suggests Fifteen Novel Species.

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.

Engineering a Balanced Acetyl Coenzyme A Metabolism in Saccharomyces cerevisiae for Lycopene Production through Rational and Evolutionary Engineering.

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 (ACC1) 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.

Upstream Activation Sequence Can Function as an Insulator for Chromosomal Regulation of Heterologous Pathways Against Position Effects in Saccharomyces cerevisiae.

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.

Flavobacterium proteolyticum sp. nov., isolated from aquaculture water.

A novel aerobic, yellow, and rod-shaped bacterial isolate, designated as 1Y8AT, was isolated from aquaculture water sampled in Jiangmen, Guangdong province, P. R. China. Here, the taxonomic position of strain 1Y8AT was conducted based on phenotypic, genomic, and chemotaxonomic characteristics. Strain 1Y8AT 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 1Y8AT was closely related to "Flavobacterium sasangense" YC6274T (99.3%), Flavobacterium aquaticum JC164T (98.4%), Flavobacterium cucumis R2A45-3T (98.0%), Flavobacterium celericrescens TWA-26T (98.0%), and Flavobacterium cheniae NJ-26T (97.2%). The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain 1Y8AT 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 1Y8AT formed an independent branch distinct from known species. The major cellular fatty acids of strain 1Y8AT were iso-C15:0, iso-C15:1 G and C15:0; 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 1Y8AT 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 1Y8AT (= GDMCC 1.1933T = KACC 22081T).

An in-cluster Sfp-type phosphopantetheinyl transferase instead of the holo-ACP synthase activates the granaticin biosynthesis under natural physiological conditions.

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 Δgra-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 Δgra-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 Δgra-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.

Croceicoccus gelatinilyticus sp. nov., isolated from a tidal flat sediment.

A novel Gram-staining-negative and short-rod-shaped bacterial strain designated as 1NDH52T was isolated from a tidal flat sediment and characterized using a polyphasic taxonomic approach. The predominant cellular fatty acids of strain 1NDH52T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) and C14:0 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 1NDH52T was closely related to type strains Croceicoccus sediminis S2-4-2 T (98.9%), Croceicoccus bisphenolivorans H4T (98.9%) and Croceicoccus pelagius Ery9T (98.7%). Phylogenomic analysis indicated that strain 1NDH52T 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 1NDH52T and the three close relatives above indicated that strain 1NDH52T should represent a novel genospecies. The genomic DNA G + C content was 62.6%. Strain 1NDH52T could produce carotenoids and its genome contained the complete carotenoids biosynthetic gene cluster. Based on the phenotypic and genotypic characteristics, strain 1NDH52T 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 1NDH52T (= GDMCC 1.2381 T = KCTC 82668 T).

Phylogenomic Analysis Substantiates the gyrB Gene as a Powerful Molecular Marker to Efficiently Differentiate the Most Closely Related Genera Myxococcus, Corallococcus, and Pyxidicoccus.

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.

Salipiger mangrovisoli sp. nov., isolated from mangrove soil and the proposal for the reclassification of Paraphaeobacter pallidus as Salipiger pallidus comb. nov.

A novel Gram-stain-negative, aerobic and rod-shaped bacterial strain designated as 6D45AT was isolated from mangrove soil and characterized using a polyphasic taxonomic approach. Strain 6D45AT 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 6D45AT fell into the genus Salipiger and shared 99.1 % identity with the closest type strain Salipiger pacificus CGMCC 1.3455T 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 6D45AT and the closest relative above were well below recognized thresholds of 70 % DDH and 95-96 % ANI for species definition, implying that strain 6D45AT should represent a novel genospecies. The phylogenomic analysis indicated that strain 6D45AT formed an independent branch distinct from reference strains. The predominant cellular fatty acid of strain 6D45AT was summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c, 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 6D45AT 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 6D45AT (=GDMCC 1.1960T=KCTC 82334T). We also propose the reclassification of Paraphaeobacter pallidus as Salipiger pallidus comb. nov. and 'Pelagibaca abyssi' as a species of the genus Salipiger.

Chitinilyticum piscinae sp. nov., isolated from aquaculture water.

A novel Gram-stain-negative and rod-shaped bacterial strain, designated as 4Y14T, was isolated from aquaculture water and characterized by using a polyphasic taxonomic approach. Strain 4Y14T 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 4Y14T belonged to the genus Chitinilyticum with high levels of similarity to Chitinilyticum litopenaei c1T (97.8 %) and Chitinilyticum aquatile c14T (97.2 %). Phylogenomic analysis indicated that strain 4Y14T formed an independent branch distinct from the two type strains above. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain 4Y14T 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 4Y14T should represent a novel genospecies. The predominant cellular fatty acids of strain 4Y14T were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and iso-C16 : 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 4Y14T 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 4Y14T (=GDMCC 1.1934T=KACC 22080T).

Qipengyuania soli sp. nov., Isolated from Mangrove Soil.

A novel Gram-stain-negative, non-motile, and rod-shaped bacterial strain, designated as 6D36T, was isolated from mangrove soil and characterized by using a polyphasic taxonomic approach. Strain 6D36T 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 6D36T were summed feature 8 (C19:1 ω7c and/or C18:1 ω6c) and C17:1 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 6D36T fell into the genus Qipengyuania and was closely related to "Erythrobacter mangrovi" MCCC 1K03690T (98.5%), Qipengyuania citrea CGMCC 1.8703T (97.6%), and Qipengyuania pelagi JCM 17468T (97.4%). The phylogenomic analysis indicated that strain 6D36T 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 6D36T 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 6D36T 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 6D36T (= GDMCC 1.1977T = KCTC 82333T).

Inhella proteolytica sp. nov. and Inhella gelatinilytica sp. nov., two novel species of the genus Inhella isolated from aquaculture water.

The two novel bacterial strains designated 1Y17T and 4Y10T from aquaculture water were characterized using a polyphasic taxonomic approach. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains 1Y17T and 4Y10T belonged to the genus Inhella and were close to Inhella crocodyli CCP-18T, Inhella inkyongensis IMCC1713T and Inhella fonticola TNR-25T. Strains 1Y17T and 4Y10T 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 (C16:1 ω7c and/or C16:1 ω6c) and C16:0; 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-ß-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 1Y17T and 4Y10T are concluded to represent two novel species of the genus Inhella, for which the names Inhella proteolytica sp. nov. (type strain 1Y17T = GDMCC 1.1830T = KACC 21948T) and Inhella gelatinilytica sp. (type strain 4Y10T = GDMCC 1.1829T = KCTC 82338T) are proposed.

Comparative genomic analysis of the genus Novosphingobium and the description of two novel species Novosphingobium aerophilum sp. nov. and Novosphingobium jiangmenense sp. nov.

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 4Y4T, 4Y9, and 1Y9AT, 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 C14:0 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 4Y4T and 1Y9AT are concluded to represent two novel species of the genus Novosphingobium, for which the names Novosphingobium aerophilum sp. nov. (type strain 4Y4T = GDMCC 1.1828 T = KACC 21946 T) and Novosphingobium jiangmenense sp. nov. (type strain 1Y9AT = GDMCC 1.1936 T = KACC 22085 T) are proposed.

Transcriptome Analysis Reveals a Promotion of Carotenoid Production by Copper Ions in Recombinant Saccharomyces cerevisiae.

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 CuSO4 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.

Roseibium litorale sp. nov., isolated from a tidal flat sediment and proposal for the reclassification of Labrenzia polysiphoniae as Roseibium polysiphoniae comb. nov.

A novel Gram-stain-negative, facultatively anaerobic, rod-shaped and non-motile bacterial strain, designated as 4C16AT, was isolated from a tidal flat sediment and characterized by using a polyphasic taxonomic approach. Strain 4C16AT 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 4C16AT fell into the genus Roseibium, and shared the highest identity of 98.9 % with the closest type strain Roseibium suaedae KACC 13772T and less than 98.0 % identity with other type strains of recognized species within this genus. The phylogenomic analysis indicated that strain 4C16AT formed an independent branch within this genus. The 28.6 % digital DNA-DNA hybridization estimate and 85.0 % average nucleotide identity between strains 4C16AT and R. suaedae KACC 13772T were the highest, but still far below their respective threshold for species definition, implying that strain 4C16AT 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 4C16AT 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 4C16AT (=GDMCC 1.1932T=KACC 22078T). We also propose the reclassification of Labrenzia polysiphoniae as Roseibium polysiphoniae comb. nov. and 'Labrenzia callyspongiae' as Roseibium callyspongiae sp. nov.

Discovery of Mycothiogranaticins from Streptomyces vietnamensis GIMV4.0001 and the Regulatory Effect of Mycothiol on the Granaticin Biosynthesis.

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 ΔmshA 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 Δmst. 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.