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.

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).

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.

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).

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).

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.

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).

Cryptic and Stereospecific Hydroxylation, Oxidation, and Reduction in Platensimycin and Platencin Biosynthesis.

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, ß-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 ß-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 ß-hydroxyl to α-hydroxyl via a C7 ketone intermediate. PtmO3 and PtmO6 represent the first dedicated C7 ß-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.

Characterization and Crystal Structure of a Nonheme Diiron Monooxygenase Involved in Platensimycin and Platencin Biosynthesis.

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 ß-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.

Discovery of the Tiancilactone Antibiotics by Genome Mining of Atypical Bacterial Type†II Diterpene Synthases.

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.

Acinetobacter guangdongensis sp. nov., isolated from abandoned lead-zinc ore.

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)).

Description of a Gram-negative bacterium, Sphingomonas guangdongensis sp. nov.

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).

Sphingomonas gimensis sp. nov., a novel Gram-negative bacterium isolated from abandoned lead-zinc ore mine.

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.

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.

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.

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.].

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.

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.

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.