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The natural soil environment of Streptomyces is characterized by variations in the availability of nitrogen, carbon, phosphate and sulfur, leading to complex primary and secondary metabolisms. Their remarkable ability to adapt to fluctuating nutrient conditions is possible through the utilization of a large amount of substrates by diverse intracellular and extracellular enzymes. Thus, Streptomyces fulfill an important ecological role in soil environments, metabolizing the remains of other organisms. In order to survive under changing conditions in their natural habitats, they have the possibility to fall back on specialized enzymes to utilize diverse nutrients and supply compounds from primary metabolism as precursors for secondary metabolite production. We aimed to summarize the knowledge on the C-, N-, P- and S-metabolisms in the genus Streptomyces as a source of building blocks for the production of antibiotics and other relevant compounds. [ABSTRACT FROM AUTHOR]

Precursor supply plays a significant role in the production of secondary metabolites. In Streptomyces bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl-and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl-and propionyl- CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in Streptomyces albus B4, which binds directly to the promoter region of the neighboring pccAB operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to pccAB is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between pccAB and bkdR. When bkdR was deleted, we found an enhanced supply of malonyland methylmalonyl-CoA precursors in S. albus B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into S. albus B4. When supplemented with propionate to provide propionyl- CoA, the novel bkdR-deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium. [ABSTRACT FROM AUTHOR]

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5′-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. Key points: • Optimizing 5′-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far. [ABSTRACT FROM AUTHOR]

The natural soil environment of Streptomyces is characterized by variations in the availability of nitrogen, carbon, phosphate and sulfur, leading to complex primary and secondary metabolisms. Their remarkable ability to adapt to fluctuating nutrient conditions is possible through the utilization of a large amount of substrates by diverse intracellular and extracellular enzymes. Thus, Streptomyces fulfill an important ecological role in soil environments, metabolizing the remains of other organisms. In order to survive under changing conditions in their natural habitats, they have the possibility to fall back on specialized enzymes to utilize diverse nutrients and supply compounds from primary metabolism as precursors for secondary metabolite production. We aimed to summarize the knowledge on the C-, N-, P- and S-metabolisms in the genus Streptomyces as a source of building blocks for the production of antibiotics and other relevant compounds.

Streptomyces can produce numerous antibiotics and many other bioactive compounds. Recently, the molecular mechanisms of transcriptional regulators in control of antibiotic production by influencing the expression of biosynthetic gene clusters (BGCs) have been extensively studied. However, for regulators that affect both antibiotic production and cell growth, the way to influence antibiotic production may be diverse, but related studies are limited. Here, based on time-course transcriptome analysis, a four-component system, SbrH1-R, consisting of the two-component system SbrKR (SBI_03479/3478) and two hypothetical proteins SbrH1 (SBI_03481) and SbrH2 (SBI_03480) potentially related with the biosynthesis of milbemycins was identified in Streptomyces bingchenggensis BC-101-4. Deletion of sbrH1-R resulted in weakened cell growth but a 110% increase of milbemycin production compared with that in BC-101-4. Comparative transcriptome analyses of the sbrH1-R mutant and BC-101-4 revealed that SbrH1-R not only indirectly represses milbemycin BGC expression, but also inhibits milbemycin production by modulating expression levels of genes related to precursor supply and antibiotic efflux. Further genetic experiments identified several new targets, including five precursor supply-associated reactions/pathways (e.g., the reaction from pyruvate to acetyl-CoA, the reaction from acetyl-CoA to citrate, the fatty acid β-oxidation process, and the branched chain amino acid and phenylalanine acid degradation pathways) and a milbemycin exporter system (MilEX2) that can be engineered for milbemycin overproduction. These results shed new light on the understanding of regulation of milbemycin biosynthesis and provide useful targets for future metabolic engineering of the native host to improve milbemycin production.

The TetR family of regulators are an important group of transcription regulators that regulate diverse cellular processes in prokaryotes. In this study, we found that XNR_0706, a TetR family regulator, controlled the expression of XNR_0345 , XNR_0454 , XNR_0513 and XNR_1438 putatively involved in fatty acid β-oxidation by interacting with the promoter regions in Streptomyces albus B4. The transcription level of these four genes was downregulated in XNR_0706 deletion strain (ΔXNR_0706) and restored by XNR_0706 complementation in Δ0706/pIB- 0706 , demonstrating that XNR_0706 was a positive transcriptional regulator of the genes. With toxic long-chain fatty acids addition in TSB media, deletion of XNR_0706 caused significantly poor growth, whereas XNR_0706 complementation increased the utilization of additional fatty acids, resulting in restored growth. Fatty acid β-oxidation is one source of acetyl- and malonyl-CoA precursors for polyketides biosynthesis in actinobacteria. Overexpression of XNR_0706 in B4/spnNEW, a spinosad heterologous expression strain derived from S. albus B4, increased spinosad yield by 20.6 %. Additionally, supplement of 0.3 g/L fatty acids resulted in a further 42.4 % increase in spinosad yield. Our study reveals a regulatory mechanism in long-chain fatty acids metabolism in S. albus and these insights into the molecular regulation of β-oxidation by XNR_0706 are instrumental for increasing secondary metabolites in actinobacteria., Competing Interests: 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., (© 2024 The Authors.)

The natural compound ( R )-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 ( pks8 ) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous ( R )-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in β-oxidation ( fadD , fadE , fadB , and fadA ). Cell growth inhibition by ( R )-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced ( R )-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous ( R )-(-)-mellein synthesis in microorganism with a high titer. ( R )-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.

Precursor supply plays a significant role in the production of secondary metabolites. In Streptomyces bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl- and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl- and propionyl-CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in Streptomyces albus B4, which binds directly to the promoter region of the neighboring pccAB operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to pccAB is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between pccAB and bkdR . When bkdR was deleted, we found an enhanced supply of malonyl- and methylmalonyl-CoA precursors in S. albus B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into S. albus B4. When supplemented with propionate to provide propionyl-CoA, the novel bkdR -deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium., Importance: In this study, we describe a pccAB operon involved in short-chain acyl-CoA carboxylation in S. albus B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for S. albus chassis engineering to enhance heterologous polyketide yield., Competing Interests: The authors declare no conflict of interest.

Pamamycins, a group of polyketides originally discovered in Streptomyces alboniger , induce sporulation in Streptomyces and inhibit the growth of Gram-positive bacteria, Mycobacterium tuberculosis and fungi. The pamamycin biosynthetic gene cluster encodes 6 ketosynthases that utilize a variety of three-carbon to five-carbon CoA thioesters as starter and extender units. This promiscuity in production results in an up to 18 different derivatives during fermentation. For more-selective production and simplified purification, we aimed to modify the precursor supply to narrow the spectrum of the produced derivatives. Eight genes potentially responsible for the supply of two major precursors, 2-S-methylmalonyl-CoA and 2-S-ethylmalonyl-CoA, were identified using the NCBI Basic Local Alignment Search Tool (BLAST) against the genome of the heterologous host S. albus J1074. Knockout mutants of the identified genes were constructed and their impact on intracellular CoA ester concentrations and on the production of pamamycins was determined. The created mutants enabled us to conclusively identify the ethylmalonyl-CoA supplying routes and their impact on the production of pamamycin. Furthermore, we gained significant information on the origin of the methylmalonyl-CoA supply in Streptomyces albus. • An engineering of precursor supply is an efficient approach to tune the pamamycin polyketide production. • The biosynthetic routes to ethylmalonyl-CoA and methylmalonyl-CoA in Streptomyces albus J1074 have been elucidated. • The panel of S. albus J1074 mutants with various intracellular levels of acyl-CoA has been generated. [ABSTRACT FROM AUTHOR]

Efficient production of fuels and chemicals by metabolically engineered micro-organisms requires availability of precursor molecules for product pathways. In eukaryotic cell factories, heterologous product pathways are usually expressed in the cytosol, which may limit availability of precursors that are generated in other cellular compartments. In Saccharomyces cerevisiae , synthesis of the precursor molecule succinyl-Coenzyme A is confined to the mitochondrial matrix. To enable cytosolic synthesis of succinyl-CoA, we expressed the structural genes for all three subunits of the Escherichia coli α-ketoglutarate dehydrogenase (αKGDH) complex in S. cerevisiae. The E. coli lipoic-acid scavenging enzyme was co-expressed to enable cytosolic lipoylation of the αKGDH complex, which is required for its enzymatic activity. Size-exclusion chromatography and mass spectrometry indicated that the heterologously expressed αKGDH complex contained all subunits and that its size was the same as in E. coli. Functional expression of the heterologous complex was evident from increased αKGDH activity in the cytosolic fraction of yeast cell homogenates. In vivo cytosolic activity of the αKGDH complex was tested by constructing a reporter strain in which the essential metabolite 5-aminolevulinic acid could only be synthetized from cytosolic, and not mitochondrial, succinyl-CoA. To this end HEM1 , which encodes the succinyl-CoA-converting mitochondrial enzyme 5-aminolevulinic acid (ALA) synthase, was deleted and a bacterial ALA synthase was expressed in the cytosol. In the resulting strain, complementation of ALA auxotrophy depended on activation of the αKGDH complex by lipoic acid addition. Functional expression of a bacterial αKGDH complex in yeast represents a vital step towards efficient yeast-based production of compounds such as 1,4-butanediol and 4-aminobutyrate, whose product pathways use succinyl-CoA as a precursor. • Expression of E. coli α-ketoglutarate dehydrogenase genes in S. cerevisiae. • Proteomics reveal assembled and lipoylated complex in the S. cerevisiae cytosol. • Aminolevulinic acid auxotroph complemented by cytosolic succinyl-CoA synthesis. [ABSTRACT FROM AUTHOR]

Neomycin, an aminoglycoside antibiotic, is widely used in the livestock husbandry due to its higher antimicrobial activity and availability of feed additives in animals. However, its production yield is relatively low and cannot meet the needs of developing market and clinical application. Here, the entire natural neo cluster was cloned from Streptomyces fradiae CGMCC 4.576 by φBT1 integrase-mediated site-specific recombination. Then, the rational reconstruction of the neo cluster was performed by using λ-Red-mediated PCR targeting for improving neomycin production. In order to coordinate with this attempt, the supplementation of suitable precursors was carried out. The constructed recombinant strain Sf/pKCZ03 has multi-copy of the neo cluster modified by disrupting the negative regulatory gene neoI and replacing the native promoter of the neoE-D with PkasO*. Compared to the yield (1282 mg/L) of Streptomyces fradiae CGMCC 4.576, the engineered strain Sf/pKCZ03 had a 36% enhancement of neomycin production. Quantitative real-time PCR analysis revealed the increased transcription of structural genes (neoE, neoB, neoL, aacC8) and regulatory genes (neoR, neoH) in Sf/pKCZ03. Additionally, under the supplementation of 1 g/L N-acetyl-D-glucosamine and 5 g/L L-glutamine, the yield of engineered strain Sf/pKCZ03 showed 62% and 107% improvements compared to that of the wild-type strain in the original medium, respectively. These findings demonstrated that engineering the antibiotic gene cluster in combination with precursors feeding was an effective approach for strain improvement, and would be potentially extended to other Streptomyces for large-scale production of commercialized antibiotics. [ABSTRACT FROM AUTHOR]

Branched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis in Saccharopolyspora erythraea. In this study, we found that the bkd operon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of the bkd operon was upregulated 5-fold in a pccD gene deletion strain (ΔpccD strain) and decreased 3-fold in a pccD overexpression strain (WT/pIB-pccD), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion of pccD significantly improved the ΔpccD strain's growth rate, whereas pccD overexpression repressed WT/pIB-pccD growth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion of gdhA1 and the BcdhE1 gene (genes in the bkd operon) resulted in lower growth rates of ΔgdhA1 and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that the bkd operon is involved in BCAA degradation. Both bkd overexpression (WT/pIB-bkd) and pccD inactivation (ΔpccD strain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB-pccD was decreased by 48%. Lastly, we explored the applications of engineering pccD and bkd in an industrial higherythromycin- producing strain. pccD deletion in industrial strain S. erythraea E3 (E3pccD) improved erythromycin production by 20%, and the overexpression of bkd in E3ΔpccD (E3ΔpccD/pIB-bkd) increased erythromycin production by 39% compared with S. erythraea E3 in an industrial fermentation medium. Addition of 30 mM valine to industrial fermentation medium further improved the erythromycin production by 23%, a 72% increase from the initial strain S. erythraea E3. IMPORTANCE We describe a bkd operon involved in BCAA degradation in S. erythraea. The genes of the operon are repressed by a TetR regulator, PccD. The results demonstrated that PccD controlled the supply of precursors for biosynthesis of erythromycin via regulating the BCAA degradation and propionyl-CoA assimilation and exerted a negative effect on erythromycin production. The findings reveal a regulatory mechanism in feeder pathways and provide new strategies for designing metabolic engineering to increase erythromycin yield. [ABSTRACT FROM AUTHOR]

Terpenoids represent the largest family of natural products. Their structural diversity is largely due to variable skeletons generated by terpene synthases. However, terpene skeletons found in nature are much more than those generated from known terpene synthases. Most promiscuous terpene synthases ( i.e. those that can generate more than one product) have not been comprehensively characterised. Here, we first demonstrated that the promiscuous terpene synthases can produce more variable terpenoids in vivo by converting precursor polyisoprenoid diphosphates of different lengths (C 10 , C 15 , C 20 , C 25 ). To release the synthetic potential of these enzymes, we integrated the engineered MVA pathway, combinatorial biosynthesis, and point mutagenesis to depict the comprehensive product profiles. In total, eight new terpenoids were characterised by NMR and three new skeletons were revealed. This work highlights the key role of metabolic engineering for natural product discovery. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald- strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald- A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity-VmaxKm for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields. [ABSTRACT FROM AUTHOR]

Abstract: Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1Δ acs2Δ S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27h−1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.20h−1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While demonstrating that yeast ACS can be fully replaced, this study demonstrates that further modifications are needed to achieve optimal in vivo performance of the alternative reactions for supply of cytosolic acetyl-CoA as a product precursor. [Copyright &y& Elsevier]

Natamycin is a safe and efficient antimycotics which is widely used in food and medicine industry. The polyene macrolide compound, produced by several bacterial species of the genus Streptomyces, is synthesized by type Ⅰ polyketide synthases using acetyl-CoA, malonyl-CoA, and methylmalonyl-CoA as substrates. In this study, four pathways potentially responsible for the supply of the three precursors were evaluated to identify the effective precursor supply pathway which can support the overproduction of natamycin in Streptomyces gilvosporeus , a natamycin-producing wild-type strain. The results showed that over-expressing acetyl-CoA synthetase and methylmalonyl-CoA mutase increased the yield of natamycin by 44.19% and 20.51%, respectively, compared with the wild type strain under shake flask fermentation. Moreover, the yield of natamycin was increased by 66.29% compared with the wild-type strain by co-overexpression of acetyl-CoA synthetase and methylmalonyl-CoA mutase. The above findings will facilitate natamycin strain improvement as well as development of strains for producing other polyketide compounds.

Actinomycetes are a rich source for the synthesis of medically and technically useful natural products. The genes encoding the enzymes for their biosynthesis are normally organized in gene clusters, which include also the information for resistance (in the case of antibacterial compounds), regulation, and transport. This facilitates the manipulation of such pathways by molecular genetic techniques. Recent advances in DNA sequencing and analytical chemistry revealed that not only new strains isolated from yet unexplored habitats, but also already known strains possess a large potential for the synthesis of novel compounds. Synthetic Biology now offers a new perspective to exploit this potential further by generating novel pathways, and thereby novel products, by combining different biosynthetic steps originating from different bacteria. The supply of precursors, which are subsequently incorporated into the final product, is often already organized in a modular manner in nature and may directly be exploited for Synthetic Biology. Here we report examples for the synthesis of building blocks and possibilities to modify and optimize antibiotic biosynthesis, exemplary for the synthesis of the manipulation of the synthesis of the glycopeptide antibiotic balhimycin. [ABSTRACT FROM AUTHOR]

Abstract: Pristinamycin I (PI), a streptogramin type B antibiotic produced by Streptomyces pristinaespiralis, contains the aproteinogenic amino acid l-phenylglycine. Recent sequence analysis led to the identification of a set of putative phenylglycine biosynthetic genes. Successive inactivation of the individual genes resulted in a loss of PI production. Production was restored by supplementation with externally added l-phenylglycine, which demonstrates that these genes are involved in phenylglycine biosynthesis and thus probably disclosing the last essential pristinamycin biosynthetic genes. Finally, a putative pathway for phenylglycine synthesis is proposed. [Copyright &y& Elsevier]

Abstract: Amycolatopsis balhimycina produces the vancomycin-analogue balhimycin. The strain therefore serves as a model strain for glycopeptide antibiotic production. Previous characterisation of the balhimycin biosynthetic cluster had shown that the border sequences contained both, a putative 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dahp), and a prephenate dehydrogenase (pdh) gene. In a metabolic engineering approach for increasing the precursor supply for balhimycin production, the dahp and pdh genes from the biosynthetic cluster were overexpressed both individually and together and the resulting strains were subjected to quantitative physiological characterisation. The constructed strains expressing an additional copy of the dahp gene and the strain carrying an extra copy of both dahp and pdh showed improved specific glycopeptide productivities by approximately a factor three, whereas the pdh overexpression strain showed a production profile similar to the wild type strain. In addition to the overexpression strains, corresponding deletion mutants, Δdahp and Δpdh, were constructed and characterised. Deletion of dahp resulted in significant reduction in balhimycin production whereas the Δpdh strain had production levels similar to the parent strain. Based on these results the relation between primary and secondary metabolism with regards to Dahp and Pdh is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Streptomyces cinnamonensis C730.1 and C730.7, are industrially mutagenized strains that produce moderate and high levels of the polyketide polyether antibiotic monensin A, respectively, in an oil-based fermentation medium. The possibility that these strains could be used for high titer production of a heterologous polyketide product was investigated by expression of the entire tetracenomycin (TCM) biosynthetic pathway using an integrative plasmid, pSET154. Expression in C730.1 led to stable production of ~0.44g/l TCM C (the final biosynthetic product) and ~2.69g/l TCM A2 (the penultimate biosynthetic product), and resulted in a 40% decrease in monensin production. Expression in the C730.7 led to higher levels of TCMs, ~0.6g/l TCM C and ~4.35g/l TCM A2, without any detectable decrease in the higher titer monensin production. Abrogation of monensin production in this strain through deletion of the corresponding biosynthetic genes did not lead to higher levels of TCM products. In the case of the C730.7 host, 85% of the TCM C and virtually all of the TCM A2 were intracellular, suggesting feedback inhibition leads to the accumulation of the final pathway intermediate. These observations contrast those made for the native producer Streptomyces glaucescens where the predominant product is TCM C and TCM titers are significantly lower levels (~0.3g/l), and demonstrate the potential utility of S. cinnamonensis strains as heterologous hosts for high level expression of a variety of polyketide synthase derived products. [Copyright &y& Elsevier]

An Escherichia coli strain, JM109, was successfully engineered into an efficient hyaluronic acid (HA) producer by co-expressing the only known class-II HA synthase from a Gram-negative bacterium ( Pasteurella multocida) and uridine diphosphate-glucose dehydrogenase from E. coli K5 strain. The engineered strain produced about 0.5 g/L HA in shake flask culture and about 2.0–3.8 g/L in a fed-batch fermentation process in a 1-L bioreactor. The sharp increase in viscosity associated with HA accumulation necessitated pure oxygen supplement to maintain fermentation in aerobic regime. Precursor supply during HA synthesis was probed by glucosamine supplement, which shortens biosynthesis pathway and eliminates one step requiring ATP. HA synthesis was increased with glucosamine supplement from 2.7 to 3.7 g/L (37%), which was mirrored with a concomitant 42% decrease in pure oxygen input, suggesting a close connection between energy metabolism and precursor supply. Decoupling HA synthesis from cell growth by using fosfomycin (an inhibitor for cell wall synthesis) led to a 70% increase in HA synthesis, suggesting detrimental effects on HA synthesis from cell growth via precursor competition. This study demonstrates a potentially viable process for HA based on a recombinant E. coli strain. In addition, the precursor supply limitation identified in this study suggests new engineering targets in subsequent metabolic engineering efforts. [ABSTRACT FROM AUTHOR]

Abstract: The heterologous production of fungal polyketides was investigated using 6-methylsalicylic acid synthase (6-MSAS) as a model polyketide synthase and Saccharomyces cerevisiae as a host. In order to improve the production of 6-MSA by enhancing the supply of precursors, the promoter of the gene (ACC1) encoding acetyl-CoA carboxylase, which catalyzes the conversion of acetyl-CoA to malonyl-CoA, was replaced with a strong, constitutive promoter (TEF1p) in a strain harboring two plasmids carrying the genes encoding 6-MSAS from Penicillium patulum and PPTase from Aspergillus nidulans, respectively. The strain was characterized in batch cultivations with a glucose minimal media (20g/L), and a 60% increase in 6-MSA titer was observed compared to a strain having the native promoter in front of ACC1. The production of 6-MSA was scaled up by the cultivation in minimal media containing 50g/L of glucose, and hereby a final titer of 554±26mg/L of 6-MSA was obtained. [Copyright &y& Elsevier]

Streptomyces can produce numerous antibiotics and many other bioactive compounds. Recently, the molecular mechanisms of transcriptional regulators in control of antibiotic production by influencing the expression of biosynthetic gene clusters (BGCs) have been extensively studied. However, for regulators that affect both antibiotic production and cell growth, the way to influence antibiotic production may be diverse, but related studies are limited. Here, based on time-course transcriptome analysis, a four-component system, SbrH1-R, consisting of the two-component system SbrKR (SBI_03479/3478) and two hypothetical proteins SbrH1 (SBI_03481) and SbrH2 (SBI_03480) potentially related with the biosynthesis of milbemycins was identified in Streptomyces bingchenggensis BC-101-4. Deletion of sbrH1-R resulted in weakened cell growth but a 110% increase of milbemycin production compared with that in BC-101-4. Comparative transcriptome analyses of the sbrH1-R mutant and BC-101-4 revealed that SbrH1-R not only indirectly represses milbemycin BGC expression, but also inhibits milbemycin production by modulating expression levels of genes related to precursor supply and antibiotic efflux. Further genetic experiments identified several new targets, including five precursor supply-associated reactions/pathways (e.g., the reaction from pyruvate to acetyl-CoA, the reaction from acetyl-CoA to citrate, the fatty acid β-oxidation process, and the branched chain amino acid and phenylalanine acid degradation pathways) and a milbemycin exporter system (MilEX2) that can be engineered for milbemycin overproduction. These results shed new light on the understanding of regulation of milbemycin biosynthesis and provide useful targets for future metabolic engineering of the native host to improve milbemycin production., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wensheng Xiang, Yanyan Zhang, Lan Ye, Shanshan Li have filed two patents related to the data in this manuscript. All other authors declare that they have no competing interests., (© 2022 The Authors.)

Bacitracin, a type of cyclic dodecapeptide antibiotic mainly produced by Bacillus , is widely used in fields of veterinary drug and feed additive. Modularization of metabolic pathways based on the concept of synthetic biology has been widely used in the efficient synthesis of target products. Here, we want to improve bacitracin production through strengthening aspartic acid (Asp) supply in B. licheniformis DW2. First, exogenous Asp addition assays implied that strengthening Asp supply benefited bacitracin production. Second, Asp synthetic pathways were strengthened via overexpressing aspartate dehydrogenase AspD and asparaginase AnsB, attaining recombinant strain DW2-ASP2, and bacitracin yield produced by DW2-ASP2 was 862.81 U/mL, increased by 14.05% compared with that of DW2 (756.49 U/mL). Then, to improve precursor oxaloacetate (OAA) accumulation for Asp synthesis, pyruvate carboxylase PycA and carbonic anhydrase EcaA were co-overexpressed in DW2-ASP2, and malic enzyme gene malS was deleted to weak overflow metabolism of tricarboxylic acid, and the attained strain DW2-ASP7 showed further increased bacitracin production from 862.81 to 989.23 U/mL. Subsequently, transporter YveA was identified as an Asp exporter, and bacitracin yield was increased to 1025.26 U/mL via deleting yveA , attaining strain DW2-ASP9. Finally, Asp ammonia-lyase gene aspA was disrupted to weaken Asp degradation, and bacitracin yield of attained strain DW2-ASP10 reached 1059.86 U/mL, increased by 40.10% compared to DW2. Taken together, this research demonstrated that metabolic engineering of Asp metabolic modules is an efficient strategy for enhancing bacitracin production, and these strategies could also be applied in the production of other peptide-related metabolites.

Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R. capsulatus . The above strategies enabled a 35-fold higher titer of bisabolene than that of the starting strain, reaching 1089.7 mg/L from glucose in a shake flask. The engineered strain produced 9.8 g/L bisabolene with a yield of >0.196 g/g-glucose under the two-phase fed-batch fermentation, which corresponds to >78% of theoretical maximum. Taken together, our work represents one of the pioneering studies to demonstrate PNSB as a promising platform for terpene-based advanced biofuel production.

Erythromycin is necessary in medical treatment and known to be biosynthesized with propionyl-CoA as direct precursor. Oversupply of propionyl-CoA induced hyperpropionylation, which was demonstrated as harmful for erythromycin synthesis in Saccharopolyspora erythraea. Herein, we identified three propionyl-CoA synthetases regulated by propionylation, and one propionyl-CoA synthetase SACE_1780 revealed resistance to propionylation. A practical strategy for raising the precursor (propionyl-CoA) supply bypassing the feedback inhibition caused by propionylation was developed through two approaches: deletion of the propionyltransferase AcuA, and SACE_1780 overexpression. The constructed Δ acuA strain presented a 10% increase in erythromycin yield; SACE_1780 overexpression strain produced 33% higher erythromycin yield than the wildtype strain NRRL2338 and 22% higher erythromycin yield than the industrial high yield Ab strain. These findings uncover the role of protein acylation in precursor supply for antibiotics biosynthesis and provide efficient post-translational modification-metabolic engineering strategy (named as PTM-ME) in synthetic biology for improvement of secondary metabolites.

Branched-chain amino acid (BCAA) degradation is a major source of propionyl coenzyme A (propionyl-CoA), a key precursor of erythromycin biosynthesis in Saccharopolyspora erythraea In this study, we found that the bkd operon, responsible for BCAA degradation, was regulated directly by PccD, a transcriptional regulator of propionyl-CoA carboxylase genes. The transcriptional level of the bkd operon was upregulated 5-fold in a pccD gene deletion strain (Δ pccD strain) and decreased 3-fold in a pccD overexpression strain (WT/pIB- pccD ), demonstrating that PccD was a negative transcriptional regulator of the operon. The deletion of pccD significantly improved the Δ pccD strain's growth rate, whereas pccD overexpression repressed WT/pIB- pccD growth rate, in basic Evans medium with 30 mM valine as the sole carbon and nitrogen source. The deletion of gdhA1 and the BcdhE1 gene (genes in the bkd operon) resulted in lower growth rates of Δ gdhA1 and ΔBcdhE1 strains, respectively, on 30 mM valine, further suggesting that the bkd operon is involved in BCAA degradation. Both bkd overexpression (WT/pIB- bkd ) and pccD inactivation (Δ pccD strain) improve erythromycin production (38% and 64%, respectively), whereas the erythromycin production of strain WT/pIB- pccD was decreased by 48%. Lastly, we explored the applications of engineering pccD and bkd in an industrial high-erythromycin-producing strain. pccD deletion in industrial strain S. erythraea E3 (E3 pccD ) improved erythromycin production by 20%, and the overexpression of bkd in E3Δ pccD (E3Δ pccD /pIB- bkd ) increased erythromycin production by 39% compared with S. erythraea E3 in an industrial fermentation medium. Addition of 30 mM valine to industrial fermentation medium further improved the erythromycin production by 23%, a 72% increase from the initial strain S. erythraea E3. IMPORTANCE We describe a bkd operon involved in BCAA degradation in S. erythraea The genes of the operon are repressed by a TetR regulator, PccD. The results demonstrated that PccD controlled the supply of precursors for biosynthesis of erythromycin via regulating the BCAA degradation and propionyl-CoA assimilation and exerted a negative effect on erythromycin production. The findings reveal a regulatory mechanism in feeder pathways and provide new strategies for designing metabolic engineering to increase erythromycin yield., (Copyright © 2018 American Society for Microbiology.)

Natural products are still key sources of current clinical drugs and innovative therapeutic agents. Since wild-type microorganisms only produce natural products in very small quantities, yields of production strains need to be improved by breaking down the precise genetic and biochemical circuitry. Herein, we use avermectins as an example of production improvement and chemical structure diversification by synthetic biology. Avermectins are macrocyclic lactones produced by Streptomyces avermitilis and are well known and widely used for antiparasitic therapy. Given the importance of this molecule and its derivatives, many efforts and strategies were employed to improve avermectin production and generate new active analogues. This review describes the current status of synthetic strategies successfully applied for developing natural-product-producing strains and discusses future prospects for the application of enhanced avermectin production., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)