Enhancing and monitoring spore production in Clostridium butyricum using pH-based regulation strategy and a robust soft sensor based on back-propagation neural networks.

Clostridium butyricum is a probiotic that forms anaerobic spores and plays a crucial role in regulating gut microbiota. However, the total viable cell count and spore yield of C. butyricum in industrial production are comparatively low. To this end, we investigated the metabolic characteristics of the strain and proposed three distinct pH regulation strategies for enhancing spore production. In addition, precise measurement of fermentation parameters such as substrate concentration, total viable cell count, and spore concentration is crucial for successful industrial probiotics production. Nevertheless, online measurement of these intricate parameters in the fermentation of C. butyricum poses a considerable challenge owing to the complex, nonlinear, multivariate, and strongly coupled characteristics of the production process. Therefore, we analyzed the capacitance and conductivity acquired from a viable cell sensor as the core parameters for the fermentation process. Subsequently, a robust soft sensor was developed using a seven-input back-propagation neural network model with input variables of fermentation time, capacitance, conductivity, pH, initial total sugar concentration, ammonium ion concentration, and calcium ion concentration. The model enables the online monitoring of total viable biomass count, substrate concentrations, and spore yield, and can be extended to similar fermentation processes with pH changes as a characteristic feature.

An industrial perspective on metabolic responses of Penicillium chrysogenum to periodic dissolved oxygen feast-famine cycles in a scale-down system.

While traveling through different zones in large-scale bioreactors, microbes are most likely subjected to fluctuating dissolved oxygen (DO) conditions at the timescales of global circulation time. In this study, to mimic industrial-scale spatial DO gradients, we present a scale-down setup based on dynamic feast/famine regime (150 s) that leads to repetitive cycles with rapid changes in DO availability in glucose-limited chemostat cultures of Penicillium chrysogenum. Such DO feast/famine regime induced a stable and repetitive pattern with a reproducible metabolic response in time, and the dynamic response of intracellular metabolites featured specific differences in terms of both coverage and magnitude in comparison to other dynamic conditions, for example, substrate feast/famine cycles. Remarkably, intracellular sugar polyols were considerably increased as the hallmark metabolites along with a dynamic and higher redox state (NADH/NAD+) of the cytosol. Despite the increased availability of NADPH for penicillin production under the oscillatory DO conditions, this positive effect may be counteracted by the decreased ATP supply. Moreover, it is interesting to note that not only the penicillin productivity was reduced under such oscillating DO conditions, but also that of the unrecyclable byproduct ortho-hydroxyphenyl acetic acid and degeneration of penicillin productivity. Furthermore, dynamic flux profiles showed the most pronounced variations in central carbon metabolism, amino acid (AA) metabolism, energy metabolism and fatty acid metabolism upon the DO oscillation. Taken together, the metabolic responses of P. chrysogenum to DO gradients reported here are important for elucidating metabolic regulation mechanisms, improving bioreactor design and scale-up procedures as well as for constructing robust cell strains to cope with heterogenous industrial culture conditions.

An inducible CRISPRi circuit for tunable dynamic regulation of gene expression in Saccharopolyspora erythraea.

Actinomyces are gram-positive bacteria known for their valuable secondary metabolites. Redirecting metabolic flux towards desired products in actinomycetes requires precise and dynamic regulation of gene expression. In this study, we integrated the CRISPR interference (CRISPRi) system with a cumate-inducible promoter to develop an inducible gene downregulation method in Saccharopolyspora erythraea, a prominent erythromycin-producing actinobacterium. The functionality of the cumate-inducible promoter was validated using the gusA gene as a reporter, and the successful inducible expression of the dCas9 gene was confirmed. The developed inducible CRISPRi strategy was then employed to downregulate the expression of target genes rppA in the wild-type strain NRRL2338 and sucC in the high erythromycin-producing strain E3. Through dynamic control of sucC expression, a significant enhancement in erythromycin production was achieved in strain E3. This study demonstrated the effectiveness of an inducible gene downregulation approach using CRISPRi and a cumate-inducible promoter, providing valuable insights for optimizing natural product production in actinomyces.

Ammonium sulfate supplementation enhances erythromycin biosynthesis by augmenting intracellular metabolism and precursor supply in Saccharopolyspora erythraea.

In this study, the cellular metabolic mechanisms regarding ammonium sulfate supplementation on erythromycin production were investigated by employing targeted metabolomics and metabolic flux analysis. The results suggested that the addition of ammonium sulfate stimulates erythromycin biosynthesis. Targeted metabolomics analysis uncovered that the addition of ammonium sulfate during the late stage of fermentation resulted in an augmented intracellular amino acid metabolism pool, guaranteeing an ample supply of precursors for organic acids and coenzyme A-related compounds. Therefore, adequate precursors facilitated cellular maintenance and erythromycin biosynthesis. Subsequently, an optimal supplementation rate of 0.02 g/L/h was determined. The results exhibited that erythromycin titer (1311.1 µg/mL) and specific production rate (0.008 mmol/gDCW/h) were 101.3% and 41.0% higher than those of the process without ammonium sulfate supplementation, respectively. Moreover, the erythromycin A component proportion increased from 83.2% to 99.5%. Metabolic flux analysis revealed increased metabolic fluxes with the supplementation of three ammonium sulfate rates.

An Efficient High-Throughput Screening of High Gentamicin-Producing Mutants Based on Titer Determination Using an Integrated Computer-Aided Vision Technology and Machine Learning.

The "design-build-test-learn" (DBTL) cycle has been adopted in rational high-throughput screening to obtain high-yield industrial strains. However, the mismatch between build and test slows the DBTL cycle due to the lack of high-throughput analytical technologies. In this study, a highly efficient, accurate, and noninvasive detection method of gentamicin (GM) was developed, which can provide timely feedback for the high-throughput screening of high-yield strains. First, a self-made tool was established to obtain data sets in 24-well plates based on the color of the cells. Subsequently, the random forest (RF) algorithm was found to have the highest prediction accuracy with an R2 value of 0.98430 for the same batch. Finally, a stable genetically high-yield strain (998 U/mL) was successfully screened out from 3005 mutants, which was verified to improve the titer by 72.7% in a 5 L bioreactor. Moreover, the verified new data sets were updated on the model database in order to improve the learning ability of the DBTL cycle.

Comparative genomic and transcriptomic analysis guides to further enhance the biosynthesis of erythromycin by an overproducer.

Omics approaches have been applied to understand the boosted productivity of natural products by industrial high-producing microorganisms. Here, with the updated genome sequence and transcriptomic profiles derived from high-throughput sequencing, we exploited comparative omics analysis to further enhance the biosynthesis of erythromycin in an industrial overproducer, Saccharopolyspora erythraea HL3168 E3. By comparing the genome of E3 with the wild type NRRL23338, we identified fragment deletions inside 56 coding sequences and 255 single-nucleotide polymorphisms over the genome of E3. A substantial number of genomic variations were observed in genes responsible for pathways which were interconnected to the biosynthesis of erythromycin by supplying precursors/cofactors or by signal transduction. Furthermore, the transcriptomic data suggested that genes involved in the biosynthesis of erythromycin were significantly upregulated constantly, whereas some genes in biosynthesis clusters of other secondary metabolites contained nonsense mutations and were expressed at extremely low levels. Through comparative transcriptomic analysis, l-glutamine/l-glutamate and 2-oxoglutarate were identified as reporter metabolites. Around the node of 2-oxoglutarate, genomic mutations were also observed. Based on the omics association analysis, readily available strategies were proposed to engineer E3 by simultaneously overexpressing sucB (coding for 2-oxoglutarate dehydrogenase E2 component) and sucA (coding for 2-oxoglutarate dehydrogenase E1 component), which increased the erythromycin titer by 71% compared to E3 in batch culture. This study provides more promising molecular targets to engineer for enhanced production of erythromycin by the overproducer.

Cephalosporin C biosynthesis and fermentation in Acremonium chrysogenum.

Cephalosporins are currently the most widely used antibiotics in clinical practice. The main strain used for the industrial production cephalosporin C (CPC) is Acremonium chrysogenum. CPC has the advantages of possessing a broad antibacterial spectrum and strong antibacterial activity. However, the yield and titer of cephalosporins obtained from A. chrysogenum are much lower than penicillin, which is also a ß-lactam antibiotic produced by Penicillium chrysogenum. Molecular biology research into A. chrysogenum has focused on gene editing technologies, multi-omics research which has provided information on the differences between high- and low-yield strains, and metabolic engineering involving different functional genetic modifications and hierarchical network regulation to understand strain characteristics. Furthermore, optimization of the fermentation process is also reviewed as it provides the optimal environment to realize the full potential of strains. Combining rational design to control the metabolic network, high-throughput screening to improve the efficiency of obtaining high-performance strains, and real-time detection and controlling in the fermentation process will become the focus of future research in A. chrysogenum. This minireview provides a holistic and in-depth analysis of high-yield mechanisms and improves our understanding of the industrial value of A. chrysogenum. KEY POINTS: • Review of the advances in A. chrysogenum characteristics improvement and process optimization • Elucidate the molecular bases of the mechanisms that control cephalosporin C biosynthesis and gene expression in A. chrysogenum • The future development trend of A. chrysogenum to meet industrial needs.

Engineering of succinyl-CoA metabolism in view of succinylation regulation to improve the erythromycin production.

As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metabolism regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resolution mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metabolism, and antibiotics biosynthesis in S. erythraea. Enzymatic experiments in vivo and in vitro were further conducted to determine the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermentation. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metabolism changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. KEY POINTS: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the production of erythromycin by using CRISPRi to regulate the transcription of gene sucC.

Knockout and functional analysis of BSSS-related genes in Acremonium chrysogenum by novel episomal expression vector containing Cas9 and AMA1.

OBJECTIVE: The target sorB gene, related to sorbicillinoid production, and the free expression element, AMA1, were used to verify the methodological approach in Acremonium chrysogenum. RESULT: CRISPR-Cas9 episomal expression system was used to introduce a point mutation into the sorB gene and the addition of sorB donor DNA achieved complete knockout of target genes. Four BSSS (yeast bud site selection system)-related genes, axl1, axl2, bud3, and bud4 were knocked out without impact on yield, dry weight, or pH. Relationships between morphology and stress tolerance in knockout strains were analyzed. CONCLUSION: The gene-editing system used in the current study exceeded 80% efficiency and arthrospores development was found to differ from that in wild-type strain.

Engineering the methyltransferase through inactivation of the genK and genL leads to a significant increase of gentamicin C1a production in an industrial strain of Micromonospora echinospora 49-92S.

In this study, a single-component high-yielding Micromonospora echinospora strain 49-92S-KL01 was constructed by deleting methyltransferase-encoding genes genK and genL. In 5-L fermentation trials, gentamicin C1a titers in the mutant strain were 3.22-fold higher than that in the parental strain (211 U/mL vs. 50 U/mL). The glycolysis pathway and tricarboxylic acid cycle fluxes were reduced by 26.8% and 26.6%, respectively, compared to the parental strain according to the metabolic flux analysis during the stationary phase, resulting in lower levels of energy supplements required for the cellular maintenance. Meanwhile, a significant enhancement in precursor (paromamine) accumulation and availability was observed in 49-92S-KL01 compared to parental strain. These results indicate that genK and genL significantly affect the synthesis of gentamicin C1a. In addition, this study provides a more rational strategy for gentamicin C1a production.

Effect of temperature on synthesis of clavulanic acid and impurity substance G during fermentation by Streptomyces clavuligerus.

Effect of temperature on synthesis of Clavulanic acid (CA) and impurity substance G during fermentation by Streptomyces clavuligerus were investigated. Results show that fermentation at 24 °C is the most favorable for CA synthesis though the fermentation duration was 20-30 hours longer than fermentation at 26 and 28 °C. Meanwhile, the impurity substance G was only 110 mg/L in the end broth of fermentation at 24 °C, which was significantly lower than 148 and 180 mg/L of fermentation at 26 and 28 °C, respectively. Correlation of specific growth rate and CA synthesis was statistically analyzed based on data of 10 batches of industrial fermentation. Two temperature-shift strategies were investigated in 50 L fermenter. Fermentation with 26-24 °C temperature strategy achieved 5097 mg/L CA titer, meanwhile the fermentation duration was shortened 24 hours comparing with fermentation at constant 24 °C. Fermentation with 26-24 °C control strategy was validated in a 60 m3 industrial fermenter, in which 4960 mg/L of CA was achieved while impurity G substance was decreased to titer 65 mg/L from 200 to 300 mg/L of normal production.

Exploring the metabolic fate of propanol in industrial erythromycin-producing strain via 13C labeling experiments and enhancement of erythromycin production by rational metabolic engineering of Saccharopolyspora erythraea.

Propanol had been widely used as a precursor for erythromycin synthesis in industrial production. However, the knowledge on the exact metabolic fate of propanol was still unclear. In the present study, the metabolic fate of propanol in industrial erythromycin-producing strain Saccharopolyspora erythraea E3 was explored via 13C labeling experiments. An unexpected pathway in which propanol was channeled into tricarboxylic acid cycle was uncovered, resulting in uneconomic catabolism of propanol. By deleting the sucC gene, which encodes succinyl-CoA synthetase that catalyse a reaction in the unexpected propanol utilization pathway, a novel strain E3-ΔsucC was constructed. The strain E3-ΔsucC showed a significant enhancement in erythromycin production in the chemically defined medium compared to E3 (786.61 vs 392.94 mg/L). Isotopically nonstationary 13C metabolic flux analysis were employed to characterize the metabolic differences between Saccharopolyspora erythraea E3 and E3-ΔsucC. The results showed that compared with the starting strain E3, the fluxes of pentose phosphate pathway in E3-△sucC increased by almost 200%. The flux of the metabolic reaction catalyzed by succinyl-CoA synthetase in E3-ΔsucC was almost zero, while the glyoxylate bypass flux significantly increased. These new insights into the precursor utilization of antibiotic biosynthesis by rational metabolic engineering in Saccharopolyspora erythraea provided the new vision in increasing industrial production of secondary metabolites.

Development of a novel noninvasive quantitative method to monitor Siraitia grosvenorii cell growth and browning degree using an integrated computer-aided vision technology and machine learning.

The rapid, accurate and noninvasive detection of biomass and plant cell browning can provide timely feedback on cell growth in plant cell culture. In this study, Siraitia grosvenorii suspension cells were taken as an example, a phenotype analysis platform was successfully developed to predict the biomass and the degree of cell browning based on the color changes of cells in computer-aided vision technology. First, a self-made laboratory system was established to obtain images. Then, matrices were prepared from digital images by a self-developed high-throughput image processing tool. Finally, classification models were used to judge different cell types, and then a semi-supervised classification to predict different degrees of cell browning. Meanwhile, regression models were developed to predict the plant cell mass. All models were verified with a good agreement by biological experiments. Therefore, this method can be applied for low-cost biomass estimation and browning degree quantification in plant cell culture.

Exploration and characterization of hypoxia-inducible endogenous promoters in Aspergillus niger.

Aspergillus niger is widely used for the efficient production of organic acids and enzyme preparations. However, this organism lacks basic genetic elements for dynamic control, especially inducible promoters that can respond to specific environmental signals. Since these are desirable for better adaptation of fermentation to large-scale industrial production, herein, we have identified the two first hypoxia-inducible promoters in A. niger, PsrbB and PfhbA. Their performance under high or low oxygen conditions was monitored using two reporter proteins, green fluorescent protein (EGFP) and ß-glucuronidase (GUS). For comparison, basal expression of the general strong promoter PgpdA was lower than PsrbB but higher than PfhbA. However, under hypoxia, both promoters showed higher expression than under hyperoxia, and these values were also higher than those observed for PgpdA. For PsrbB, strength under hypoxia was ~2-3 times higher than under hyperoxia (for PfhbA, 3-9 times higher) and ~2.5-5 times higher than for PgpdA (for PfhbA, 2-3 times higher). Promoter truncation analysis showed that the PsrbB fragment -1024 to -588 bp is the core region that determines hypoxia response. KEY POINTS: The first identification of two hypoxia-inducible promoters in A. niger is a promising tool for modulation of target genes under hypoxia. Two reporter genes revealed a different activity and responsiveness to hypoxia of PfhbA and PsrbB promoters, which is relevant for the development of dynamic metabolic regulation of A. niger fermentation. PsrbB promoter truncation and bioinformatics analysis is the foundation for further research.

In vitro CRISPR/Cas9 system for genome editing of Aspergillus niger based on removable bidirectional selection marker AmdS.

We established an in vitro clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided DNA endonucleases (Cas9) system to efficiently produce specific genome editing in Aspergillus niger, using a novel recyclable, bidirectional selection marker gene amdS without the need of prior production of an amdS mutant. The donor DNA plasmid consisted of amdS open reading frame, promoter, terminator, and directional repeats (DRs) flanking sequences. It was cotransformed with recombinant nuclease Cas9 and the sgRNA, which targets to the pigment gene olvA of A. niger strain CBS513.88. The positive olive transformants, other than the wild-type strain, were able to grow on the media containing acetamide as the sole nitrogen source and cesium chloride. Furthermore, culturing the transformants on media with fluoroacetamide and urea allowed a loop-out of the amdS expression cassette by recombining the flanking DRs. This study confirmed the facts that the endogenous amdS can be used as a dominant marker and that it can be removed by counter-selection in gene editing of A. niger. The proposed in vitro CRISPR/Cas9 method offers a powerful tool for marker-free genetic manipulation of filamentous fungi industrial-specific strains.

Coupled metabolic-hydrodynamic modeling enabling rational scale-up of industrial bioprocesses.

Metabolomics aims to address what and how regulatory mechanisms are coordinated to achieve flux optimality, different metabolic objectives as well as appropriate adaptations to dynamic nutrient availability. Recent decades have witnessed that the integration of metabolomics and fluxomics within the goal of synthetic biology has arrived at generating the desired bioproducts with improved bioconversion efficiency. Absolute metabolite quantification by isotope dilution mass spectrometry represents a functional readout of cellular biochemistry and contributes to the establishment of metabolic (structured) models required in systems metabolic engineering. In industrial practices, population heterogeneity arising from fluctuating nutrient availability frequently leads to performance losses, that is reduced commercial metrics (titer, rate, and yield). Hence, the development of more stable producers and more predictable bioprocesses can benefit from a quantitative understanding of spatial and temporal cell-to-cell heterogeneity within industrial bioprocesses. Quantitative metabolomics analysis and metabolic modeling applied in computational fluid dynamics (CFD)-assisted scale-down simulators that mimic industrial heterogeneity such as fluctuations in nutrients, dissolved gases, and other stresses can procure informative clues for coping with issues during bioprocessing scale-up. In previous studies, only limited insights into the hydrodynamic conditions inside the industrial-scale bioreactor have been obtained, which makes case-by-case scale-up far from straightforward. Tracking the flow paths of cells circulating in large-scale bioreactors is a highly valuable tool for evaluating cellular performance in production tanks. The "lifelines" or "trajectories" of cells in industrial-scale bioreactors can be captured using Euler-Lagrange CFD simulation. This novel methodology can be further coupled with metabolic (structured) models to provide not only a statistical analysis of cell lifelines triggered by the environmental fluctuations but also a global assessment of the metabolic response to heterogeneity inside an industrial bioreactor. For the future, the industrial design should be dependent on the computational framework, and this integration work will allow bioprocess scale-up to the industrial scale with an end in mind.

Engineering Pichia pastoris to improve S-adenosyl- l-methionine production using systems metabolic strategies.

S-adenosyl-l-methionine (SAM) is a highly valued chemical that can be used as a dietary supplement and has been used to treat depression, osteoarthritis, and liver problems as well. We adopted systems metabolic engineering strategies to improve SAM production in a high-producing strain (GS115/DS56). First, the cystathionine ß-synthase gene CYS4 was downregulated using a weak promoter PG12 to reduce the removal of homocysteine from SAM cycle, thus leading to a 48.8% increase in the SAM titer (1.68 g/L) from the strain G12-CBS, while preventing cysteine auxotrophy induced by deletion of this essential gene. Subsequently, the SAM titer of G12-CBS was improved to 13.01 g/L in 15-L fed-batch fermentation using the optimal l-methionine feeding strategy. Finally, based on comparative transcriptomics, five genes were chosen and overexpressed for further enhancement of SAM production. Among them, GDH2 and ACS2 exhibited positive effects, and the additional overexpression of GDH2 led to a 52.3% increase of titer (2.71 g/L) in shake flask culture. Therefore, the engineered Pichia pastoris strains can be utilized in industrial production of SAM using a simple and cost-effective process, and these approaches could be employed for improving the production of other chemicals by P. pastoris.

Improving cytosolic aspartate biosynthesis increases glucoamylase production in Aspergillus niger under oxygen limitation.

BACKGROUND: Glucoamylase is one of the most industrially applied enzymes, produced by Aspergillus species, like Aspergillus niger. Compared to the traditional ways of process optimization, the metabolic engineering strategies to improve glucoamylase production are relatively scarce. RESULTS: In the previous study combined multi-omics integrative analysis and amino acid supplementation experiment, we predicted four amino acids (alanine, glutamate, glycine and aspartate) as the limited precursors for glucoamylase production in A. niger. To further verify this, five mutants namely OE-ala, OE-glu, OE-gly, OE-asp1 and OE-asp2, derived from the parental strain A. niger CBS 513.88, were constructed respectively for the overexpression of five genes responsible for the biosynthesis of the four kinds of amino acids (An11g02620, An04g00990, An05g00410, An04g06380 and An16g05570). Real-time quantitative PCR revealed that all these genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production in shake flask cultivation. Notably, the results demonstrated that mutant OE-asp2 which was constructed for reinforcing cytosolic aspartate synthetic pathway, exhibited significantly increased glucoamylase activity by 23.5% and 60.3% compared to CBS 513.88 in the cultivation of shake flask and the 5 L fermentor, respectively. Compared to A. niger CBS 513.88, mutant OE-asp2 has a higher intracellular amino acid pool, in particular, alanine, leucine, glycine and glutamine, while the pool of glutamate was decreased. CONCLUSION: Our study combines the target prediction from multi-omics analysis with the experimental validation and proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis. In short, this systematically conducted study will surely deepen the understanding of resources allocation in cell factory and provide new strategies for the rational design of enzyme production strains.

Advances in sophorolipid-producing strain performance improvement and fermentation optimization technology.

Sophorolipids (SLs), currently one of the most promising biosurfactants, are secondary metabolites produced by many non-pathogenic yeasts, among which Candida bombicola ATCC 22214 is the main sophorolipid-producing strain. SLs have gained much attention since they exhibit anti-tumor, anti-bacterial, anti-inflammatory, and other beneficial biological activities. In addition, as biosurfactants, SLs have a low toxicity level and are easily degradable without polluting the environment. However, the production cost of SLs remains high, which hinders the industrialization process of SL production. This paper describes SL structure and the metabolic pathway of SL synthesis firstly. Furthermore, we analyze factors that contribute to the higher production cost of SLs and summarize current research status on the advancement of SL production based on two aspects: (1) the improvement of strain performance and (2) the optimization of fermentation process. Further prospects of lowering the cost of SL production are also discussed in order to achieve larger-scale SL production with a high yield at a low cost. KEY POINTS: • Review of advances in strain performance improvement and fermentation optimization. • High-throughput screening and metabolic engineering for high-performance strains. • Low-cost substrates and semi-continuous strategies for efficient SL production.

A preliminary study on the impact of exogenous A-Factor analogue 1,4-butyrolactone on stimulating bitespiramycin biosynthesis.

Bitespiramycin is composed of nine main acylated spiramycin components with isovaleryspiramycin as the major component. However, even with excellent therapeutic effects, its application and industrialization are restricted due to its low titer. In this study, the exogenous addition of A-Factor analogue 1,4-butyrolactone (1,4-BL) stimulated an improvement in bitespiramycin biological titer by 29% with a tiny influence on concentration of major component. Moreover, the mechanism of 1,4-BL stimulating effect was preliminarily explored by the analyses of three key enzyme activities, intracellular metabolite profiling and metabolic flux distribution. All results coordinately revealed that the extensive accumulation of methylmalonyl-CoA and acetyl-CoA was the direct reason for the enhanced bitespiramycin biosynthesis. This study would provide theoretical and technical basis for the application of 1,4-BL addition strategy to industrial bitespiramycin production.