Enhancement of vitamin B6 production driven by omics analysis combined with fermentation optimization.

BACKGROUND: Microbial engineering aims to enhance the ability of bacteria to produce valuable products, including vitamin B6 for various applications. Numerous microorganisms naturally produce vitamin B6, yet the metabolic pathways involved are rigorously controlled. This regulation by the accumulation of vitamin B6 poses a challenge in constructing an efficient cell factory. RESULTS: In this study, we conducted transcriptome and metabolome analyses to investigate the effects of the accumulation of pyridoxine, which is the major commercial form of vitamin B6, on cellular processes in Escherichia coli. Our omics analysis revealed associations between pyridoxine and amino acids, as well as the tricarboxylic acid (TCA) cycle. Based on these findings, we identified potential targets for fermentation optimization, including succinate, amino acids, and the carbon-to-nitrogen (C/N) ratio. Through targeted modifications, we achieved pyridoxine titers of approximately 514 mg/L in shake flasks and 1.95 g/L in fed-batch fermentation. CONCLUSION: Our results provide insights into pyridoxine biosynthesis within the cellular metabolic network for the first time. Our comprehensive analysis revealed that the fermentation process resulted in a remarkable final yield of 1.95 g/L pyridoxine, the highest reported yield to date. This work lays a foundation for the green industrial production of vitamin B6 in the future.

Rational construction of a high-quality and high-efficiency biosynthetic system and fermentation optimization for A82846B based on combinatorial strategies in Amycolatopsis orientalis.

BACKGROUND: Oritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production. RESULTS: Firstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities. CONCLUSION: Under approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.

Process optimized for production of iturin A in biofilm reactor by Bacillus velezensis ND.

In this research, to provide an optimal growth medium for the production of iturin A, the concentrations of key amino acid precursors were optimized in shake flask cultures using the response surface method. The optimized medium were applied in a biofilm reactor for batch fermentation, resulting in enhanced production of iturin A. On this basis, a step-wise pH control strategy and a combined step-wise pH and temperature control strategy were introduced to further improve the production of iturin A. Finally, the fed-batch fermentation was performed based on combined step-wise pH and temperature control. The titer and productivity of iturin A reached 7.86 ± 0.23 g/L and 65.50 ± 1.92 mg/L/h, respectively, which were 37.65 and 65.20% higher than that before process optimization.

High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.

Optimization of glutathione production in Saccharomyces cerevisiae HBSD-W08 using Plackett-Burman and central composite rotatable designs.

BACKGROUND: Glutathione is an important bioactive tripeptide and is widely used in the food, medicine, and cosmetics industries. The aim of this study was to provide an efficient method for producing GSH and to explore its synthesis mechanism. Saccharomyces cerevisiae strain HBSD-W08 was screened for GSH production, and its fermentation medium was optimized using single-factor experiments of the Plackett-Burman and central composite rotatable designs. This method was used to analyze the effects of the presence and concentration of various carbon sources, organic and inorganic nitrogen sources, metal ions, and precursor amino acids on GSH production and catalase, superoxide dismutase, and γ-glutamylcysteine synthetase activity. RESULTS: The three most significant factors affecting GSH production were peptone (optimal concentration [OC]: 2.50 g L- 1), KH2PO4 (OC: 0.13 g L- 1), and glutamic acid (OC: 0.10 g L- 1). GSH productivity of HBSD-W08 was obtained at 3.70 g L- 1 in the optimized medium. The activity of γ-GCS, which is a marker for oxidative stress, was found to be highly positively correlated with GSH production. CONCLUSIONS: This finding revealed an underlying relationship between GSH synthesis and oxidative stress, providing useful information for developing effective GSH fermentation control strategies.

Metabolic engineering strategies for naringenin production enhancement in Streptomyces albidoflavus J1074.

BACKGROUND: Naringenin is an industrially relevant compound due to its multiple pharmaceutical properties as well as its central role in flavonoid biosynthesis. RESULTS: On our way to develop Streptomyces albidoflavus J1074 as a microbial cell factory for naringenin production, we have significantly increased the yields of this flavanone by combining various metabolic engineering strategies, fermentation strategies and genome editing approaches in a stepwise manner. Specifically, we have screened different cultivation media to identify the optimal production conditions and have investigated how the additive feeding of naringenin precursors influences the production. Furthermore, we have employed genome editing strategies to remove biosynthetic gene clusters (BGCs) associated with pathways that might compete with naringenin biosynthesis for malonyl-CoA precursors. Moreover, we have expressed MatBC, coding for a malonate transporter and an enzyme responsible for the conversion of malonate into malonyl-CoA, respectively, and have duplicated the naringenin BGC, further contributing to the production improvement. By combining all of these strategies, we were able to achieve a remarkable 375-fold increase (from 0.06 mg/L to 22.47 mg/L) in naringenin titers. CONCLUSION: This work demonstrates the influence that fermentation conditions have over the final yield of a bioactive compound of interest and highlights various bottlenecks that affect production. Once such bottlenecks are identified, different strategies can be applied to overcome them, although the efficiencies of such strategies may vary and are difficult to predict.

Enhancement of herbicolin A production by integrated fermentation optimization and strain engineering in Pantoea agglomerans ZJU23.

BACKGROUND: The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering. RESULTS: Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett-Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King's B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain. CONCLUSIONS: This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization.

BACKGROUND: Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. RESULTS: In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. CONCLUSIONS: This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Daptomycin production enhancement by ARTP mutagenesis and fermentation optimization in Streptomyces roseosporus.

AIMS: We evaluated whether the randomness of mutation breeding can be regulated through a double-reporter system. We hope that by establishing a new precursor feeding strategy, the production capacity of industrial microorganisms after pilot scale-up can be further improved. METHODS AND RESULTS: In this study, the industrial strain Streptomyces roseosporus L2796 was used as the starter strain for daptomycin production, and a double-reporter system with the kanamycin resistance gene Neo and the chromogenic gene gusA was constructed to screen for high-yield strain L2201 through atmospheric and room temperature plasma (ARTP). Furthermore, the composition of the culture medium and the parameters of precursor replenishment were optimized, resulting in a significant enhancement of the daptomycin yield of the mutant strain L2201(752.67 mg/l). CONCLUSIONS: This study successfully screened a high-yield strain of daptomycin through a double-reporter system combined with ARTP mutation. The expression level of two reporter genes can evaluate the strength of dptEp promoter, which can stimulate the expression level of dptE in the biosynthesis of daptomycin, thus producing more daptomycin. The developed multi-stage feeding rate strategy provides a novel way to increase daptomycin in industrial fermentation.

Engineering of Shikimate Pathway and Terminal Branch for Efficient Production of L-Tryptophan in Escherichia coli.

L-tryptophan (L-trp), produced through bio-manufacturing, is widely used in the pharmaceutical and food industries. Based on the previously developed L-trp-producing strain, this study significantly improved the titer and yield of L-trp, through metabolic engineering of the shikimate pathway and the L-tryptophan branch. First, the rate-limiting steps in the shikimate pathway were investigated and deciphered, revealing that the combined overexpression of the genes aroE and aroD increased L-trp production. Then, L-trp synthesis was further enhanced at the shaking flask level by improving the intracellular availability of L-glutamine (L-gln) and L-serine (L-ser). In addition, the transport system and the competing pathway of L-trp were also modified, indicating that elimination of the gene TnaB contributed to the extracellular accumulation of L-trp. Through optimizing formulas, the robustness and production efficiency of engineered strains were enhanced at the level of the 30 L fermenter. After 42 h of fed-batch fermentation, the resultant strain produced 53.65 g/L of L-trp, with a yield of 0.238 g/g glucose. In this study, the high-efficiency L-trp-producing strains were created in order to establish a basis for further development of more strains for the production of other highly valuable aromatic compounds or their derivatives.

Revealing the Mechanisms of Enhanced ß-Farnesene Production in Yarrowia lipolytica through Metabolomics Analysis.

ß-Farnesene is an advanced molecule with promising applications in agriculture, the cosmetics industry, pharmaceuticals, and bioenergy. To supplement the shortcomings of rational design in the development of high-producing ß-farnesene strains, a Metabolic Pathway Design-Fermentation Test-Metabolomic Analysis-Target Mining experimental cycle was designed. In this study, by over-adding 20 different amino acids/nucleobases to induce fluctuations in the production of ß-farnesene, the changes in intracellular metabolites in the ß-farnesene titer-increased group were analyzed using non-targeted metabolomics. Differential metabolites that were detected in each experimental group were selected, and their metabolic pathways were located. Based on these differential metabolites, targeted strain gene editing and culture medium optimization were performed. The overexpression of the coenzyme A synthesis-related gene pantothenate kinase (PanK) and the addition of four mixed water-soluble vitamins in the culture medium increased the ß-farnesene titer in the shake flask to 1054.8 mg/L, a 48.5% increase from the initial strain. In the subsequent fed-batch fermentation, the ß-farnesene titer further reached 24.6 g/L. This work demonstrates the tremendous application value of metabolomics analysis for the development of industrial recombinant strains and the optimization of fermentation conditions.

Transcriptome and Metabolome Analysis Revealing the Improved ε-Poly-l-Lysine Production Induced by a Microbial Call from Botrytis cinerea.

ε-Poly-l-lysine (ε-PL) is a wide-spectrum antimicrobial agent, while its biosynthesis-inducing signals are rarely reported. This study found that Botrytis cinerea extracts could act as a microbial call to induce a physiological modification of Streptomyces albulus for ε-PL efficient biosynthesis and thereby resulted in ε-PL production (34.2 g/liter) 1.34-fold higher than control. The elicitors could be primary isolated by ethanol and butanol extraction, which resulted in more vibrant, aggregate and stronger mycelia. The elicitor-derived physiological changes focused on three aspects: ε-PL synthase, energy metabolism, and lysine biosynthesis. After elicitor addition, upregulated sigma factor hrdD and improved transcription and expression of pls directly contributed to the high ε-PL productivity; upregulated genes in tricarboxylic acid (TCA) cycle and energy metabolism promoted activities of citrate synthase and the electron transport system; in addition, pool enlargements of ATP, ADP, and NADH guaranteed the ATP provision for ε-PL assembly. Lysine biosynthesis was also increased based on enhancements of gene transcription, key enzyme activities, and intracellular metabolite pools related to carbon source utilization, the Embden-Meyerhof pathway (EMP), the diaminopimelic acid pathway (DAP), and the replenishment pathway. Interestingly, the elicitors stimulated the gene transcription for the quorum-sensing system and resulted in upregulation of genes for other antibiotic production. These results indicated that the Botrytis cinerea could produce inducing signals to change the Streptomyces mycelial physiology and accelerate the ε-PL biosynthesis. IMPORTANCE This work identified the role of microbial elicitors on ε-PL production and disclosed the underlying mechanism through analysis of gene transcription, key enzyme activities, and intracellular metabolite pools, including transcriptome and metabolome analysis. It was the first report for the inducing effects of the "microbial call" to Streptomyces albulus and ε-PL biosynthesis, and these elicitors could be potentially obtained from decayed fruits infected by Botrytis cinerea; hence, this may be a way of turning a biohazard into bioproduct wealth. This study provided a reference for application of microbial signals in secondary metabolite production, which is of theoretical and practical significance in industrial antibiotic production.

Enhancement of antibacterial and growth-promoting effects of Paenibacillus polymyxa by optimizing its fermentation process.

AIMS: We aimed to enhance the antibacterial and growth-promoting effects of Paenibacillus polymyxa by improving the yield of spores, lipopeptides and indole-3-acetic acid (IAA) in the fermentation process. METHODS AND RESULTS: Through medium optimization by the response surface method and feeding fermentation, the number of spores reached 2.37 × 109  cfu ml-1 with an increase of 38%, the content of lipopeptides reached 60.8 mg L-1 with an increase of 89%, and the content of IAA reached 24.3 mg L-1 with an increase of 176%, respectively, comparing with the original (un-optimized) culture conditions. The fermentation culture of P. polymyxa from the optimized medium and feeding fermentation resulted in higher colonization of P. polymyxa in soils than that from the original culture during the 49 days for testing. Comparing with the supernatant of the original culture, the supernatant of the P. polymyxa culture from the optimized medium and feeding fermentation showed enhanced antibacterial effects and plant growth-promoting effects. The enhanced antibacterial effect was shown as the increase of the inhibition zone by 59%, 45% and 26% against Ralstonia solanacearum, Erwinia carotovora and Xanthomonas campestris. The enhanced growth-promoting effects on tomato and strawberry plants were the increase of plant height by 47% and 5%, root length by 23% and 15% and root weight by 65% and 110%. CONCLUSIONS: The combination of medium optimization and feeding fermentation effectively improved the yield of spores, lipopeptides and IAA. Lipopeptides and IAA lead to enhanced antibacterial and plant growth-promoting effects of the P. polymyxa product. SIGNIFICANCE AND IMPACT OF THIS STUDY: The optimized fermentation method significantly improved the yield of spores, lipopeptides and IAA, thus providing theoretical and technical support for enhancing the antibacterial and growth-promoting effects of P. polymyxa products in agriculture.

Fermentation optimization and amylase activity of endophytic Bacillus velezensis D1 isolated from corn seeds.

AIMS: To acquire quality amylase adopted in practical applications, endophytic bacteria were identified as Bacillus velezensis strain D1 which was isolated from corn seeds. The fermentation conditions and amylase properties of the strain were investigated. METHODS AND RESULTS: The strain D1 was identified via morphological, physiological and 16S rDNA phylogenetic analysis. The fermentation conditions of secreting amylase were optimized by single-factor and orthogonal experiments. The α-amylase gene was expressed in E. coli and purified by means of immobilized metal ion affinity chromatography (IMAC), upon which the enzyme activity of purified recombinant α-amylase was determined. The results outlined that (1) The strain D1 was identified as Bacillus velezensis. (2) The optimized fermentation conditions for maximum amylase yields included 44°C for 48 h at pH 7.5. (3) The enzyme had an optimal reaction temperature of 60°C with the highest activity at 50°C and tolerance to 4-h incubation at 70°C. (4) The enzyme was strong acid resistant and tolerated at pH 5.0-6.0 while the optimal pH was 8.0. (5) Besides, the amylase activity was elevated by the presence of Ca2+ and Cu2+ . (6) The activity of purified recombinant amylase was 20.59 U/ml under optimal conditions, nearly seven times that of crude amylase preparations. CONCLUSIONS: The amylase produced by Bacillus velezensis D1 is strongly tolerant towards acid and high temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: Amylases with thermophilic and acid-resistant characteristics are useful for a wide range of applications in food, brewing, textile, starch, paper and deterrent industries. The enzyme from Bacillus velezensis D1 can be effectively used in different areas of industries.

Optimization of fermentation conditions for production of neutral metalloprotease by Bacillus subtilis SCK6 and its application in goatskin-dehairing.

In this study, a high protease-producing strain was screened by spread plate method and identified by molecular biology and morphological identification. It was identified as Bacillus sp. LCB14. A neutral protease gene was cloned and heterologous expressed by B. subtilis SCK6. Then, the recombinant protease was used to dehair the goat skins. The fermentation conditions of neutral protease production by B. subtilis SCK6 were optimized. The single factor experiments, Plackett-Burma experiment, and response surface method were conducted to determine fermentation medium and culture conditions. The optimized medium contained corn meal 49 g/L, soluble starch 28 g/L, soybean meal 17 g/L, corn steep liquor powder 8 g/L, yeast extract 10 g/L, Na2HPO4 2.3 g/L, KH2PO4 1.9 g/L, MgSO4 0.5 g/L, MnCl2 0.1 g/L and ZnSO4 0.05 g/L. The optimized culture conditions were 35 °C and pH 7.0. Under the optimum conditions, the recombinant strain reached 33467.28 U/mL after 72 hr ferment. Moreover, by fed batch in 30 L fermenters, neutral protease production reached 39,440.78 U/mL and shortened fermentation time from 72 hr to 46 hr. Finally, the crude enzyme was utilized to replace sodium sulfide for dehairing of goatskins. The enzymatic dehaired pelts were white, smooth, and soft; the grain side of enzymatic dehaired pelts were clear; there was no obvious damage to the grain side of enzymatic dehaired pelts by visual observation and tactile test. Furthermore, there were no hair roots, hair follicles and other glands in enzymatic dehaired belts, and the collagen fibers of enzymatic dehaired belt were dispersed well by histological analysis.

Avilamycin production enhancement by mutagenesis and fermentation optimization in Streptomyces viridochromogenes.

Avilamycin, an excellent growth-promoting feed additive, produced by Streptomyces viridochromogenes, was widely used to promote the growth of poultry by inhibiting Gram-positive bacteria. In this work, the methods of combinational mutagenesis of UV (Ultraviolet) and ARTP (Atmospheric and room temperature plasma), and rational screening by high concentrations of CaCl2 were utilized to promote the production of avilamycin. The avilamycin high-yielding mutant strains of Z-6 (29.31 mg/L), A-9 (36.84 mg/L) and F-23 (45.73 mg/L) were screened out, with yields of avilamycin improved by 57.92%, 98.49% and 146.39%, respectively, compared with the wild strain (WT). The performance comparison showed that Z-6, A-9 and F-23 mutant strains had stronger abilities of substrate consumption, cell growth and antibiotic synthesis than WT. Furthermore, the composition of fermentation medium, inoculation parameters, supplementation strategies of oxygen vectors, glucose and precursors (L-valine, D-xylose and sodium acetate) had been optimized and the avilamycin yield of the mutant strain F-23 was significantly enhanced by 41.87% by fermentation optimization. In summary, the strategy of increasing the production of avilamycin in S. viridochromogenes in this work might provide an alternative method to enhance the synthesis of secondary metabolites in other Streptomyces.

Stepwise metabolic engineering of Candida tropicalis for efficient xylitol production from xylose mother liquor.

BACKGROUND: Commercial xylose purification produces xylose mother liquor (XML) as a major byproduct, which has become an inexpensive and abundant carbon source. A portion of this XML has been used to produce low-value-added products such as caramel but the remainder often ends up as an organic pollutant. This has become an issue of industrial concern. In this study, a uracil-deficient Candida tropicalis strain was engineered to efficiently convert XML to the commercially useful product xylitol. RESULTS: The xylitol dehydrogenase gene was deleted to block the conversion of xylitol to xylulose. Then, an NADPH regeneration system was added through heterologous expression of the Yarrowia lipolytica genes encoding 6-phosphate-gluconic acid dehydrogenase and 6-phosphate-glucose dehydrogenase. After process optimization, the engineered strain, C. tropicalis XZX-B4ZG, produced 97.10 g L- 1 xylitol in 120 h from 300 g L- 1 XML in a 5-L fermenter. The xylitol production rate was 0.82 g L- 1 h- 1 and the conversion rate was 92.40 %. CONCLUSIONS: In conclusion, this study performed a combination of metabolic engineering and process optimizing in C. tropicalis to enhance xylitol production from XML. The use of C. tropicalis XZX-B4ZG, therefore, provided a convenient method to transform the industrial by-product XML into the useful material xylitol.

Enhanced O-succinyl-l-homoserine production by recombinant Escherichia coli ΔIJBB*TrcmetL/pTrc-metAfbr -Trc-thrAfbr -yjeH via multilevel fermentation optimization.

AIMS: Constructing a strain with high yield of O-succinyl-l-homoserine (OSH) and improving the titre through multilevel fermentation optimization. METHODS AND RESULTS: OSH high-yielding strain was first constructed by deleting the thrB gene to block the threonine biosynthesis. Single-factor experiment was carried out, where a Plackett-Burman design was used to screen out three factors (glucose, yeast and threonine) from the original 11 factors that affected the titre of OSH. The Box-Behnken response surface method was used to optimize the fermentation conditions. Through gene editing and medium optimization, the titre of OSH increased from 7·20 to 8·70 g l-1 in 500 ml flask. Furthermore, the fermentation process and fed-batch fermentation conditions including pH, temperature, feeding strategy and feeding medium were investigated and optimized. Under the optimal conditions, the titre of OSH reached 102·5 g l-1 , which is 5·6 times higher than before (15·6 g l-1 ). CONCLUSIONS: O-succinyl-l-homoserine fermentation process was established and the combination of response surface methodology and metabolic pathway analysis effectively improved the titre of OSH. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the titre of OSH reached the needs for industrial production and the metabolic pathway of OSH was demonstrated for further optimization.

Fusarium solani G6, a novel vitexin-producing endophytic fungus: characterization, yield improvement and osteoblastic proliferation activity.

The study aimed to characterize a novel vitexin-producing endophytic fungus Fusarium solani G6 from Cajanus cajan, improve its capability for producing vitexin and evaluate its osteoblastic proliferation activity. A total of 153 endophytic fungi, classified into 6 genera, were isolated from C. cajan. Among them, only one strain, endophyte G6 identified as Fusarium solani, was found to produce vitexin. After the optimization of fermentation conditions, the highest vitexin yield (18.72 mg/L) for the strain was observed in PDB liquid medium containing 20.54 g/L of glucose and 8.90 g/L of ammonium sulfate, at an initial medium pH of 5.1 and at 28 °C for 6 days of cultivation. Moreover, the fungal vitexin exhibited notable osteoblastic proliferation stimulating activity. A novel vitexin-producing endophytic fungus F. solani G6 was characterized from C. cajan for the first time. The findings highlighted its potential use for large-scale production of vitexin and might have a promising use as therapeutic agent for osteoporosis.

Engineering Bafilomycin High-Producers by Manipulating Regulatory and Biosynthetic Genes in the Marine Bacterium Streptomyces lohii.

Bafilomycin A1 is the representative compound of the plecomacrolide natural product family. This 16-membered ring plecomacrolide has potent antifungal and vacuolar H+-ATPase inhibitory activities. In our previous work, we identified a bafilomycin biosynthetic gene cluster (baf) from the marine bacterium Streptomyces lohii ATCC BAA-1276, wherein a luxR family regulatory gene orf1 and an afsR family regulatory gene bafG were revealed based on bioinformatics analysis. In this study, the positive regulatory roles of orf1 and bafG for bafilomycin biosynthesis are characterized through gene inactivation and overexpression. Compared to the wild-type S. lohii strain, the knockout of either orf1 or bafG completely abolished the production of bafilomycins. The overexpression of orf1 or bafG led to 1.3- and 0.5-fold increased production of bafilomycins, respectively. A genetically engineered S. lohii strain (SLO-08) with orf1 overexpression and inactivation of the biosynthetic genes orf2 and orf3, solely produced bafilomycin A1 with the titer of 535.1 ± 25.0 mg/L in an optimized fermentation medium in shaking flasks. This recombinant strain holds considerable application potential in large-scale production of bafilomycin A1 for new drug development.