Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering.

Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel "PEACES" system by (1) expressing the NAD + kinase gene ppnk from Clostridium glutamicum and the NADP + -dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.

Deciphering Multidrug-Resistant Plasmids in Disinfection Residual Bacteria from a Wastewater Treatment Plant.

Current disinfection processes pose an emerging environmental risk due to the ineffective removal of antibiotic-resistant bacteria, especially disinfection residual bacteria (DRB) carrying multidrug-resistant plasmids (MRPs). However, the characteristics of DRB-carried MRPs are poorly understood. In this study, qPCR analysis reveals that the total absolute abundance of four plasmids in postdisinfection effluent decreases by 1.15 log units, while their relative abundance increases by 0.11 copies/cell compared to investigated wastewater treatment plant (WWTP) influent. We obtain three distinctive DRB-carried MRPs (pWWTP-01-03) from postdisinfection effluent, each carrying 9-11 antibiotic-resistant genes (ARGs). pWWTP-01 contains all 11 ARGs within an ∼25 Kbp chimeric genomic island showing strong patterns of recombination with MRPs from foodborne outbreaks and hospitals. Antibiotic-, disinfectant-, and heavy-metal-resistant genes on the same plasmid underscore the potential roles of disinfectants and heavy metals in the coselection of ARGs. Additionally, pWWTP-02 harbors an adhesin-type virulence operon, implying risks of both antibiotic resistance and pathogenicity upon entering environments. Furthermore, some MRPs from DRB are capable of transferring and could confer selective advantages to recipients under environmentally relevant antibiotic pressure. Overall, this study advances our understanding of DRB-carried MRPs and highlights the imminent need to monitor and control wastewater MRPs for environmental security.

Simple Genomic Traits Predict Rates of Polysaccharide Biodegradation.

Natural and chemically modified polysaccharides are extensively employed across a wide array of industries, leading to their prevalence in the waste streams of industrialized societies. With projected increasing demand, a pressing challenge is to swiftly assess and predict their biodegradability to inform the development of new sustainable materials. In this study, we developed a scalable method to evaluate polysaccharide breakdown by measuring microbial growth and analyzing microbial genomes. Our approach, applied to polysaccharides with various structures, correlates strongly with well-established regulatory methods based on oxygen demand. We show that modifications to the polysaccharide structure decreased degradability and favored the growth of microbes adapted to break down chemically modified sugars. More broadly, we discovered two main types of microbial communities associated with different polysaccharide structures─one dominated by fast-growing microbes and another by specialized degraders. Surprisingly, we were able to predict biodegradation rates based only on two genomic features that define these communities: the abundance of genes related to rRNA (indicating fast growth) and the abundance of glycoside hydrolases (enzymes that break down polysaccharides), which together predict nearly 70% of the variation in polysaccharide breakdown. This suggests a trade-off, whereby microbes are either adapted for fast growth or for degrading complex polysaccharide chains, but not both. Finally, we observe that viral elements (prophages) encoded in the genomes of degrading microbes are induced in easily degradable polysaccharides, leading to complex dynamics in biomass accumulation during degradation. In summary, our work provides a practical approach for efficiently assessing polymer degradability and offers genomic insights into how microbes break down polysaccharides.

Interval Split Covariance Intersection Filter: Theory and Its Application to Cooperative Localization in a Multi-Sensor Multi-Vehicle System.

The data incest problem causes inter-estimate correlation during data fusion processes, which yields inconsistent data fusion results. Especially in the multi-sensor multi-vehicle (MSMV) system, the data incest problem is serious due to multiple relative position estimations, which not only lead to pessimistic estimation but also cause additional computational overhead. In order to address the data incest problem, we propose a new data fusion method termed the interval split covariance intersection filter (ISCIF). The general consistency of the ISCIF is proven, serving as supplementary proof for the split covariance intersection filter (SCIF). Moreover, a decentralized MSMV localization system including absolute and relative positioning stages is designed. In the absolute positioning stage, each vehicle uses the ISCIF algorithm to update its own position based on absolute measurements. In the relative position stage, the interval constraint propagation (ICP) method is implemented to preprocess multiple relative position estimates and initially prepare input data for ISCIF. Then, the proposed ISCIF algorithm is employed to realize relative positioning. In addition, comparative simulations demonstrate that the proposed method can achieve both accurate and consistent results compared with the state-of-the-art methods.

Risk factors for pegylated liposomal doxorubicin-induced moderate to severe hand-foot syndrome in breast cancer patients: assessment of baseline clinical parameters.

BACKGROUND: Hand-foot syndrome (HFS) is a side effect of skin related to pegylated liposomal doxorubicin (PLD) application. Moderate to severe hand-foot syndrome (MSHFS) might have a serious impact on patients' quality of life and treatment. However, information on risk factors for the development of MSHFS is still limited. To analyze the risk factors for PLD-induced MSHFS in breast cancer patients and constructed a logistic regression prediction model. METHODS: We conducted a retrospective analysis of breast cancer patients who were treated with a PLD regimen in the Tumor Hospital of Harbin Medical University from January 2017 to August 2019. A total of 26 factors were collected from electronic medical records. Patients were divided into MSHFS (HFS > grade 1) and NMHFS (HFS ≤ grade 1) groups according to the NCI classification. Statistical analysis of these factors and the construction of a logistic regression prediction model based on risk factors. RESULTS: A total of 44.7% (206/461) of patients developed MSHFS. The BMI, dose intensity, and baseline Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) levels in the MSHFS group, as well as good peripheral blood circulation, excessive sweat excretion, history of gallstones, and tumour- and HER2-positive percentages, were all higher than those in the NMHFS group (P < 0.05). The model for predicting the occurrence of MSHFS was P = 1/1 + exp. (11.138-0.110*BMI-0.234*dose intensity-0.018*baseline ALT+ 0.025*baseline AST-1.225*gallstone history-0.681* peripheral blood circulation-1.073*sweat excretion-0.364*with or without tumor-0.680*HER-2). The accuracy of the model was 72.5%, AUC = 0.791, and Hosmer-Lemeshow fit test P = 0.114 > 0.05. CONCLUSIONS: Nearly half of the patients developed MSHFS. The constructed prediction model may be valuable for predicting the occurrence of MSHFS in patients.

Sirtuin3 deficiency exacerbates carbon tetrachloride-induced hepatic injury in mice.

Sirtuin3 (SIRT3) plays an important role in maintaining normal mitochondrial function and alleviating oxidative stress. After carbon tetrachloride (CCl4 ) administration, the expression of SIRT3 decreased in the liver of mice, which indicated that the SIRT3 might play a crucial role during chemical-induced acute hepatic injury. To verify the hypothesis, CCl 4 was given to induce acute hepatic injury in SIRT3 knockout (KO) mice and wild-type (WT) mice. CCl 4 -induced liver injury was more severe in SIRT3 KO mice compared with the WT mice. In addition, the oxidative stress induced by CCl 4 was enhanced in the SIRT3 KO mice. Furthermore, the increased expression of dynamin-related protein 1 was also aggravated in SIRT3 KO mice after CCl 4 administration. In conclusion, our study demonstrated that SIRT3 deficiency exacerbated CCl 4 -induced impairment of the liver in mice, and the mechanism might be related to enhanced oxidative stress.

De Novo Biosynthesis of Dihydroquercetin in Saccharomyces cerevisiae.

Dihydroquercetin is a vital flavonoid compound with a wide range of physiological activities. However, factors, such as metabolic regulation, limit the heterologous synthesis of dihydroquercetin in microorganisms. In this study, flavanone 3-hydroxylase (F3H) and flavanone 3'-hydroxylase (F3'H) were screened from different plants, and their co-expression in Saccharomyces cerevisiae was optimized. Promoter engineering and redox partner engineering were used to optimize the corresponding expression of genes involved in the dihydroquercetin synthesis pathway. Dihydroquercetin production was further improved through multicopy integration pathway genes and systems metabolic engineering. By increasing NADPH and α-ketoglutarate supply, the catalytic efficiency of F3'H and F3H was improved, thereby effectively increasing dihydroquercetin production (235.1 mg/L). Finally, 873.1 mg/L dihydroquercetin titer was obtained by fed-batch fermentation in a 5-L bioreactor, which is the highest dihydroquercetin production achieved through de novo microbial synthesis. These results established a pivotal groundwork for flavonoids synthesis.

[Effects of N, P, and K application rates on distribution of 13C assimilates, starch accumulation in grains and fertilizer utilization of wheat]. / 氮磷钾施用量对小麦旗叶13CåŒåŒ–ç‰©åˆ†é ã€ç±½ç²’æ·€ç²‰ç§¯ç´¯å’Œè‚¥æ–™åˆ©ç”¨çš„å½±å“.

Clarifying the appropriate application rates of N, P, and K fertilizers and the physiological mechanisms of wheat under water-saving recharge irrigation in the North China Plain would provide a theoretical basis for formulating reasonable fertilization plans for high-yield and high-efficiency wheat production. We established four treatments with different amounts of nitrogen (N), phosphorus (P2O5), and potassium (K2O) application: 0, 0, and 0 kg·hm-2 (F0), 180, 75, and 60 kg·hm-2 (F1), 225, 120, and 105 kg·hm-2 (F2), and 270, 165, and 150 kg·hm-2 (F3). During the jointing and anthesis stages of wheat, the relative water content of each treatment in the 0-40 cm soil layer was replenished to 70%, to investigate the differences in wheat flag leaf photosynthetic characteristics, distribution of 13C assimilates, grain starch accumulation, and fertilizer utilization. The results showed that the relative chlorophyll content of flag leaves, photosynthetic and chlorophyll fluorescence parameters, 13C assimilate allocation in each organ, enzyme activities involved in starch synthesis, and starch accumulation in the F1 treatment were significantly higher than that in F0 treatment, which was an important physiological basis for the 20.9% increase in grain yield. The above parameters and yield in the F2 and F3 treatments showed no significant increase compared to F1 treatment, while fertilizer productivity and agronomic efficiency of N, P, and K decreased by 17.5%-58.4% and 12.7%-50.7%, respectively. Therefore, F1 could promote flag leaf photosynthetic assimilate production and grain starch accumulation under water-saving supplementary irrigation conditions, resulting in higher grain yield and fertilizer utilization efficiency.

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

A 16S RNA Analysis of Yangzhou Geese with Varying Body Weights: Gut Microbial Difference and Its Correlation with Body Weight Parameters.

China is a major goose-raising country, and the geese industry plays a significant role in animal husbandry. Therefore, goose growth performance (body weight) is a critical topic. Goose gut microbiota influences weight gain by regulating its energy metabolism and digestion. Additionally, the impact of cecal microbial community structure on goose growth and development, energy metabolism, and immunity has been examined. However, most studies have used different additives or feeds as variables. Improving the understanding of the dynamic changes in gut microbial communities in geese of different body weights during their growth and development and their correlation with the host's body weight is necessary. In this study, the cecal microbiota of healthy Yangzhou geese with large (L) and small (S) body weights, all at the same age (70 days old) and under the same feeding conditions, were sequenced using 16S rRNA. The sequencing results were annotated using QIIME2 (classify-sklearn algorithm) software, and the linkET package was used to explore the correlation between intestinal microorganisms and the body weight of the Yangzhou goose (Spearman). At the phylum level, the Firmicutes/Bacteroidetes ratio in the large body weight group was approximately 20% higher than that in the small body weight group, with Bacteroidetes and Firmicutes exhibiting a highly significant negative correlation. At the genus level, Bacteroides constituted the most abundant microbial group in both groups, although the Prevotellaceae_Ga6A1_group exhibited a higher abundance in the large than the small weight group. Spearman correlation analysis and the linkET package were used to analyze the correlation between cecal microflora and production performance indicators that showed significant differences between the two groups and showed that birth weight was significantly positively correlated with Deferribacterota at the phylum level. At the genus level, leg and chest muscle weights exhibited significant positive correlations with Prevotellace-ae_Ga6A1_group, suggesting its critical role in promoting the growth and development of goose leg and chest muscles. A significant negative correlation was observed between [Ruminococ-cus]_torque and Prevotellaceae_Ga6A1_group. These findings offer a crucial theoretical foundation for the study of gastrointestinal microorganisms and provide insights into the development and formulation of poultry probiotics.

Engineering Saccharomyces cerevisiae for synthesis of ß-myrcene and (E)-ß-ocimene.

Monoterpenes are among the important natural plant terpenes. Monoterpenes usually have the characteristics of volatility and strong aroma. ß-Myrcene and its isomer (E)-ß-ocimene are typical acyclic monoterpenes. They are high-value monoterpenes that have been widely applied in foods, cosmetics, and medicines. However, large-scale commercial production of ß-myrcene and (E)-ß-ocimene is restricted by their production method that mainly involves extraction from plant essential oils. Currently, an alternative synthetic route utilizing an engineered microbial platform was proposed for effective production. This study used a Saccharomyces cerevisiae strain previously constructed for squalene production as the starting strain. Farnesyl diphosphate synthase (Erg20) expression was weakened by promoter replacement and screened for optimal myrcene synthase (MS) and ocimene synthase (OS) activities. In the resulting S. cerevisiae engineered for ß-myrcene and (E)-ß-ocimene synthesis, titers of ß-myrcene and (E)-ß-ocimene were enhanced by a fusion expressing a mutant Erg20* with the obtained monoterpene synthase and optimizing the added solvent in a two-phase fermentation system. Finally, by scaling up in a 5-L fermenter, 8.12 mg/L of ß-myrcene was obtained, which was first reported in yeast, and 34.56 mg/L of (E)-ß-ocimene was obtained, which is the highest reported to date. This study provides a new synthesis route for ß-myrcene and (E)-ß-ocimene. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03818-2.

Annotation-free discovery of functional groups in microbial communities.

Recent studies have shown that microbial communities are composed of groups of functionally cohesive taxa whose abundance is more stable and better-associated with metabolic fluxes than that of any individual taxon. However, identifying these functional groups in a manner that is independent of error-prone functional gene annotations remains a major open problem. Here we tackle this structure-function problem by developing a novel unsupervised approach that coarse-grains taxa into functional groups, solely on the basis of the patterns of statistical variation in species abundances and functional read-outs. We demonstrate the power of this approach on three distinct datasets. On data of replicate microcosms with heterotrophic soil bacteria, our unsupervised algorithm recovered experimentally validated functional groups that divide metabolic labour and remain stable despite large variation in species composition. When leveraged against the ocean microbiome data, our approach discovered a functional group that combines aerobic and anaerobic ammonia oxidizers whose summed abundance tracks closely with nitrate concentrations in the water column. Finally, we show that our framework can enable the detection of species groups that are probably responsible for the production or consumption of metabolites abundant in animal gut microbiomes, serving as a hypothesis-generating tool for mechanistic studies. Overall, this work advances our understanding of structure-function relationships in complex microbiomes and provides a powerful approach to discover functional groups in an objective and systematic manner.

Mutation-induced infections of phage-plasmids.

Phage-plasmids are extra-chromosomal elements that act both as plasmids and as phages, whose eco-evolutionary dynamics remain poorly constrained. Here, we show that segregational drift and loss-of-function mutations play key roles in the infection dynamics of a cosmopolitan phage-plasmid, allowing it to create continuous productive infections in a population of marine Roseobacter. Recurrent loss-of-function mutations in the phage repressor that controls prophage induction leads to constitutively lytic phage-plasmids that spread rapidly throughout the population. The entire phage-plasmid genome is packaged into virions, which were horizontally transferred by re-infecting lysogenized cells, leading to an increase in phage-plasmid copy number and to heterozygosity in a phage repressor locus in re-infected cells. However, the uneven distribution of phage-plasmids after cell division (i.e., segregational drift) leads to the production of offspring carrying only the constitutively lytic phage-plasmid, thus restarting the lysis-reinfection-segregation life cycle. Mathematical models and experiments show that these dynamics lead to a continuous productive infection of the bacterial population, in which lytic and lysogenic phage-plasmids coexist. Furthermore, analyses of marine bacterial genome sequences indicate that the plasmid backbone here can carry different phages and disseminates trans-continentally. Our study highlights how the interplay between phage infection and plasmid genetics provides a unique eco-evolutionary strategy for phage-plasmids.

Efficient Production of Chlorogenic Acid in Escherichia coli Via Modular Pathway and Cofactor Engineering.

Chlorogenic acid is a natural phenolic compound widely used in the food and daily chemical industries. Compared to plant extraction, microbial cell factories provide a green and sustainable production method for the production of chlorogenic acid. However, complex metabolic flux distribution and potential byproducts limited the biosynthesis of chlorogenic acid in microorganisms. A de novo biosynthesis pathway for chlorogenic acid was constructed in Escherichia coli via modular engineering. Increasing the shikimate pathway flux greatly promoted chlorogenic acid production, and the influence of pyruvate metabolism on chlorogenic acid synthesis was also explored. The supply of cofactors for the key enzymes quinate/shikimate 5-dehydrogenase (YdiB) and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC) was enhanced by a cofactor regeneration system. Furthermore, mutants of YdiB were verified for chlorogenic acid production in vivo. Chlorogenic acid browning occurred when the buffer pH of the buffer exceeded 6.0, but two-stage pH control achieved a chlorogenic acid titer of 2789.2 mg/L in a 5 L fermenter, the highest reported to date. This study provided a strategy for the efficient production of chlorogenic acid from simple carbon sources.

Enhancement of phycocyanobilin biosynthesis in Escherichia coli by strengthening the supply of precursor and artificially self-assembly complex.

Phycocyanobilin (PCB) is widely used in healthcare, food processing, and cosmetics. Escherichia coli is the common engineered bacterium used to produce PCB. However, it still suffers from low production level, precursor deficiency, and low catalytic efficiency. In this study, a highly efficient PCB-producing strain was created. First, chassis strains and enzyme sources were screened, and copy numbers were optimized, affording a PCB titer of 9.1 mg/L. Most importantly, the rate-limiting steps of the PCB biosynthetic pathway were determined, and the supply of precursors necessary for PCB synthesis was increased from endogenous sources, affording a titer of 21.4 mg/L. Then, the key enzymes for PCB synthesis, HO1 and PcyA, were assembled into a multi-enzyme complex using the short peptide tag RIAD-RIDD, and 23.5 mg/L of PCB was obtained. Finally, the basic conditions for PCB fermentation were initially determined in 250 mL shake flasks and a 5-L bioreactor to obtain higher titers of PCB. The final titer of PCB reached 147.0 mg/L, which is the highest reported titer of PCB so far. This research provided the foundation for the industrial production of PCB and its derivatives.

Identification of key genes through the constructed CRISPR-dcas9 to facilitate the efficient production of O-acetylhomoserine in Corynebacterium glutamicum.

O-Acetylhomoserine (OAH) is an important platform chemical for the synthesis of L-methamidophos and l-methionine. It has been produced efficiently in Corynebacterium glutamicum. However, a wider range of key factors had not been identified, limiting further increases in OAH production. This study successfully identified some limiting factors and regulated them to improve OAH titer. Firstly, an efficient clustered regularly interspaced short palindromic repeats/dead CRISPR associated protein 9 (CRISPR-dCas9) system was constructed and used to identify the key genes in central metabolism and branch pathways associated with OAH biosynthesis. Then, the gltA gene involved in TCA cycle was identified as the most critical gene. A sequential promoter PNCgl2698, which showed different transcriptional intensity in different strain growth periods, was used to control the expression of gltA gene, resulting in OAH production of 7.0 g/L at 48 h. Finally, the OAH titer of the engineered strain reached 25.9 g/L at 72 h in a 5-L bioreactor. These results show that the identification and regulation of key genes are critical for OAH biosynthesis, which would provide a better research basis for the industrial production of OAH in C. glutamicum.

Production of pyruvic acid with Candida glabrata using self-fermenting spent yeast cell dry powder as a seed nitrogen source.

Pyruvic acid is an important organic acid and a key industrial raw material. It is widely used in the chemical, agricultural, and food fields. Candida glabrata is the preferred strain for pyruvic acid production. The waste yeast cell for pyruvic acid fermentation with C. glabrata are rich in protein, amino acid, nucleic acid, and vitamins, as potential and cost-effective nitrogen source raw material. In this study, the potential of C. glabrata to produce pyruvic acid using spent yeast cell dry powder was evaluated. When 30 g/L of spray-dried spent yeast cell powder was used as the seed nitrogen source, a high titer of pyruvic acid was obtained. The pyruvic acid production reached 63.4 g/L with a yield of 0.59 g/g in a 5 L bioreactor. After scale-up to a 50 L bioreactor using the fermented spent yeast cell dry powder as a seed nitrogen source, 65.1 g/L of pyruvic acid was harvested, with a yield of 0.61 g/g. This study proposes a promisingapproach for increasing the pyruvic acid titer and reducing the costs.

Bioproduction of quercetin using recombinant thermostable glycosidases from Dictyoglomus thermophilum.

Quercetin is an essential ingredient in functional foods and nutritional supplements, as well as a promising therapeutic reagent. Also, the green technique to produce quercetin via rutin biotransformation is attractive. Genes encoding two thermostable glycosidases from Dictyoglomus thermophilum were cloned and expressed in Escherichia coli, which were applied in rutin biotransformation to produce highly pure quercetin at a high temperature. The production of biocatalysts were scaled up in a 5-L bioreactor, yielding a several-fold increase in total enzyme activity and a quercetin production of 14.22 ± 0.26 g/L from 30 g/L of rutin. Feeding strategies were optimized to boost biomass and enzyme production, achieving an activity of 104,801.80 ± 161.99 U/L for rhamnosidase and 12,637.23 ± 17.94 U/L for glucosidase, and a quercetin yield of 20.24 ± 0.27 g/L from the complete conversion of rutin. This study proposes a promising approach for producing high-quality quercetin in an industrial setting.

Efficient 1,3-dihydroxyacetone biosynthesis in Gluconobacter oxydans using metabolic engineering and a fed-batch strategy.

1,3-Dihydroxyacetone (DHA) is a commercially important chemical and widely used in cosmetics, pharmaceuticals, and food industries as it prevents excessive water evaporation, and provides anti-ultraviolet radiation protection and antioxidant activity. Currently, the industrial production of DHA is based on a biotechnological synthetic route using Gluconobacter oxydans. However, achieving higher production requires more improvements in the synthetic process. In this study, we compared DHA synthesis levels in five industrial wild-type Gluconobacter strains, after which the G. oxydans WSH-003 strain was selected. Then, 16 dehydrogenase genes, unrelated to DHA synthesis, were individually knocked out, with one strain significantly enhancing DHA production, reaching 89.49 g L-1 and 42.27% higher than the wild-type strain. By optimizing the culture media, including seed culture and fermentation media, DHA production was further enhanced. Finally, using an established fed-batch fermentation system, DHA production reached 198.81 g L-1 in a 5 L bioreactor, with a glycerol conversion rate of 82.84%.

Efficient biosynthesis of exopolysaccharide in Candida glabrata by a fed-batch culture.

Polysaccharides are important natural biomacromolecules. In particular, microbial exopolysaccharides have received much attention. They are produced by a variety of microorganisms, and they are widely used in the food, pharmaceutical, and chemical industries. The Candida glabrata mutant 4-C10, which has the capacity to produce exopolysaccharide, was previously obtained by random mutagenesis. In this study we aimed to further enhance exopolysaccharide production by systemic fermentation optimization. By single factor optimization and orthogonal design optimization in shaking flasks, an optimal fermentation medium composition was obtained. By optimizing agitation speed, aeration rate, and fed-batch fermentation mode, 118.6 g L-1 of exopolysaccharide was obtained by a constant rate feeding fermentation mode, with a glucose yield of 0.62 g g-1 and a productivity of 1.24 g L-1 h-1. Scaling up the established fermentation mode to a 15-L fermenter led to an exopolysaccharide yield of 113.8 g L-1, with a glucose yield of 0.60 g g-1 and a productivity of 1.29 g L-1 h-1.