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.

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.

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.

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.

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.

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.

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.

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.

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.

Efficient Production of 2,5-Diketo-D-gluconic Acid by Reducing Browning Levels During Gluconobacter oxydans ATCC 9937 Fermentation.

D-Glucose directly generates 2-keto-L-gulonic acid (2-KLG, precursor of vitamin C) through the 2,5-diketo-D-gluconic acid (2,5-DKG) pathway. 2,5-DKG is the main rate-limiting factor of the reaction, and there are few relevant studies on it. In this study, a more accurate quantitative method of 2,5-DKG was developed and used to screen G. oxydans ATCC9937 as the chassis strain for the production of 2,5-DKG. Combining the metabolite profile analysis and knockout and overexpression of production strain, the non-enzymatic browning of 2,5-DKG was identified as the main factor leading to low yield of the target compound. By optimizing the fermentation process, the fermentation time was reduced to 48 h, and 2,5-DKG production peaked at 50.9 g/L, which was 139.02% higher than in the control group. Effectively eliminating browning and reducing the degradation of 2,5-DKG will help increase the conversion of 2,5-DKG to 2-KLG, and finally, establish a one-step D-glucose to 2-KLG fermentation pathway.

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%.

High dose and hepatobiliary dysfunction are associated with hand-foot syndrome in patients with lymphoma using pegylated liposomal doxorubicin: a retrospective study.

PURPOSE: In clinical practice, the risk factors for pegylated liposomal doxorubicin-related hand-foot syndrome remain unclear. The purpose of this study was to determine the risk factors associated with hand-foot syndrome in patients with lymphoma using pegylated liposomal doxorubicin. METHODS: This retrospective descriptive analysis included patients with lymphoma who received PLD treatment (≥ 2 cycles of chemotherapy) at our cancer centre and had complete follow-up data from January 2016 to February 2020. Clinical, laboratory data, as well as the occurrence of hand-foot syndrome (incidence, location, severity, impact on follow-up chemotherapy) were obtained. The primary end point was the incidence of hand-foot syndrome, which was classified according to the "Common Terminology Criteria for Adverse Events" (Version 4.0). A multivariate logistic regression analysis was used to identify risk factors for hand-foot syndrome in patients with lymphoma using doxorubicin liposomes. FINDINGS: A total of 167 patients met the inclusion criteria. 58 developed HFS, of which 45 occurred after the second course of chemotherapy. The multivariate logistic regression analysis revealed that a dose increase of pegylated liposomal doxorubicin and hepatobiliary dysfunction were significantly associated with an increased risk for hand-foot syndrome(dose intensity, OR = 6.479; 95% CI, 1.431-29.331 [P = 0.015]; history of gallstones, OR = 14.144, 95% CI, 1.512-132.346 [P = 0.020]; alanine aminotransferase, OR = 1.194, 95% CI, 1.056-1.350 [P = 0.005]; aspartate aminotransferase, OR = 1.162, 95% CI, 1.010-1.336 [P = 0.035]; and glutamine transpeptidase, OR = 1.092, 95% CI, 1.016-1.174 [P = 0.018]). IMPLICATIONS: These findings contribute to the risk assessment of patients with lymphoma before using pegylated liposomal doxorubicin. For patients with the above risk factors, preventive measures should be taken in advance to reduce the incidence of HFS.

Efficient Production of Scleroglucan by Sclerotium rolfsii and Insights Into Molecular Weight Modification by High-Pressure Homogenization.

Scleroglucan is a non-ionic water-soluble polysaccharide, and has been widely used in the petroleum, food, medicine and cosmetics industries. Currently, scleroglucan is mainly produced by Sclerotium rolfsii. A higher level of scleroglucan (42.0 g/L) was previously obtained with S. rolfsii WSH-G01. However, the production of scleroglucan was reduced despite a higher glucose concentration remaining. Additionally, the molecular weight of scleroglucan was large, thus restricted its application. In this study, by adjusting the state of seeds inoculated, the degradation issue of scleroglucan during the fermentation process was solved. By comparing different fed-batch strategies, 66.6 g/L of scleroglucan was harvested by a two-dose fed-batch mode, with 53.3% glucose conversion ratio. To modify the molecular weight of scleroglucan, a combination method with HCl and high-pressure homogenization treatment was established. Finally, scleroglucan with molecular weight of 4.61 × 105 Da was obtained. The developed approaches provide references for the biosynthesis and molecular weight modification of polysaccharides.

Potential of anammox process towards high-efficient nitrogen removal in wastewater treatment: Theoretical analysis and practical case with a SBR.

Anammox process has been widely applied in the wastewater nitrogen removal for its high rate and low cost. However, few researches reported the process potential in treating low-strength nitrogen wastewater. In this study, a sequencing batch reactor (SBR) was taken to explore the feasibility of low-strength nitrogen wastewater treatment by anammox process in theory and practice. The practical operation indicated that the effluent with satisfactory quality (1.90 ± 0.70 mg-TN·L-1) could be achieved, when the SBR was fed with low-strength nitrogen influent (6.20 ± 0.45 mg-NH4+-N·L-1 and 7.96 ± 0.59 mg-NO2--N·L-1). The hydraulic retention time (HRT), nitrogen removal efficiency, nitrogen removal rate (NRR) and hydraulic loading rate of SBR were 5.42 h, 86.5%, 0.054 kg-N·m-3·d-1 and 4.43 m3·m-3·d-1 during the 79-day operation, respectively. The theoretical analysis revealed the potential of anammox SBR. When SBR is stably operated, the maximum NRR would be 0.062 kg-N·m-3·d-1 if the effluent nitrogen was required to be as low as 3 mg·L-1. The NRR value is feasible for engineering. However, considering the lower specific substrates utilization rate in practice, the maximum stable NRR was calibrated and found inefficient afterwards. In order to improve the potential of anammox process, the reactors without back mixing and with periodic bioaugmentation should be taken in priority for the engineering applications. In particular, the bioaugmentation frequency and single addition amount were calculated as 7 d and 0.3 g-VSS·L-1, respectively. The results may provide guidance for the development of high-efficient and stable nitrogen removal process under low-strength condition.

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.

Genome-based analysis to understanding rapid resuscitation of cryopreserved anammox consortia via sequential supernatant addition.

Simple cryopreservation of anaerobic ammonium-oxidation (anammox) consortia has become a promising preservation technology for the fast start-up of the anammox process. Here, we use genome-resolved metagenomics and metatranscriptomics to understand of the microbial interaction in a simple and effective resuscitation process for long-term cryopreserved anammox consortia by sequential addition of anammox SBR supernatant. Performance results showed that sequential addition of anammox supernatant significantly reduced the resuscitation time of the granule-based anammox process from 40 to 20 days. Genome-centric metagenomics were used to recover 19 high-quality draft genomes of anammox and heterotrophic bacteria. Comparative metatranscriptomic analysis was conducted to examine the gene expression of Candidatus Kuenenia stuttgartiensis, the dominant anammox bacterium, and heterotrophic bacteria to better understand their potential interactions. Proteobacteria-affiliated bacteria found in the supernatant were highly active in producing the secondary metabolites molybdopterin cofactor and folate which are needed for growth of the auxotrophic anammox bacteria. In addition, the significantly higher expression levels of hzsA and CO2-fixtion genes in the Candidatus Kuenenia genome indicated the anammox bacteria were likely more active and growing faster after sequential anammox supernatant addition during the resuscitation process. The resuscitation treatment pulse assays confirmed that sequential addition of supernatant was an effective way for the rapid resuscitation of anammox consortia. Our findings offer the first evidence of cross-feeding during the rapid resuscitation of cryopreserved anammox consortia.

High Throughput Screening Platform for a FAD-Dependent L-Sorbose Dehydrogenase.

2-Keto-L-gulonic acid (2-KLG) is the direct precursor for the production of L-ascorbic acid (L-Asc) on industrial scale. Currently, the production of L-Asc in the industry is a two-step fermentation process. Owing to many unstable factors in the fermentation process, the conversion rate of L-sorbose to 2-KLG has remained at about 90% for many years. In order to further improve the production efficiency of 2-KLG, a FAD-dependent sorbose dehydrogenase (SDH) has been obtained in our previous research. The SDH can directly convert L-sorbose to 2-KLG at a very high efficiency. However, the enzyme activity of the SDH is relatively low. In order to further improve the enzyme activity of the SDH, a high throughput screening platform the dehydrogenase is essential. By optimizing the promoter, host and sorbosone dehydrogenase (SNDH), knockout of the aldosterone reductases and PTS related genes, a reliable platform for high-throughput screening of more efficient FAD-dependent SDH has been established. By using the high-throughput screening platform, the titer of the 2-KLG has been improved by 14.1%. The method established here could be useful for further enhancing the FAD-dependent SDH, which is important to achieve the efficient one-strain-single-step fermentation production of 2-KLG.

Progressive Microbial Community Networks with Incremental Organic Loading Rates Underlie Higher Anaerobic Digestion Performance.

Although biotic interactions among members of microbial communities have been conceived to be crucial for community assembly, it remains unclear how changes in environmental conditions affect microbial interaction and consequently system performance. Here, we adopted a random matrix theory-based network analysis to explore microbial interactions in triplicate anaerobic digestion (AD) systems, which is widely applied for organic pollutant treatments. The digesters were operated with incremental organic loading rates (OLRs) from 1.0 g volatile solids (VS)/liter/day to 1.3 g VS/liter/day and then to 1.5 g VS/liter/day, which increased VS removal and methane production proportionally. Higher resource availability led to networks with higher connectivity and shorter harmonic geodesic distance, suggestive of more intense microbial interactions and quicker responses to environmental changes. Strikingly, a number of topological properties of microbial network showed significant (P < 0.05) correlation with AD performance (i.e., methane production, biogas production, and VS removal). When controlling for environmental parameters (e.g., total ammonia, pH, and the VS load), node connectivity, especially that of the methanogenic archaeal network, still correlated with AD performance. Last, we identified the Methanothermus, Methanobacterium, Chlorobium, and Haloarcula taxa and an unclassified Thaumarchaeota taxon as keystone nodes of the network.IMPORTANCE AD is a biological process widely used for effective waste treatment throughout the world. Biotic interactions among microbes are critical to the assembly and functioning of the microbial community, but the response of microbial interactions to environmental changes and their influence on AD performance are still poorly understood. Using well-replicated time series data of 16S rRNA gene amplicons and functional gene arrays, we constructed random matrix theory-based association networks to characterize potential microbial interactions with incremental OLRs. We demonstrated striking linkage between network topological features of methanogenic archaea and AD functioning independent of environmental parameters. As the intricate balance of multiple microbial functional groups is responsible for methane production, our results suggest that microbial interaction may be an important, previously unrecognized mechanism in determining AD performance.