Isobutanol is a promising next-generation biofuel with a high energy density and is considered a sustainable aviation fuel precursor. Klebsiella pneumoniae is an industrial workhorse microorganism used for the production of various chemicals. An endogenous isobutanol synthesis pathway, interconnected with the branched-chain amino acid and 2,3-butanediol biosynthesis pathways, has been identified in K. pneumoniae. However, isobutanol production by K. pneumoniae is inefficient due to the substantial formation of by-products. In this study, we enhanced isobutanol synthesis by systematically engineering K. pneumoniae to reduce by-product accumulation. The synthesis of 2-hydroxyisovalerate was reduced by knocking out panE. Formate synthesis was eliminated by disrupting pflB. The production of ethanol, acetate, and succinate was reduced by deleting aceE. Pyruvate accumulation was alleviated by overexpressing budB. In batch fermentation, the engineered strain produced 13.97 g/L of 2-ketoisovalerate, a key intermediate in the isobutanol pathway, with a conversion ratio of 0.43 mol/mol glucose. Fed-batch fermentation further increased the 2-ketoisovalerate titer to 26.63 g/L after 54 h of cultivation. The decarboxylation of 2-ketoisovalerate to isobutyraldehyde is catalysed by IpdC. Overexpression of the ipdC T290L variant shifted the major product from 2-ketoisovalerate to isobutanol, resulting in a batch fermentation titer of 5.32 g/L isobutanol and a conversion ratio of 0.28 mol/mol glucose. Fed-batch fermentation further increased the final isobutanol titer to 10.32 g/L after 52 h of cultivation. This study establishes an efficient strategy for high-level production of isobutanol or 2-ketoisovalerate in K. pneumoniae.

1,3-Propanediol (1,3-PDO) is a bulk chemical that can be produced by Klebsiella pneumoniae using glycerol as a substrate. In the 1,3-PDO synthesis pathway, part of the glycerol is oxidised to maintain intracellular NADH balance. Consequently, the theoretical maximum yield of 1,3-PDO from glycerol was lower than 1 mol/mol. In this study, engineered K. pneumoniae strains were constructed to direct all glycerol toward 1,3-PDO synthesis, with NADH being supplied through the catabolism of glucose. However, glycerol utilisation was inhibited in the presence of glucose. To alleviate this carbon catabolite repression (CCR), ptsG and crr were individually knocked out. The dha pathway is responsible for 1,3-PDO synthesis. Key genes in the oxidation branch of this pathway, including dhaK, dhaL, dhaD, and gldA, were knocked out to block this pathway. However, the expression of the dha operon was impaired in these strains, resulting in low 1,3-PDO production. In contrast, knocking out dhaM, which encodes a subunit of dihydroxyacetone kinase II, effectively blocked the glycerol oxidation pathway while maintaining the activity of the dha operon. Additionally, glpK was knocked out to block the sn-glycerol-3-phosphate formation from glycerol. Glucose to glycerol with the ratio of 0.5:1 mol/mol was the optimal value for 1,3-PDO production by K. pneumoniae ∆dhaM∆ptsG∆glpK, leading to a balance of NADH generation and consumption. Microaerobic conditions were favourable for 1,3-PDO production than anaerobic or aerobic conditions. In fed-batch fermentations, this strain produced 58.6 g/L of 1,3-PDO after 70 h, achieving a yield of 0.93 mol/mol glycerol, 2 mol/mol glucose, 0.63 mol/mol substrate. A highly efficient 1,3-PDO production technology that using glycerol and glucose as co-substrates was established.

Klebsiella pneumoniae is a commonly known 2,3-butanediol producer. 2,3-Butanediol synthesis and branched-chain amino acid (BCAA) synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. Those two pathways do not interfere with each other in the wild-type strain. Knocking out budA (encoding α-acetolactate decarboxylase) blocks the 2,3-butanediol synthesis pathway. Meanwhile, metabolites of the BCAA synthesis pathway (valine, 2-ketoisovalerate, 2,3-dihydroxyisovalerate and 2-hydroxyisovalerate) are accumulated. However, the mechanism underlying the metabolite changes resulting from the inactivation of budA remains unclear. In this study, both ex vivo and in vitro experiments were conducted to elucidate this mechanism. Kinetic parameters of BudA and acetohydroxy acid isomeroreductase (IlvC) were determined. BudA has a higher affinity toward α-acetolactate and has a higher catalytic constant (Km = 3.66 mM, kcat = 7.8 s-1) compared to IlvC (Km = 17.98 mM, kcat = 0.68 s-1). ex vivo experiments showed that IlvC activities were not influenced by knocking out budA and vice versa. IlvC activities were improved in the cells in which ilvC was overexpressed, but this did not lead to the accumulation of metabolites of the BCAA synthesis pathway. The activities of IlvC in the cell were not affected by the accumulation of 2,3-dihydroxyisovalerate, 2-ketoisovalerate, or valine in the broth. These results indicated that the competitiveness of BudA and IlvC in the cell determines the metabolites distribution between those two pathways. The inactivation of BudA and intact IlvC led to the exceeded α-acetolactate flow into the BCAA synthesis pathway, which caused the accumulation of metabolites of the BCAA synthesis pathway.

Methanol, which can come from methane or carbon dioxide, is a valuable renewable one-carbon (C1) feedstock for the production of biofuels and food chemicals. A new method was developed to create a multienzyme complex by combining methanol dehydrogenase (Mdh), 3-hexulose-6-phosphate synthase (Hps), and 6-phospho-3-hexuloseisomerase (Phi) in equal parts using SpyTag/Catcher and DogTag/Catcher systems. This self-assembly of multiple enzymes improves the conversion of methanol to fructose-6-phosphate (F6P) and was used to engineer a synthetic methylotroph from B. subtilis 168 that could efficiently utilize methanol. Various metabolic regulations related to key carbon pathways were tested and integrated to boost methanol consumption in this engineered strain. The final strain, B. subtilis SM6, could consume 3.87 g/L of methanol, marking the highest level of coutilization with xylose to date. The strategies employed in this research optimized the distribution of metabolic flow for formaldehyde and xylose, offering valuable insights for future studies on synthetic methylotrophs.

Gene expression in electroactive bacteria (EAB), which sense electrochemical stimuli and elicit genetic response, can be regulated through electrogenetic methods. Although differential gene expression patterns have been observed in biofilms grown on polarized electrodes, little is known about the molecular mechanisms that sense electrochemical potential and bring about the genetic regulation in response to this potential. Here, we observe that the molecular weight of poly gamma glutamic acid (gamma-PGA) produced in Bacillus subtilis PB5760 biofilms changed with the applied electrochemical potential. The application of 0.2 and 0.4 V vs. Ag/AgCl reduced the molecular weight of gamma-PGA in the biofilm from 3800 to 4070 kDa at open circuit potential (OCP) to 1040-1590 kDa and 2170-2730 kDa, respectively. Under the same conditions, the relative levels of expression of pgdS, a gene that codes for a gamma-PGA-specific endo-hydrolase in cells growing at OCP, 0.2 and 0.4 V vs. Ag/AgCl were 1.12 +/- 0.57, 2.74 +/- 0.54, 1.61 +/- 0.57, respectively, which correlates with the molecular weight reduction. This is the first biotechnological application of electrogenetics for producing gamma-PGA with a tunable molecular weight. Results show the feasibility of electrogenetics approach for gene regulation in EAB.

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-gamma-glutamic acid (gamma-PGA)-producing strains is an essential strategy for improving gamma-PGA production. Bacillus tequilensis BL01 Delta pgdS Delta ggt Delta sucA Delta gudB:P43-ppc-pyk-gdhA for the efficient synthesis of gamma-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of gamma-PGA by 2.18-fold (3.24 +/- 0.22 g/L) compared to that of B. tequilensis BL01 Delta pgdS Delta ggt Delta sucA Delta gudB (1.02 +/- 0.11 g/L). To further improve the titer of gamma-PGA and decrease the production of byproducts, three enzymes (Ppc, Pyk, and AceE) were assembled to a complex using SpyTag/Catcher pairs. The results showed that the gamma-PGA titer of the assembled strain was 31.31% higher than that of the unassembled strain. To further reduce the production cost, 25.73 +/- 0.69 g/L gamma-PGA with a productivity of 0.48 g/L/h was obtained from cheap molasses. This work provides new metabolic engineering strategies to improve the production of gamma-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of gamma-PGA from molasses.

As artificial extracellular matrix-like materials, silk-elastin-like protein (SELP) hydrogels, with excellent mechanical properties, high tunability, favorable biocompatibility, and controlled degradability, have become an important candidate in biomedical materials. In this study, SELP is composed of silk-like (GAGAGS) and elastin-like (GXGVP) tandem repeats, in which X residues are set as tyrosine and lysine. Furthermore, SELP polymers are prepared via SpyTag/SpyCatcher. To explore a gentler and more efficient enzymatic crosslinking method, an innovative method was invented to apply laccase to catalyze the formation of SELP hydrogels. Gelation could be successfully achieved in 2-5 min . SELP hydrogels mediated by laccase had the characteristic of low swelling rate, which could maintain a relatively stable shape even when immersed in water, and hence had the potential to be further developed into injectable biomaterials. Additionally, SELP hydrogels cross-linked by laccase showed excellent biocompatibility verified by L929 and HEK 293 T cells with cell viability >93.8 %. SELP hydrogels also exhibit good properties in sustained drug release and cell encapsulation in vitro. This study demonstrates a novel method to construct SELP hydrogels with excellent biocompatibility and expands the possibility of SELP-based material applications in biomedical fields.

Poly γ-glutamic acid (γ-PGA) is a promising biopolymer for various applications. For glutamic acid-independent strains, the titer of γ-PGA is too low to meet the industrial demand. In this study, we isolated a novel γ-PGA-producing strain, Bacillus tequilensis BL01, and multiple genetic engineering strategies were implemented to improve γ-PGA production.First, the one-factor-at-a-time method was used to investigate the influence of carbon and nitrogen sources and temperature on γ-PGA production. The optimal sources of carbon and nitrogen were sucrose and (NH4)2SO4 at 37 °C, respectively. Second, the sucA, gudB, pgdS, and ggt genes were knocked out simultaneously, which increased the titer of γ-PGA by 1.75 times. Then, the titer of γ-PGA increased to 18.0 ± 0.3 g/L by co-overexpression of the citZ and pyk genes in the mutant strain. Furthermore, the γ-PGA titer reached 25.3 ± 0.8 g/L with a productivity of 0.84 g/L/h and a yield of 1.50 g of γ-PGA/g of citric acid in fed-batch fermentation. It should be noted that this study enables the synthesis of low (1.84 × 105 Da) and high (2.06 × 106 Da) molecular weight of γ-PGA by BL01 and the engineering strain.The application of recently published strategies to successfully improve γ-PGA production for the new strain B. tequilensis BL01 is reported. The titer of γ-PGA increased 2.17-fold and 1.32-fold compared with that of the wild type strain in the flask and 5 L fermenter. The strain shows excellent promise as a γ-PGA producer compared with previous studies. Meanwhile, different molecular weights of γ-PGA were obtained, enhancing the scope of application in industry.

Strain KSNA2 T , a Gram-negative, moderately halophilic, facultatively anaerobic, motile, rod-shaped bacterium, was isolated from the surface-sterilized stem tissue of a beach morning glory ( Cafystegia soldanella ) plant in Chuja Island, Jeju-do, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences revealed that strain KSNA2 T formed a distinct lineage within the family Enterobacteriaceae , with the highest 16S rRNA gene sequence similarity to Izhakiella australiensis KCTC 72143 (96.2%) and Izhakiella capsodis KCTC 72142 T (96.0%), exhibited 95.5–95.9% similarity to other genera in the family Enterobacter-iaceae and Erwiniaceae. Conserved signature indels analysis elucidated that strain KSNA2 T was delimited into family Enterobacteriaceae. KSNA2 genome comprises a circular chromosome of 5,182,800 bp with 56.1% G + C content Digital DNA-DNA relatedness levels between strain KSNA2 T and 18 closely related species were 19.3 to 21.1%. Average nucleotide identity values were between 72.0 and 76.7%. Growth of strain KSNA2 T was observed at 4 to 45°C (optimum, 25°C) and pH 5.0 to 12.0 (optimum, pH 7.0) in the presence of 0 to 11% (w/v) NaCl (optimum, 0–7%). The major cellular fatty acids (> 10%) were C 16 :o followed by summed feature 8 (C 18 ω7 c and/or C 18 ω 6c) , summed feature 3 (C 16:1 ω 7c and/or C 16:1 ω6 c) , C 17:0 cyclo , and C 14:0 . The major isoprenoid quinone was ubiquinone-8 (Q-8). With combined phylogenetic, genomic, phenotypic, and chemotaxo-nomic features, strain KSNA2 T represents a novel species of a new genus in the family Enterobacteriaceae , for which the name Jejubacter calystegiae gen. nov., sp. nov. is proposed. The type strain is KSNA2 T (= KCTC 72234 T = CCTCC AB 2019098 T ).

Gellan gum is a water-soluble exopolysaccharide, it has applications in the food, pharmaceutical and chemical industries. In this study, a gellan gum producing strain was isolated from rice root, and this strain was identified be the species of Sphingomonas azotifigens. The Plackett-Burman design was applied to investigate the main factors affecting gellan gum production by S. azotifigens GL-1 in a molasses and cheese whey based medium; the medium compositions were optimized by response surface methodology. The optimum cheese whey based medium consisted of cheese whey 68.34 g/L, Na2HPO4 14.58 g/L and KH2PO4 7.66 g/L, and the maximum gellan gum production that using this medium was 33.75 +/- 1.55 g/L. 14.75 +/- 0.65 g/L gellan gum was obtained with an optimized molasses medium, which consisted of molasses 50 g/L, Na2HPO4 9.71 g/L and KH2PO4 5.92 g/L. The molecular weight of gellan gum obtained from two medias were 1.06 x 10(6) and 0.89 x 10(6) Da, respectively. The cheese whey-derived gellan gum showed a higher rhamnose, lower glucuronic acid and higher glycerate content compared to the molasses-derived gellan gum. S. azotifigens GL-1 has a high gellan gum production capacity in a cheap medium suggesting it has great potential as an industrial gellan gum producer.

Abstract Background 1,3-propanediol (1,3-PDO) is the most widely studied value-added product that can be produced by feeding glycerol to bacteria, including Lactobacillus sp. However, previous research reported that L. reuteri only produced small amounts and had low productivity of 1,3-PDO. It is urgent to develop procedures that improve the production and productivity of 1,3-PDO. Results We identified a novel L. reuteri CH53 isolate that efficiently converted glycerol into 1,3-PDO, and performed batch co-fermentation with glycerol and glucose to evaluate its production of 1,3-PDO and other products. We optimized the fermentation conditions and nitrogen sources to increase the productivity. Fed-batch fermentation using corn steep liquor (CSL) as a replacement for beef extract led to 1,3-PDO production (68.32 ± 0.84 g/L) and productivity (1.27 ± 0.02 g/L/h) at optimized conditions (unaerated and 100 rpm). When CSL was used as an alternative nitrogen source, the activity of the vitamin B12-dependent glycerol dehydratase (dhaB) and 1,3-propanediol oxidoreductase (dhaT) increased. Also, the productivity and yield of 1,3-PDO increased as well. These results showed the highest productivity in Lactobacillus species. In addition, hurdle to 1,3-PDO production in this strain were identified via analysis of the half-maximal inhibitory concentration for growth (IC50) of numerous substrates and metabolites. Conclusions We used CSL as a low-cost nitrogen source to replace beef extract for 1,3-PDO production in L. reuteri CH53. These cells efficiently utilized crude glycerol and CSL to produce 1,3-PDO. This strain has great promise for the production of 1,3-PDO because it is generally recognized as safe (GRAS) and non-pathogenic. Also, this strain has high productivity and high conversion yield.

Background Poly-γ-glutamic acid (γ-PGA) is a promising biopolymer and has been applied in many fields. Bacillus siamensis SB1001 was a newly isolated poly-γ-glutamic acid producer with sucrose as its optimal carbon source. To improve the utilization of carbon source, and then molasses can be effectively used for γ-PGA production, 60 cobalt gamma rays was used to mutate the genes of B. siamensis SB1001. Results Bacillus siamensis IR10 was screened for the production of γ-PGA from untreated molasses. In batch fermentation, 17.86 ± 0.97 g/L γ-PGA was obtained after 15 h, which is 52.51% higher than that of its parent strain. Fed-batch fermentation was performed to further improve the yield of γ-PGA with untreated molasses, yielding 41.40 ± 2.01 g/L of γ-PGA with a productivity of 1.73 ± 0.08 g/L/h. An average γ-PGA productivity of 1.85 g/L/h was achieved in the repeated fed-batch fermentation. This is the first report of such a high γ-PGA productivity. The analysis of the enzyme activities showed that they were affected by the carbon sources, enhanced ICDH and GDH, and decreased ODHC, which are important for γ-PGA production. Conclusion These results suggest that untreated molasses can be used for economical and industrial-scale production of γ-PGA by B. siamensis IR10.

Bacillus subtilisnaturally produces large amounts of 2,3-butanediol (2,3-BD) as a main by-product during poly-gamma-glutamic acid (gamma-PGA) production. 2,3-BD is a promising platform chemical in various industries, and co-production of the two chemicals has great economic benefits. Co-production of gamma-PGA and 2,3-BD by a newly isolatedB. subtilisCS13 was investigated here. The fermentation medium and culture parameters of the process were optimized using statistical methods. It was observed that sucrose,l-glutamic acid, ammonium citrate, and MgSO4 center dot 7H(2)O were favorable for gamma-PGA and 2,3-BD co-production at culture pH of 6.5 and 37 degrees C. An optimal medium composed of 119.8 g/L sucrose, 48.8 g/Ll-glutamic acid, 21.1 g/L ammonium citrate, and 3.2 g/L MgSO4 center dot 7H(2)O was obtained by response surface methodology (RSM). The results show that the titers of gamma-PGA and 2,3-BD reached 27.8 +/- 0.9 g/L at 24 h and 57.1 +/- 1.3 g/L at 84 h with the optimized medium, respectively. gamma-PGA and 2,3-BD production byB. subtilisCS13 was significantly enhanced in fed-batch fermentations. gamma-PGA (36.5 +/- 1.1 g/L, productivity of 1.22 +/- 0.04 g/L/h) and 2,3-BD concentrations (119.6 +/- 2.8 g/L, productivity of 2.49 +/- 0.66 g/L/h) were obtained in the optimized medium with feeding sucrose. The co-production of 2,3-BD and gamma-PGA provides a new perspective for industrial production of gamma-PGA and 2,3-BD.

The genus Izhakiella was established and designated as a member of the family Enterobacteriaceae in 2016. Although the taxonomical classification of most members in this family has been relatively resolved after two reclassifications in 2016 and 2017, the classification of the genus Izhakiella remains ambiguous. In this study, a polyphasic approach was used to provide evidence supporting the fact that the genus Izhakiella should no longer be considered a member of Enterobacteriaceae and proposes its reclassification into the family Erwiniaceae. The phylogenetic tree of type species in the families Enterobacteriaceae and Erwiniaceae based on the sequences of the 16S rRNA gene, rpoB housekeeping gene, and the whole-genome comprising the 92 core genes revealed that the genus Izhakiella forms a phylogenetic lineage within the family Erwiniaceae. The average nucleotide identity (ANI) value of the type species with genus Izhakiella was found to be higher for the family Erwiniaceae than that for the family Enterobacteriaceae. Notably, 12 conserved signature indels (CSIs) that are exclusively shared among the Erwiniaceae clade members were found in the type strains of the genus Izhakiella. Based on these analyses, this study suggests the reclassification of I. capsodis and I. australiensis into the family Erwiniaceae.

Poly-gamma-glutamic acid (gamma-PGA) is a retaining agent; it has applications in the food, medicine, agriculture, cosmetics and wastewater treatment industries. Most of the gamma-PGA producing strains belong to the genus Bacillus. This study reports on a novel gamma-PGA producing species. Bacillus siamensis SB1001 was screened and isolated from organically cultivated soybeans exhibiting a high gamma-PGA producing ability. The fermentation medium and culture parameters for gamma-PGA production by Bacillus siamensis SB1001 were optimized by statistical methods. The sucrose, L-glutamic acid and dipotassium phosphate in the medium were shown to be the significant factors of the gamma-PGA production, and the optimum medium obtained consisted of the following: 106.86 g/L sucrose, 69.84 g/L L-glutamic acid and 2.39 g/L dipotassium phosphate. Using the optimized medium, 25.22 g/L gamma-PGA were produced with a productivity of 1.05 g/L/h. The gamma-PGA obtained had a molecular weight of 7.9 x 10(5) Da and a polydispersity index of 2.34, and the ratio of D-/L-glutamic acid was 89.71%:10.29%. To the best of our knowledge, this is the first report of gamma-PGA production by B. siamensis strain. B. siamensis SB1001 has great potential as an industrial gamma-PGA producer.