Dexin Wang

Background: 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. Results: 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. Conclusions: A highly efficient 1,3-PDO production technology that using glycerol and glucose as co-substrates was established.

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.

Poly-γ-glutamic acid (γ-PGA) is a promising biopolymer for various applications. In this study, we isolated a novel γ-PGA-producing strain, Bacillus halotolerans F29. The one-factor-at-a-time method was used to investigate the influence of carbon sources, nitrogen sources, and culture parameters on γ-PGA production. The optimal carbon and nitrogen sources were sucrose and (NH₄)₂SO₄, respectively. The optimal culture conditions for γ-PGA production were determined to be 37 °C and a pH of 5.5. Response surface methodology was used to determine the optimum medium components: 77.6 g/L sucrose, 43.0 g/L monosodium glutamate, and 2.2 g/L K₂HPO₄. The γ-PGA titer increased significantly from 8.5 ± 0.3 g/L to 20.7 ± 0.7 g/L when strain F29 was cultivated in the optimized medium. Furthermore, the γ-PGA titer reached 50.9 ± 1.5 g/L with a productivity of 1.33 g/L/h and a yield of 2.23 g of γ-PGA/g of L-glutamic acid with the optimized medium in fed-batch fermentation. The maximum γ-PGA titer reached 45.3 ± 1.1 g/L, with a productivity of 1.06 g/L/h when molasses was used as a carbon source. It should be noted that the γ-PGA yield in this study was the highest of all reported studies, indicating great potential for the industrial production of γ-PGA.

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-γ-glutamic acid (γ-PGA)-producing strains is an essential strategy for improving γ-PGA production. Bacillus tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-ppc-pyk-gdhA for the efficient synthesis of γ-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of γ-PGA by 2.18-fold (3.24 ± 0.22 g/L) compared to that of B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB (1.02 ± 0.11 g/L). To further improve the titer of γ-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 γ-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 γ-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 γ-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of γ-PGA from molasses.

Background: 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. Results: 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. Conclusion: 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.

Background: Bacillus subtilis CS13 was previously isolated for 2,3-butanediol (2,3-BD) and poly-γ-glutamic acid (γ-PGA) co-production. When culturing this strain without L-glutamic acid in the medium, 2,3-BD is the main metabolic product. 2,3-BD is an important substance and fuel with applications in the chemical, food, and pharmaceutical industries. However, the yield and productivity for the B. subtilis strain should be improved for more efficient production of 2,3-BD. Results: The medium composition, which contained 281.1 g/L sucrose, 21.9 g/L ammonium citrate, and 3.6 g/L MgSO₄·7H₂O, was optimized by response surface methodology for 2,3-BD production using B. subtilis CS13. The maximum amount of 2,3-BD (125.5 ± 3.1 g/L) was obtained from the optimized medium after 96 h. The highest concentration and productivity of 2,3-BD were achieved simultaneously at an agitation speed of 500 rpm and aeration rate of 2 L/min in the batch cultures. A total of 132.4 ± 4.4 g/L 2,3-BD was obtained with a productivity of 2.45 ± 0.08 g/L/h and yield of 0.45 g_2,3-BD/g_sucrose by fed-batch fermentation. The meso-2,3-BD/2,3-BD ratio of the 2,3-BD produced by B. subtilis CS13 was 92.1%. Furthermore, 89.6 ± 2.8 g/L 2,3-BD with a productivity of 2.13 ± 0.07 g/L/h and yield of 0.42 g_2,3-BD/g_sugar was achieved using molasses as a carbon source. Conclusions: The production of 2,3-BD by B. subtilis CS13 showed a higher concentration, productivity, and yield compared to the reported generally recognized as safe 2,3-BD producers. These results suggest that B. subtilis CS13 is a promising strain for industrial-scale production of 2,3-BD.

Bacillus subtilis naturally produces large amounts of 2,3-butanediol (2,3-BD) as a main by-product during poly-γ-glutamic acid (γ-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 γ-PGA and 2,3-BD by a newly isolated B. subtilis CS13 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 MgSO₄·7H₂O were favorable for γ-PGA and 2,3-BD co-production at culture pH of 6.5 and 37 °C. An optimal medium composed of 119.8 g/L sucrose, 48.8 g/L l-glutamic acid, 21.1 g/L ammonium citrate, and 3.2 g/L MgSO₄·7H₂O was obtained by response surface methodology (RSM). The results show that the titers of γ-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. γ-PGA and 2,3-BD production by B. subtilis CS13 was significantly enhanced in fed-batch fermentations. γ-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 γ-PGA provides a new perspective for industrial production of γ-PGA and 2,3-BD. Key points • A strategy for co-production of γ-PGA and 2,3-BD was developed. • The culture parameters for the co-production of γ-PGA and 2,3-BD were studied. • RSM was used to optimize the medium for γ-PGA and 2,3-BD co-production. • 36.5 g/L γ-PGA and 119.6 g/L 2,3-BD were obtained from the optimum medium in fed-batch fermentation.