Advances in regulating vitamin K2 production through metabolic engineering strategies.

Vitamin K2 (menaquinone, VK2, MK) is an essential lipid-soluble vitamin that plays critical roles in inhibiting cell ferroptosis, improving blood clotting, and preventing osteoporosis. The increased global demand for VK2 has inspired interest in novel production strategies. In this review, various novel metabolic regulation strategies, including static and dynamic metabolic regulation, are summarized and discussed. Furthermore, the advantages and disadvantages of both strategies are analyzed in-depth to highlight the bottlenecks facing microbial VK2 production on an industrial scale. Finally, advanced metabolic engineering biotechnology for future microbial VK2 production will also be discussed. In summary, this review provides in-depth information and offers an outlook on metabolic engineering strategies for VK2 production.

Enhanced vitamin K2 production by engineered Bacillus subtilis during leakage fermentation.

Menaquinone-7 (MK-7), a valuable member of the vitamin K2 series, is an essential nutrient for humans. It is used for treating coagulation disorders, and osteoporosis, promoting liver function recovery, and preventing cardiovascular diseases. In this study, to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of surfactants on the metabolic synthesis of MK-7 by the mutant strain Bacillus subtilis 168 KO-SinR (BS168 KO-SinR) was analyzed. The scanning electron microscopy and flow cytometry results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. Quantitative real-time PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the electron microscopy results showed that the addition of surfactant changed the permeability of the cell membrane. The research results of this paper can serve as a reference for the industrial development of MK-7 prepared by fermentation.

Poly-γ-Glutamic Acid Production by Engineering a DegU Quorum-Sensing Circuit in Bacillus subtilis.

As a natural biological macromolecule, γ-polyglutamic acid (γ-PGA) plays a significant role in medicine, food, and cosmetic industries owing to its unique properties of biocompatibility, biodegradability, water solubility, and viscosity. Although many strategies have been adopted to increase the yield of γ-PGA in Bacillus subtilis, the effectiveness of these common approaches is not high because the strong viscosity affects cell growth. However, dynamic regulation based on quorum sensing (QS) has been extensively applied as a fundamental tool for fine-tuning gene expression in reaction to changes in cell density without adding expensive inducers. A modular PhrQ-RapQ-DegU QS system is developed based on promoter PD4, which is upregulated by phosphorylated DegU (DegU-P). In this study, first, we analyzed the DegU-based gene expression regulation system in B. subtilis 168. We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters and degU region. Furthermore, we constructed a PhrQ-RapQ-DegU QS system to dynamically control the synthesis of γ-PGA in BS168. Cell growth and efficient synthesis of the target product can be dynamically balanced by the QS system. Our dynamic adjustment approach increased the yield of γ-PGA to 6.53-fold of that by static regulation in a 3 L bioreactor, which verified the effectiveness of this strategy. In summary, the PhrQ-RapQ-DegU QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which can be stretched to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

[A multicenter survey of antibiotic use in very and extremely low birth weight infants in Hunan Province].

OBJECTIVE: To investigate the current status of antibiotic use for very and extremely low birth weight (VLBW/ELBW) infants in neonatal intensive care units (NICUs) of Hunan Province. METHODS: The use of antibiotics was investigated in multiple level 3 NICUs of Hunan Province for VLBW and ELBW infants born between January, 2017 and December, 2017. RESULTS: The clinical data of 1 442 VLBW/ELBW infants were collected from 24 NICUs in 2017. The median antibiotic use duration was 17 days (range: 0-86 days), accounting for 53.0% of the total length of hospital stay. The highest duration of antibiotic use was up to 91.4% of the total length of hospital stay, with the lowest at 14.6%. In 16 out of 24 NICUs, the antibiotic use duration was accounted for more than 50.0% of the hospitalization days. There were 113 cases with positive bacterial culture grown in blood or cerebrospinal fluid, making the positive rate of overall bacterial culture as 7.84%. The positive rate of bacterial culture in different NICUs was significantly different from 0% to 14.9%. The common isolated bacterial pathogens Klebsiella pneumoniae was 29 cases (25.7%); Escherichia coli 12 cases (10.6%); Staphylococcus aureus 3 cases (2.7%). The most commonly used antibiotics were third-generation of cephalosporins, accounting for 41.00% of the total antibiotics, followed by penicillins, accounting for 32.10%, and followed by carbapenems, accounting for 13.15%. The proportion of antibiotic use time was negatively correlated with birth weight Z-score and the change in weight Z-score between birth and hospital discharge (rs=-0.095, -0.151 respectively, P<0.01), positively correlated with death/withdrawal of care (rs=0.196, P<0.01). CONCLUSIONS: Antibiotics used for VLBW/ELBW infants in NICUs of Hunan Province are obviously prolonged in many NICUs. The proportion of routine use of third-generation of cephalosporins and carbapenems antibiotics is high among the NICUs.

[Risk factors for neonatal pulmonary hemorrhage in the neonatal intensive care unit of a municipal hospital].

OBJECTIVE: To investigate the risk factors for neonatal pulmonary hemorrhage (NPH) in the neonatal intensive care unit (NICU) of a municipal hospital, and to provide a basis for the early identification and treatment of NPH. METHODS: A total of 112 neonates who were admitted to the NICU of Shaoyang Central Hospital of Hunan Province and diagnosed with NPH were enrolled as the case group. A nested case-control method was used to select, as a control group (n=224), the neonates who underwent the treatment with an assisted mechanical ventilator and did not experience pulmonary hemorrhage. Univariate analysis and unconditional logistic regression analysis were used to identify the high risk factors for NPH. RESULTS: The univariate analysis showed that compared with the control group, the case group had significantly higher incidence rates of gestational diabetes and cholestasis in mothers, cesarean delivery, gestational age <34 weeks, 5-minute Apgar score ≤5, birth weight <2 500 g, heart failure and disseminated intravascular coagulation (DIC) before the development of NPH, partial pressure of oxygen/fraction of inspired oxygen (oxygenation index, OI) ≤100, and a reduction in mean platelet volume. The multivariate logistic regression analysis showed that DIC, heart failure, and OI ≤100 were independent risk factors for NPH (OR=33.975, 3.975, 1.818 respectively; P<0.05). CONCLUSIONS: Heart failure, OI ≤100, and DIC are risk factors for the development of NPH in the NICU of the municipal hospital.

[Clinical effectiveness of INSURE method in the treatment of neonatal respiratory distress syndrome].

OBJECTIVE: To evaluate the clinical effectiveness and safety of intubation-surfactant-extubation (INSURE) method in the treatment of neonatal respiratory distress syndrome (NRDS), and to investigate its possible mechanisms. METHODS: Sixty-four premature infants, who were admitted for NRDS and treated with pulmonary surfactant from March 2010 to March 2012, were enrolled in the study. They were randomly divided into INSURE (n=32) and conventional mechanical ventilation (CMV) groups (n=32). The two groups were compared in terms of respiratory function, ventilation time, duration of oxygen therapy, complications, and prognosis, as well as expression of interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and serum ferritin (SF). RESULTS: Oxygenation index in the INSURE group was significantly higher than in the CMV group at 48 hours after treatment (P<0.05). Compared with the CMV group, the INSURE group showed significantly lower incidence of ventilator-associated pneumonia (VAP) and significantly shorter duration of oxygen therapy (P<0.05 for all comparisons). There were no significant differences in ventilation time and the incidence of pneumothorax, intracranial hemorrhage, necrotizing enteroolitis, bronchopulmonary dysplasia, and pneumorrhagia between the two groups (P>0.05). The levels of TNF-α and SF were significantly lower in the INSURE group than in the CMV group at 6, 24, 48, and 72 hours after treatment (P<0.05), while the level of IL-10 was significantly higher in the INSURE group than in the CMV group (P<0.05). CONCLUSIONS: INSURE method can improve the oxygenation function of the lung, decrease the incidence of VAP and shorten the duration of oxygen therapy in neonates with NRDS, which is probably due to the fact that this method can reduce the production of TNF-α and SF and inhibit the decrease of IL-10.

[Clinical application of high-frequency oscillatory ventilation for the treatment of neonatal pneumothorax].

OBJECTIVE: To evaluate the clinical effect of high-frequency oscillatory ventilation (HFOV) for the treatment of neonatal pneumothorax. METHODS: Retrospective analysis was performed on the clinical data of 23 neonates with pneumothorax who received HFOV from January 2007 to June 2011. Of the 23 cases, 19 cases were treated by HFOV as soon as they were diagnosed with pneumothorax, and 4 cases were treated by HFOV after the occurrence of pneumothorax during conventional mechanical ventilation (CMV) or continuous positive airway pressure (CPAP) ventilation. Another 23 neonates with pneumothorax who received CMV in the same period were selected as controls. The HFOV group and control group were compared with respect to oxygenation index (OI) and arterial/alveolar oxygen tension ratio (a/APO(2)) before and after 1, 12, 24, and 48 hours of ventilation as well as mechanical ventilation time, gas absorption time, complication, and prognosis. RESULTS: Both groups showed significantly decreased OI and significantly increased a/APO(2) after ventilation (P<0.05). Compared with the control group, the HFOV group had significantly lower OI and significantly higher a/APO(2) after 1, 12, 24, and 48 hours of ventilation (P<0.05). Mechanical ventilation and gas absorption times were significantly shorter in the HFOV group than in the control group (P<0.05). Twenty-two cases were cured in the HFOV group and 21 in the control group. Each group included one case of ventilator-associated pneumonia that was later cured with antibiotics. CONCLUSIONS: Compared with CMV, HFOV performs better in improving the pulmonary oxygenation function of neonates with pneumothorax and can shorten both mechanical ventilation time and gas absorption time without increasing the incidence of adverse effects.