Search

Bioproduction of renewable chemicals is considered as an urgent solution for fossil energy crisis. However, despite tremendous efforts, it is still challenging to generate microbial strains that can produce target biochemical to high levels. Here, we report an example of biosynthesis of high-value and easy-recoverable derivatives built upon natural microbial pathways, leading to improvement in bioproduction efficiency. By leveraging pathways in solventogenic clostridia for co-producing acyl-CoAs, acids and alcohols as precursors, through rational screening for host strains and enzymes, systematic metabolic engineering-including elimination of putative prophages, we develop strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl butyrate. Techno-economic analysis results suggest the economic competitiveness of our developed bioprocess. Our principles of selecting the most appropriate host for specific bioproduction and engineering microbial chassis to produce high-value and easy-separable end products may be applicable to other bioprocesses.

BACKGROUND: Clostridium saccharoperbutylacetonicum N1-4 (HMT) is a strictly anaerobic, spore-forming Gram-positive bacterium capable of hyper-butanol production through the well-known acetone-butanol-ethanol fermentation process. Recently, five putative RRNPP-type QSSs (here designated as QSS1 to QSS5) were predicted in this bacterial strain, each of which comprises a putative RRNPP-type regulator (QssR1 to QssR5) and a cognate signaling peptide precursor (QssP1 to QssP5). In addition, both proteins are encoded by the same operon. The functions of these multiple RRNPP-type QSSs are unknown. RESULTS: To elucidate the function of multiple RRNPP-type QSSs as related to cell metabolism and solvent production in N1-4 (HMT), we constructed qssR-deficient mutants ΔR1, ΔR2, ΔR3 and ΔR5 through gene deletion using CRISPR-Cas9 and N1-4-dcas9-R4 (with the QssR4 expression suppressed using CRISPR-dCas9). We also constructed complementation strains by overexpressing the corresponding regulator gene. Based on systematic characterization, results indicate that QSS1, QSS2, QSS3, and QSS5 positively regulate the sol operon expression and thus solvent production, but they likely negatively regulate cell motility. Consequently, QSS4 might not directly regulate solvent production, but positively affect cell migration. In addition, QSS3 and QSS5 appear to positively regulate sporulation efficiency. CONCLUSIONS: Our study provides the first insights into the roles of multiple RRNPP-type QSSs of C. saccharoperbutylacetonicum for the regulation of solvent production, cell motility, and sporulation. Results of this study expand our knowledge of how multiple paralogous QSSs are involved in the regulation of essential bacterial metabolism pathways.

BACKGROUND: Sucrose is an naturally abundant and easily fermentable feedstock for various biochemical production processes. By now, several sucrose utilization pathways have been identified and characterized. Among them, the pathway consists of sucrose permease and sucrose phosphorylase is an energy-conserving sucrose utilization pathway because it consumes less ATP when comparing to other known pathways. Bacillus amyloliquefaciens NK-1 strain can use sucrose as the feedstock to produce poly-γ-glutamic acid (γ-PGA), a highly valuable biopolymer. The native sucrose utilization pathway in NK-1 strain consists of phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-P hydrolase and consumes more ATP than the energy-conserving sucrose utilization pathway. RESULTS: In this study, the native sucrose utilization pathway in NK-1 was firstly deleted and generated the B. amyloliquefaciens 3Δ strain. Then four combination of heterologous energy-conserving sucrose utilization pathways were constructed and introduced into the 3Δ strain. Results demonstrated that the combination of cscB (encodes sucrose permease) from Escherichia coli and sucP (encodes sucrose phosphorylase) from Bifidobacterium adolescentis showed the highest sucrose metabolic efficiency. The corresponding mutant consumed 49.4% more sucrose and produced 38.5% more γ-PGA than the NK-1 strain under the same fermentation conditions. CONCLUSIONS: To our best knowledge, this is the first report concerning the enhancement of the target product production by introducing the heterologous energy-conserving sucrose utilization pathways. Such a strategy can be easily extended to other microorganism hosts for reinforced biochemical production using sucrose as substrate.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production. RESULTS: In this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain. CONCLUSIONS: We proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher γ-PGA production than the NK-1 strain. This work proposed a new strategy for improving the target product in microbial cell factories.

Megaregion has emerged as a global urban form, typically based on the polycentric strategy to enhance regional development. How to measure megaregional spatial structure and discriminate different roles of cities has become increasingly important to enrich the knowledge of the formation of a megaregion. Meanwhile, various indices have been used to identify vital nodes in the field of complex network. Which indices, however, are suitable for megaregion analysis remain unsolved. To address this requirement, this study first reviewed the typical indices for identifying vital nodes in the complex network theory, and pointed out that in a weighted city network scenario, weighted degree centrality, hub & authority score, and S-core decomposition (which represent network centrality, connectivity, and structures, respectively) are suitable for analyzing megaregional spatial structures. Then, we explored the city hierarchies and spatial structure in Guangdong Province, China, using the three indices. The hierarchical structure of the weighted city network in Guangdong Province had been identified using S-core decomposition. From the perspective of polycentric structure, Guangzhou and Shenzhen have the strongest node degrees and strength of mobility flows, while the Guangzhou-Dongguan-Shenzhen corridor has been identified via the hub & authority score which is designed to evaluate the connectivity in a weighted network. Moreover, we conducted a comparison analysis of three indices. The findings of this study not only enrich the understanding of city hierarchies and the structure of a megaregion, but also highlight that although various indices are available, they should be applied selectively in accordance with the study context. [ABSTRACT FROM AUTHOR]

Based on panel data of 31 provinces in China from 2000 to 2019, this study explores the complex dynamic interaction between scientific and technological innovation, industrial structure transformation and economic development by building PVAR model. After GMM estimation, impulse response function analysis and variance decomposition analysis, the following conclusions are drawn: Technological innovation has a significant promoting effect on economic development, and economic development can also promote technological innovation; Technological innovation has a positive impact on industrial structure transformation, while industrial structure transformation has a slight negative impact on technological innovation; Economic development can lead to industrial structure transformation, but the impact of industrial structure transformation on economic development is not significant. [ABSTRACT FROM AUTHOR]