A pyruvate-centered metabolic regulation mechanism for the enhanced expression of exogenous genes in Escherichia coli.

A novel Escherichia coli efficient expression system had been constructed in our previous study. The system was based on the overexpression of endogenous genes prpD and malK to enhance the expression of exogenous genes. In this study, a general regulatory mechanism of prpD and malK was first revealed through transcriptome analysis and many experimental verifications. We surprisingly proved that overexpression of malK could up-regulate the expression of prpD and propanoate metabolism, which leads to increased expression of exogenous genes. More importantly, the overexpression of prpD or malK could arouse a complex set of pyruvate-centered metabolic networks that mainly increase the energy supply (ATP), by-product recycling (acetate), and amino acids for the efficient expression of exogenous genes. This novel theory for promoting the efficient expression of exogenous genes will be useful in a wide range of fields. It also opens up a new perspective on the regulation of metabolism in E. coli cells.

Multiplex genetic engineering improves endogenous expression of mesophilic α-amylase gene in a wild strain Bacillus amyloliquefaciens 205.

A wild strain Bacillus amyloliquefaciens 205 was screened for its high activity of α-amylase. A mesophilic α-amylase encoding gene amyE-205 was revealed and analyzed by genome sequencing. In order to facilitate plasmid transformation to strain 205, an interspecific plasmid transformation method was improved with 5-13 times higher in transformants than that of electronic transformation. A series of CRISPR genome editing tools have been successfully constructed for gene knockout, transcript repression and activation in 205 genome. At this basis, sporulation related genes spo0A and spoIIAC were knockout and suppressed with CRISPR/Cas9 and CRISPR/dCas9 respectively. The double knockout strain 205spo- was eliminated sporulation with 22.8% increasing of α-amylase activity. The optimal binding site G8 for dCas9-ω has been confirmed in the transcript activation. When amyE-205 was over-expressed with high copy plasmid pUC980-2, its whole upstream sequences containing G8 were also cloned. Whereafter, dCas9-ω was used to activate amyE-205 expression both at genome and plasmid. The final engineered strain 205PG8spo- achieved 784.3% promotion on α-amylase activity than the starting strain 205. The novel genetic tool box containing an efficient interspecific transformation method and functional CRISPR systems, superadded the multiplex regulation strategies used in strain modification would be also applicative in many Bacillus species.

Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside.

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

High copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980.

BACKGROUND: pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli-B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. RESULTS: The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. CONCLUSION: The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

High-level extracellular production of an alkaline pectate lyase in E. coli BL21 (DE3) and its application in bioscouring of cotton fabric.

A high heterologous expression of an alkaline pectate lyase (APL) pelNK93I in E. coli was obtained through optimizing the lactose feeding and fed-batch fermentation. The highest soluble APL activity produced by E. coli BL21 (pET22b-pelNK93I) was 10,181 U/mL which is the highest level so far. On this basis, to improve the extracellular yield of APL, optimized glycine feeding was used to achieve elevated extracellular production of pelNK93I. The highest extracellular APL activity produced by E. coli BL21 (pET22b-pelNK93I) was 6357 U/mL which was also relatively higher than that in previous reports. The final productivity of APL was 282.8 U/mL/h in the fermentation of E. coli BL21 (pET22b-pelNK93I) in a 10 L fermenter. Thus the current study has provided a cost-effective method for the over-expression and preparation of alkaline pectate lyase pelNK93I for its industrial applications. Moreover, pelNK93I (4 U/mL) used for bioscouring increased cottonseed husk removal and radial capillary effect of cotton fabric by 37.63% and 47.06%, respectively, making it a promising enzyme in green textile technology.

Ethanol effects on the overexpression of heterologous catalase in Escherichia coli BL21 (DE3).

A novel method involving ethanol-induced increase in the heterologous recombinant protein expression in E. coli cells was commonly used in recent studies. However, the detailed mechanism of this method is still to be revealed. This work used comparative transcriptomic analysis and numerous experiments to uncover the mechanism of ethanol effects on the expression of heterologous catalase in the recombinant strain E. coli BL21 (pET26b-katA). The key regulatory genes malK and prpD were found to have the most significant effects on the expression of heterologous catalase. Thus, the maltose ABC transporter and carbon metabolism from propanoate metabolism to citrate cycle were found to be the main regulatory pathways activated by ethanol to enhance the synthesis of heterologous proteins. Based on these mechanisms, a universally applicable E. coli expression host strain for improving the expression of heterologous proteins might be constructed.