A prokaryotic-like polycistronic expression system was developed in Pichia pastoris and Acremonium chrysogenum. With simplicity and higher effectiveness, this polycistronic expression system is suitable for introducing multiple genes or entire metabolic pathways into industrial eukaryotic microorganisms.

A rapid in situ immobilization process was developed based on conventional separation technique of immobilized metal affinity chromatography (IMAC) and was studied in the case of d-amino acid oxidase (DAAO) with binding–enhancing Heli-tag (His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly). A recombinant Escherichia coli strain JM105 (Δase)/pGEMK-R-DAAO-Heli was successfully constructed to synthesize chimeric protein DAAO-Heli. Without additional purification procedure, the tagged enzyme DAAO-Heli could be directly immobilized to EP-IDA-Ni2+ support with purity of 90 % and DAAO activity of over 70 U/g support. Experimental results showed that the immobilized DAAO-Heli was 73 times more thermally stable than free enzyme. Besides, it remained 67 % of initial activity after 100 cycles of batch catalysis and its operational stability was improved 36 times than that of the previously IMAC-immobilized DAAO-His. Furthermore, the epoxy (EP) support could be easily recovered and repeatedly used with simple steps, which could reduce the immobilization costs significantly.

To express high-active soluble d-amino acid oxidase (DAAO), a constitutive plasmid that is regulated by a native hydantoinase promoter (PHase), was constructed. A d-amino acid oxidase gene (dao) was ligated with the PHase and cloned into pGEMKT to constitutively express protein of DAAO without the use of any inducer such as isopropyl β-d-1-thiogalactopyranoside which is poisonous to the cells and environment. The ribosome binding site region, host strain, and fermentation conditions were optimized to increase the expression level. When cultivated in a 5-m3 fermenter, the enzyme activity of JM105/pGEMKT-R-DAAO grown at 37 °C was found to be 32 U/mL and increase 16-fold over cells of BL21(DE3)/pET-DAAO grown at 28 °C. These results indicate the success of our approaches to overproducing DAAO in soluble form in Escherichia coli.

Two categories of expression systems with different promoters and substitutive ribosome binding site region (RBS) were constructed in order to improve the soluble expression of d-amino acid oxidase (DAAO) basing on the hypothesis that the optimal promoter and suitable RBS would provide the recombinant expression system with better matched expression rate, which served as key factor to the heterogenous synthesis of soluble protein. The results showed that with rational promoter recombination and delicate RBS substitution, significant increase of DAAO activity (20-fold) was obtained for strain JM105/pGEMKT-Tac-R-DAAO over the previously constructed strain BL21(DE3)/pET-DAAO. Furthermore, similar optimization strategy proved feasible in the active expression of other enzymes such as glutaryl-7-aminocephalosporanic acid acylase (GCA) and N-Carbamyl-d-amino acid amidohydrolase (d-Case).Highlights► With RBS substitution, soluble DAAO expression was increased over 2-fold under T7 promoter. ► Constitutive expression system with Tac promoter and optimal RBS improved DAAO activity 20-fold. ► Expression rate optimization proved effective in the expression of other emzymes GCA and D-case.

•We integrate T7 RNA Polymerase gene and T7 Promoter into a single plasmid.•We use small antisense RNA to regulate T7 RNA Polymerase expression on plasmid.•We construct a novel T7 expression system independent on DE3 lysogenic hosts.•We prove the expression ability of this system in 4 non-DE3 E. coli strains.•We enhance the protein expression in an industrial wild-type strain by this system.It is desirable to build a universal and efficient protein expression system for wild-type prokaryotic strains in biotechnology industry and the outstanding T7 expression system could be a good candidate. However, the current utilization of T7 system depends on the specific DE3 lysogenic hosts, which severely limits its application in wild-type strains. In this study, a host-independent T7 expression system without relying on DE3 lysogenic hosts to provide T7 RNA Polymerase was developed. T7 RNA Polymerase gene (Gene1) and T7 Promoter were successfully integrated into a single plasmid with the regulation of proper antisense RNA to limit T7 RNA Polymerase expression at a non-lethal level. This host-independent T7 expression system realized efficient protein expression in 4 non-DE3 Escherichia coli strains and a wild-type Sinorhizobium strain TH572.

By use of PCR, the genes encoding d-carbamoylase from A. radiobacter TH572 were cloned in plasmid pET30a and transformed into Escherichia coli BL21 (DE3) to overexpress d-carbamoylase. However, almost all of the protein remained trapped in inclusion bodies. To improve the expression of the properly folded active enzyme, a constitutive plasmid of pGEMT-DCB was constructed using the native hydantoinase promoter (PHase) whose optimal length was confirmed to 209 bp. Furthermore, the RBS region in the downstream of PHase was optimized to increase the expression level, so the plasmid pGEMT-R-DCB was constructed and transformed into E. coli strain Top10F′. The enzyme activity of Top10F′/pGEMT-R-DCB grown at 37 °C was found to be 0.603 U/mg (dry cell weight, DCW) and increase 58-fold over cells of BL21 (DE3) harboring the plasmid pET-DCB grown at 28 °C.