No abstract

The gene encoding D-amino acid oxidase (DAAO) from qêáÖçåçéëáë=î~êá~Äáäáë CBS 4095 has been cloned and expressed in bëÅÜÉêáÅÜá~= Åçäá BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose, which was used as an inducer, and DO concentration appeared to be critical for the cell growth of bK= Åçäá BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50% until specific stage of bacterial growth (OD 600 30-40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD 600 81, respectively. By reproducing these optimized conditions in a 12-m 3 fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD 600 80. © KSBB hÉóïçêÇëW=aJ~ãáåç=~ÅáÇ=çñáÇ~ëÉI=Escherichia coli=_iON=EabPFI=ÑÉêãÉåí~íáçåI=ÇáëëçäîÉÇ=çñóÖÉå= = = = =

Genomic analysis of the clavulanic acid (CA)-high-producing Streptomyces clavuligerus strains, OL13 and OR, developed through random mutagenesis revealed a frameshift mutation in the cas1 gene-encoding clavaminate synthase 1. Overexpression of the intact cas1 in S. clavuligerus OR enhanced the CA titer by approximately 25%, producing ~ 4.95 g/L of CA, over the OR strain in the flask culture. Moreover, overexpression of the pathway-specific positive regulatory genes, ccaR and claR, in the OR strain improved CA yield by approximately 43% (~ 5.66 g/L) in the flask. However, co-expression of the intact cas1 with ccaR-claR did not further improve CA production. In the 7 L fermenter culture, maximum CA production by the OR strain expressing the wild-type cas1 and ccaR-claR reached approximately 5.52 g/L and 6.01 g/L, respectively, demonstrating that reverse engineering or simple rational metabolic engineering is an efficient method for further improvement of industrial strains.

No abstract

Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.