Cyanobacteria are potentially useful photosynthetic microorganisms for bioremediation under oligotrophic environments. Here, the biphenyl degradation pathway genes of b-proteobacterium Acidovorax sp. strain KKS102 were co-expressed in cyanobacterium Synechocystis sp. PCC6803 cells under control of the photo-inducible psbE promoter. In the KKS102 cells, biphenyl is dioxygenated by bphA1 and bphA2 gene products complex using electrons supplied from NADH via phA4 and bphA3 gene products (BphA4 and BphA3，respectively), and converted to benzoic acid by bphB , BphC and bphD gene products. Unexpectedly, biphenyl was effectively hydroxylated in oligotrophic BG11 medium by co-expressing the bphA3 , bphA1 and bphA2 genes without the bphA4 gene, suggesting that endogenous cyanobacteria-derived protein(s) can supply electrons to reduce BphA3 at the start of the biphenyl degradation pathway. Furthermore, biphenyl was converted to benzoic acid by cyanobacterial cells co-expressing bphA3 , bphA1 , bphA2 , bphB , bphC and bphD . Structural gene-screening using recombinant Escherichia coli cells co-expressing bphA3 , bphA1 , bphA2 , bphB and bphC suggested that petH , which encodes long- and short-type NADP-ferredoxin oxidoreductase isomers (FNR L and FNR S , respectively), and slr0600 , which is annotated as an NADPH-thioredoxin reductase gene in CyanoBase, were BphA3-reducible proteins. Purified FNR L and FNR S , and the slr0600 gene product showed BphA3 reductase activity dependent on NADPH and the reduced form of glutathione, respectively, potentially shedding light on the physiological roles of the slr0600 gene product in cyanobacterial cells. Collectively, our results demonstrate the utility of Synechocystis sp. PCC6803 cells as a host for bioremediation of biphenyl compounds under oligotrophic environments without an organic carbon source.

The coenzyme specificity of enzymes is one of the critical parameters for the engineered production of biological compounds using bacteria. Since NADPH is produced abundantly in photosynthetic organisms, conversion of an NADH-specific enzyme into an NADPH-specific one is a useful approach for the efficient carbon-neutral production of biological compounds in photosynthetic organisms. In the present study, an NADH-specific ferredoxin reductase component, BphA4 of biphenyl dioxygenase BphA from Acidovorax sp. strain KKS102, was changed to an NADPH-dependent form using a method combining structure-based systematic mutations and site-directed random mutagenesis. The resultant CRG mutant, in which Glu 175 -Thr 176 -Gln 177 of an NADH-recognition loop in the wild-type BphA4 was replaced with Cys 175 -Arg 176 -Gly 177 , was highly specific and active for NADPH, and its biochemical and structural properties for NADPH were nearly the same as those of the wild-type BphA4 for NADH. In addition, this mutation project was assessed by a semi-empirical prediction method of mutation effects, and the results suggested that the CRG mutant was one of the best NADPH-specific mutants.