Engineering Neurospora crassa for cellobionate production directly from cellulose without any enzyme addition

In a cellulosic biorefinery, the cellulase enzymes needed for hydrolysis are one of the major contributors to high processing costs, while the hydrolysis product, cellobiose, has strong inhibition to the cellulases. In this study, we report engineering recombinant Neurospora crassa strains which are able to produce cellobionate, an organic acid, from cellulose without any enzyme addition. Recombinant strains were constructed by heterologously expressing laccase genes from different sources under different promoters in N. crassa F5Δmus-51Δace-1Δcre-1ΔndvB which has six out of seven β-glucosidase (bgl), two transcription factor (cre1 and ace-1), and the cellobionate phosphorylase (ndvB) genes deleted. The strain expressing laccase from Botrytis aclada under a copper metallothionein promoter (HL10) produced the highest laccase activity. N. crassa HL10 produced 47.4mM cellobionate from cellulose without any enzyme addition. The yield of cellobionate from hydrolyzed cellulose was about 94.5%. Conversion of cellobiose to cellobionate improved cellulose conversion and increases product yield.