One barrel of oil contains 81% fuels (46% gasoline, 9% jet fuel, 26% diesel) and 19% petrochemical co-products (lubricants, paints, solvents, adhesives, wax, greases, tar, asphalt, paraffin wax and more). For cellulosic biofuels (ethanol and butanol) to become commercially profitable, the efforts should not only be to improve the efficiency and economics of biofuels production, but also to mimic the petro-industry model by self-producing of high value co-products including microbial heterologous cellulases and recombinant bio-pharmaceuticals in the non-food portions of cellulosic bioenergy crops. Cellulases are needed to convert plant cellulose into fermentable sugars for biofuels. At present microbial cellulase mix are produced at cost of $1.0 per gallon of ethanol. This dissertation will present co-production of biologically active Acidothermus cellulolyticus (E1) endo-cellulase, Trichoderma reesei 1,4-â-cellobiohydrolases I (CBH I) exo-cellulase and cow rumen Butyrivibrio fibrisolvens microbial cellobiase in corn (corn) plant endoplasmic reticulum, apoplast (cell wall area) and vacuole respectively. It is estimated that in this research the heterologous E1 and cellobiase are respectively produced at 0.8 and 1.2 kilograms per ton of corn silage. Another high-value co-product is human saliva secretory leukocyte protease inhibitor (hsSLPI) which is well known as an anti-HIV protein and as the reason that HIV is not transmitted via human saliva. Biologically active human SLPI can not be produced in E. coli due to SLPI's anti-microbial activity. This dissertation will present production of biologically active human SLPI in plant apoplast for preclinical testing. Pretreatment of lignocellulosic biomass is required to breakdown lignin in order to expose crop cellulose to cellulases. Depending of the method used, pretreatment processes cost $1.0 to $2.15 per gallon of ethanol. This dissertation will also present down-regulation of corn stover (leaf and stem) lignin content via RNA interference (RNA1) of corn Cinnamoyl-CoA reductase (CCR). Such lignin down-regulation concomitantly increased corn crystalline cellulose. In addition, enzymatic hydrolysis of ammonia fiber expansion (AFEX)-pretreated corn stover of low lignin transgenic corn lines had a higher percent glucan conversion rate. Following the petro-industry model, co-production of high value recombinant co-products, and lignin down-regulation of bioenergy crops are expected to expedite commercialization of cellulosic biofuels.
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