Complete hydrolysis of cellulose has been a key characteristic of biomass technology because of the limitation of industrial production hosts to use cellodextrin, the partial hydrolysis product of cellulose. Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin of the enzymatic hydrolysis (via endoglucanase and exoglucanase) of cellulose. Conversion of cellobiose to glucose is executed by β-glucosidase. The complete extracellular hydrolysis of celluloses has several critical barriers in biomass technology. An alternative bioengineering strategy to make the bioprocessing less challenging is to engineer microbes with the abilities to hydrolyze and assimilate the cellulosic-hydrolysate cellodextrin. Microorganisms engineered to metabolize cellobiose rather than the monomeric glucose can provide several advantages for lignocellulose-based biorefineries. This review describes the recent advances and challenges in engineering efficient intracellular cellobiose metabolism in industrial hosts. This review also describes the limitations of and future prospectives in engineering intracellular cellobiose metabolism.

Background

As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors.

Results

Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number.

Conclusions

The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.