2.1

Increased Substrate Range Through Expression of a Single Heterologous Gene

One of the possible disadvantages of Z. mobilis is that it has a limited carbon substrate range as it can only use the simple C6 sugars glucose, fructose and sucrose. As a result early studies on its genetic manipulation focused on ex­tending its substrate range for ethanol production. Skotnicki et al. [18] first reported high frequency conjugal transfer of plasmids from Escherichia coli and Pseudomonas aeruginosa, and this was followed by expression of the lac Z gene and production of ^-galactosidase in strains of Z. mobilis [21,22]. How­ever, the strain ZM6100 (RP1:Tn 951) derived from this work was shown to progressively lose all plasmid markers in batch culture under non-selective conditions. Subsequently a new strain, ZM6306, was developed in contin­uous culture which showed 100% stability for all plasmid markers when grown without selection pressure. Synthesis of ^-galactosidase was induced in continuous culture by addition of lactose resulting in increased ethanol production and unutilized galactose [23].

Further studies to extend the substrate range were reported which involved the cloning and expression of a в-glucosidase gene from Xanthomonas al — bineans [24] and a-glucosidase gene from a Bacillus sp [44], however enzyme expression levels were low.

2.2