An early attempt was made to construct a xylose-utilizing strain of Z. mo — bilis by Liu et al. [45,46] involving expression of genes for xylose isomerase (XI), xylulokinase (XK) and the xylose transport protein from X. albilineans XA1-1. Although the recombinant strain was shown to possess both XI and XK activity, it was unable to grow on xylose as the sole carbon source. Subse­quently, Feldmann et al. [47] constructed a recombinant strain of Z. mobilis ZM4 (pZY228) that expressed the xylA and xylB genes from Klebsiella pneu­moniae for XI and XK, respectively, and the tktA gene for transketolase (TKT) activity from Escherischia coli. However, this recombinant strain was also un­able to grow on xylose.

On the basis of these earlier studies, Zhang et al. [10] constructed a recom­binant strain that successfully converted xylose to ethanol by expression of a transaldolase (talB) gene from E. coli in addition to those expressing XI, XK and TKT activity. This recombinant strain encoded genes for enzymes both for xylose assimilation (XI, XK) and for completion of the pentose phosphate pathway (TKT, TAL) in Z. mobilis. The transformation of wild-type strains of Z. mobilis with the 14.4 kb expression vector (pZB5) was then shown to fa­cilitate the efficient conversion of xylose to ethanol via a completed pentose phosphate pathway (Fig. 1).

This research at NREL was continued further by Deanda et al. [11] who successfully developed a strain capable of arabinose utilization. This recombi­nant strain harbored a plasmid (pZB206) expressing five heterologous genes from E. coli encoding L-arabinose isomerase (araA), L-ribulokinase (araB), L-ribulose-5-phosphate-4-epimerase (araD), transaldolase (talB) and trans- ketolase (tktA).

In related studies on the development of a xylose utilizing strain of Z. mo­bilis, De Graaf et al. [48] built on the earlier research by Feldmann et al. [47] and introduced a further plasmid (pZY228) into strain ZM4 (pXY228). This former plasmid harbored talB from E. coli thereby facilitating expression of all the requisite additional enzymes in Z. mobilis for xylose assimilation and metabolism.

Although there were differences in their construction of these two recom­binant strains, the metabolic pathway for both recombinant strains resulting from expression of xylA, xylB, tktA and talB was the same as shown pre-

viously in Fig. 1. Xylose enters the Entner-Doudoroff pathway via fructose — 6-phosphate and glyceraldehyde-3-phosphate and is converted into ethanol. The following balance equations represent the metabolism of glucose and xy­lose by these recombinant xylose-metabolizing strains of Z. mobilis.

Glucose + ADP + Pi ^ 2Ethanol + 2CO2 + ATP,

3Xylose + 3ADP + 3Pi ^ 5Ethanol + 5CO2 + 3ATP,

Theoretical ethanol yield = 0.51 gethanol/g sugar (glucose or xylose).

Further studies on recombinant strains created at NREL have involved the construction of integrant xylose-utilizing strains [36-38] and additionally an
integrant xylose/arabinose-utilizing strain designated AX101 [39]. This strain was produced using random insertion and site-specific insertion via homolo­gous recombination.

The specific enzyme activities of the various xylose-utilizing recombinant strains have been determined as a means of identifying possible rate limita­tions. For the strains developed at NREL, the specific activity associated with XI was the lowest [49,50]. However, based on calculation of the metabolic fluxes associated with the each enzyme introduced for xylose metabolism, De Graaf et al. [48] concluded for their strain that the flux associated with XK was significantly lower than that of others. This suggested that a metabolic bot­tleneck may exist in their strain, ZM4 (pZY228) (pZY557 tal), due to the low expression of xylulokinase.

Subsequent kinetic studies involving the over-expression of XK (Fig. 2) in an acetate-resistant mutant of the NREL-derived strain ZM4 (pZB5) showed no increase in the maximum specific growth rate or specific rate of xylose metabolism, although there was evidence of a small increase (0.4 gL-1) in production of xylitol for the over-expressing strain [51]. Further research on

these recombinant strains is likely to focus on potential rate-limiting sites, as well as expression of heterologous enzymes from other microbial sources for increased ethanol tolerance.

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