By Pseudomonas

PHAs are biocompatible and biodegradable thermoplastic polyesters that are made by many bacteria when these are grown on ample supplies of carbon sources and

Renewable Resources Collage
FIGURE 3.5 Freeze fracture electron micrograph of P. oleovorans cells growing in a two-liquid-phase medium containing 85% aqueous phase and 15% octane. (EM photo by Jaap Kingma and Hans Preusting, University of Groningen, The Netherlands, 1990.)

limiting amounts of nitrogen, sulfur, phosphorus, or magnesium (de Smet et al. 1983b; Hartmann et al. 2006; Kessler and Witholt 1998; Lageveen et al. 1988; Zinn et al. 2001). The composition of the PHAs formed under such conditions depends on the growth substrates, culture conditions, and the specificity of the PHA-synthe-sizing enzymes (Huisman et al. 1989; Lageveen et al. 1988). Pseudomonas oleovorans produces two PHA polymerases that show a preference for C7 to C9 3-hydroxyacyl (CoA) monomers, probably formed by beta-oxidation of fatty acids and oxidized alkanes or by de novo fatty acid biosynthesis during growth on carbohydrates (Ren et al. 2005a, 2005b). Many other (CoA activated) fatty acyl derivatives can also be utilized as cosubstrates by P. oleovorans, permitting the biosynthesis of a wide range of PHA copolymers (Kessler et al. 2001; van der Walle et al. 2001). PHA chains form intracellular granules of 200- to 500-nm diameter. Figure 3.5 shows some of these granules as they develop in the cytoplasm of P. oleovorans. PHAs are of interest to synthetic and polymer chemists because it is still difficult, if not impossible, to produce synthetic biodegradable polymers, and because the monomers may be useful chiral synthons for further chemical synthesis (de Roo et al. 2002). The bacterial polymer granules are also of interest to polymer chemists and materials scientists as doping materials in polymer blends because it is difficult to create small synthetic polymer granules.

The production of useful amounts of a variety of PHAs with specific chemical, physical, and mechanical properties requires adequate intracellular PHA accumulation as well as simultaneous high cell-mass production on the different substrates used (Kellerhals 1999; Schmid et al. 1998b).

3.3.2 Small Molecules: Organic Chemistry with Whole-Cell Biocatalysts

We have examined the oxidation of aliphatic (Chang et al. 2000; Duetz et al. 2003; Smits et al. 1999, 2003; van Beilen et al. 2001, 2002, 2003a, 2003b; Whyte et al. 2002) and aromatic (Buhler et al. 2000; Li et al. 2002; Panke et al. 2000; Wubbolts et al. 1994) compounds by bacterial enzymes such as monooxygenases, oxidases, and dehydrogenases.

To extend the number of potentially interesting biocatalysts, we have developed a strain library of alkane- and aromatic-compound-oxidizing organisms as well as a rapid screening system to identify desired enzymatic activities (Duetz et al. 2000; Duetz and Witholt 2001, 2004).* Using this library, we have explored the genetics of alkane oxidation by a large number of environmental microorganisms in more detail (Smits et al. 2002; van Beilen et al. 2002; van Beilen 2003), which provides a basis for the development of additional biocatalytic systems for the oxidation of alkanes and alkane derivatives.

We have also used this strain library to develop biocatalysts for the oxidation and hydroxylation of heterocyclic alkanes, including substituted pyrrolidines and pyrrolidinones (Li 1999; Li et al. 2001, 2002). Similarly, we have studied the conversion of D-limonene, widely available as a by-product of the orange juice industry, to (+) trans-carveol (Duetz et al. 2001b, 2003) and perillyl alcohol (van Beilen et al. 2005). Examples of biooxidations of aromatic compounds include the epoxidation of styrene to styrene epoxide (Panke et al. 1999; Wubbolts 1994) and the production of benzaldehyde (Buhler et al. 2003a, 2003b).

The enzymes involved in the above bioconversions generally require cofactors (microtimamide aolenine olinucleotide (phosphate) [NAD(P)], flavin aolenine dinu-cleotide [FAD], pyrroloquinoline quinone [PQQ]) or electron-transfer proteins, which is the reason why such reactions must be carried out in whole cells (Duetz et al. 2001a). The performance of these enzymes depends on the state of the host cells (Chen et al. 1996; de Smet et al. 1983; Staijen et al. 2000). Thus, we have studied the characteristics of intact cells in two-liquid-phase media, and we have determined how these affect the performance of relevant enzymes (Favre-Bulle and Witholt 1992). Many of these studies have utilized P. oleovorans, the organism first isolated by Lee and Chandler in Houston, which, as might be expected given the source of this organism (Lee 1941), performs very nicely in two-liquid-phase media (Witholt et al. 1990).

3.4 INDUSTRIAL POTENTIAL OF TWO-LIQUID-PHASE biocatalysis

Biocatalysis has long been taken to be a promising tool in synthesis because of the expected regio- and enantioselectivity of enzymes. Synthetic catalysts with steadily improving selectivities are meanwhile emerging, based in part on lessons

* See www.enzyscreen.com for more information on the strain library and screening equipment and approaches.

learned from protein structure and enzyme catalytic mechanisms (Ligtenbarg et al. 2003).

What then is the industrial and economic potential of biocatalytic processes? Are they limited to expensive fine chemicals only, or can they be expected to spread into other areas? What is the lower limit of production and processing costs per unit product manufactured in a two-liquid-phase bioconversion system?

Answers to these questions require more complete information than available at present. A partial answer is that several major chemical companies, including BASF, DSM, and Lonza, have begun to explore two-liquid-phase bioconversions of apolar compounds in the past one or two decades (Schmid et al. 2001). Together with DuPont, Dow, Cargill, and several other companies, these companies have defined white biotechnology as the next frontier of the developing biotechnology sector (see footnote 1).

Another partial answer is that there is considerable experience with large-scale installations for the production of single-cell protein (SCP) by microorganisms from various bulk carbon sources, including agricultural wastes, starches, and oils, both vegetable and mineral (Scrimshaw 1966). Until 20 years ago, SCP was seen as a potentially interesting food supplement (Kharatyan 1978; Kihlberg 1972). Work on SCP led to the development of large-scale two-liquid-phase systems for the utilization of «-paraffins and mineral oil by yeast, both by Western oil companies and by companies in the former Soviet Union and former East Germany (Calvert 1976). SCP ultimately failed to develop because the economics of the process were disappointing and the need for food from oil was less pressing than believed in the 1960s and 1970s. The experience with these installations will, however, be useful in the development of industrial-scale two-liquid-phase bio-catalytic processes with organic compounds.

Thus, as biocatalyst- and synthetic-catalyst-based processes improve, differences in process performance or product quality may, in many cases, fade away. Increasingly, multistep synthesis routes combine both of these approaches (Schoemaker et al. 2003), with the choice of specific catalytic steps being guided by product life-cycle analysis (Saling et al. 2005; Sugiyama et al. 2006) and the economics of overall reaction sequences.

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