Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-05-10
2002-08-13
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S252300, C435S320100
Reexamination Certificate
active
06432686
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the field of molecular biology and the use of recombinant organisms for the production of 1,3-propanediol. More specifically it describes the expression of cloned genes that affect the intracellular transport of vitamin B
12
in conjunction with genes that effectively convert a carbon substrate to 1,3-propanediol.
BACKGROUND
1,3-Propanediol is a monomer having utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds.
A variety of chemical routes to 1,3-propanediol are known. For example, 1,3-propanediol is prepared 1) from ethylene oxide over a catalyst in the presence of phosphine, water, carbon monoxide, hydrogen and an acid; 2) by the catalytic solution phase hydration of acrolein followed by reduction; or 3) from hydrocarbons such as glycerol, reacted in the presence of carbon monoxide and hydrogen over catalysts having atoms from Group VIII of the periodic table. Although it is possible to generate 1,3-propanediol by these methods, they are expensive and generate waste streams containing environmental pollutants.
It has been known for over a century that 1,3-propanediol can be produced from the fermentation of glycerol. Bacterial strains able to produce 1,3-propanediol have been found, for example, in the groups Citrobacter, Clostridium, Enterobacter, Ilyobacter, Klebsiella, Lactobacillus, and Pelobacter. In each case studied, glycerol is converted to 1,3-propanediol in a two-step, enzyme-catalyzed reaction sequence. In the first step, a dehydratase catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HP) and water (Equation 1). In the second step, 3-HP is reduced to 1,3-propanediol by a NAD
+
-linked oxidoreductase (Equation 2).
Glycerol→3-HP+H
2
O (Equation 1)
3-HP+NADH+H
+
→1,3-Propanediol+NAD
+
(Equation 2)
The 1,3-propanediol is not metabolized further and, as a result, accumulates in high concentration in the media. The overall reaction consumes a reducing equivalent in the form of a cofactor, reduced &bgr;-nicotinamide adenine dinucleotide (NADH), which is oxidized to nicotinamide adenine dinucleotide (NAD
+
).
The production of 1,3-propanediol from glycerol is generally performed under anaerobic conditions using glycerol as the sole carbon source and in the absence of other exogenous reducing equivalent acceptors. Under these conditions, in strains of Citrobacter, Clostridium, and Klebsiella, for example, a parallel pathway for glycerol operates which first involves oxidation of glycerol to dihydroxyacetone (DHA) by a NAD
+
- (or NADP
+
-) linked glycerol dehydrogenase (Equation 3). The DHA, following phosphorylation to dihydroxyacetone phosphate (DHAP) by a DHA kinase (Equation 4), becomes available for biosynthesis and for supporting ATP generation via, for example, glycolysis.
Glycerol+NAD
+
→DHA+NADH+H
+
(Equation 3)
DHA+ATP→DHAP+ADP (Equation 4)
In contrast to the 1,3-propanediol pathway, this pathway may provide carbon and energy to the cell and produces rather than consumes NADH.
In
Klebsiella pneumoniae
and
Citrobacter freundii
, the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon. The dha regulons from Citrobacter and Klebsiella have been expressed in
Escherichia coli
and have been shown to convert glycerol to 1,3-propanediol.
The biological production of 1,3-propanediol requires glycerol as a substrate for a two step sequential reaction in which a dehydratase enzyme (typically a coenzyme B
12
-dependent dehydratase) converts glycerol to an intermediate, 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by a NADH-(or NADPH) dependent oxidoreductase. These cofactor requirements are complex and necessitate the use of a whole cell catalyst for an industrial process incorporating this reaction sequence for the production of 1,3-propanediol. A process for the production of 1,3-propanediol from glycerol using an organism containing a coenzyme B
12
-dependent diol dehydratase is described in U.S. Pat. No. 5,633,362 (Nagarajan et al.). However, the process is not limited to the use of glycerol as feedstock. Glucose and other carbohydrates are suitable substrates and, recently, these substrates have been shown to be substrates for 1,3-propanediol production. Carbohydrates are converted to 1,3-propanediol using mixed microbial cultures where the carbohydrate is first fermented to glycerol by one microbial species and then converted to 1,3-propanediol by a second microbial species. U.S. Pat. No. 5,599,689 (Haynie et al.). For reasons of simplicity and economy, a single organism able to convert carbohydrates to 1,3-propanediol is preferred. Such an organism is described in U.S. Pat. No. 5,686,279 (Laffend et al.).
Some bacteria, such as Salmonella or Klebsiella, are able to synthesize coenzyme B
12
to enable a diol or glycerol dehydratase to operate, but other species must transport B
12
from outside of the cell. The term “B
12
” is used to refer collectively to coenzyme B
12;
derivatives of coenzyme B
12
where the upper axial 5′-deoxyadenosyl ligand is replaced with another ligand (for example, an aquo-, cyano- or methyl group); and the radical species, cob(II)alamin.
B
12
transport into bacteria presents two major problems. First, the B
12
molecule is too large for passage through outer membrane porins, thus requiring a specific outer membrane transport system. Second, owing to the scarcity of B
12
in the environment, the outer membrane transport system must have a high affinity for B
12
and move it into the periplasm for subsequent transport by another system across the inner membrane. For
E. coli
, which is unable to synthesize the corrin ring of B
12
, an external supply of B
12
is required for growth under certain conditions. These requirements may be modest; when a functional 5-methyltetrahydrofolate-homocysteine methyltransferase (MetH) is present ~25 B
12
molecules (methylcobalamin) are required and ~500 coenzyme B
12
molecules are needed for ethanolamine ammonia-lyase dependent growth.
Several proteins are required for the transport process. The 66 kDa outer membrane protein BtuB serves as the high affinity (K
d
=0.3 nM) receptor for adenosyl-, aquo-, cyano- and methyl cobalamins and the corresponding cobinamides. When grown in the absence of B
12
or at low levels (<1 nM) ~200 copies of BtuB are present per cell. However, the growth of cells in media containing high levels of B
12
(>0.1 uM) represses synthesis of BtuB, and even at levels of 5 nM uptake activities are repressed 80-90%. Unlike Salmonella, the
E. coli
BtuB is not repressed by aerobiosis. Transport into the periplasm requires the interaction of BtuB with a 26 kDa inner membrane protein TonB in an energy-dependent process that also requires co-transport of calcium. In fact, the high affinity binding of B
12
to BtuB is calcium dependent and there is evidence for a reciprocal B
12
dependent calcium binding site with a K
d
for calcium of ~30 nM at pH 6.6 at saturating levels of B
12
. This affinity for calcium decreases with decreasing pH. TonB uses proton motive force to drive a structural alteration needed for transport. In the absence of TonB, B
12
penetrates die outer membrane with very low efficiency. TonB also energizes outer-membrane transport systems for iron, including the FepA and FhuA systems. Thus BtuB competes with these systems for TonB activity. In the absence of protein synthesis, the rate of B
12
transport decreases with a half life of ~20 min and is attributable to a loss of TonB activity. Transfer of B
12
from BtuB to the periplasmic binding protein is poorly characterized and may involve a protein encoded by the btuF locus, at least in Salmonella.
Transport across the inner membrane is
Bulthuis Ben A.
Gatenby Anthony A.
Trimbur Donald E.
Whited Gregory M.
E. I. du Pont de Nemours and Company
Monshipouri Maryam
Prouty Rebecca E.
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