The document discusses the commercial synthesis of l-ascorbic acid (vitamin C) from d-glucose, highlighting inefficiencies in the current microbial fermentation method. It proposes a new approach by engineering a single microorganism, E. herbicola, to convert d-glucose into 2-keto-l-gulonic acid using a cloned reductase gene from Corynebacterium, which significantly simplifies the process. Additionally, it explores the enhancement of 2,5-dkg reductase activity through directed mutagenesis and substitution of NADPH with the cheaper cofactor NADH, leading to potential cost-effective production of ascorbic acid.

1. Molecular biology Seminar Notes.
Synthesis of L-Ascorbic Acid
 L-Ascorbic acid (vitamin C) is currently synthesized commercially by an expensive
process starting with d-glucose that includes one microbial fermentation step and a
number of chemical steps (Fig. 13.5).
 The last step in this process is the acid-catalysed conversion of 2-keto-l-gulonic acid
(2-KLG) to l-ascorbic acid.
 Biochemical studies of the metabolic pathways of a number of different
microorganisms have shown that it may be possible to synthesize 2-KLG by a different
pathway. For example, some bacteria (Acetobacter, Gluconobacter, and Erwinia) can
convert glucose to 2, 5-diketo-d-gluconic acid (2,5-DKG), and others
(Corynebacterium, Brevibacterium, and Arthrobacter) have the enzyme 2,5-DKG
reductase, which converts 2,5-DKG to 2-KLG.
 The current procedure for synthesizing ascorbic acid could be improved by producing
2-KLG from glucose by cofermentation with suitable organisms. Unfortunately,
cocultivation has problems of its own.
 For example, the two fermenting organisms might have different temperature and pH
optima. The medium requirements and growth rates also might differ in such a way that
the fermentation conditions are optimal for one organism and suboptimal for the other.
This situation leads to the eventual “washout” (depletion or loss) of one of the
organisms. Some of these incompatibilities may be overcome by utilizing a tandem
fermentation process in which the two organisms are cultivated in succession (Fig.
13.6).
 Of course, this approach requires two separate fermentations rather than one, and if the
organisms have different growth requirements, it is difficult to run the process on a
continuous basis.
 Therefore, the best way to convert glucose into 2-KLG would be to engineer a single
microorganism that carried all of the required enzymes. The conversion of d-glucose to
2, 5-DKG by Erwinia herbicola includes several enzymatic steps, whereas the
transformation of 2, 5- DKG to 2-KLG by a Corynebacterium sp. requires only one.

2.  Consequently, the simplest strategy for constructing a single organism that is able to
convert d-glucose to 2-KLG is to isolate the 2, 5-DKG reductase gene from the
Corynebacterium sp. and express it in E. herbicola.
Steps involved are as follows:
 Purification: The first step in cloning the 2, 5-DKG reductase gene from the
Corynebacterium sp. involved purifying the enzyme and determining the sequence of
the first 40 amino acids from the N-terminal end of the molecule.
 Hybridisation: Onthe basis of the known amino acid sequence, two 43-nucleotide DNA
hybridization probes, each corresponding to a different portion of the protein molecule,
were synthesized. Because 71% of the nucleotides in the Corynebacterium sp. are either
G or C, the probes were designed to include, where possible, a G or C in the third
position of all codons, thereby minimizing the extent of the mismatch between the
probe and the target DNA. This approach was taken because at the time that this work
was done, mixed probes were not readily available.
 Screening: A Corynebacterium DNA clone bank was screened with these two probes.
Any clones that hybridized with only one of the probes were discarded. It was assumed
that any DNA that interacted with only one probe was probably not the target DNA. A
clone that hybridized with both probes was isolated and then sequenced; it contained
the 2, 5-DKG reductase gene. The DNA sequences that were upstream of the ATG start
signal were deleted and replaced with transcriptional and translational signals that
function in E. coli, because the regulatory sequences from gram-positive
microorganisms, such as Corynebacterium spp., are not efficiently utilized by E. coli.
 Transformation in E.coli: This construct expressed 2, 5-DKG reductase activity in E.
coli and subsequently was sub cloned onto a broad-host-range vector, which was used
to transform E. herbicola, which is able to use E. coli transcriptional and translational
signals.

3.  The transformed Erwinia cells were able to convert d-glucose directly to 2-KLG. The
endogenous Erwinia enzymes, localized in the inner membrane of the bacterium,
converted glucose to 2, 5-DKG, and the cloned 2, 5- DKG reductase, localized in the
cytoplasm, catalysed the conversion of 2, 5-DKG to 2-KLG (Fig. 13.7). Thus, by
genetic manipulation, the metabolic capabilities of two very dissimilar microorganisms
were combined into one organism, which was able to produce the end product of the
engineered metabolic pathway. This recombinant organism should be useful as a source
of 2-KLG for the production of l-ascorbic acid, thereby replacing the first three steps
of the currently used process (Fig. 13.5).
 The commercial utility of the cloned 2, 5-DKG reductase gene product might be
improved by replacing certain amino acids of the enzyme to create mutants with
increased catalytic activity and enhanced thermal stability.
Strain Development by recombinant DNA technology
 When the 2, 5-DKG reductase gene was first isolated, the amino acid residues that
contributed to the active site of this enzyme were not known. However, from the

4. primary amino acid sequence, computer modelling predicted an enzyme structure with
an eight-stranded β-barrel (Fig. 13.8). This structure consisted of eight twisted parallel
β-strands arranged close together, surrounded by eight α-helices that were joined to the
α-strands through loops of various lengths. This folding pattern had previously been
observed for 17 other enzymes whose crystal structures were known.
 By comparison with the structures of these other proteins, three of the loops that might
be involved in substrate binding were identified (Fig. 13.8).
 Using oligonucleotide-directed mutagenesis: 12 different mutants, each with a single
amino acid change in one of these loops, were constructed. Of the 12 mutants, 11
produced enzymes with a lower 2, 5-DKG reductase specific activity than that of the
native form of the enzyme. The 12th mutant, in which amino acid residue 192 was
changed from glutamine to arginine, had approximately twice the activity of the native
enzyme.
 Kinetic studies revealed that this increase in activity resulted from a 1.8-fold increase
in the maximal rate of the enzyme-catalysed reaction (Vmax) and a 25% decrease in the
Michaelis constant (Km) of the enzyme-catalysed reaction.
 Modification in the cofactor: The reaction catalysed by 2, 5-DKG reductase utilizes
reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor.
However, the cellular concentration of reduced nicotinamide adenine dinucleotide
(NADH) is usually about 10-fold greater than that of NADPH, while the financial cost
of NADPH is about 10 times higher than that of NADH. To lower the cost of the
bacterial production of ascorbic acid, it would be beneficial to engineer a version of 2,
5-DKG reductase that used NADH instead of NADPH.
 The only structural difference between NADH and NADPH is the presence or absence
of a phosphate group attached to the 2′ site of the adenine moiety. From the three-
dimensional structure of 2, 5-DKG reductase complexed with NADPH, it appears that
5 amino acid residues interact directly with the 2′ phosphate residue of NADPH.
 Using cassette mutagenesis (Fig. 13.9), a total of 40 different mutants of this enzyme
were constructed; in each constructed mutant, 1 of the 5 amino acid residues that
computer models suggested interacted with the 2′ phosphate residue was changed to a
different amino acid. Following the expression, purification, and kinetic
characterization of the 40 mutants in E. coli, it was observed that changing three of the
five selected amino acids resulted in increases in 2, 5- DKG reductase activity with
NADH as the cofactor.

5.  In the best case, when the arginine residue at position 238 was changed to histidine,
there was a sevenfold improvement over the wild type with NADH as a cofactor.
Moreover, after two amino acid alterations were combined in one protein, an enzyme
that showed even more activity with NADH included a change of the lysine residue at
position 232 to glycine, as well as the change of arginine at position 238 to histidine.
Also, when the best NADH-active mutant was combined with a double mutant which
increased the binding of the substrate, even further improvements in activity with
NADH were observed.
 The activity of the enzyme isolated from the final construct was 72 times higher than
the activity of the wild-type enzyme. It now remains to be seen whether this engineered
enzyme can be used as the basis for the economically efficient biological synthesis of
ascorbic acid.