Modification of bacteria

Details Modification of bacteria Modification of bacteria

2001-05-09

2003-12-16

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S320100, C435S132000, C435S161000, C435S183000, C435S243000, C435S252100, C435S252300, C435S252310, C435S252330

Reexamination Certificate

active

06664076

ABSTRACT:

This invention relates to a novel method of in vivo methylation of nucleic acids. In particular, the invention relates to thermophilic Bacillus strains transformed using a plasmid transformation system based on the method of in vivo methylation. The invention can be used to increase ethanol production.
Many bacteria have the ability to ferment simple hexose sugars into a mixture of acidic and pH-neutral products via the process of glycolysis. The glycolytic pathway is universal and comprises a series of enzymatic steps whereby a six carbon glucose molecule is broken down, via multiple intermediates, into two molecules of the three carbon compound pyruvate. This process results in the net generation of ATP (biological energy supply) and the reduced cofactor NADH.
Pyruvate is an important intermediary compound of metabolism. Under aerobic conditions (oxygen available), pyruvate is first oxidised to acetyl CoA and then enters the tricarboxylic acid cycle (TCA) which generates synthetic precursors, CO
2
and reduced cofactors. The cofactors are then oxidised by donating hydrogen equivalents, via a series of enzymatic steps, to oxygen resulting in the formation of water and ATP. This process of energy formation is known as oxidative phosphorylation.
Under anaerobic conditions (no available oxygen), fermentation occurs in which the degradation products of organic compounds serve as hydrogen donors and acceptors. Excess NADH from glycolysis is oxidised in reactions involving the reduction of organic substrates to products such as lactate and ethanol. In addition, ATP is regenerated from the production of organic acids such as acetate in a process known as substrate level phosphorylation. Therefore, the fermentation products of glycolysis and pyruvate metabolism include a variety of organic acids, alcohols and CO
2
.
The majority of facultatively anaerobic bacteria do not produce high yields of ethanol either under aerobic or anaerobic conditions. Most faculatative anaerobes metabolise pyruvate aerobically via pyruvate dehydrogenase (PDH) and the tricarboxylic acid cycle (TCA).
Under anaerobic conditions, the main energy pathway for the metabolism of pyruvate is via pyruvate-formate-lyase (PFL) pathway to give formate and acetyl-CoA. Acetyl-CoA is then converted to acetate, via phosphotransacetylase (PTA) and acetate kinase (AK) with the co-production of ATP, or reduced to ethanol via acetalaldehyde dehydrogenase (AcDH) and alcohol dehydrogenase (ADH). In order to maintain a balance of reducing equivalents, excess NADH produced from glycolysis is re-oxidised to NAD
+
by lactate dehydrogenase (LDH) during the reduction of pyravate to lactate. NADH can also be re-oxidised by AcDH and ADH during the reduction of acetyl-CoA to ethanol but this is a minor reaction in cells with a functional LDH. Theoretical yields of ethanol are therefore not achieved since most acetyl CoA is converted to acetate to regenerate ATP and excess NADH produced during glycolysis is oxidised by LDH.
Ethanologenic organisms, such as
Zymomonas mobilis
and yeast, are capable of a second type of anaerobic fermentation, commonly referred to as alcoholic fermentation, in which pyruvate is metabolised to acetaldehyde and CO
2
by pyruvate decarboxylase (PDC). Acetaldehyde is then reduced to ethanol by ADH regenerating NAD
+
. Alcoholic fermentation results in the metabolism of 1 molecule of glucose to two molecules of ethanol and two molecules of CO
2
. DNA which encodes both of these enzymes in
Z. mobilis
has been isolated, cloned and expressed recombinantly in hosts capable of producing high yields of ethanol via the synthetic route described above.
A key improvement in the production of ethanol using biocatalysts can be achieved if operating temperatures are increased to levels at which the ethanol is conveniently removed in a vaporised form from the fermentation medium. However, at the temperatures envisioned, traditional mesophilic microorganisms, such as yeasts and
Z. mobilis,
are incapable of growth. This has led researchers to consider the use of thermophilic, ethanologenic bacteria such as Bacillus sp as a functional alternative to traditional mesophilic organisms. See EP-A-0370023.
The use of thermophilic bacteria for ethanol production offers many advantages over traditional processes based upon mesophilic ethanol producers. Such advantages include the ability to ferment a wide range of substrates, utilising both cellobiose and pentose sugars found within the dilute acid hydrolysate of lignocellulose, as well as, the reduction of ethanol inhibition by continuous removal of ethanol from the reaction medium using either a mild vacuum or gas sparging. In this way, the majority of the ethanol produced may be automatically removed in the vapour phase at temperatures above 50° C. allowing the production phase to be fed with high sugar concentrations without exceeding the ethanol tolerance of the organism, thereby making the reaction more efficient. The use of thermophilic organisms also provides significant economic savings over traditional process methods based upon lower ethanol separation costs.
The use of facultative anaerobes also provides advantages in allowing a mixed aerobic and anaerobic process. This facilitates the use of by-products of the anaerobic phase to generate further catalytic biomass in the aerobic phase which can then be returned to the anaerobic production phase.
It is possible that organisms which carry out glycolysis or a variant thereof can be engineered to divert as much as 50% of the carbon in a sugar molecule via glycolysis and a synthetic, metabolic pathway which comprises enzymes encoded by heterologous genes. The result is an engineered organism which produces ethanol as its primary fermentation product.
The inventors have produced sporulation deficient variants of a thermophilic, facultatively anaerobic, Gram-positive bacterium which exhibit improved ethanol production-related characteristics. This has been achieved through the development of a plasmid transformation system based on a novel method of in vivo methylation.
The production of recombinant Bacillus sp, engineered to express a heterologous gene, has previously been hampered by a Hae III type restriction system that limited plasmid transformation.
In vivo methylation has been used previously to overcome different restriction problems in other bacteria such as
Xanthomonas campestris.
For example, De Feyter and Gabriel (De Feyter, R, Gabriel, D. W.) Journal of Bacteriology 173 (1991) (20): 6421-7 have shown that where cosmid libraries of DNA from the bacterium
X. campestris
were restricted when introduced into strains of
Escherichia coli,
the use of cloned DNA methylase genes increased the frequency of transfer of foreign genes into
X. campestris
pv.
malracearum.
In this instance, restriction was associated with the mcrBC+ gene in
E. coli.
Restriction was overcome using a plasmid (pUFRO52) encoding the XmaI and XmaIII DNA methylases isolated from
X. campestris
pv
malracearum.
Subsequent plasmid transfer from
E. coli
strains to
X. campestris
pv.
malvacearum
by conjugation was significantly enhanced.
Similarly, Mermelstein and Papoutsakis (Mermelstein, L. D, and Papoutsakis, E. T) Appl. Environ. Biology 59(4) (1993) have shown that in vivo methylation in
E. coli
by
B.subtilis
phage phi 3TI methyltransferase can be used to protect plasmids from restriction upon transformation of
Clostridium acetobutylicum.
Transformation efficiency in Bacillus strains was initially limited by a HaeIII-type restriction system, previously identified in Bacillus strain LLD-R. Bacillus strain LLD-R possesses a powerful HaeIII type restriction-modification system similar to that found in
Haemophilus aegyptius
(Zaidi S. H. E. (1991) PhD thesis, Imperial College, London). The HaeIII restriction endonuclease methylates the inner cytosine residues in the recognition site S-GGCC-3 which occurs frequently in the GC rich genome of LLD-R. HaeIII restriction of heterologous plasmid DNA in strain LLD-R prese

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