Yeast strains for the production of lactic acid transformed...

Details Yeast strains for the production of lactic acid transformed... Yeast strains for the production of lactic acid transformed...

2000-06-29

2002-08-06

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

Reexamination Certificate

active

06429006

ABSTRACT:

The invention refers to yeast strains transformed with at least one copy of a gene coding for lactic dehydrogenase (LDH) and further modified for the production of lactic acid with high yield and productivity.
BACKGROUND OF THE INVENTION
The applications of lactic acid and its derivatives encompass many fields of industrial activities (i.e., chemistry, cosmetic, and pharmacy), as well as important aspects of food manufacture and use. Furthermore, today there is growing interest in the production of such an organic acid to be used directly for the synthesis of biodegradable polymer materials.
Lactic acid may be produced by chemical synthesis or by fermentation of carbohydrates using microorganisms. The latter method is now commercially preferred because microorganisms have been developed that produce exclusively one isomer, as opposed to the racemic mixture generated by chemical synthesis. The most important industrial microorganisms, such as species of the genera Lactobacillus, Bacillus, and Rhizopus, produce L(+)-lactic acid. Production by fermentation of D(−)-lactic acid or mixtures of L(+)- and D(−)-lactic acid are also known.
During a typical lactic acid fermentation, there is an inhibitory effect caused by lactic acid produced on the metabolic activities of the producing microorganism. Besides the presence of lactic acid, lowering the pH value also inhibits cell growth and metabolic activity. As a result, the extent of lactic acid production is greatly reduced.
Therefore, the addition of Ca(OH)
2
, CaCO
3
, NaOH, or NH
4
OH to neutralise the lactic acid and to thereby prevent the pH decrease is a conventional operation in industrial processes to counteract the negative effects of free lactic acid accumulation.
These processes allow the production of lactate(s) by maintaining the pH at a constant value in the range of about 5 to 7; this is well above the pK
a
of lactic acid, 3.86.
Major disadvantages are connected to the neutralisation of lactic acid during the fermentation. Mainly, additional operations are required to regenerate free lactic acid from its salt and to dispose of or recycle the neutralising cation; this is an expensive process. All the extra operations and expense could be eliminated if free lactic acid could be accumulated by microorganisms growing at low pH values, thus minimising the production of lactate(s).
It has been proposed the use of recombinant yeasts expressing the lactate dehydrogenase gene so as to shift the glycolytic flux towards the production of lactic acid.
FR-A-2 692 591 (Institut Nationale la Recherche Agronomique) discloses yeast strains, particularly Saccharomyces strains, containing at least one copy of a gene coding for a lactate dehydrogenase from a lactic bacterium, said gene being under the control of sequences regulating its expression in yeasts.
Said strains may give both the alcoholic and the lactic fermentation and this so called “intermediate” or “balanced” fermentation could be exploited in areas such as brewing, enology, and baking.
Porro et al., (Biotechnol. Prog. 11, 294-298, 1995) have also reported the transformation of
S. cerevisiae
with a gene coding for bovine lactate dehydrogenase.
However, because of the high production of ethanol, the yield in the production of lactic acid for both the processes described was not considered to be competitive with that obtainable by the use of lactic bacteria.
In the past decade, “non conventional yeasts” other than
S. cerevisiae
have gained considerable industrial interest as host for the expression of heterologous proteins. Examples are the methanol-utilising yeasts such as
Hansenula polimorpha
and
Pichia Pastoris
, the lactose-utilizing yeasts such as
Kluyveromyces lactis
. In addition to enabling the use of a wider range of substrates as carbon and energy sources, other arguments have been put forward to the industrial use of “non conventional yeasts”. Generally speaking, biomass and product-yield are less affected, in some of these yeasts, by extreme conditions of the cellular environment. High-sugar-tolerant (i.e., 50-80% w/v glucose medium; Torulaspora-syn.
Zygosaccharomyces
-
delbrueckii, Zygosaccharomyces rouxii
and
Zygosaccharomyces bailii
; Ok T and Hashinaga F., Journal of General & Applied Microbiology 43(1): 39-47, 1997) and acid- and lactic-tolerant (
Zygosaccharomyces rouxii
and
Zygosaccharomyces bailii
; Houtsma P C, et al., Journal of Food Protection 59(12), 1300-1304, 1996.) “non conventional yeasts” are available. As already underlined the cost of down stream processing could be strongly reduced if the fermentation process is carried out under one or more of the above mentioned “extreme conditions”.
SUMMARY OF THE INVENTION
According to a first embodiment, this invention provides yeast strains lacking ethanol production ability or having a reduced ethanol production ability and transformed with at least one copy of a gene coding for lactic dehydrogenase (LDH) functionally linked to promoter sequences allowing the expression of said gene in yeasts.
More particularly, this invention provides yeast strains having a reduced pyruvate dehydrogenase activity and a reduced pyruvate decarboxylase activity and transformed with at least one copy of a gene coding for lactic dehydrogenase (LDH) functionally linked to promoter sequences allowing the expression of said gene in yeasts.
According to another embodiment, this invention provides yeast strains of Kluyveromyces, Torulaspora and Zygosaccharomyces species, transformed with at least one copy of a gene coding for lactic dehydrogenase (LDH) functionally linked to promoter sequences allowing the expression of the gene in said yeasts.
According to a further embodiment, the invention also provides yeast cells transformed with a heterologous LDH gene and overexpressing a lactate transporter.
Other embodiments are the expression vectors comprising a DNA sequence coding for a lactic dehydrogenase functionally linked to a yeast promoter sequence and a process for the preparation of DL-, D- or L-lactic acid by culturing the above described metabolically engineered yeast strains in a fermentation medium containing a carbon source and recovering lactic acid from the fermentation medium.
Furthermore, the invention provides processes for improving the productivity (g/l/hr), production (g/l) and yield (g/g) on the carbon source of lactic acid by culturing said yeast strains in a manipulated fermentation medium and recovering lactic acid from the fermentation medium.
DESCRIPTION OF THE INVENTION
It has been found that production of lactic acid can be obtained by metabolically modified yeasts belonging to the genera Kluyveromyces, Saccharomyces, Torulaspora and Zygosaccharomyces.
More particularly, it has been found that very high yields in the production of lactic acid are obtained by engineered yeast strains so as to replace at least the ethanolic fermentation by lactic fermentation.
Even higher yields (>80% g/g) in the production of lactic acid may be obtained by engineered yeast strains so as to replace both the ethanolic fermentation and the use of pyruvate by the mitochondria by lactic fermentation.
To this purpose, the invention also provides transformed yeast cells having an increased LDH activity, for instance as a consequence of an increased LDH copy number per cell or of the use of stronger promoters controlling LDH expression.
An increased LDH copy number per cell means at least one copy of a nucleic acid sequence encoding for lactic dehydrogenase protein, preferably at least two copies, more preferably four copies or, even more preferably, at least 10-50 copies of said nucleic acid sequence.
In order to have the highest production of lactic acid, yeast cells transformed according to the invention preferably overexpress a lactate transporter. This can be obtained by transforming yeast cells with one or more copies of a gene required for lactate transport.
The strains according to the invention can be obtained by several methods, for instance by genetic engineering techniques aiming at

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