Characterization of LDH Genes for L-Lactic Acid Production in Pichia Pastoris

Recently, the demand for plastic material is growing substantially. Since most of it nowadays is not recyclable, the amount of plastic deposited in the environment reaches 140 million tons annually⁽¹⁾. Those are estimated to degrade in about a thousand years when derived from petrochemical sources. Therefore due to environmental and economical issues, efforts turned into the development of green alternatives to petrochemical derived plastics.

Biomaterials are natural products that are synthesised and catabolised by different organisms and that have found broad biotechnological applications. Bioplastics, also known as green-plastic, are a special type of biomaterial made from renewable biomass feedstock such as corn and sugar-cane⁽²⁾. They are polyesters, produced by a range of microbes, cultured under different nutrient and environmental conditions⁽³⁾. During its synthesis less carbon is emitted and lower energy is required. Furthermore bioplastics are biodegradable which facilitates recycling thus reducing the amount of plastic deposited in the environment.

The development of a more sustainable society is expected to gradually replace petroleum derived plastics by bioplastics. Thus, the demand for this type of material is estimated to grow at 20-30% by 2016. L-Lactic acid (2- hidroxy propionic acid) is widely used in food, chemical, cosmetics and pharmaceutical industries. Also, it is the monomer of polylactic acid (PLA), which is a bioplastic. PLA products can be used in a wide variety of applications ranging from packaging to fibers and foams ⁽⁴⁾.

 The purity of the lactic acid is important as it influences the physico-chemical properties of the poly (lactic acid), for example, the polymer L-lactic acid is the desired form for the production of packages⁽⁵⁾. Consequently the ideal microorganism for the production of lactic acid is one that produces pure enantiomeric forms, with high yield, and low cost substrate.

The development of an efficient process for the industrial production of poly (lactic acid) directly depends on the economically viable production of lactic acid. Currently, the production of lactic acid is mainly made from starch⁽⁶⁾. However with the increasing production of biodiesel there is an overproduction of glycerol. This is the major waste produced during the conversion of vegetable oil into biodiesel. Furthermore, the use of glycerol is considered advantageous to reduce the cost of biodiesel production in the biorefinery ⁽⁷⁾.

Different microorganisms have been genetically modified to produce lactic acid, but in most cases sugar is used as substrate. Pichia pastoris,  a metilotrophic yeast, can reach high cell density using crude glycerol as carbon source, survive in low pH levels, although it is not able to produce lactic acid. Genetic engineering can make possible this production, from overexpression of the gene that encode an enzyme able to catalyze the conversion of pyruvate to lactate. The enzyme lactate dehydrogenase (LDH) is encoded by specific genes from different species. It determines yield and purity of produced lactic acid.

In this study, different strains are constructed to enable high yield and productivity of lactic acid from crude glycerol. At least 4 different genes encoding for LDH have been tested for l-lactic acid production in P.pastoris. In parallel genetic engineering strategies for increased pyruvate inside de the cell and secreted lactic acid have also been tested. After strain development, fermentations are carried out to determine which recombinant strains results in higher yield and productivity of L-lactic acid.

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