Production of D-lactate in Geobacillus spp App No 50484-338192

Bioplastic, as a replacement for hydrocarbon plastic, is a rapidly expanding market that is predicted to grow from 1.4M Tonnes in 2012 to over 6M Tonnes by 2017 (Source: European Bioplastics). Within this market, Polylactic Acid (PLA) is an alternative material for existing products in packaging, food serviceware and textiles together with enabling new products to be developed for high value markets where desired variations in the properties of the PLA can be achieved by mixing of D and L isomers of lactic acid in different proportions.
Current lactic acid production is mainly in the more common L isomer from 1st generation feedstocks, which compete with food crops for agricultural land and produce waste that requires processing prior to disposal. The D isomer is mainly produced from the L isomer through a combination of chemical and biological processes. This proposal seeks to develop a fully biological process to create an innovative and efficient manufacturing route for producing D lactic acid that reduces the dependency on fossil oil, leading to the de-carbonisation of the industrial process for the production of PLA. The collaboration brings together Rebio Technologies Ltd, the University of Bath and the Centre for Process Innovation (CPI). Rebio have expertise in the development of industrial strains based on a Geobacillus host for the production of 2nd generation bioethanol from a wide range of feedstocks such as cane bagasse, corn stover and fractions derived from municipal solid waste. This is combined with knowledge of the development of different biopolymers and their potential markets, and they are in a position to take a successful project forward into production. The University of Bath have considerable expertise in the genetic engineering of a wide range of organisms but specifically have a background in Geobacillus and will carry out the necessary strain engineering to convert an L-lactate to a D-lactate producing strain. CPI brings knowledge of the downstream processing of lactic acid and in the area of process scale up and will be able to evaluate market potential from data arising from from scale-up experiments .

Geobacillus spp are natural producers of L-lactate. In fermentative strains such as G. thermoglucosidasius the transition from aerobic to anaerobic conditions is characterised by the initial production of lactate as respiration becomes limited, followed by the combined production of acetate, formate, ethanol and lactate as cells enter full fermentative conditions. Therefore, either by use of low aeration or inactivation of alternative fermentation pathways, it would be feasible to turn various Geobacillus species into homo-(L)-lactate producers.
To convert the strains from L-lactate to D-lactate producers will require replacement of the L-lactate dehydrogenase (ldh) with a D-ldh. Inactivation of the natural L-ldh by gene disruption is straightforward, being one of the first steps used to develop ethanologenic strains. However, there are no reports of natural homo D-lactate producing thermophiles, which could act as a source of a suitable heterologous gene replacement. However, work at Rebio Technologies has shown that under stressful conditions, such as in media with a high concentration of mixed sugars, small amounts of D-lactate were produced. A search of the proprietary genome sequence showed that, in addition to the fermentative L-ldh, this strain encodes a 'flavocytochrome D-lactate dehydrogenase', a class of enzymes that appear to be D-lactate specific and which normally function in the aerobic catabolism of D-lactate. Similar enzymes are also present in a few other Geobacillus spp, such as G. kaustophilus.
The aim of this project is therefore to upregulate the native, or express a heterologous D-ldh in G thermoglucosidasius and knockout the L-ldh to produce a strain which can be evaluated for production of D-lactate from renewable feedstocks. Evaluation of scale-up performance will be used to generate process data to allow decisions to be made on commercialisation.

As described in proposal submitted to IUK