Production of D-lactate in Geobacillus spp App No 50484-338192
Lead Research Organisation:
University of Bath
Department Name: Biology and Biochemistry
Abstract
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 .
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 .
Technical Summary
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.
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.
Planned Impact
As described in proposal submitted to IUK
People |
ORCID iD |
David Jonathan Leak (Principal Investigator) |
Description | The project involved try to find and express an enzyme that will produce D-lactate that will work in a thermophilic bacterium. We found and characterised 3 genes encoding specific D-lactate dehydrogenases (ie they reduce pyruvate to D lactate and not L-lactate). One of them was shown to work in Geobacillus thermoglucosidasius ( a thermophilic bacterium) and placed in an appropriate genetic background to evaluate in bioreactors. This was passed on to project partners, CPI, for final evaluation. |
Exploitation Route | Having constructed a strain to produce D-lactate it will then be possible to produce either L-lactate (the natural product) or D-lactate at will, from renewable lignocellulosic feedstocks. These are precursors for making the plastic, poly-lactic acid (PLA), production of which will be taken up by our industrial partner. |
Sectors | Manufacturing, including Industrial Biotechology |
Description | The preliminary data provided in this project allowed our partners to prepare and submit an application for an IBCatalyst award to scale up this process. This was not successful, but one of the consortium (Fiberight) has since secured Innovate funding for an extension of this work. |
First Year Of Impact | 2017 |
Sector | Manufacturing, including Industrial Biotechology |
Impact Types | Economic |
Description | P2P NIBB |
Amount | £63,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 05/2016 |
End | 11/2016 |
Description | Evaluation of thermophilic bacilli for production of lactate at low pH |
Organisation | Fiberight Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | Evaluation of pH tolerance and lactate production from municipal solid waste-derived sugars in various strains of Geobacillus |
Collaborator Contribution | Provision of MSW-derived sugar concentrate |
Impact | Initial report - currently in further negotiations |
Start Year | 2016 |
Description | Identification of Suitable Geobacillus spp for the Commercial Production of Lactic Acid from MSW Derived Platform Sugars |
Organisation | Fiberight Ltd |
Country | United Kingdom |
Sector | Private |
PI Contribution | Prepared a double knockout strain of Parageobacillus thermoglucosidasius which produces exclusively L-lactate |
Collaborator Contribution | Provided enzyme hydrolysed feedstock from municipal solid waste derived cellulose fraction |
Impact | Homolactate producing strain produced and supplied to partners at Aberystwyth for evaluating |
Start Year | 2017 |
Description | Popular Science (Bath) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Pint of Science - interactive 30 min presentation followed by Q &A in a local pub |
Year(s) Of Engagement Activity | 2016 |
URL | http://www.bath.ac.uk/news/2016/04/18/pint-of-science-2016/ |