The biomass used may be sugars from food crops such as maize (corn) or can be cellulosic or lignocellulosic (non-edible parts of the plant) in nature. While ethanol has been the major focus, as a fuel produced from biomass, more recently researchers have shifted their focus to longer chain-length molecules such as biodiesel and isobutanol, due to their higher energy density and compatibility with existing motor vehicle engines and infrastructure for refining, housing and dispensing of these liquid fuels. The majority of biofuels processes can be classified in four different categories: First -, second -, third - and fourth generation biofuels (Figure 1).

Geobacillus thermoglucosidasius is a promising “platform” organism to use in the production of a range of useful metabolites with demonstrated ability to produce ethanol, isobutanol and polylactic acid for bio-degradable plastics. This Gram positive thermophile, capable of growth at 60°C, has been heavily engineered for ethanol production. It naturally produces large amounts of lactic acid from pyruvate (a product of glycolysis) under microaerobic conditions. The organism has been engineered to redirect its metabolism towards ethanol production by knocking out (interrupting) the genes responsible for lactate production (Lactate dehydrogenase; ldh) and conversion of pyruvate to formate (Pyruvate formatelyase; pfl), as well as improving expression of a gene for conversion of pyruvate to acetyl-CoA (Pyruvate dehydrogenase; pdh). These changes meant that most, if not all, pyruvate was directed towards ethanol production. Although extensive work has been done in engineering the organism for enhanced ethanol production one very effective pathway for redirecting carbon flow to ethanol has not yet been demonstrated in G. thermoglucosidasius.

Researchers at the University of the Western Cape’s Institute for Microbial Biotechnology and Metagenomics (IMBM) explored the possibility of using pyruvate decarboxylase (PDC) to further improve the organisms’ ability to produce ethanol. This enzyme is responsible for the non-oxidative conversion of pyruvate to acetaldehyde, which in turn is used by alcohol dehydrogenase as substrate to convert to ethanol. They first characterized two novel bacterial PDC’s then attempted to express the more thermostable of these enzymes from Gluconobacter oxydans in G. thermoglucosidasius to improve ethanol yields. Initial expression was unsuccessful. Analysis of the codon usage pattern for the gene revealed that the codon usage was suboptimal in the heterologous host G. thermoglucosidasius and after codon harmonization, they could demonstrate successful expression of the enzyme at 52°C, however not at the bacterium’s optimum growth temperature of 60°C. Successful expression was concomitant with enhanced ethanol production close to the theoretical yield possible (0.5 g/l).

Codon harmonization is a seldom used technique of codon optimization first described in 2008. Unlike techniques for codon optimization previously described, harmonization seeks to adjust the translation frequency of the gene sequence in it’s heterologous host, to match that in it’s native host. Although enhanced ethanol production was observed, the main benefit to come from the study was perhaps the realization of how to codon optimize genes for expression in G. thermoglucosidasius. If it is to be used as a platform organism for metabolite production, successful heterologous expression of genes derived from other bacteria will be a key and this method shows one way to enable it.

Part 2: Bacteriophage resistance engineering
In Part 1, we looked at how G. themoglucosidasius is being engineered to produce ethanol. Here we will look at the effect viruses have on G. thermoglucosidasius and how researchers at the University of the Western Cape’s Institute for Microbial Biotechnology and Metagenomics (IMBM) have found ways to overcome this issue.

Bacteriophages are viruses that specifically infect bacteria. Recent estimates suggest that these are the most abundant biological entities on the planet with roughly 10 virus particles to every bacterial cell and a total estimate of 10³¹ virus particles. With the advent of next generation sequence technology, which allows for the study of whole virus populations, it has become apparent that there is a very wide diversity of bacteriophages. They also appear to be ubiquitous in nature having been found in many extreme environments, including soda lakes, terrestrial hot springs, deep sea hydrothermal vents, hot/cold deserts and hypersaline environments.

Since the early days of industrial bacterial fermentations to produce acetone and butanol in the 1920’s, it has been known that bacteriophages (phages for short) negatively impact these processes. Phages which infect the bacterium, can either lyse the host organism leading to failed fermentation and the economic loss associated with it, or they can integrate into the hosts genome as a prophage, where they can cause slow fermentations with reduced yield. Over time, researchers have developed several ways in which this risk can be mitigated. In the dairy industry researchers have turned to molecular biology to engineer bacterial strains to be resistant against particular phages, and they use these on a rotation basis to avoid a phage population being established in the production facility. They also make use of growth media (phage inhibitory media) low in calcium to prevent phages from developing properly, should they infect starter cultures.

It is generally accepted that fermentations employing thermophilic bacteria such as G. thermoglucosidasius and Bacillus coagulans are less prone to contamination and even immune to phage-related failure or it may be that they are not often reported. However, phages that infect thermophiles are known and are expected to eventually become a problem for industries making use of such organisms. As a commercial platform, G. thermoglucosidasius would be expected to ferment a range of globally sourced feedstocks and could be exposed to phages from a variety of environments which may lead to failed or stuck fermentations and associated financial loss. This also means that it will be an on-going problem for those wishing to employ this organism for large scale metabolite production. Although several Geobacillus species phages have been described, sequenced, and one in particular GVE2, well studied, none have been found that infect G. thermoglucosidasius. IMBM researchers identified a novel virus (GVE3) that infects G. thermoglucosidasius from a failed industrial ethanol fermentation. Following characterization of the GVE3 genome, they identified a protein that, when expressed in G. thermoglucosidasius, rendered the bacterium resistant against GVE3 infection.

A second resistance mechanism was discovered when “naturally” resistant isolate were selected for. Following genome sequencing, a mutation in the polysaccharide pyruvyl transferase (csaB) was identified. The role of this protein in bacterial cells is thought to be the addition of a pyruvate moiety to cell wall lipopolysaccharide which is in turn used by surface layer homology domain (SLHD) containing proteins to adhere to and display on the cell surface.

This is only the second time that this protein (CsaB) has been shown to be involved in phage infection, and although not thought to be directly involved as phage attachment point on the cell, may well affect the display of SLHD-containing proteins on the surface. This points to one of these proteins likely being involved in attachment of the phage to the outside of the cell during infection.

References:

1. Van Zyl, LJ, Taylor, MP, Eley, K, Tuffin, M, Cowan, D. 2014. Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius. Appl. Micro. Biotechnol. 98:1247-1259
2. Van Zyl, LJ., Schubert, W-D., Tuffin, IM., Cowan, D. 2014. Structure and Functional Characterization of Pyruvate decarboxylase from Gluconacetobacter diazotrophicus. BMC Struct. Biol. DOI:10.1186/s12900-014-0021-1
3. Van Zyl LJ, Sunda F, Taylor MP, Cowan DA, Trindade MI. 2015. Identification and characterization of a novel Geobacillus thermoglucosidasius bacteriophage, GVE3. Arch. Virol. doi:10.?1007/?s00705-015-2497-9
4. Van Zyl LJ, Taylor MP, Trindade M. 2015. Engineering resistance to phage GVE3 in Geobacillus thermoglucosidasius. Appl. Microbiol. Biotechnol. doi: 10.1007/s00253-015-7109-9