Question

You intend to use bacteria to produce ethanol for your spin-off business in Palapye. The bacteria (strain: Ethanol Blue) you have, is a very poor ethanol producer and is very sensitive to ethanol. However, an industrial strain (strain: Ethanol Red), which has both an elevated ethanol producing and a high ethanol tolerance trait, is available in your laboratory collections for use as a control. Both bacteria sporulate with relative ease. Note that an elevated ethanol producer is always resistant to elevated amounts of ethanol. a) Isn’t it strange that natural bacteria isolates produce ethanol? Explain. [2 marks] b) Briefly describe 2 methods (taking advantage of their sporulation ability only) you would use to improve the fermentative capacity of Ethanol Blue. [4 marks] c) State any other methods other than evolutionary engineering and recombinant DNA technology that can be used to develop the strain. [2 marks] d) Is there a correlation between ethanol production and tolerance? [2 marks] Total [10 marks]

Answer 1

a)Ethanol fermentation is one of the oldest and most important fermentation processes used in the biotechnology industry. ... Many microorganisms, including bacteria and yeasts, can produce ethanol as the major fermentation product from carbohydrates

b)progress has been made in engineering pentose fermentation capacity into the yeast S. cerevisiae.For that purpose, two heterologous pathways for D-xylose utilization have been utilized. First, the genes encoding D-xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces (Pichia) stipitis have been expressed in S. cerevisiae. This resulted in D-xylose fermentation, but also in significant production of xylitol under anaerobic conditions, which is due to NADH/NADPH cofactor imbalance of XR and XDH .The performance of these strains has been improved considerably by addressing the cofactor imbalance and by over-expression of endogenous xylulokinase (XK) and enzymes of the non-oxidative part of the pentose phosphate pathway .

The second pathway allows direct isomerization of D-xylose to xylulose through heterologous expression of xylose isomerase (XI). After the first successful attempt to express the thermophilic bacterium Thermus thermophilus XI into S. cerevisiae ,recombinant strains expressing the fungal Piromyces sp. strain E2 xylose isomerase have been reported with better enzymatic activity .By using an isomerization instead of a reduction/oxidation conversion of D-xylose to xylulose, the problem of co-factor imbalance is avoided. However, the rate of D-xylose utilization in XI expressing strains was found to be inferior to that in strains harboring the XR/XDH pathway . This was mostly attributed to the low activity of the XI enzyme in S. cerevisiae and its inhibition by xylitol, generated from reduction of D-xylose by the endogenous enzymes encoded by GRE3, GCY1, YPR1, YDL124W and YJR096W .The level of xylitol produced is much lower, however, than in the strains expressing the XR/XDH pathway. Deletion of GRE3 in an XI expressing strain improved both the rate of D-xylose consumption and ethanol production .The aldose reductase, encoded by GRE3, plays a role in stress protection and its deletion is therefore not desirable in industrial yeast strains .To overcome these problems, Brat et al., constructed the first recombinant S. cerevisiae strain demonstrating high activity of prokaryotic XI, using codon-optimized XylA gene from Clostridium phytofermentans. This enzyme was much less inhibited by xylitol compared to the enzyme from Piromyces. Nevertheless, the rate of D-xylose consumption and ethanol production by this recombinant strain was still slow.

Different metabolic and evolutionary engineering strategies have been used successfully to improve D-xylose utilization in a yeast strain expressing Piromycesxylose isomerase. Overexpression of genes encoding xylulokinase and enzymes of the non-oxidative part of the pentose phosphate pathway, combined with deletion of GRE3to reduce xylitol formation, considerably improved the D-xylose consumption rate.This finally resulted in strains with strong pentose fermentation capacity and partial cofermentation of glucose and D-xylose Moreover, the xylose isomerase pathway was compatible with the bacterial L-arabinose utilization pathway, in contrast to the XR/XDH pathway [These results suggested that the xylose isomerase pathway might be the pathway of choice for constructing superior industrial yeast strains with optimal fermentation performance in lignocellulose hydrolysates .However, all these engineered strains were still made in a haploid laboratory yeast strain background, displaying in general suboptimal fermentation performance and poor robustness and stress tolerance, which makes them unsuitable for use in industrial fermentations. Since previous work showed that XI expressing strains displayed higher yield of ethanol per consumed D-xylose compared to strains harboring the XR/XDH pathway and since they profit from direct isomerization of D-xylose to xylulose without cofactor requirement, the XI pathway seemed to be most promising to engineer into a robust industrial yeast strain.

In this work, we have selected Ethanol Red as industrial host strain to engineer high-capacity pentose-fermentation, because it is one of the most widely used yeast strains for first-generation bioethanol production. The strain has excellent fermentation capacity, high robustness and stress tolerance, and also displays excellent performance in fed-batch production on molasses, is tolerant to dehydration and retains high vitality during storage and transport. Using this strain, we have developed the first industrial S. cerevisiae strain that converts D-xylose to ethanol with a yield close to the theoretical maximum yield and with a very high specific rate of fermentation. For that purpose, a recombinant strain was first constructed by chromosomal integration of codon-optimized XylA from C. phytofermentans in an over-expression gene cassette containing genes of the non-oxidative pentose phosphate pathway and the D-xylose transporting hexose transporter HXT7. Subsequently, we have used Ethyl Methanesulfonate (EMS) mutagenesis, genome shuffling and selection in lignocellulose hydrolysate, enriched with D-xylose, and subsequent evolutionary adaptation in complex medium with D-xylose, to greatly enhance both D-xylose utilization efficiency and inhibitor tolerance. The activity of XI was dramatically increased in the evolved strain, but other genetic changes were also required for its superior D-xylose fermentation capacity in lignocellulose hydrolysates.