Introduction to Industrial Biotechnology

Transcription

1 Introduction to Industrial Biotechnology Lecture 2 - Getting things in and out mechanisms of solute transport across membranes and their application to IBBE

2 Learning outcomes Think about the transporter repertoire of different microbes what could it mean? Understand some examples of where the energetics of uptake has made a different to product formation Appreciate the diversity of efflux systems in biology, some examples of their exploitation & the great potential for future manipulation.

3 Transport profiles Database Statistics March 2016 Total microbes included: 2840 (only closed genomes) 32 phyla of Bacteria, comprising 2615 individuals 6 phyla of Archaea, comprising 168 individuals 11 phyla of Eukaryota, comprising 57 individuals Total transporter proteins: 1,028,038 Last analysis of this type in 2005 with 141 genomes by Ren and Paulsen

4 The transporter anatomy of E. coli K-12 PTS MFS (70) APC (22) ABC (217*) [68 systems 57 uptake, 11 export] Escherichia coli K-12 MG Mb 573 TPs RND (10) MOP(8) In terms of distinct systems, has more secondary transporters that any other type

5 The transporter anatomy of Clostridium botulinum PTS PTS (30) MFS Escherichia coli Clostridium botulinum K-12 MG Mb 4.6 Mb 446 TPs 573 TPs ABC (214*) MFS (7) MOP(17) APC (17)

6 The transporter anatomy of S. cerevisiae PTS (0) MFS MFS (85) Escherichia coli S. cerevisiae K-12 MG Mb 4.6 Mb 318 TPs 573 TPs ABC Broader use of secondary transporters

7 Families do bear some relationship to substrates For small families, often named/defined by their ability to transport a particular biochemical For larger families like ABC that have sub-families that recognise different substrates, it is possible for predict to a general class of substrate most of the time. For MFS this is even more difficult, and many substrates simply not known. Much more functional work still needed.

8 Looking at this variation by taxonomic groupings Across most taxonomic groups there is large variation in transporter concentration 25% 20% 15% 10% 5% 0% γ ε δ β α Clostridia Bacilus ClostriaiC Proteobacteria Firmicutes Actinobacteria

9 Uptake

10 Transport of oligosaccharides to increase yield Louise Glass & Jamie Cate added whole new functionalities to yeast at the level of transport. Also improved efficiency of simultaneous saccharification & fermentation.

11 A clean deletion strain, TDXylose, cannot grow on D-xylose mM of xylose OD 650nm Constructed BW25113 strains with clean deletions in xyle or xylh or both Time (Hours) Can observe subtle growth phenotypes. Wild-type strain has a clear diauxie when growing on xylose. The xyle mutant has no growth delay, but grows the slowest The xylh mutant has a lag in growth, then grows like the wild-type Can measure small changes in OD 650 very accurately using the Tecan plate reader. Each strain grown in quintuplicate!

12 A 650nm 10mM xylobiose Discovery of xylobiose secondary carriers E. coli cannot grow on the disaccharide xylobiose (X 2 ) as a sole carbon source. Lonnie Ingram found xyntb in Klebsiella oxytoca that enable growth on X 2. We engineered in a chromosomal β-xylosidase gene to the chromosome of our TDXylose strain to enable a growth-based screen for xylobiose transporters in trans TDXyl xynb + 0 Time (hours)

13 Efflux in IBBE

14 Multidrug efflux Large amount of knowledge of many different families of multidrug & antibiotic efflux proteins (including structures). 1) ABC efflux (primary) ABC MATE OUT MFS 2) The RND multidrug efflux pumps (secondary) 3) Other families of secondary transporters MATE, SME and MFS SMR IN RND

15 What do these proteins normally do? but more recently discovering what these proteins normally do MdtJI in E. coli (SMR family) spermidine AcrAB-TolC phospholipids, BpeAB-OprB homoserine lactone secretion (quorum sensing). QacA also confers resistance to cationic antimicrobial peptides. Roles in IBBE? Already emerging roles for AcrAB-TolC in E. coli particularly in improving tolerance to alcohols, ethanol and butanol, and other organic solvents, like hexane, cyclohexane & octane. Butanol Hexane Cyclohexane Octane

16 New efflux systems directly related to IBBE In parallel other more biotechnology-minded groups in Europe & Japan found random mutants in their small molecule-producing bacteria that upregulated expression of various uncharacterised membrane transporters. Discovery of the LysE L-lysine exporter in Corynebacterium glutamicum led this field. Also the ThrE and BrnFE systems discovered for L-Thr and L-Iso, respectively. L-Glu appears to be exported via a totally novel route through a MscS-like mechanosensitive channel. LysE 2.A.75 H + ThrE 2.A.79 LIV-E 2.A.78 AlaE 2.A.104 OUT 10 TMS 6-8 TM S 4 L-Lys L-Thr L-Met & BCAA L-Ala IN There are now known to be efflux systems for all 20 amino acids!

17 Some examples of expansion of transporter families across the microbial genomes MscS Mean = Medium = Halothece (α-proteobacteria) - 17 Pseudoanabeana (Cyanobacteria) - 13 Krokinobacter (Bacteroidetes) - 10 Halorubrum (Euryarchaeota) - 11 Escherichia - ~ 7 Corynebacteria - ~ 2 Clostridium sp. ~1

18 Using L-valine efflux to increase yield Prof. Sang Yup Lee KAIST As part of an elegant series of experiments to engineer E. coli to produce L-valine, he thought about transporters. Deleted the uptake system. Put the ygazh genes in multicopy. See yield increase from 4.34 g/l to 5.25 g/l. When he overexpressed the natural activator for ygazh, the Lrp protein, yield increased to 7.61 g/l (75% yield increase by thinking about the membrane). Park et al., (2007) PNAS 104:

19 What else is going to be effluxed? Exciting times for effluxers! Now clearly roles for sugar efflux in some bacteria, including C. glutamicum and E. coli. Also, there are efflux systems for organic acid, such as succinate. And there are efflux systems for nucleosides. What else might be effluxed? Also, until now we have been thinking about the inner (cytoplasmic) membrane only, but Gram ve bacteria have another barrier to cross. Could the key molecule that your company is interested be effluxed to improve your bioprocess?

20 Recap of learning outcomes Think about the transporter repertoire of different microbes what could it mean? Understand some examples of where the energetics of uptake has made a different to product formation Appreciate the diversity of efflux systems in biology, some examples of their exploitation & the great potential for future manipulation.