Lec 8 - Metabolic Engineering for Lys production Flashcards
What is metabolic engineering?
Redesigning of a biochemical pathway to either enhance the synthesis of a product or produce a new product
What are the 3 main ways that metabolic engineering is carried out? (think generally)
- insertion of new sets of genes to create a new product
- redirecting metabolism for the synthesis of specific products
- stopping the production of other unwanted products
Give the five strategies for improving chemical production buy metabolic engineering
- directly altering metabolic pathway (altering enzyme w/ highest flux coefficient of control, altering flux for production of certain product, replacing enzymes w/ better enzymes eg ones with lower Km, higher Vmax)
- modifying transporters to increase specificity for substrates
- uncouple growth and production phase
- faster regeneration of precursors, energy supplies and cofactors (over expressing enzymes for NADH, ATP production)
- increase the tolerance of the cell to the toxic (potentially) product @ high concns
Give examples of amino acids being used in the food industry.
- Lys - added to soy products/bread for feeds for pigs etc
- essential aa / those not present in plant based foods (eg lys, met) added to foods to increase nutritional quality
- flavour enhancers - eg Monosodium glutamate (MSG) and glycine
What is the name of the bacteria that is used to produce L-Lys and L-Glu in industry?
What is its special feature that enables it to be used for Lys production?
Corynebacterium glutamicum
cannot break down L-Lys therefore excretes it
Under what fermentation conditions is C. glutamicum grown in?
fed batch fermenters
Draw the branched biosynthesis pathway for the production of Lys and other products from C. glutamicum.
see Kelly 1 notes
What yields (in terms of g/L) do we see today due the metabolic engineering processes we apply?
180g/L
What enzyme catalyses the production of aspartate -> asp semi aldehyde?
Aspartakinase (LysC)
Why can we simply not block the enzyme responsible for asp semi-aldehyde conversion to homoserine ?
because then cell would die because cannot synthesis Met, Thr and these aa would have to be added to medium (expensive)
Describe the enzyme aspartakinase and how it is inhibited
inhibited by both Lys and Thr that act TOGETHER at allosteric sites on the B subunit
a2B2 heterodimer
feedback inhibition
How do we use antimetabolites to establish feedback resistant aspartakinases?
antimetabolites can be substrate analogues that interrupt specific metabolic processes by replacing the natural substrate from the AS
aminoethylcysteine (substrate analogue to L-Lys) used to identify aspartakinases that are resistant to feedback inhibition once AEC is present
mutagenesis of the aspartakinase can mean that its site no longer fits that of AEC/Lys therefore no feedback inhibition and more production of Lys
What are the 3 other metabolic engineering methods that increase Lys production from Corynebacterium glutamicum?
- point mutations of dapA promoter therefore increased expression of dapA. encodes synthase enzyme that is directly related to Lys biosynthesis after the branch point of the pathway. small increases in yield but on industrial scale = worthwhile
- over expression of pntAB genes that encode transhydrogenase (NADH + NADP = NAD + NADPH). increases in NADPH content. conversion of oxaloacetate -> lysine involves 4 moles of NADPH
- over expression of LysE therefore causing more LysE transport proteins in the membrane (cant be too many because cell death)
What ions are sym/antiported with the export of Lys and what is the overall net change in charge?
- one Lys and 2 H+s ANTIPORT
- one Lys and 2OH- s SYMPORTED
- overall net loss of 1 -ve charge to the environment
