This document discusses recombinant protein production in E. coli and challenges related to inhibitory byproducts like acetate. It summarizes that (1) acetate accumulation reduces protein expression yields and quality by interfering with processes like disulfide bond formation, (2) introducing a heterologous acetolactate synthase enzyme redirects carbon flux from acetate production toward less harmful byproducts like butanediol, and (3) this metabolic engineering approach maintained acetate levels below 20mM and increased recombinant beta-galactosidase expression yields by 50% for batch and 220% for fed-batch cultivations.

1. Presented by
Aathilakshmi U
(810021509001)
1st year M.Tech Biotechnology

2. rDNA protein production
• Technical challenges in recombinant protein production processes employing E.
coli is to maintain high intracellular product levels at high cell
concentrations.
• This dual goal is difficult to achieve due to the accumulation of inhibitory
culture byproducts.
• During both aerobic and anaerobic growth, carbon and reductant fluxes are
balanced by the excretion of acidic byproducts, the most abundant of which is
acetate. This weak acid is a well-known growth inhibitor.
• Most importantly, it reduces the cellular efficiency for the expression of
recombinant products and affects the quality of intracellular proteins,
apparently by interfering with disulfide bond formation.

3. Influence of pH
• Organic acids have been shown to influence cell growth at concentrations that are
low in comparison to inhibitory levels of mineral acids.
• The undissociated forms of short-chain fatty acids produced intracellularly, such as
acetic acid, can freely permeate the cell membrane and accumulate in the medium.
• Subsequently, a fraction of the undissociated acid that is present extracellularly re-
enters the cell, where it dissociates given the relatively higher intracellular pH. This
means that, in effect, weak acids act as proton conductors.
• If this process continues undiminished, the intracellular pH will approach the
external pH and hence the ∆pH component of the protonmotive force will collapse .
• In addition, a low external pH (<5) can cause almost complete growth stasis (without
cell lysis) presumably due to the irreversible denaturation of DNA and protein.

4. Experimental study
• In addition to the preceding effects on cellular energetics, there are many other
factors contributing to the inhibitory nature of weak organic acids that make
minimization of acetate excretion a prerequisite for optimizing the production
yields of recombinant processes.
• The chemostat data of Jensen and Carlsen (1990), who studied the effects of
acetate on the production of human growth hormone in E. coli, clearly
illustrate the significance of acetate in this recombinant system.
• By varying the amount of acetate in the feed, it was determined that acetate
levels of 40mM reduce recombinant protein yields by approximately 35%
without having any effect on the biomass yield.

5. • This result agrees with the general observation that the acetate
threshold that influences recombinant protein yields is usually lower
than that causes notable growth inhibition.
• In the same study, increasing the acetate level to 100 mM caused a
reduction in biomass yield by more than 70%, whereas recombinant
product yield declined by a factor of 2.
• Several other investigators have implicated acetate as an important
factor in the deterioration of recombinant process productivities.

6. Butanediol formation
• It is widely accepted that acetate excretion results from an imbalance
between the glycolytic flux and the cell's actual requirements for metabolic
precursors and energy.
• Pyruvate, which is the end product of glycolysis as well as the precursor of
acetate, provides a suitable junction for effecting acetate accumulation.
• The strategy involves the introduction of a heterologous enzyme to catalyze
the redirection of surplus carbon flux to a less harmful byproduct than acetate.
• The B. subtilis acetolactate synthase (ALS) enzyme was selected for this
purpose on the basis of the fermentation characteristics of this group of
microorganisms.

8. • Butanediol producers normally have two distinct enzymes that convert
pyruvate to acetolactate:
• Acetolactate synthase (ALS)
• Acetohydroxy acid synthetase (AHAS)
• AHAS, an anabolic enzyme found in many microorganisms, also
catalyzes the initial steps from pyruvate in the formation of the
branched-chain amino acids valine, leucine, and isoleucine.
• AHAS is also a flavoprotein that is regulated by end product feedback
inhibition, such as by valine.
• ALS does not require FAD for activity, nor is it inhibited by the
presence of branched amino acids.

10. • The alsS gene from B. subtilis encoding the ALS enzyme was successfully
expressed in E. coli.
• This enzyme acts at the pyruvate branch point, redirecting excess carbon flux
away from acetate and toward the non inhibitory byproduct α-acetolactate.
• Characterization of the resulting strain indicated that acetate excretion can be
maintained below 20mM even in dense cultures employing rich glucose
medium.
• Moreover, the engineered strain is a more efficient host for the production of
recombinant proteins.
• The volumetric expression of recombinant ꞵ-galactosidase was found to
increase by about 50% in batch cultivations and by about 220% in high cell
density fed-batch cultivations.
• These results demonstrate the successful application of metabolic engineering
for the improvement of cellular characteristics.

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