Seung-Sub’s paper

Glycogen is the most widely used as an energy storage compound.

a-1,4 and a-1,6 linkage

branch chains is 8-12 glucose units, particle diameter 20nm-50nm

molecular size is about 107-108Da

In present, glycogen is known to have a lot of bio-activities as well as its role as an energy
storage compound.

-antitumor effects, reduced fa accumulation et al,,

TBP38 shows functional properties.

-high water solubility, stable solubility at RT

-lower digestibility to amyloglucosidase than amylopectin

-strong immunostimulating on RAW2346.7 cell

1. Glycogen metabolism in E.coli

The synthesis of glycogen occurs when C is abundant, but another nutrients such as nitrogen,
phosphate are limited.

Gene cluster consist tow operons glgBX & glgCAP

<First operon>

glgB(glycogen branching enzyme)

glgX(debranching enzyme)

<second operon>

glgA(Glycogen synthase)

glgC(ADP-glucose pyrophosphorylase)

glgP(glycogen phosphorylase)

glgC catalyze ADP glucose using G-1-P as substrate  formed ADP glucose catalyzed glgA 

formed a-1,4-glucosyl units branching enzyme catalyzed formation of the branched a-1,6-glucosyl
units

Degradation of glycogen is performed glgX & glgP

glgP cleaves nonreducing ends of a-1,4 linkages to producing G-1-P

glgP reduces the length of the maltotriosyl and maltotetraosyl

glgX(debranching enzyme) uses them as substrates and release maltotriose and maltotetrose

2. Maltodextrin metabolism in E.coli

Regulated by 10 genes that have role as transport, synthesis and degradation.

The regulon of the maltodextrin sys is controlled by malT which is acting activator

Maltodextrins longer than maltoheptaose are unable to enter the cytoplasm

MalS(periplasmic a-amylase) cleaves the maltodextrins

Enter the cytoplasm through the ABC transporter

MalE take a role like maltose binding protein.]

MalQ(amylomaltase) polymerizes the maltodextrin(n-1) of various chain length as a substrate

entering in cytoplasm through the ABC transporter

MalP & malZ

MalP maltodextrins( n-1)  maltodextrins(n-2) + G-1-P

MalZ maltodextrins( n-1)  maltodextrins(n-2) + G

G-1-P, G can used as substrates of glycogen synthesis or glycolysis

3. TBP38 mutant

Maltodextrins are polymerized by MalQ(amylomaltase) to long maltodextrins

Which are substrate of glycogen synthesis by glgB

malP mutant accumulated highly long chain maltodextrins

glgA mutant showed no glycogen accumulation

but TPB38 accumulated more than 20-fold than WT

homogeneous 2.6x106-4.6x106Da

DP≤12 greater than 50%

4. High cell density culture

-reduced volume
-improve productivity

-lower production costs and reduced investment equipment

The nutrients such as carbon, nitrogen, phosphorus, sulfur, magenseium, potassium, iron,
manganese, zinc, and copper can affect growth of E.coli

Precipitation of media compound  affect purification and recovery sys

-combination of anions(phosphate, sulfates and chloride)

-cations(sodium, calcium, ammonium and magnesium)

-acetate // E.coli excretes acetate aerobic or anaerobic condition with excess carbonthis makes
inhibit the growth of cell as well as production of metabolites

Many studies suggest reduced acetate accumulation

controlling acetatekinase/phosphotransacetylase (AckA/Pta) and pyruvate oxidase(PoxB)

the ackA-pta pathway which consists of two genes in one operon

generate acetate from acetyl-CoA at exponential phase

elimination of these region can strongly inhibit acetate production but mutant can produce
acetate through poxB pathway under aerobic conditions

poxB mutant lower maximum growth than parental strain

preventing overflow of acetate is to reduce the growth rate by supplying carbon source slowly

In this study developing strain that accumulates glycogen by inactivating the debranching enzyme
in TBP38
Sung-Joon’s paper

1. Introduction

Glycogen has similar structure like AP, but glycogen has more random chain length distribution of
branch linkages and shows monomodal chain length whereas polymodal chain.

Maltose and maltodextrins enter the cytoplasm through ABC transporter.

glgB catalyses formation of a-1,6-glucosidic bond by cleaving a-1,4-glucosidic linkages of

substrate

Microorganisms have been engineered to produce ethanol, bulk chemicals, and valuable drugs
form inexpensive starting materials

E.coli because of their fast growth rates and facultive anaerobes

Can produce tailor chemeicals, fatty alcohol

In this study, to establish a glycogen biosynthesis sys for producing tailor-type glycogen,
metabolic pathways of E.coli was engineered glgB of origin was disrupted and replaced by
recombinant plasmid with other branching enzyme genes