PROGRESS REPORT
PROJECT TITLE: Nutritional improvement of corn ethanol coproducts via yeast engineering
PROJECT NUMBER: Yanmei Zhang, Aravindan Rajendran, Cristiano E. Rodrigues Reis
REPORTING PERIOD: quarterly reports
PRINCIPAL INVESTIGATOR: Bo Hu
ORGANIZATION: University of Minnesota, Department: Bioproducts and Biosystems Engineering
PHONE NUMBER: 612-625-4215
EMAIL: [email protected]
1.) PROJECT ACTIVITIES COMPLETED DURING THE REPORTING PERIOD. (Describe project
progress specific to goals, objectives, and deliverables identified in the project workplan.)
We encountered a challenge that the lysine content in the corn ethanol coproducts was not improved as
the ratio of yeast in the coproducts was decreased, although the lysine concentration was improved in the
engineered cells. Thus, the specific goal during this period is to figure out how to increase the yeast cell
growth and biomass accumulation of the lysine engineered strains, thus increasing the total lysine content
during fermentation. Firstly, we retest the free lysine and the total lysine content in the lysine engineered
yeast cells to make sure if the cell specific lysine production was indeed improved. The amino acids
profile was also determined. Secondly, the protease expression system was designed to introduce into the
yeast cell to improve the cell growth. Thirdly, the strategies for enriching tryptophan and arginine in yeast
cells was also discussed.
a) Free and total lysine test in engineered strains
To detect the intracellular free lysine and total lysine content in the cells of engineered strains, the
cells grown at different time points were harvested. The intracellular metabolites were extracted in boiling
ethanol after quickly quenching in cold methanol, then the free amino acids were tested. The total amino
acids were tested after acid hydrolysis of protein samples. The amino acids were analyzed by GC-FID
using ez:faast kits.
b) Protease expression in the lysine engineered yeast cell
The use of certain proteases in ethanol fermentation has been proven to enhance cell growth and
fermentation. So we propose to introduce protease into the engineered lysine strains and try to improve
the cell growth and biomass accumulation. The protease from Neurospora crassa, Trichoderma ressei or
corn was chosen to express in the cell surface of the yeast cells. The expression vector was designed and
constructed.
c) Tryptophan and arginine engineering in yeast
For increasing the intracellular content of tryptophan and arginine, we design a short 44 residues
peptide rich in tryptophan and arginine for expression in Yeast. That peptide contains 14 residues of
tryptophan and 14 residues of arginine across its sequence. Moreover, a prolonged peptied containing
multiple copies of that 44 residues unit in sequence will be expressed as well. The prolonged peptied may
exhibit a property with increased stability and lowered degradation in vivo.
2.) IDENTIFY ANY SIGNIFICANT FINDINGS AND RESULTS OF THE PROJECT TO DATE.
a) The intracellular free and total lysine content was increased in yeast cells of engineered strains
The cell specific free lysine production in the strains ΔLYS80:O20,
a
ΔLYS80:O14 and ΔLYS80:O14/20 was improved by 2-4 times
compared with wild type, while the total lysine in engineered cells was
improved by 20-100% compared with wild type (Fig.1a). Therefore, the
ratio of the free lysine to the lysine integrated into protein was increased
in the engineered cells/ The free and total lysine content in yeast cells
b
can be improved by engineering of lysine biosynthesis pathway in S.
cerevisiae.
As we can tell from Fig.1b, the total lysine level was significantly
decreased in ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20 when
compared with wild type. Although higher total lysine content in yeast
cell was found in ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20,
Fig. 1 Lysine determination in engineered yeast cells
(a) and total lysine production (b)
the significant decrease in lysine production was mainly due to the
less yeast cell biomass accumulated.
When the amino acids profile is expressed as relative% (based on total AA), the free lysine ratio was
increased compared to wild type. However, the total lysine ratio was barely changed compared to the wild
type. Similarly, most of amino acids tested here showed similar level as the wild type. This was
reasonable as the total amino acids was reduced in the mutants ΔLYS80:O20, ΔLYS80:O14 and
ΔLYS80:O14/20.
b) Expression of aspartic protease from N. crassa in S. cerevisiae
To construct the protease expression vector, the gene encoding aspartic protease N. crassa was
synthesized and constitutively expressed under the PGK1 promoter. The
N terminal of the protease was fused to secretion signal sequence of ∂factor. The protease was fused to ∂-agglutinin peptide at C terminal in
order to anchor the protease to the cell wall. A partial rDNA fragment
was inserted before the gene expression cassette and used as the
homologous integration site (Fig. 2) Now vector construction is in
process.
The strains ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20
contain Geneticin resistance gene KanMX, which needs to be
Fig. 2 Construction of protease expression plasmid
removed before further manipulation. The Cre expression plasmids
pSH66 was transformed to the engineered strains, the transformants without Geneticin resistance is being
screened. The protease expression vector can be introduced into the gene modified strains without maker.
Intracellular total & free lysine
(nmol/10 8 cell)
700
CEN.PK 113-7D
ΔLYS80:O20
ΔLYS80:O14
ΔLYS80:O14-20
600
500
400
300
200
100
0
6
24
Time (h)
48
1400
72
CEN.PK 113-7D
ΔLYS80:O20
1000
ΔLYS80:O14-20
Total intracellular lysine
(nmol/mL)
1200
ΔLYS80:O14
800
600
400
200
0
6
24
rDNA
Time (h)
PGK1
promoter
48
Secretion
signal
72
Protease
3’ half of � agglutinin
3.) CHALLENGES ENCOUNTERED. (Describe any challenges that you encountered related to project
progress specific to goals, objectives, and deliverables identified in the project workplan.)
The overall goal is to significantly increase the feeding value of corn ethanol co-products. The major
challenges till now is the total lysine content was decreased in the mutants showing higher total lysine in
yeast cells due to less cell biomass accumulation. The methods to increase the cell growth was developed
by engineering protease in the strains we have.
The strategies for tryptophan and arginine are being developed in alternative ways. Its challenge
could be that the expression of tryptophan and arginine rich peptide may decrease the growth rate of the
yeast cell, resulting from the positive charge of arginine in neutral pH conditon could interfere the normal
membranal system function of cell membrane and other internal membranal organelle.
4.) FINANCIAL INFORMATION (Describe any budget challenges and provide specific reasons for
deviations from the projected project spending.)
No. We have a second postdoc researcher Jingyu Wang joining our group to work on this project.
5.) EDUCATION AND OUTREACH ACTIVITES. (Describe any conferences, workshops, field days,
etc attended, number of contacts at each event, and/or publications developed to disseminate project
results.)
We attended the Ag Expo, 2017 and present a poster “Nutritional Value Improvement of Corn Ethanol
Coproducts” in Mankato, MN. January 26, 2017.
Now the manuscript is being drafted for genetic engineering of lysine biosynthesis for improved lysine
production.