PROGRESS REPORT

PROJECT TITLE: Nutritional improvement of corn ethanol coproducts via yeast engineeringPROJECT NUMBER: 1081-16EUREPORTING PERIOD: quarterly reportsPRINCIPAL INVESTIGATOR: Bo HuORGANIZATION: University of Minnesota, Department: Bioproducts and Biosystems EngineeringPHONE NUMBER: 612-625-4215EMAIL: bhu@umn.edu

1.) PROJECT ACTIVITIES COMPLETED DURING THE REPORTING PERIOD. (Describe project progress specific to goals, objectives, and deliverables identified in the project workplan.)The specific goal during this period is to determine whether the total lysine content is improved in yeast cells and in the corn ethanol coproducts. a) Total lysine test in engineered strains

To detect the total lysine content in the cells of engineered strains, the cells grown at exponential phase were harvested and washed twice with sterilized water. The cells were then immediately lyophilized. The frozen dried samples were then hydrolyzed by 6 N HCl for 24 h at 110 oC. The amino acids were analyzed by HPLC-MS. The lysine content in yeast cells was analyzed.b) Total lysine determination in coproducts of corn ethanol fermentation

To determine if the lysine level is improved in corn ethanol coproducts, the fermentation of the liquefied corn mash from Absolute Energy by engineered lysine yeast has been carried out. Briefly, after ethanol fermentation, the fermentation broth was taken and immediately frozen dried. Then after hydrolysis in 6 N HCl at 110 oC for 24 h, the lysine content as well as the amino acids profile was tested and compared with the coproducts generated from wild strains to evaluate the nutritional improvements of DDGS generated at our proposed process. The fermentation productivity was determined by detection of glucose, ethanol, glycerol and acetic acid.

2.) IDENTIFY ANY SIGNIFICANT FINDINGS AND RESULTS OF THE PROJECT TO DATE. a) The total lysine content was increased in yeast cells of engineered strains

As we can tell from Fig.1, the lysine pool in yeast cells of double mutants ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20 was increased by 51%, 95% and 42%, respectively. Accordingly, the free lysine content in yeast cells was increased by 279%, 135% and 247%. As the free lysine was not significantly improved in OLYS20, OLYS14 and ΔLYS80, the total lysine was not affected in theses mutants. As we proposed, the total lysine content in yeast cells can be improved by engineering of lysine biosynthesis pathway in S. cerevisiae.

b) The total lysine was not improved in corn ethanol coproducts caused by growth defectAfter the corn ethanol fermentation, the total lysine level in the fermentation broth was shown in Fig.

2. The lysine content in mutant OLYS20, OLYS14 and ΔLYS80 was nearly unchanged compared with the wild type. However, the lysine content was significantly decreased in ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20, by 11%, 17% and 30% when compared with wild type. Although higher total lysine content in yeast cell was found in ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20, the

Fig. 1 Total lysine determination in yeast cells

significant decrease in lysine in fermentation media was probably due to the less yeast cell biomass accumulated.

When the AA profile is expressed as relative% (based on total AA), the lysine ratio was barely changed compared to the wild type. Similarly, all amino acids tested here showed similar level as the wild type (Fig. 3). This was reasonable as the total amino acids was reduced in the mutants ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20.

The fermentation of the lysine engineered strains using corn mash was also analyzed. The results (Fig. 4) showed that the ethanol production was delayed in engineered strains ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20 with slower glucose consumption rate. This may be caused by the decreased growth of the engineered strains. The ethanol concentration was 6%, 7% and 12% lower than the wild type at 96 h. In addition, the glycerol content was increased in these three mutants, which also indicates the growth defect. In

contrast, the acetic acids production was decreased in these mutants. The reason

needs to be examined.

3.) CHALLENGES ENCOUNTERED. (Describe any challenges that you encountered related to project progress specific to goals, objectives, and deliverables identified in the project workplan.)Our first objectives during this period is to increase the total lysine content in yeast biomass, and to date we obtained the engineered strains that showed increased total lysine level in cells. The second objective is to increase the total lysine during ethanol fermentation with not affecting ethanol production. But in our study, the total lysine content was decreased in the mutant showed higher total lysine in yeast cells. As the lysine in fermentation media is composed of two part: corn and yeast. It was reported that the yeast protein contributed about 20% of the total protein upon fermentation. Although the total lysine was improved in yeast cells of ΔLYS80:O20, ΔLYS80:O14 and ΔLYS80:O14/20, the lysine in fermented

Fig. 2 Total lysine determination in fermented corn mash

Fig. 3 Total amino acids profile in fermented corn mash

Fig. 4 The fermentability test during corn mash fermentation

corn mash was decreased as the cell growth was significantly affected. Thus, the nutritional value may be not increased in corn ethanol coproducts as the ratio of yeast in coproducts is decreased.

Two strategies are being developed to overcome the problem here. The first one is to engineer the lysine transporter in vacuole, which make more lysine transport into vacuole and reduce the lysine feedback inhibition of its biosynthesis. This will further increase the lysine production in S. cerevisiae. The second one is the secondary fermentation of thin stillage to directly increase the key amino acids content. The preliminary result using Mucor sp. showed that the average amino acids percentage nearly doubled in the fermented products, indicating rectified feeding value in animal feeding industry. Based on these result, we can perform secondary fermentation using amino acids overproducing strains, such as Corynebacterium glutamicum and Bacillus subtilis, which aim to increase the lysine content in the final products.

4.) FINANCIAL INFORMATION (Describe any budget challenges and provide specific reasons for deviations from the projected project spending.)No

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 presented a poster “Genetic Engineering of Yeast to Improve Feeding Value of Corn Ethanol Co-product DDGS” at showcase of Department of Bioproducts and Biosystems Engineering in University of Minnesota, Saint Paul, MN. October 27, 2016. We attended the Ag Expo, 2017 and present a poster “Nutritional Value Improvement of Corn Ethanol Coproducts” in Mankato, MN. January 26, 2017.Now we are drafting the manuscript for genetic engineering of lysine biosynthesis for improved lysine production.

Fig. 5 Secondary fermentation of thin stillage for amino acids improvement