scholarly journals A teaching protocol demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae and Yarrowia lipolytica

2019 ◽  
Author(s):  
N Milne ◽  
L R R Tramontin ◽  
I Borodina
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Yarrowia Lipolytica ◽  
Yeast Species ◽  
Production Capacity ◽  
Value Added ◽  
Phenotypic Screen ◽  
Rate Limiting ◽  
Β Carotene

ABSTRACT We present a teaching protocol suitable for demonstrating the use of EasyClone and CRISPR/Cas9 for metabolic engineering of industrially relevant yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, using β-carotene production as a case study. The protocol details all steps required to generate DNA parts, transform and genotype yeast, and perform a phenotypic screen to determine β-carotene production. The protocol is intended to be used as an instruction manual for a two-week practical course aimed at MSc and PhD students. The protocol details all necessary steps for students to engineer yeast to produce β-carotene and serves as a practical introduction to the principles of metabolic engineering including the concepts of boosting native precursor supply and alleviating rate-limiting steps. It also highlights key differences in the metabolism and heterologous production capacity of two industrially relevant yeast species. The protocol is divided into daily experiments covering a two week period and provides detailed instructions for every step meaning this protocol can be used ‘as is’ for a teaching course or as a case study for how yeast can be engineered to produce value-added molecules.

scholarly journals 5-Aminolevulinic acid fermentation using engineered Saccharomyces cerevisiae

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Kiyotaka Y. Hara ◽  
Masaru Saito ◽  
Hiroko Kato ◽  
Kana Morikawa ◽  
Hiroshi Kikukawa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Aminolevulinic Acid ◽  
Value Added ◽  
Acid Fermentation ◽  
Rate Limiting Step ◽  
Ala Synthetase ◽  
Engineered Saccharomyces Cerevisiae ◽  
Rate Limiting ◽  
Ala Biosynthesis

Abstract Background 5′-Aminolevulinic acid (ALA) is widely used in the pharmaceutical industry, healthcare, and food production, and is a substrate for the biosynthesis of heme, which is required for respiration and photosynthesis. Enhancement of ALA biosynthesis has never been developed in Saccharomyces cerevisiae, which is a well-known model microorganism used for bioproduction of many value-added compounds. Results We demonstrated that metabolic engineering significantly improved ALA production in S. cerevisiae. First, we found that overexpression of HEM1, which encodes ALA synthetase, increased ALA production. Furthermore, addition of an optimal amount of glycine, a substrate for ALA biosynthesis, or levulinic acid, an inhibitor of ALA dehydrogenase, effectively increased ALA production. Next, we developed an assay for multiple metabolites including ALA and found that aconitase, encoded by ACO1 and ACO2, is the rate-limiting enzyme of ALA biosynthesis when sufficient glycine is supplied. Overexpression of ACO2 further enhanced ALA production in S. cerevisiae overexpressing HEM1. Conclusions In this study, ALA production in S. cerevisiae was enhanced by metabolic engineering. This study also shows a strategy to identify the rate-limiting step of a target synthetic pathway by assay for multiple metabolites alongside the target product. This strategy can be applied to improve production of other valuable products in the well-studied and well-industrialized microorganism S. cerevisiae.

Metabolic engineering of β-carotene biosynthesis in Yarrowia lipolytica

2020 ◽  
Vol 42 (6) ◽  
pp. 945-956 ◽  
Author(s):  
Xin-Kai Zhang ◽  
Dan-Ni Wang ◽  
Jun Chen ◽  
Zhi-Jie Liu ◽  
Liu-Jing Wei ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Yarrowia Lipolytica ◽  
Β Carotene

scholarly journals Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance

2009 ◽  
Vol 75 (22) ◽  
pp. 7205-7211 ◽  
Author(s):  
Ken Ukibe ◽  
Keisuke Hashida ◽  
Nobuyuki Yoshida ◽  
Hiroshi Takagi
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Cytochrome P450 ◽  
Metabolic Engineering ◽  
Stress Tolerance ◽  
Yeast Cells ◽  
Cytochrome P450 Enzyme ◽  
Oxidative Stress Tolerance ◽  
Astaxanthin Production ◽  
Β Carotene

ABSTRACT The red carotenoid astaxanthin possesses higher antioxidant activity than other carotenoids and has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. In this study, we produced astaxanthin in the budding yeast Saccharomyces cerevisiae by introducing the genes involved in astaxanthin biosynthesis of carotenogenic microorganisms. In particular, expression of genes of the red yeast Xanthophyllomyces dendrorhous encoding phytoene desaturase (crtI product) and bifunctional phytoene synthase/lycopene cyclase (crtYB product) resulted in the accumulation of a small amount of β-carotene in S. cerevisiae. Overexpression of geranylgeranyl pyrophosphate (GGPP) synthase from S. cerevisiae (the BTS1 gene product) increased the intracellular β-carotene levels due to the accelerated conversion of farnesyl pyrophosphate to GGPP. Introduction of the X. dendrorhous crtS gene, encoding astaxanthin synthase, assumed to be the cytochrome P450 enzyme, did not lead to astaxanthin production. However, coexpression of CrtS with X. dendrorhous CrtR, a cytochrome P450 reductase, resulted in the accumulation of a small amount of astaxanthin. In addition, the β-carotene-producing yeast cells transformed by the bacterial genes crtW and crtZ, encoding β-carotene ketolase and hydroxylase, respectively, also accumulated astaxanthin and its intermediates, echinenone, canthaxanthin, and zeaxanthin. Interestingly, we found that these ketocarotenoids conferred oxidative stress tolerance on S. cerevisiae cells. This metabolic engineering has potential for overproduction of astaxanthin and breeding of novel oxidative stress-tolerant yeast strains.

Management of the learning curve: a case of overseas production capacity expansion

2016 ◽  
Vol 36 (1) ◽  
pp. 42-60 ◽  
Author(s):  
Peter-Christian Pedersen ◽  
Dmitrij Slepniov
Keyword(s):  
Learning Curve ◽  
Wind Turbine ◽  
Lead Time ◽  
Qualitative Methodology ◽  
Production Capacity ◽  
Value Added ◽  
Capacity Expansion ◽  
Learning Curves ◽  
Content Type

Purpose – This paper focuses on the management of the learning curve in overseas capacity expansions. The purpose of this paper is to unravel the direct as well as indirect influences on the learning curve and to advance the understanding of how these affect its management. Design/methodology/approach – The paper builds on the offshoring, capacity expansion and learning curve literature. The existing scholarship often lacks detailed insights into the factors surrounding the globalisation of production, and how constructing and operationalising new capacities overseas should be implemented. The paper employs qualitative methodology and draws on a longitudinal, factory-level analysis of an in-depth case study of a Danish wind turbine manufacturer. Findings – This study goes beyond a simplistic treatment of the lead time and learning required to establish a new capacity. The authors examined the dimensions of the learning process involved in a capacity expansion project and identified the direct and indirect labour influences on the production learning curve. On this basis, the study proposes solutions to managing learning curves in overseas capacity expansions. Furthermore, the paper concludes with measures that have the potential to significantly reduce the non-value-added time when establishing new capacities overseas. Originality/value – The paper uses a longitudinal in-depth case study of a Danish wind turbine manufacturer and goes beyond a simplistic treatment of the lead time and learning required to establish a new capacity.

scholarly journals Yarrowia lipolytica as an Oleaginous Platform for the Production of Value-Added Fatty Acid-Based Bioproducts

2021 ◽  
Vol 11 ◽  
Author(s):  
Huhu Liu ◽  
Yulan Song ◽  
Xiao Fan ◽  
Chong Wang ◽  
Xiangyang Lu ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Metabolic Engineering ◽  
Yarrowia Lipolytica ◽  
Fermentation Process ◽  
Acid Metabolism ◽  
Fatty Acid Biosynthesis ◽  
Alternative Pathway ◽  
Value Added ◽  
Acid Biosynthesis ◽  
Current Production

The microbial fermentation process has been used as an alternative pathway to the production of value-added natural products. Of the microorganisms, Yarrowia lipolytica, as an oleaginous platform, is able to produce fatty acid-derived biofuels and biochemicals. Nowadays, there are growing progresses on the production of value-added fatty acid-based bioproducts in Y. lipolytica. However, there are fewer reviews performing the metabolic engineering strategies and summarizing the current production of fatty acid-based bioproducts in Y. lipolytica. To this end, we briefly provide the fatty acid metabolism, including fatty acid biosynthesis, transportation, and degradation. Then, we introduce the various metabolic engineering strategies for increasing bioproduct accumulation in Y. lipolytica. Further, the advanced progress in the production of fatty acid-based bioproducts by Y. lipolytica, including nutraceuticals, biofuels, and biochemicals, is summarized. This review will provide attractive thoughts for researchers working in the field of Y. lipolytica.

scholarly journals Biomanufacturing of Value-Added Products from Oils or Fats: A Case Study of Yarrowia lipolytica

2020 ◽  
Author(s):  
Na Liu ◽  
Ya-Hue Soong ◽  
Iman Mirzaee Kakhki ◽  
Andrew Olson ◽  
Peng Yu ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Value Added ◽  
Value Added Products
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