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Method for uncoupling yield and productivity of non-catabolic compounds produced by host cells

A catabolism, host cell technology, applied in the field of decoupling between yield and productivity, and can solve problems such as yield reduction

Pending Publication Date: 2022-03-18
AMYRIS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to rate-yield coupling, any attempt to increase productivity by increasing oxygen and / or sugar transfer rates will result in a concomitant decrease in yield, negating the cost-effectiveness of increased productivity

Method used

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  • Method for uncoupling yield and productivity of non-catabolic compounds produced by host cells
  • Method for uncoupling yield and productivity of non-catabolic compounds produced by host cells
  • Method for uncoupling yield and productivity of non-catabolic compounds produced by host cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0129] Example 1: Yield as a function of cell-specific rate

[0130] Inoculate a single colony in 15ml of 2% sucrose, 1% maltose, 2g / L lysine LGM and 50mM succinate pH 5.0 in a 125ml flask, then grow at 28°C and shake at 200r.p.m. to OD600 at 4 Between 9 and 9, with residual glucose between 3 and 6 g, 1-1.50% glycerol was added to the culture to a concentration of 20%, and then the cell suspension was stored in a 1 ml vial at -80°C. Thaw 1-2 flasks of cells in a solution containing 3gl-1 yeast extract, 7gl-1NH4H2PO4, 1gl-1KH2PO4, 0.5gl-1MgSO4·7H2O, 50mM succinate pH 5.0, 4% sucrose, 2% maltose, 2g / L Lysine and trace metal and vitamin solutions were grown for 24 h, and then subcultured in the same medium for 24 h to an OD600 reading of 0.1. 25 ml of culture was used to inoculate a 0.5 liter fermenter (Sartorius, Germany) containing 225ml fermentation medium containing 15g1-1 NH4H2PO4, 20g1-1 total reducing sugars (TRS) from sucrose syrup (Florida Crystals, West Palm Beach), an...

example 2

[0139] Example 2: Computational Modeling and Metabolomics Assays Demonstrate ATP-Driven Coupling Between Rate and Yield

[0140] To assess possible mechanistic reasons for the rate-yield coupling, we analyzed 2 / L / h) or high OTR (180mmol O 2 / L / hr) Absolute concentrations of central metabolites from glycolysis, TCA cycle, pentose phosphate pathway and isoprenoid pathway in fermentation samples of a single strain run. Sampling from the bioreactor was performed using a rapid sampling protocol, in which the sample was quenched immediately after removal from the tank to capture the metabolic state. While most central metabolites were determined at similar absolute concentrations under low and high OTR conditions, two metabolites (isocitrate and α-ketoglutarate) stood out as having very different concentrations (see Figure 5 ). Interestingly, these two metabolites represent successive steps in the TCA cycle: isocitrate can be converted to α-ketoglutarate by the action of isocit...

example 3

[0197] Example 3 - Waste ATP Combustion Increases Product Yield Relative to Biomass

[0198] We hypothesized that excess ATP might affect the rate-yield coupling if biomass is the preferred sink for excess ATP. ATP expenditure for cellular maintenance is constant regardless of the specific ATP generation rate. Thus, at low cell-specific rates, the ATP available for the production of biomass or non-catabolic compounds decreases proportionally, while at high cell-specific rates, the ATP available for the production of biomass or non-catabolic compounds increases proportionally . If the most efficient way to consume excess ATP is for cells to take it up into biomass, then less carbon is available for the production of non-catabolic compounds, and yields at high cell-specific rates decrease. Conversely, at low cell-specific rates, less excess ATP is used to force biomass formation and proportionally more carbon can be shunted to the production of non-catabolic compounds. The hy...

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Abstract

The present invention provides compositions and methods for uncoupling the yield and productivity of an isoprenoid compound produced in a host cell. In some embodiments, yield and productivity are uncoupled by genetically modifying host cells to reduce flux through citric acid circulation (TCA). In other embodiments, yield and productivity are uncoupled by reducing ATP levels in host cells.

Description

Background technique [0001] Yield and productivity are the two main cost drivers of any biomanufacturing process. For the biomanufacture of non-catabolic compounds such as isoprenoids, productivity and yield are generally a function of other cell-related rates, eg, sugar and / or oxygen consumption rates. Fermentation process design that achieves the best combination of productivity and yield at the lowest cost requires close characterization of this relationship. For isoprenoid production, under standard fermentation conditions, yield is consistently inversely correlated with cell-specific oxygen and sugar uptake rates and productivity, i.e., the faster the isoprenoid-producing cell takes up oxygen and sugar , the lower the yield of isoprenoid. This relationship is called "rate-yield coupling". This inverse coupling of yield and production rate is problematic because yield and production rate are two key cost drivers in isoprenoid production. Due to rate-yield coupling, any...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/14C12P19/20C12N1/18C12N1/20C12N15/52C12N15/81
CPCC12N1/18C12N15/81C12N9/00C12N9/14C12Y306/01005C12Y106/03001C12N9/0036C12N9/0057C12Y110/03011C12N15/52C12P5/007Y02E50/10
Inventor 佩内洛普·R·处约书亚·亚当·勒曼托马斯·乔恩·谢尔巴特钱德雷什·塔克安妮·恩宁·东江汉笑
Owner AMYRIS INC
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