Methods and materials for the synthesis of organic products

Abstract

This invention provides biocatalysts that are recombinant yeast cells comprising recombinant expression vectors encoding heterologous lactate dehydrogenase genes for producing lactate.

Description

[0001] This application is a divisional of U.S. Ser. No. 09 / 992,430, filed Nov. 23, 2001 which claims priority to U.S. Provisional Application Ser. No. 60 / 252,541, filed Nov. 22, 2000, the entire disclosures of which are explicitly incorporated by reference herein.BACKGROUND OF THE INVENTION [0002] 1. Technical Field [0003] The invention relates to methods and materials involved in the production of organic products. [0004] 2. Background Information [0005] Organic products such as lactic acid have many important industrial uses. For example, organic acids can be used to synthesize plastic materials as well as other products. To meet the increasing need for organic products, more efficient and cost effective production methods are being developed. One such method involves the use of bacteria. Specifically, certain bacteria can produce large quantities of particular organic products under certain fermentation conditions. The use of living bacteria as factories, however, is limited by ...

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Benefits of technology

[0131] It will be appreciated that the methods and materials described herein can be adapted and used in any type of culturing process including, without limitation, the processes commonly referred to as “continuous fermentation” and “batch fermentation” processes. In addition, the microorganisms used during one production process can be recovered and reused in subsequent production processes. For example, the microorganisms can be reused multiple times to produce a desired organic product. Further, any carbon source can be used. For example, allose, altrose, glucose, mannose, gulose, iodose, galactose, talose, melibiose, sucrose, fructose, raffinose, stachyose, ribose, arabinose, xylose, lyxose, starches such as corn starch and wheat starch, and hydrolysates such as corn fiber hydrolysates and other cellulosic hydrolysates can be used as a carbon source for the production of either biomass or the desired organic product. Moreover, any medium can be used. For example, standard culture media (e.g., yeast minimal medium and YP medium (yeast extract 10 g / L, peptone broth 20 g / L)) as well as media such as corn steep water and corn steep liquor can be used. A significant advantage of the present invention is that the preferred microorganisms, especially when grow

[0132] In under aerobic conditions, can utilize minimal media. The anaerobic production typically will not require additional nutrients, so the final product can be isolated from a relatively clean fermentation broth using any of a variety of separation techniques. Liquid-liquid extraction is a well-known technique for the separation of organic acids from fermentation broths, and results in considerable purification. With the present invention it is believed that simpler, less costly, less energy-consuming systems may also be useful.

[0133] In one embodiment, the present invention uses genetically modified yeast having a crabtree-negative phenotype in a train-type process that induces a “switch” in the metabolic pathway after a critical cell density has been reached and at which time it is desired to dramatically increase the specific productivity of the desired organic product. A typical method for inducing the metabolic pathway switch is by moving the biomass from a highly aerated vessel to a substantially anaerobic vessel, causing oxygen starvation. It is noted that a common carbohydrate (e.g., glucose or xylose) can be used as the carbon source during both the growth phase and the production phase. The use of a genetically modified yeast cell having a crabtree-negative phenotype can be critical to the success of this embodiment. In addition, the specific productivity of the desired organic product can be critical to success. The term “specific productivity” as used herein reflects the amount of product produced and is represented as the number of grams of organic product produced per gram of biomass (dry weight) per hour, i.e., g / (g*hour). Typically, the specific productivity for organic products such as lactate and acrylate is greater than about 0.1 g / (g*hour), for example, greater than about 0.2 g / (g*hour), or greater than about 0.5 g / (g*hour). By providing a high specific productivity as described herein, the energy required for cell maintenance may be obtained via the fermentative product pathway under substantially anaerobic conditions, rather than relying on aeration to generate high amounts of energy via the respiratory pathway.

[0134] It is noted that substantially anaerobic vessels are aerated at a rate of less than about 0.1 VVM. Under certain production situations, no aeration will be used. In addition, the yield (i.e., g organic product / g carbon source consumed) in this embodiment typically is greater than about 70 wt %, and is produced without the addition of carbon sources such as ethanol and acetate. In some cases, in order to achieve the specific productivity required to generate the required energy for cell maintenance, it may be necessary to enhance the pathway from glucose to pyruvate in addition to providing the necessary enzymes to produce the desired product.

[0135] In another embodiment, the train-type process can be designed such that only the highly aerated growth vessel is equipped with sterilization capability. The anaerobic production vessel is typically operated at temperatures greater than about 35° C. (e.g., greater than about 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45° C.). Few wild-type yeast will be able to survive and compete with the genetically modified yeast at such temperatures as the pH drops during product production, especially since they will not have an enhanced fermentation pathway that can generate energy for cell maintenance, in addition, the yeast can be engineered to contain “killer plasmids” as described herein, which can prevent yeast from other species from surviving. The invention also provides various methods for culturing yeast cells. For example, a yeast cell having a crabtree-negative phenotype can be cultured with culture medium either having an organic ph value less than about 3.0, or containing a corn fiber hydrolysate. Other methods for culturing yeast cells include, without limitation, culturing yeast cells having a crabtree-negative phenotype at a temperature greater than about 35° C. with culture medium either having an inorganic pH value less than about 3.0, or containing a pentose carbon or corn fiber hydrolysate.

[0136] Further, the invention provides a process for making an organic product. This process includes growing a microorganism under culture conditions, and changing the culture conditions to promote production of the organic product. In this process, the microorganism has reduced pyruvate decarboxylase, alcohol dehydrogenase, aldehyde dehydrogenase, and / or acetyl-CoA synthase activity, and exhibits a growth rate in the absence of ethanol and acetate that is at least about 30 percent (e.g., about 35, 40, 50, 75, 100, 150, 200 percent, or more) of that observed in a corresponding microorganism not having reduced pyruvate decarboxylase, alcohol dehydrogenase, aldehyde dehydrogenase, and / or acetyl-CoA synthase activity. Typically, culture conditions that promote cellular respiration are used in situations where rapid growth is needed, or where the organic product to be produced cannot be produced without cellular respiration, while culture conditions that reduce cellular respiration are used in situations where rapid growth is not needed, or where the organic product to be produced can be produced without cellular respiration.

Problems solved by technology

Specifically, the ability of a microorganism to tolerate low pH obviates the need to maintain a neutral pH environment, which can be difficult and expensive during large-scale production processes.

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