CN113614221A - Lactobacillus bioconversion process - Google Patents

Abstract

A method for producing a cell metabolite by culturing lactobacillus cells in a cell culture, comprising feeding the cell culture in an aerobic reactor system during a production phase using a feed medium comprising a carbon source for bioconversion into the cell metabolite and isolating the cell metabolite from the cell culture, and the lactobacillus strains deposited as DSM33056, DSM33057, DSM33058, DSM33059, and DSM 33060, or progeny or derivatives of any of the foregoing strains.

Description

Lactobacillus bioconversion process

Technical Field

The present invention relates to a method for producing cellular metabolites by culturing Lactobacillus (Lactobacillus diolivorans), and to novel Lactobacillus strains.

Background

Chemical modification of small molecules by microorganisms is commonly referred to as biotransformation or biosynthesis. For the production of materials for industrial use, extensive research has been carried out on bioconversion. Microbial production of alcohols, sugars or organic acids is a promising approach to obtain end-products or building-block chemicals (building-block chemicals) from renewable carbon sources (e.g., monomers for polymer synthesis). The most common chemicals produced by the bioconversion process are 1, 3-propanediol, citric acid, lactic acid or succinic acid, and sugar alcohols, such as mannitol. Biotechnological conversion of glycerol to 1, 3-propanediol is usually accomplished by bacteria under anaerobic conditions.

Willke et al (eur.j.lipid sci.technol.2008,110,831-840) reviewed the process of bioconversion of glycerol to 1, 3-propanediol without the use of fossil resources. Microorganisms of the clostridiaceae, enterobacteriaceae and lactobacillus families have been reported to be promising candidates for the industrial production of 1, 3-propanediol.

Although microbial processes for the microbial production of valuable chemicals from renewable resources are known, most production organisms rely on one or several carbon sources and most known production organisms require high purity carbon sources. Therefore, carbon sources are a major cost factor.

WO 2013064682 a2 discloses lactobacilli for use in a bioconversion process wherein the raw material used in the bioconversion process is a complex mixture of organic carbon sources comprising carbohydrates to be bioconverted, such as raw glycerol, having a purity lower than industrial grade. In any case, the cell culture uses nitrogen to provide anaerobic conditions in the reactor.

It is desirable to reduce the cost of producing cellular metabolites in lactobacillus (l.diolvorans) cell cultures.

Lactobacillus (Lactobacillus) has been widely used in the food and feed industry. WO2010/122165a1 relates to a method for producing sourdough and baked goods with an extended shelf life by biological preservation by co-fermenting selected lactobacilli, such as lactobacillus buchneri (l.buchneri), lactobacillus parachui (l.parabruchneri) and lactobacillus lactis (l.diolvorans).

Lactobacillus novelta (DSMZ 14421, LMG 19667) was isolated from aerobically stabilized corn silage and identified as a1, 2-propanediol degrading bacterium (Kroneman et al, International Journal of Systematic and evolution Microbiology 2002,52, 639-substituted 646).

WO2006/007395a1 and WO2007/103032a2 describe the use of inocula including lactobacilli to add bacterial additives to (sugar cane) silage, in particular in order to reduce the dry matter content of the silage.

Vivek et al (Bioresource Technology 2016,213: 222-.

Jolly et al (Journal of Bioscience and Bioengineering 2014,1182: 188-.

Disclosure of Invention

It is an object of the present invention to provide a biotransformation process suitable for producing cell metabolites in lactobacillus cell cultures, which process is simple and inexpensive, in particular to obtain cell metabolites in high yields.

The objects are solved by the claimed subject matter and are further set forth herein.

The present invention provides a method for producing a cell metabolite by culturing lactobacillus cells in a cell culture, comprising feeding the cell culture in an aerobic reactor system with a feed medium (feed medium) comprising a carbon source for bioconversion into the cell metabolite during the production phase, and isolating the cell metabolite from the cell culture.

According to a particular aspect, the aerobic reactor system does not include or employ any device or method capable of protecting the cell culture from the supply of oxygen. Specifically, the aerobic reactor system does not include or use components for purging (or supplying or otherwise actively introducing) nitrogen or any anaerobic gas (particularly air or a gas other than oxygen). Although oxygen may be present in the feed medium and thus supplied to the cell culture by feeding the cell culture during the production phase, the results demonstrate that the methods described herein successfully produce bioconverted products in high yield without the need for any means to limit the oxygen content of the cell culture.

In particular, the cell culture is carried out without oxygen protection, for example without anaerobic gas supply during the production phase. Specifically, the anaerobic gas supply is below 0.1 (volume/minute (vol/vol/min)).

It has been demonstrated that lactobacillus cell cultures used in the methods described herein successfully produce cell metabolites by bioconversion of carbon sources despite the absence of oxygen limiting means in the reactor system. Although the prior art describes the use of organisms under oxygen protection (under strictly anaerobic conditions) during the production phase, it has been found that the cell cultures described herein readily consume any oxygen present in the cell cultures during the production phase, and therefore produce anaerobic bacteria in situ without any external measures to limit oxygen, and are surprisingly insensitive to any such oxygen present in the aerobic reactor system described herein, and still produce bioconversion products in high yield.

In particular, the dissolved oxygen in the production phase cell culture does not exceed 80% of the dissolved oxygen in the oxygen-saturated liquid phase (oxygen saturation is 100%) and may be below the detection limit (according to the manufacturer's instructions, as determined by standard assays, e.g. using luminescence quenching assays, e.g. using VisiFerm^TMOD Sensor HAMILTON). In an exemplary standard method, the luminescence of certain organic pigments (luminophores) is quenched in the presence of oxygen. The luminophores absorb the excitation light and release a portion of the absorbed energy by emitting fluorescent light. In the presence of oxygen, energy is transferred from the excited luminophores to the oxygen. The luminophores do not emit fluorescence and the measurable fluorescence signal decreases.

Specifically, the dissolved oxygen is in the range of 0.1 to 5%, preferably 0.1 to 1%.

According to a particular aspect, during the production phase, the cell culture is maintained at a pH value in the range of 4 to 7, while the temperature is in the range of 25 to 40 ℃. Preferably, the pH is about 5.7 ("about" is understood to be +/-0.1) and the temperature is about 30 ℃ ("about" is specifically understood to be +/-1 ℃).

According to a particular aspect, the duration of the production phase is in the range of 0.1-200 hours. Preferably, the duration of the production phase is at least any one of 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. Preferably, the duration of the production phase is less than 180 hours, more preferably even less than 100 hours.

According to a particular aspect, the carbon source comprised in the feed medium comprises one or more carbohydrates selected from the group consisting of glycerol, sugars and sugar acids. Sugars include hexoses and pentoses. Preferred hexoses are glucose, mannose, galactose and rhamnose, and preferred pentoses are xylose, arabinose and ribose. Both D-and D-forms of the sugar can be used; furthermore, the saccharides may also be derivatized, for example acetylated. Preferred sugar acids are uronic acids, such as glucuronic acid, galacturonic acid, or derivatives of such acids, such as methyl glucuronic acid.

According to a particular aspect, the carbon source comprised in the feed medium is selected from the group consisting of: glycerol, raw glycerol, glucose, fructose, lactose, sucrose, starch, biomass hydrolysate, cellulose, lignocellulose hydrolysate, sugar beet extract, molasses, organic acids, organic salts, and combinations of any of the foregoing, preferably wherein the carbon source comprises glycerol or carbohydrates having a purity below technical grade and an ash content of at least 0.1% (w/w).

The methods described herein specifically employ a starting material as the carbon source that comprises a complex mixture of organic carbon sources including one or more carbohydrates to be bioconverted and impurities, such as one or more carbohydrates of less than industrial grade purity.

In particular, the starting material is a complex mixture of organic carbon sources comprising an ash content of at least 0.1% (w/w), in particular at least 0.2% or at least 0.25% (w/w).

In particular embodiments the starting material is selected from the group consisting of: raw glycerol (also referred to herein as raw glycerol), sugar cane, sugar beets, starch plants, cellulose, hemicellulose, lignocellulose (including lignocellulosic plant material or lignocellulosic hydrolysates), and chitin (e.g., chitin-containing material from shellfish). Raw glycerol may be obtained from fatty acid production, from biodiesel production, from bioethanol production or soap production. Other raw materials may come from waste streams of the paper industry, sugar industry or wood industry.

In particular, the carbon source is raw glycerol and is used in the feed medium at a concentration of up to 90% (w/w), in particular at least any one of 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% (w/w) in the feed, preferably in the range of 40% to 60%, or about 50%.

In particular, the raw glycerol comprises glycerol of a purity lower than technical grade and comprises an ash content of at least 0.1% (w/w), in particular at least 0.2% or at least 0.25% (w/w).

In particular, crude glycerol is obtained as a by-product of fatty acid production, soap production, bioethanol production or biodiesel production.

The methods described herein specifically produce target cell metabolites. In particular, cellular metabolites are not constitutively produced (or primary metabolites), i.e. supporting bacterial growth, but secondary metabolites or such metabolites produced by biosynthesis by the addition of a carbon source to be bioconverted in culture.

In particular, cellular metabolites are chemical substances or low molecular weight organic molecules (small molecules).

In particular, the cell metabolite is selected from the group consisting of: 1, 3-propanediol, 1, 2-propanediol, 2-amino-1, 3-propanediol, 3-hydroxybutyrate, poly-3-hydroxybutyrate, ethanol, 1-butanol, 2-butanol, isobutanol, 2, 3-butanediol, butanone, lactic acid, citric acid, propionic acid, 3-hydroxypropanal, 3-hydroxypropionic acid, butyric acid, valeric acid, caproic acid, adipic acid, succinic acid, fumaric acid, malic acid, 2, 5-furandicarboxylic acid, aspartic acid, glucaric acid, gluconic acid, glutamic acid, itaconic acid, levulinic acid, acrylic acid, propanol, isopropanol, 1-butanol, 2-butanol, pentanol, hexanol, heptanol, octanol, butanediol, 2, 3-butanediol, 3-hydroxybutyrolactone, xylitol, arabitol, and the like, Sorbitol, mannitol, vitamin C, riboflavin, thiamine, tocopherol, cobalamin, pantothenic acid, biotin, pyridoxine, nicotinic acid, folic acid, 3-hydroxybutyrolactone, diaminohexane, and dihydroxyacetone.

Specifically, the chemical is an organic acid or alcohol; exemplary chemicals produced are 1, 3-propanediol, lactic acid, 3-hydroxypropionic acid, and mannitol.

Specifically, glycerol (especially crude glycerol) is bioconverted to produce at least one or more of 1, 3-propanediol, 3-hydroxypropanal, and/or 3-hydroxypropionic acid.

Embodiments of the invention relate to methods wherein

(i) Producing 1, 3-propanediol from crude glycerol; or

(ii) Producing 3-hydroxypropionic acid from crude glycerol; or

(iii) Producing 3-hydroxypropanal from crude glycerol; or

(iv) Producing lactic acid from a lignocellulosic or lignocellulosic biomass hydrolysate; or

(v) Mannitol is produced from sugar beet extract or hydrolysate.

According to a particular aspect, the feed medium is continuously added to the cell culture during the production phase.

According to a particular aspect, the production phase is carried out in fed-batch mode or continuous mode.

Specifically, prior to the production phase, cell culture is performed in batch mode using growth medium to accumulate biomass.

In particular, the growth medium that allows biomass to accumulate comprises a carbon source, a nitrogen source, a sulfur source, and a phosphate source. Typically, such media also contain trace elements, vitamins and amino acids.

Typically, the growth medium comprises more than one carbon source to obtain a high concentration of biomass, e.g., at least 5g/L, preferably at least 7.5g/L, even more preferably at least 10g/L of biomass.

In particular, the growth medium comprises a carbon source selected from hexose, pentose or heptose. The carbon source is selected from one or more of glucose, sucrose, fructose, xylose, arabinose and mannose.

In particular, the carbon source in the growth medium is selected from the raw materials, for example from the group consisting of: raw glycerol, starch plant raw material hydrolysate or lignocellulose plant raw material hydrolysate.

The specific method described by the invention comprises the following steps:

a) inoculating lactobacillus into a fermentation medium;

b) culturing lactobacillus in a growth medium to accumulate biomass;

c) culturing lactobacillus in a production medium to produce a cellular metabolite or chemical; and

d) isolation and purification of cellular metabolites or chemicals.

In a particular embodiment, the fermentation medium may be a growth medium, and/or culture steps b) and c) are employed in a single batch or in separate batches.

According to a particular aspect, a method for producing 1, 3-propanediol from raw glycerol comprises,

a) inoculating lactobacillus into a fermentation medium using raw glycerol as a carbon source,

b) culturing lactobacillus in a growth medium to accumulate biomass,

c) culturing a lactic acid bacterium in a production medium to produce 1, 3-propanediol, an

d) Isolating and purifying the 1, 3-propanediol.

According to a particular aspect, lactic acid is produced from glucose and/or xylose derived from lignocellulose or lignocellulose hydrolysate, including

a) Inoculating lactobacillus into a fermentation culture medium which takes lignocellulose or lignocellulose hydrolysate as a carbon source,

b) culturing lactobacillus in a growth medium to accumulate biomass,

c) culturing a lactic acid bacterium in a production medium to produce lactic acid, and

d) separating and purifying lactic acid.

According to a particular aspect, mannitol is produced from sugar beet fructose (e.g. extract or hydrolysate), comprising

a) Inoculating lactobacillus into fermentation medium with beet extract or hydrolysate as carbon source,

b) culturing lactobacillus in a growth medium to accumulate biomass,

c) culturing a lactic acid bacterium in a production medium to produce mannitol, an

d) Isolating and purifying mannitol.

In particular, the methods described herein provide for direct inoculation of bacteria into a growth medium. The fermentation medium may be used as a growth medium.

In a particular embodiment, lactobacillus grown on raw materials (e.g., raw glycerol, lignocellulose, or lignocellulose hydrolysate) is cultured in batch to accumulate biomass, followed by feeding a purified or unpurified carbon source (e.g., containing carbohydrates such as glycerol) at a specific feed rate, optionally with or without glucose and/or xylose, to accumulate cellular metabolites or chemicals, such as 1, 3-propanediol or lactic acid.

In particular, a lactobacillus strain is a natural strain that can be isolated from natural sources or otherwise isolated from a population of strains. Any such isolated strain is provided as a pure strain without detectable cells of different origin. Such isolated strains do not occur naturally and are therefore artificial or man-made.

According to a particular aspect, the lactobacillus cell is an isolated naturally occurring lactobacillus bacterium, strain, progeny or derivative thereof.

In particular, the lactobacillus cell is any one of the lactobacillus strains from the deposit DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, or a progeny or derivative of any of the foregoing.

Specific embodiments relate to lactobacilli derived from selected strains, including the DSM14421 strain (Leibniz institute) DSMZ-German collection of microorganisms, Broensvk, Germany; (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschwei) Genbank accession No.: AF264701(16S rRNA), such as AF264701.2), and LMG 19667, LMG 68 strains (LMG from BCCM/LMG university of Netherlands (UGent) microbiological laboratories (Laboratorian voor microbiology, Universet Gent (UGent)), G77 strains (CUPV: university of collectins la university of palace washer Vasco) (Western), Journal of protein, protein 69, Marc of Protek research, Biotechnology, Volvin P7, Volvin research, Volvin technologies, Volvin research, Inc. Sorburgh, Volvin Biokulture technologies, Volvin, Sorbus research, Sorbus, the Collection of Industrial microorganisms (Waclaw Dabrowski Institute of Agricultural and Food Biotechnology)), Benef microorganisms.2014 Dec; 5(4) 471-81, e.g.using natural cell lines or recombinant production cell lines.

According to a specific embodiment, genetically engineered or recombinant lactobacillus strains are used. Particular embodiments refer to strains that have been subjected to mutagenesis and selected for improved bioconversion.

In particular, lactobacillus is used as a genetically engineered or recombinant strain, e.g., modified to produce metabolites including chemicals, preferably to process raw materials or waste materials, e.g., to produce isolated fermentation products.

In particular, genetically engineered or recombinant lactobacillus strains may be used, preferably host cells modified to overexpress homologous sequences and/or transformed with vectors comprising heterologous sequences that are specific coding and/or non-coding sequences, e.g. overexpressing homologous and/or heterologous sequences. In particular, the host cells are engineered to increase the yield or titer or productivity of the bioconversion product, e.g., by overexpressing one or more genes that form a metabolite of the target cell.

More specifically, the host cell is engineered to increase the yield of 1, 3-propanediol bioconverted from unprocessed glycerol and/or the productivity, e.g., specific productivity and/or volumetric productivity, of the host cell.

For example, host cells are engineered to overexpress NADPH-dependent 1, 3-propanediol oxidoreductase.

The invention also provides a lactobacillus (l.diolvorans) strain being any one of the lactobacillus strains deposited as DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, or a progeny or derivative of any of the foregoing.

In particular, derivatives of these strains comprise one or more mutations for over-expressing the homologous sequences and/or transforming the bacteria with vectors comprising heterologous sequences to increase the yield of cellular metabolites.

These strains, progeny or derivatives are particularly useful in the methods described herein.

According to a particular aspect, the present invention provides a method for the bioconversion of a carbon source into a cell metabolite in a cell culture by an aerobic fed-batch fermentation process using lactobacillus cells.

In particular, the carbon source is glycerol or a carbohydrate having a purity lower than technical grade and an ash content of at least 0.1% (w/w), in particular at least 0.2% or at least 0.25% (w/w).

In particular, the lactobacillus cells described herein are used for the bioconversion of glycerol to 1, 3-propanediol, preferably wherein the glycerol is biodiesel-derived raw glycerol or glycerol of lower purity than technical grade and comprising an ash content of at least 0.1% (w/w), in particular at least 0.2% or at least 0.25% (w/w).

According to a particular aspect, lactobacillus is used to process raw materials or waste materials to produce isolated fermentation products, such as cellular metabolites or chemicals, such as metabolites produced by a (natural, wild-type or recombinant) metabolic pathway of an organism.

According to a particular aspect, the cell metabolite is a chemical produced by bioconversion of a carbon source in a bacterial cell culture and is purified from the cell culture or a portion of the cell culture.

Such chemicals can be end products, industrial grade chemicals or higher purity chemicals, such as USP grade chemicals, or intermediates used in the production of derivatives (including chemicals, solvents or polymers). Preferred chemicals are produced as purified chemicals by preparative isolation and/or purification, more preferably on a large or industrial scale. In particular, chemicals may be produced in large quantities, referred to as bulk materials (bulk chemicals) or bulk chemicals. Chemicals can also be produced for use as pharmaceutical ingredients, bactericides and special chemicals for technical applications.

Cell metabolites can be obtained at high concentrations. For example, the 1, 3-propanediol concentration at the stage of production or at the end of production is preferably at least 40g/L, more preferably at least 60g/L, more preferably at least 80g/L, more preferably at least 100 g/L.

The methods described herein specifically provide high yield production. Specifically, chemicals, such as 1, 3-propanediol, are produced in high yields of at least 40% or 50%, as determined specifically below.

In particular, the product yield may be calculated based on substrate consumption, in other words, the amount of product produced on a molar basis divided by the amount of substrate consumed on a molar basis, the product yield preferably being at least 40%, more preferably at least any one of 50%, 60%, 70%, 80%, 85%, 90% or 95%.

The product yield may be calculated on the basis of substrate supply, in other words, the amount of product produced on a molar basis divided by the amount of substrate fed to the process on a molar basis, the product yield is preferably at least 40%, more preferably at least any one of 50%, 60%, 70%, 80%, 85%, 90% or 95%.

Preferably, the cell metabolite is produced as a high purity material, in particular, at least 95% pure, preferably at least 96% pure, preferably at least 97% pure, preferably at least 98% pure, even more preferably 99% pure, most preferably 99.5% pure (w/w).

Preferred purity grades are at least technical grade (generally considered to be above 97% (w/w) purity).

In particular, large scale production and isolation of cellular metabolites, e.g. on an industrial scale, is preferred. Thus, the term "isolated" as used herein refers specifically to the preparation of an isolated and purified sufficient amount of a cellular metabolite from a fermentation system for further use. Thus, this preparative isolation is in contrast to analytical determination of fermentation products, which are used only for analytical purposes.

Preferred cell lines exhibit maximum volumetric and specific productivity in the production of cell metabolites or chemicals (e.g., 1, 3-propanediol) of 0.8g/L/h and 0.15g/g cell mass/h, respectively. Preferred cell lines exhibit average volumetric and specific productivities for the production of cellular metabolites or chemicals (e.g., 1, 3-propanediol) of 0.5g/L/h and 0.1g/g cell mass/h, respectively.

According to a particular embodiment, the present invention relates to the use of lactobacilli in a series of bioconversion processes, e.g. as a platform microorganism, to convert carbohydrates (e.g. including different types of low purity carbohydrates) from raw materials of at least two different carbohydrate sources into cell metabolites or chemicals. There is evidence that the lactobacilli are tolerant to growth on different raw materials, while various carbon sources can be used to grow and/or produce cellular metabolites or chemicals.

Drawings

FIG. 1 shows a schematic view of aPreparation of agarose gels of genomic DNA from three strains of Lactobacillus (DSM 14421; LMG 19668 and Vogelbusch1050 (deposited under DSM 33056)).

Detailed Description

Specific terms used throughout the specification have the following meanings.

The term "aerobic reactor system" as used herein refers in particular to a production unit comprising one or more reactors, including a production reactor containing a production cell culture (which may be, for example, a flow-through reactor used in a fed-batch or continuous process, or a closed vessel used in a batch process), i.e. a cell culture during a production phase. Such a production reactor may be an airtight vessel. The air-tight vessel may have one or more vents to vent off-gas to be released and to ensure that pressure build-up in the vessel is minimized. The aerobic reactor system is specifically equipped with one or more means for introducing a feed medium into the production reactor to feed the cell culture during the production phase; and isolating or collecting the cell culture medium or supernatant containing the metabolite of the target cell.

The particular reactor system provides for commercial scale cell culture. In some embodiments, the size of the culture is at least about 100L, or at least about 200L, or at least about 500L, or at least about 1000L, or at least about 10000L, or at least about 100000L, or at least about 500000L. In some embodiments, the culture is about 300L to about 1000000L.

According to a low-cost cultivation strategy, the cell line is cultivated under aerobic cultivation conditions, and an aerobic fed-batch fermentation scheme is optionally adopted. To this end, aerobic reactor systems typically do not include components or devices to protect the cell culture from oxygen, such as by purging anaerobic gas.

When culturing a lactobacillus cell culture in an aerobic reactor system, the cell culture may still contain only a small amount of detectable dissolved oxygen, or less than a detection limit, as measured in the production phase cell culture medium. This is because lactobacillus may have the ability to consume oxygen contained in cell culture media or feed during the production phase. Hypoxic levels generally refer to levels of oxygen that are lower than the levels in the oxygen-saturated liquid phase of the cell culture. The hypoxic level can be any one of a saturated dissolved oxygen concentration (oxygen saturation of 100%) in the liquid phase of about 0.1% to about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or below the detection limit. In a particular embodiment, the cell culture medium comprises, for example, from about 4ppb to about 8ppb dissolved oxygen in the medium.

The process described herein specifically refers to cell culture under aerobic culture conditions, which differs particularly from anaerobic fermentation in that it uses an aerobic reactor system that does not include a vented production reactor or fermentation vessel, which introduces an inert or anaerobic gas for purging, or purges air (or oxygen) from the vessel. Anaerobic fermentation typically includes a purging step performed by introducing an anaerobic gas (e.g., a gas selected from the group consisting of helium, nitrogen, an inert gas, and combinations thereof) into the production chamber. This is especially to be avoided in aerobic reactor systems. By avoiding the use of such vented production reactors or fermentation vessels, the production costs will be significantly reduced compared to anaerobic fermentation.

According to the prior art method, lactobacillus is ventilated and purged with nitrogen under oxygen protection, and a biotransformation process is performed using an anaerobic reactor system to produce cell metabolites. This is necessary because in the cultivation of anaerobic bacteria belonging to the genus Lactobacillus, growth conditions favorable for the proliferation of anaerobic bacteria, especially for the production phase, need to be employed because the key enzyme glycerol dehydratase is highly sensitive to oxygen.

It has been shown that lactobacillus can be cultured in an aerobic reactor system without aeration with inert or anaerobic gas, and therefore, lactobacillus can be cultured under aerobic conditions while maintaining hypoxia. Surprisingly, lactobacillus can withstand such conditions when cultured in an aerobic reactor system according to the methods described herein. In spite of aeration for protection against oxygen, the biotransformation is very efficient and produces the target cell metabolites in high yield. Thus, a low-cost production method can be provided, and expensive ventilation equipment and ventilation processes can be avoided.

It is convenient to use specific strains of lactobacillus which are novel isolates and which have been deposited as further described herein, in particular strains of lactobacillus (l.diolvorans) deposited as DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060.

The deposited strains have been shown to be easy to handle and surprisingly insensitive to oxygen, which means that they can be handled in a completely ventilated environment without losing their growth or production capacity. This oxygen insensitivity is even more surprising when cell metabolites are produced during the production phase in cell culture, which provides significant advantages. The deposited strain grew well under the described conditions and was susceptible to conversion of pharmaceutical or sub-industrial grade glycerol to 1, 3-propanediol.

The aerobic reactor system used herein may take the form of a static fermentation or a continuous fermentation system that provides a favorable environment for the process described herein such that optimal biological conversion and synthesis of the target metabolites is maintained. In certain cases, the use of inert or anaerobic gases to purge the cell culture or purge the reactor is to be avoided.

In general, the culture medium used in such cell culture, e.g., a growth medium or a feed medium, can be in the form of an aqueous solution or slurry and introduced into the reactor without providing an anaerobic environment.

As used herein, "reactor" may include a fermentor or a fermentation unit, or any other reaction vessel, and the term "reactor" may be used interchangeably with "fermentor". For example, a production reactor or bioreactor unit may perform one or more, or all of the following: feeding nutrients and/or carbon sources, inlet and outlet flows of fermentation or cell culture medium, separating gas and liquid phases, maintaining reaction temperature, maintaining oxygen and CO₂Level, maintain pH level, agitation (e.g., stirring), and/or cleaning/disinfecting. Exemplary production reactors used in aerobic reactor systems are generally not injected with a suitable inert gas. For example, a reactor system may contain multiple reactors within the system, while a facility may contain multiple reactor systems. In various embodiments, the reactor may be adapted for batch, semi-batch fed, fed-batch, perfusion, and/or continuous fermentation processes. Suitable reactors may be multi-purpose, single-purpose, disposable or non-disposable, and may be made of any suitable material (including metals)Alloys (e.g., stainless steel and Inconel (Inconel), plastic, and/or glass).

In embodiments, the apparatuses, facilities, and methods described herein may also include any suitable unit operations and/or other equipment not mentioned, such as operations and/or equipment for isolating, purifying, and isolating biosynthetic products. Any suitable facility and environment may be used, such as a conventional stick-build facility, a modular, mobile, and temporary facility, or any other suitable structure, facility, and/or arrangement. Further, unless otherwise specified, the devices, systems, and methods described herein may be disposed of and/or performed in a single location or facility, or alternatively may be disposed of and/or performed in separate or multiple locations and/or facilities.

Suitable culturing techniques may include culturing in a bioreactor starting with a batch phase, followed by a short exponential feed phase at a high specific growth rate, followed by a feed phase at a low specific growth rate. Another suitable culture technique may include a batch phase followed by a fed-batch phase at any suitable specific growth rate or combination of specific growth rates, e.g., to change from a high growth rate to a low growth rate during the production phase. Another suitable culture technique may include a batch phase followed by a continuous culture phase of low dilution rate.

As used herein, the term "bioconversion" is understood to be a cellular biosynthetic process of a compound, such as an organic carbon source or a carbohydrate, to a cellular metabolite. The compounds may be biotransformed by cells in cell culture. The production of such compounds by cell culture is also referred to as "in vivo production" or "in vivo biotransformation". It is understood that this biotransformation in cell culture is "ex vivo", which means that it does not use higher organisms, animals or humans. As further described herein, specific bioconversion processes are performed in bacterial cell cultures to produce cell metabolites, such as chemicals, particularly chemicals as further described herein.

As used herein, the term "carbon source"Refers to a carbon substrate, in particular a fermentable carbohydrate, such as a carbohydrate used in a bioconversion process, specifically including a source carbohydrate capable of being metabolized by a host organism or a production cell line. As used herein, the term "carbohydrate" is used in a broad sense, e.g. including sugars, sugar acids and their derivatives, e.g. according to the general formula (CH)₂O)_nPolyhydroxy aldehydes or ketones and their derivatives. Specifically included are aldehydes (e.g., glyceraldehyde), ketones (e.g., dihydroxyacetone), and hydrogenated derivatives (e.g., glycerol). Specifically included are polyols, preferably at least three carbon polyols, such as sorbitol, mannitol or glycerol. The specific carbohydrate used as carbon source is supplied in purified form or as raw material and is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, polyols and their derivatives.

The carbon source for bioconversion as described herein may be a purified carbon source or raw material (e.g., a carbon source comprising carbohydrates in a mixture containing impurities), and may also be understood as a "fermentable carbon source" or a "fermentable carbohydrate" because it is converted to a cell metabolite by a fermenting organism.

As used herein, the term "cell line" refers to a given clone of a particular cell type that has acquired long-term proliferative capacity. The term "host cell line" refers to a cell line used to express endogenous or recombinant genes or products of metabolic pathways to produce polypeptides or cellular metabolites mediated by such polypeptides. A "production host cell line" or "production cell line" is generally understood to be a cell line that is ready for culture in a bioreactor to obtain a product of a production process. Suitable production cell lines can produce some useful intermediates and products by saccharification of the low molecular weight sugars produced by fermentation-treated biomass material. Suitable producer cell lines can also produce the necessary enzymes to saccharify the treated biomass material while producing useful intermediates and products. For example, fermentation or other biological processes may produce alcohols, organic acids, hydrocarbons, hydrogen, proteins, or mixtures of any of these.

As used herein, the terms "cell culture" or "culturing" or "cultivation" in relation to lactobacillus cells refer to the maintenance of the bacterial cells in an artificial environment (e.g., in vitro environment), under conditions conducive to cell growth, differentiation, or sustained survival, in an active or quiescent state, particularly in a controlled bioreactor, according to methods known in the art.

When a cell culture is cultured using a suitable medium, the cells are contacted with the medium or substrate in a culture vessel under conditions suitable to support culturing of the cells in the cell culture. As described herein, media useful for cell growth are provided. Standard cell culture techniques are well known in the art.

Cell cultures as described herein particularly use techniques for producing metabolites of interest by biotransformation, e.g. obtaining a product in cell culture medium that can be separated from the cell biomass (referred to herein as "cell culture supernatant"), and optionally can be isolated and purified to obtain a higher purity product.

Cell culture media provides the nutrients necessary to maintain cell survival and cell growth in a controlled, manual intervention and in vitro environment. The characteristics and composition of the cell culture medium may vary according to the particular cell requirements. Important parameters include osmotic pressure, pH and nutritional formula. The addition of nutrients can be accomplished in a continuous or discontinuous manner according to methods known in the art.

Batch culture is a cell culture mode in which all nutrients required for culturing cells are contained in an initial medium, and additional supply of other nutrients is not required during fermentation, while in a fed-batch process, one or more nutrients are supplied to a culture by feeding after the end of a batch culture phase. Although in most processes, the mode of feeding is critical, the cell culture and methods described herein are not limited to a certain mode of cell culture.

In certain embodiments, the cell culture process employs a fed batch process.

In another embodiment, the host cells described herein are cultured in a continuous mode. The continuous fermentation process is characterized in that fresh medium is added to the bioreactor at a defined, constant and continuous rate, while broth is removed from the bioreactor at the same defined, constant and continuous rate. By keeping the media, feed rate and removal rate at the same constant level, the cell culture parameters and conditions in the bioreactor are kept constant.

Stable cell culture as described herein refers to maintaining the genetic properties of the cells, in particular maintaining a higher biosynthesis rate and production level, e.g. even after about 20 passages in culture, preferably at least 30 passages, more preferably at least 40 passages, most preferably at least 50 passages. In particular, the present invention provides stable lactobacillus cell cultures with great advantages when producing target cell metabolites on an industrial scale.

As used herein, the term "cell metabolite" or "target cell metabolite" refers to a metabolite produced by a cell by bioconversion of a carbon source, e.g., production of a primary or secondary metabolite by a metabolic pathway.

The biotransformation products produced by the methods described herein are in particular secondary metabolites. Secondary metabolites refer to organic compounds produced by cells (e.g., bacteria) that are not directly involved in the normal growth, development, or reproduction of an organism. Unlike primary metabolites, the absence of secondary metabolites does not result in immediate death of the organism, but rather, can permanently impair the viability, reproductive ability, or aesthetic appearance of the organism, or may not significantly change at all. Specific secondary metabolites are generally limited to a narrow group of species within the phylogenetic group.

As used herein, the term "chemical" refers to a chemical substance or compound, particularly a low molecular weight organic molecule (referred to as "small organic molecule"), such as used in the chemical industry, pharmaceutical industry, agriculture, cosmetic industry, food industry, and feed industry.

Compounds specifically produced according to the methods described herein may include small or large amounts of chemicals of low or high purity, including bulk chemicals, fine chemicals, and specialty chemicals. The term includes the final product as well as intermediates in the production of the derivative, such as reaction products, including monomers used in polymer synthesis.

As used herein, the term "raw glycerol" is also referred to as "raw glycerol", which is a by-product from a fatty acid, bioethanol or biodiesel production process or from a soap production process, also referred to as industrial glycerol derived (derived) from biodiesel production. Crude glycerol can be used as a raw or source material, and also as a carbon substrate in some biotechnological fields. It contains glycerol (typically in the range of 40-88%, e.g., about 50% (w/w), in some cases up to 95%) as well as salts, soaps, and other impurities, including water and methanol. Typical composition of raw glycerol, for example from novalo s.r.l. (milan, italy), from

Raw glycerol in the product provided crude glycerol obtained from a biodiesel production process having the following composition: glycerol 85-95% (w/w), organic/inorganic acids 5-10% (w/w) and methanol 0-1.5% (w/w). Another example of raw glycerol is thin stillage (raw material produced by ethanol distillation, or by-products from bioethanol distillation), or thick stillage (concentrated thin stillage, such as stillage obtained by evaporating thin stillage into thick stillage).

In particular, the raw glycerol used in the processes described herein is obtained from waste materials of biodiesel, bioethanol or fatty acid production, e.g. from different seed oil feedstocks, including palm oil, jatropha, mustard, rapeseed, canola (canola), crambe, soybean and waste edible oils.

In particular, crude glycerol is recovered from the product of fatty acids, biodiesel, bioethanol or soap without further processing, e.g. separated from fatty acids, biodiesel and soap, respectively. Nevertheless, the term "crude glycerol" also includes crude glycerol that has been fractionated or processed, including, for example, by filtration, ion exchange, chemical addition, and/or fractional vacuum distillation to obtain different commercial grades of material. According to a particular embodiment, the raw glycerol may be treated to neutralize the material, for example to obtain a pH ranging between 6 and 8, preferably about 7. So as to obtain an organic phase containing fatty acids and an aqueous phase containing glycerol in addition to impurities. Although this fractionated raw glycerol still contains glycerol of a purity lower than that of technical grade glycerol, in particular containing unwanted ash, it can still be used as a carbon source in the process described herein.

According to a certain embodiment, the raw glycerol is subjected to a pasteurization or sterilization treatment, e.g. autoclaving the raw glycerol at 121 ℃ for 10 to 100 minutes, e.g. about 20 minutes, before using it as a carbon source for the bioconversion. According to another embodiment, the material is pasteurized by heating to at least 80 ℃ for 10 to 100 minutes (e.g., about 15 minutes ("about" means +/-1 or 2 ℃)) before using raw glycerol as a carbon source for bioconversion.

The purity of technical grade glycerol is typically greater than 97% (w/w).

In particular, the crude glycerol used as a carbon source for bioconversion is characterized by a glycerol purity that is lower than that of technical grade glycerol, such as a purity of less than 97%, or less than 96%, specifically less than 95%, more specifically less than 94%, more specifically less than 93%, more specifically less than 92%, more specifically less than 91%, more specifically less than 90% (w/w).

In particular, the use of crude glycerol is characterized by the content of impurities, such as ash, determined by standard measurements.

The ash content in the crude glycerol is mainly derived from sodium in the catalyst used in the biodiesel production process and is typically between 0.25% and 5.50%, and in some cases up to 8%, depending on the oil source and the biodiesel production process (Thompson et al applied Engineering in Agriculture 2006, Vol.22(2): 261-.

As used herein, the term "derivative" in relation to a lactobacillus strain refers to a strain obtained from a parent strain by e.g. mutagenesis and selection or directed mutagenesis.

In particular, derivatives of the lactobacillus strains referred to herein may be produced by methods of limited mutagenesis, e.g. maintaining a certain sequence similarity throughout the genome, e.g. at least any one of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

It is to be understood that the bioconversion products produced by the methods described herein are cellular metabolites or chemicals, or derivatives of any of the foregoing, which are conveniently produced when isolated and optionally purified. By "derivative" of a cellular metabolite or chemical substance is understood in particular a compound derived from the product of a biotransformation process, e.g. by chemical, physical or biosynthetic processes, including polymerization, acetylation, methylation or phosphorylation.

As used herein, the term "Lactobacillus (Lactobacillus diovorans)" or "Lactobacillus (l.diovorans)" refers to a species of Lactobacillus that is the isolated strain, wild-type bacterium or derivative thereof (e.g., genetically engineered producer cell line) first described by kroneman et al (International Journal of Systematic and evolution Microbiology 2002,52, 639-646).

The "deposited strain" of the lactic acid bacterium referred to herein is as follows.

The following strains of Lactobacillus (L.diolvorans) have been isolated in Austria and deposited in DSMZ-German Collection of microorganisms and strains of microorganisms on day 26 of 2.2019 by Voigler Broth, Inc., Vienna, Origan, Bronstein (DE)38124, Maschererweig 1B/Neuroff street (Inhoffenstra. beta.e) 7B.

The deposits relate to lactobacillus strains and cultures which are characterized as follows.

DSM33056 (also known as Lactobacillus WoGilbert 1050) is characterized as follows: the strain is easy to handle and insensitive to oxygen, which means that it can be handled in a completely ventilated environment without loss of growth or productivity. Especially in the mixture of glycerol and any one or more of glucose, fructose or mannose, the strain has outstanding 1, 3-propanediol production performance.

DSM33057 (also known as lactobacillus HH) is characterized as follows: the strain is easy to handle and insensitive to oxygen, which means that it can be handled in a completely ventilated environment without loss of growth or productivity. The strain has outstanding 1, 3-propanediol productivity, particularly in a mixture of glycerol and any one or more of glucose or fructose. Especially in the mixture of glycerol and sucrose, the strain is one of the best strains for producing 1, 3-propanediol.

DSM33058 (also known as lactobacillus HM) is characterized as follows: the strain is easy to handle and insensitive to oxygen, which means that it can be handled in a completely ventilated environment without loss of growth or productivity. Especially in the mixture of glycerol and any one or more of glucose, fructose or mannose, the strain has outstanding 1, 3-propanediol production performance.

DSM33059 (also known as lactobacillus HS) is characterized as follows: the strain is easy to handle and insensitive to oxygen, which means that it can be handled in a completely ventilated environment without loss of growth or productivity. Especially in the mixture of glycerol and any one or more of glucose or fructose, the strain has outstanding 1, 3-propanediol production performance. Especially in the mixture of glycerol and xylose, the strain is best performed in the performance of producing 1, 3-propanediol. In addition, it also performs best in a mixture of glycerol and mannose.

DSM 33060 (also known as lactobacillus HF) is characterized as follows: the strain is easy to handle and insensitive to oxygen, which means that it can be handled in a completely ventilated environment without loss of growth or productivity. Especially in the mixture of glycerol and any one or more of glucose, fructose or mannose, the strain has outstanding 1, 3-propanediol production performance.

As used herein, the term "end product" refers to a cell metabolite or a controlled quality chemical that is manufactured and optionally processed into a product for further manufacturing or industrial purposes or as a consumable product.

As used herein, the term "genetically engineered" refers to a recombinant organism used to produce a fermentation product. The organism is typically a production cell line, engineered to improve the production process or capable of producing new products. This term is in contrast to "wild-type" organisms, which are usually not genetically engineered, the term "wild-type" as used in the present disclosure generally refers to "isolated from a natural source", such as lactobacillus (l.diolvorans) DSM 14424 or DSM14421, or lactobacillus (l.diolvorans) LMG 19668, or lactobacillus (l.diolvorans) G77, or any deposited strain DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, including any progeny thereof.

As used herein, the term "intermediate" refers to a chemical formed as a product of a bioconversion process that can be isolated from a fermentation broth and further processed to form a derivative or final product.

As used herein, the term "isolated" in relation to a cellular metabolite, chemical substance or biosynthetic or bioconversion process refers to a substance that is isolated or purified from at least one impurity (especially by preparative methods) to obtain a product that is completely separated from the environment with which it is associated prior to isolation, and thus exists in a "substantially pure" form. "isolated" is not necessarily meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with essential activity, e.g., impurities that may be present due to incomplete purification.

The term "isolated" as used herein refers to a bacterial strain.

As used herein, the term "metabolic pathway" refers to a biochemical reaction occurring within an organism, for example, the metabolism of a particular compound or a microbial enzymatic pathway to produce a cellular metabolite, especially a small organic molecule. Metabolic pathways refer to all biosynthetic, modification and degradation pathways of compounds within cells. Metabolic engineering refers to the introduction, deletion and modification of metabolic pathways of microorganisms, particularly by employing appropriate recombinant techniques, which are widely used for the efficient production of desired metabolites and biomolecules. The new synthetic products can even be obtained by metabolic engineering of the host cell.

As described herein, the term "mutagenesis" refers to a recombinant construct or organism having a mutated nucleic acid such that a variant thereof is obtained with at least one change in a non-coding or coding region. Mutagenesis may be by random, semi-random or site-directed mutagenesis.

As used herein, the term "overexpression" as used in relation to a host cell, particularly a recombinant host cell, is intended to include increasing the expression of a polypeptide or protein (e.g., an enzyme used in a bioconversion process) to a level above that normally produced by the cell. The term is intended to include the overexpression of homologous (endogenous) as well as heterologous sequences or proteins, in particular to increase the productivity of the cell to produce a bioconversion product, e.g. at least 1.5-fold, preferably at least 2-fold, more preferably at least 3-fold compared to a wild-type host cell of the same type.

As used herein, the term "production phase" refers to a stage of cell culture where a bioconversion product is produced. The production phase may specifically be after the growth phase, where the cell culture accumulates biomass by growing the cells. The cultivation during the growth and production phases can be conveniently carried out by batch, fed-batch and continuous cultivation processes. A carbon source suitable for use as a substrate for bioconversion into a cell metabolite may be comprised in the feed of a fed batch process, e.g. to maintain a production cell line under carbon substrate limited conditions, i.e. under growth limited conditions and in production mode using a limited amount of carbon source. This limited amount can be used in a fed-batch process, where a carbon source is contained in the feed medium and is supplied to the culture at a low feed rate for sustained energy delivery, e.g., to produce a biosynthetic or fermentation product while maintaining the biomass at a low specific growth rate. During the production phase of the cell culture, a feed medium is usually added to the fermentation broth.

As used herein, the term "raw material" refers in particular to any complex carbon source or carbohydrate material, in particular carbohydrate-rich biomass, including monomeric or low-purity carbohydrates present in polymerized form, e.g. from bioenergy crops, industrial crops, agricultural residues, municipal waste, industrial waste, yard waste, wood, straw, chitin containing residues from shell-animals. Specific examples of raw materials are raw (raw) glycerol, sugar cane, sugar beet, starch plants, cellulose, hemicellulose, lignocellulose hydrolysates or chitin.

The complex carbohydrate mixture in the starting material typically comprises at least two different major organic carbon sources, e.g. each carbon source is present in an amount of at least 2%, specifically at least 3%, more specifically at least 4%, more specifically at least 5% (w/w).

In particular, the raw material used in the process described herein is characterized by a purity of fermentable carbohydrate content below technical grade (technical grade purity > 97% (w/w)), such as below 96%, in particular below 95%, more in particular below 94%, more in particular below 93%, more in particular below 92%, more in particular below 91%, more in particular below 90% purity (w/w).

In particular, the raw materials used in the processes described herein, which are characterized by the content of impurities, such as ash, are determined by standard methods. An exemplary standard method determines the residue left after ignition of the organics. Ash is generally considered to be an approximate measure of the mineral and other inorganic content of biomass.

Typical STANDARD METHODS FOR determining the ash content OF a raw material (e.g. crude glycerol) are gravimetric METHODS, such as described in the INTERNATIONAL UNION OF PURE AND applied chemistry (INTERNATIONAL UNION OF PURE AND applied chemistry AND APPLIED CHEMISTRY), department OF applied chemistry (APPLIED CHEMISTRY DIVISION), petroleum council (coincidence oil), FATS AND DERIVATIVES (FATS AND DERIVATIVES), STANDARD METHODS FOR the analysis OF OILS, FATS AND DERIVATIVES ("STANDARD METHODS FOR THE ANALYSIS OF OILS, FATS AND DERIVATIVES"), 6 th edition, 1 st supplement: section 2 (1980), section III, Glycerol, published by preparation of A.HAUTFENNE, university of Nature, Luwen, N.C., Belgium (6)^th Edition,1^st Supplement:Part 2(1980),SECTIONⅢ.GLYCERINES,Prepared for publication by A.HAUTFENNE,UniversitéCatholique de Louvain,Louvain-la-Neuve,Belgium)。

According to the method, the test consists of a test part of the combustion, the ignition of the organic substances and the weighing of the residues. The ash content is defined as the amount of ash, expressed in mass percent (n/m).

The raw material specifically used in the bioconversion process described herein is characterized by an ash content of at least 0.1% (w/w), specifically at least 0.2% or at least 0.25%, more specifically at least 0.50%, more specifically at least 0.75%, more specifically at least 1%, even more specifically at least 2% (w/w).

More specifically, the raw materials used include fermentable carbohydrates that are less pure than technical grade and have a certain impurity content (e.g., ash, as described further herein).

In particular, the raw material used is raw glycerol, which is characterized by a glycerol purity lower than that of technical-grade glycerol, and by the presence of a certain amount of impurities (as described further herein, for example ash).

In particular, the raw materials for the bioconversion of a carbon source into a cell metabolite as described herein are crude materials recovered from various sources without further processing. Nevertheless, the term "raw material" also includes crude material that has been fractionated or processed, including, for example, by filtration, centrifugation, ion exchange, chemical addition, enzymatic treatment, and distillation (e.g., fractional vacuum distillation) to obtain different commercial grades of material. Preferably, the raw materials are processed and treated as described herein before use. Preferred treatments include pasteurization, sterilization (e.g., by autoclaving), treatment with chemicals (e.g., acids or bases) (e.g., to achieve a pH of 6 to 8, preferably about 7), or fractionation (e.g., to reduce the content of unwanted organic acids or alcohols).

This fractionated or processed raw material also contains fermentable carbohydrates of less purity than technical grade and unwanted ash and can therefore be used as raw material in the process described herein.

According to a certain embodiment, the raw material is pasteurized or sterilized, for example, by heat treatment at 80 ℃ for 10 to 100 minutes (e.g., about 15 minutes ("about" means +/-1 or 2 ℃)), or by autoclaving the material at 121 ℃ for 10 to 100 minutes (e.g., about 20 minutes ("about" means +/-1 or 2 ℃).

In particular, the methods described herein include the additional step of sterilizing the raw materials prior to adding the raw materials to the cell culture.

In the present invention, the term "sequence identity" means that two or more nucleotide sequences have (to some extent, up to 100%) identical or conserved base pairs at corresponding positions.

"percent (%) identity" of a gene or genomic nucleotide sequence is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to the nucleotides in the DNA sequence to which the candidate DNA sequence is to be compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without regard to any conservative substitutions as part of the sequence identity. Alignment for determining percent nucleotide sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

As used herein, the term "recombinant" refers to "made by or the result of genetic engineering". Thus, a "recombinant microorganism" comprises at least one "recombinant nucleic acid". The recombinant microorganism may be a mutant produced by mutagenesis by suitable methods and/or specifically includes an expression vector or a cloning vector, or it has been genetically engineered to comprise a recombinant nucleic acid sequence or a recombinant metabolic pathway to produce cellular metabolites at high yield or to produce novel cellular metabolites that cannot be produced in large quantities by wild-type cells (before recombination).

As described herein, lactobacillus is surprisingly tolerant to aerobic culture conditions, and thus strains of lactobacillus can produce cellular metabolites in high yield by biotransformation even in the absence of nitrogen or other shielding gases. Various carbon sources can be used as substrates for the bioconversion, even those of raw or waste material and low purity. Thus, the strain can be used as a production cell line to produce the product of a bioconversion process in an economical and efficient manner. Specific lactobacillus strains that are preferably used are provided. In particular, lactobacillus strains readily metabolize a range of substrate sugars or polyols or other carbohydrates for conversion (bioconversion) into cellular metabolites or chemicals. A particularly preferred carbohydrate is glycerol, especially raw glycerol.

A particularly preferred method comprises the steps of:

(i) the raw material is subjected to a pre-treatment for purification and/or sterilization,

(ii) fermentation of raw materials for cell growth and cell metabolism or accumulation of chemicals,

(iii) isolating cellular metabolites or chemicals, and

(iv) purifying the cellular metabolites or chemicals.

Specifically, the production process for producing 1, 3-propanediol from raw glycerol comprises the steps of:

(i) the raw glycerol is subjected to a pre-treatment for purification and/or sterilization, for example, separation of the aqueous phase by pH adjustment and fractionation, and/or sterilization by autoclaving,

(ii) fermenting raw glycerol to promote cell growth and 1, 3-propanediol accumulation,

(iii) isolating 1, 3-propanediol, and

(iv) purifying the 1, 3-propanediol.

In particular, the starting material may be used for culturing lactobacilli for growth of the organism to increase biomass in the preparation of the bioconversion process, and may further be used during the production phase, e.g. as a substrate for bioconversion or simultaneously with another carbon source for bioconversion. Usually the accumulation of biomass and the production of cellular metabolites are performed sequentially or simultaneously. For growing organisms, the raw material is optionally pre-treated, e.g. to remove the lipid phase, and may be supplemented with one or more other carbon sources, such as glucose, xylose, arabinose, fructose, sucrose or others.

In certain cases, a large amount of more than one cellular metabolite is produced by the fermentation process, and these cellular metabolites can be isolated and purified. Thus, particular embodiments relate to the production of purified forms of at least two different cellular metabolites by bioconversion of a fermentable carbohydrate of a starting material, particularly in the same fermentation process.

Renewable resources to be converted include glycerol, such as raw (crude) glycerol, e.g. thin stillage (raw material obtained as a by-product of bioethanol distillation) or thick stillage (concentrated thin stillage, obtained e.g. by evaporation into thick stillage). Preferably, the raw glycerol is treated prior to use in the fermentation process, for example by autoclaving and separating the glycerol containing aqueous phase, thereby reducing the fatty acid content.

Other preferred renewable resources include sugars. Examples of sugars are hexoses, such as glucose, and pentoses, such as xylose or arabinose. Possible sources of glucose may be various purification grades of starch plant material or sugar cane or sugar beet.

Cellulose glucose is another preferred carbon source. A preferred source of pentose sugars is hemicellulose plant material. In particular, the degree of purification of the hemicellulose plant material may be lower.

The conversion of the carbon source is carried out in a liquid phase, preferably in an aqueous environment. The transformation of the carbon source may be carried out simultaneously with the growth of the microorganism or independently of the growth of the microorganism. Thus, cellular metabolites may accumulate in the culture medium either as the microorganism grows or after the microorganism stops growing, or both.

The microorganism may be inoculated into the fermentation medium, or directly into the production medium or feed. Wherein the fermentation medium is a medium that provides for the growth of bacteria, such as a growth medium.

Growth and/or production may suitably be carried out in batch mode, fed-batch mode or continuous mode.

Preferred embodiments include batch culture to provide biomass followed by fed-batch culture to efficiently convert the carbon source to the desired product. The carbon source for biomass production may, but need not be, the same as the carbon source for production of the cell metabolite.

For example, the strain may be grown on glucose or xylose to provide biomass, then fed with (unprocessed) glycerol or unprocessed glycerol and glucose, which is converted to 1, 3-propanediol.

The growth medium that allows biomass to accumulate typically includes a carbon source, a nitrogen source, a sulfur source, and a phosphate source. Typically, such media also contain trace elements, vitamins and amino acids. The medium may or may not contain an antifoaming agent.

Preferred nitrogen sources include ammonia; ammonium salts, e.g. NH₄Cl、(NH₄)₂SO₄、(NH₄)₂CO₃(ii) a Nitrates, e.g. NaNO₃、KNO₃(ii) a Urea; an amino acid.

Preferred sulfur sources include sulfuric acid or sulfates, e.g., (NH)₄)₂SO₄、Na₂SO₄、NaHSO₄、MgSO₄、MnSO₄Methionine, cysteine.

Preferred sources of phosphate include phosphoric acid or phosphates, e.g. Na₂HPO₄、NaH₂PO₄、K₂HPO₄、KH₂PO₄。

Other typical media components include yeast extract, peptone, meat extract, malt extract, corn steep liquor powder or fish meal.

Preferably, the medium is supplemented with vitamin B₁₂。

Typical growth media for lactobacilli include casein peptone (tryptic digest), meat extract, yeast extract, Tween 80(Tween 80), K₂HPO₄Sodium acetate (Na-acetate), (NH)₄)₂H-citrate, MgSO₄×7H₂O、MnSO₄×H₂O。

Another exemplary growth culture of LactobacillusThe nutrient group comprises K₂HPO₄、(NH₄)₂H-citrate, KH₂PO₄Sodium chloride, ascorbic acid, potassium acetate, tween 80 and MgSO₄×7H₂O、MnSO₄×H₂O、CoSO₄×7H₂O, calcium lactate, DL-alanine, DL-aminobutyric acid, glycine, L-histidine HCl, L-lysine HCl, L-phenylalanine, L-proline, L-serine, L-threonine, L-cysteine, L-arginine, L-aspartic acid, L-asparagine, L-glutamic acid, L-isoleucine, L-leucine, L-methionine, L-tyrosine, L-tryptophan, L-valine, nicotinic acid, calcium pantothenate, cyanocobalamine, p-aminobenzoic acid, inositol, pyridoxal hydrochloride (pyridoxal HCl), riboflavin, biotin, folic acid and FeSO₄×7H₂O。

Typical production media include a carbon source for bioconversion, as well as sugars and optionally vitamins.

The feed for the fermentation is typically a carbohydrate solution comprising at most 50 wt% carbohydrate. The feed rate may limit the inhibitory effect of the product on cell growth, so high product yield based on substrate supply is possible.

The fermentation is preferably carried out in a slightly acidic medium, for example at a pH of 5.5 to 6.0, preferably at a pH of about 5.7 ("about" is to be understood as +/-0.1).

Typical fermentation times are about 24 to 180 hours, temperatures in the range of 26 ℃ to 40 ℃, preferably 30 ℃ ("about" being specifically understood as +/-1 °).

The production cell line is particularly useful on a large or industrial scale.

The specific production scale preferably employs a volume of at least 50L, preferably at least 1m³More preferably at least 10m³More preferably at least 100m³Most preferably at least 500m³。

Production conditions on an industrial scale are, for example, in the range from 100L to 100m³Or a larger reactor, and using a typical process time of several days, or a continuous production process in a fermentor of about 50-1000L or more,wherein the dilution rate is about 0.05-0.15h^-1。

Suitable culturing techniques may include culturing in a bioreactor, starting with a batch phase, followed by a fed-batch phase at a low specific growth rate. Another suitable culture technique may include a batch phase followed by a continuous culture phase of low dilution rate.

In particular, lactobacillus strains may be used in homologous fermentation processes where the cellular metabolite or end product produced (which may be a derivative of the cellular metabolite) is predominantly a chemical substance, such as lactate. Or the process may be a heterologous fermentation in which some other product of a metabolic pathway is produced. Genetically engineered variants of lactobacillus may include metabolic pathways that do not naturally occur in lactobacillus, which allow for the production of a variety of cellular metabolites.

An applicable metabolic pathway that mediates the biotransformation or metabolism of a carbon source (e.g. glycerol; hexoses, e.g. glucose, fructose, galactose or pentoses, e.g. xylose or arabinose) in lactobacilli under aerobic conditions provides for efficient conversion of the carbon or carbohydrate source to cell metabolites. The cellular metabolites so produced may be typically negligible products of metabolic pathways in the wild type strain. Homologous fermentation pathways can be designed by overexpressing pathways involved in the conversion of carbohydrate sources to desired cellular metabolites, and/or blocking those pathways that lead to the synthesis of competing by-products.

For example, the specific enzyme involved is over-expressed by glycerol transporter, 1, 3-propanediol oxidoreductase, glycerol kinase, glycerol-3-phosphate dehydrogenase, glycerol dehydrogenase, dihydroxyacetone kinase and triose phosphate isomerase. In addition, synthesis of unwanted by-products (e.g., succinate, acetate, and ethanol) can be minimized by knocking out, deleting, or inhibiting the corresponding gene.

A specific example relates to the fermentation of glucose and glycerol to 1, 3-propanediol with wild-type lactobacilli during the fed-batch phase, e.g. biomass accumulates as it grows on glucose and fermentation begins as glycerol is fed.

Another specific example relates to the fermentation of glucose and glycerol to 1, 3-propanediol in a fed-batch phase with a wild-type lactobacillus, wherein biomass is accumulated when grown on glucose in the presence of glycerol and fermentation is started when glycerol and glucose are fed.

Another specific example relates to the fermentation of xylose and glycerol to 1, 3-propanediol using wild-type lactobacilli, e.g. using xylose as a batch medium for growing biomass and glycerol as a feed medium to start the bioconversion process.

According to another specific example, wild-type lactobacilli are batch cultured with glucose and raw glycerol, in a batch medium both glucose and raw glycerol are used to grow biomass and to achieve bioconversion of glycerol to 1, 3-propanediol.

Another example involves culturing wild-type lactobacilli with glucose in a batch phase, allowing it to grow biomass and bioconverting glucose to lactic acid.

Yet another example involves culturing wild-type lactobacilli with xylose in a batch phase, allowing it to grow biomass and bioconverting the xylose to lactic acid.

It was further shown that wild-type lactobacilli could be fermented with arabinose in a batch phase, allowing growth of biomass and bioconversion of arabinose to lactic acid.

According to another example, a wild-type lactobacillus is fermented with fructose in a batch stage, allowed to grow biomass, and bioconverted to mannitol.

In accordance with a further embodiment of the present invention,

a. 1, 3-propanediol can be produced from glucose and/or mannose in industrial glycerol (raw glycerol; by-products from fatty acid, biodiesel, bioethanol or soap production) in various mixtures. The production of 1, 3-propanediol is not limited to the use of raw glycerin as a raw material, and may be carried out using a mixture of raw glycerin and thin stillage (distillation from alcohol or evaporation of thin stillage into thick stillage)) as a raw material.

b. 3-hydroxypropionic acid can be produced as another chemical using any of raw materials such as raw glycerol, a mixture of raw glycerol with thin stillage or thick stillage.

c. Lactic acid can be produced as another chemical using raw materials including any of raw glycerol, a mixture of raw glycerol and thin stillage, concentrated stillage or lignocellulosic biomass hydrolysate (miscanthus hydrosalte).

d. Using crude glycerol as a raw material, 3-hydroxypropanal can be produced as another chemical.

Recombinant strains of lactobacillus LMG 19668 are described which employ expression constructs, such as plasmids containing endogenous genetic elements, including the promoter of the lactobacillus gene for glyceraldehyde-3-phosphate dehydrogenase and an origin of replication isolated from the endogenous plasmid of lactobacillus. Recombinant lactobacillus LMG 19668 has been engineered to overexpress 1, 3-propanediol oxidoreductase. Thus, the yield of biotransformation and 1, 3-propanediol biosynthesis can be improved.

According to a particular embodiment, the lactobacillus strain is subjected to mutagenesis and/or selection procedures, for example to obtain a strain with improved productivity, improved substrate range, improved product or substrate tolerance, improved product range or improved glycerol uptake.

Preferred mutagenesis methods include contacting the lactobacillus with a mutagenic substance, such as Nitrosoguanidine (NTG), 5-bromo-deoxyuridine (5BU), Ethyl Methanesulfonate (EMS), Methyl Methanesulfonate (MMS), or diethyl sulfate (DES). Another preferred mutagenesis method comprises UV irradiation of a culture of Lactobacillus.

A preferred selection method involves plating a bacterial culture on agar plates showing selective pressure options. For example, selection can be by plating the bacterial culture on agar plates containing increased amounts of product or substrate impurities to select strains that exhibit increased tolerance to the product or substrate impurities. Another preferred method of selecting the desired bacterial strain comprises a sorting procedure by flow cytometry.

According to a particular aspect, the lactobacillus (l. diolvorans) strain is genetically engineered, for example, that the natural genetic setting of the organism has been altered.

This includes, for example:

● deletion, inactivation, or attenuation of naturally occurring genes in organisms

● overexpression of naturally occurring genes in organisms

● mutation of a naturally occurring gene in an organism

● addition or expression of genes not naturally present in organisms

● or any combination thereof.

The specific method for the genetic engineering of lactobacillus (l.diolvorans) comprises:

● to provide recombinant DNA fragments, and/or

● transforming the organism with said DNA fragments.

In general, the recombinant nucleic acids or organisms mentioned herein can be produced by recombinant techniques well known to those skilled in the art. According to the present invention, conventional methods of molecular biology, microbiology and recombinant DNA techniques in the art may be employed. These techniques are explained in detail, for example, in Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982).

Examples of DNA fragments for genetic engineering of lactobacillus include plasmids, linear DNA fragments, such as PCR products, and others well known in the art. Specific plasmids contain the origin of replication of the native lactobacillus plasmid, a native promoter, a selectable marker, and an expression cassette for the gene of interest. Nucleotide sequences useful for engineering cell lines can be obtained from a variety of sources, and the nucleotide sequences used in the methods described herein will provide improved bioconversion processes. The source of the promoter is preferably genomic DNA from Lactobacillus sp DSM14421, DSM33056, DSM33057, DSM33058, DSM33059, DSM 33060, LMG 19668 or G77.

The promoter may be any suitable DNA sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host. The promoter is preferably from a gene encoding a protein homologous to the host cell. The promoter may be an endogenous promoter or heterologous to the host cell.

Promoter sequences suitable for use in prokaryotic host cells may include, but are not limited to, promoters obtained from Lactobacillus (Lactobacillus sp.), Lactococcus (Lactobacillus sp.), Staphylococcus (Staphylococcus sp.), Pediococcus (Pediococcus sp.), Enterobacter (Enterobacteria sp.), Streptococcus (Streptococcus sp.), and Oenococcus (Octoccus sp.). Promoters are not limited to any particular species, provided that they can function in prokaryotic host cells, particularly in lactobacilli.

Other suitable promoter sequences for use in bacterial host cells may include, but are not limited to, promoters obtained from genes encoding metabolic enzymes known to be present in high concentrations in cells, for example, glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, phosphofructokinase or enolase.

In preferred expression systems, the promoter is an inducible or constitutive promoter.

Suitable expression vectors typically comprise regulatory sequences suitable for expression of the DNA encoding the heterologous polypeptide or protein in prokaryotic host cells. Examples of regulatory sequences include promoters, operators and enhancers, ribosome binding sites, and sequences that control transcription and translation initiation and termination. The control sequences may be operably linked to the DNA sequence to be expressed. For example, a promoter sequence is said to be operably linked to a coding sequence if the promoter controls the transcription of the coding sequence.

A preferred method employs a plasmid, which is pSHM. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, and specifically designed plasmids. A preferred expression vector for the production of recombinant Lactobacillus may be any expression vector suitable for expressing a recombinant gene in a bacterial host cell. The recombinant expression vector may be any vector capable of replication or integration in the genome of the host organism.

Preferably, a plasmid derived from a wild-type lactobacillus is used as the expression vector.

To allow expression of the recombinant nucleotide sequence in a host cell, the expression vector may provide the recombinant nucleotide sequence with a functional promoter adjacent to the 5' end of the coding sequence. Thus, transcription is regulated and initiated by this promoter sequence.

According to a particular embodiment, the recombinant construct is obtained by ligating the relevant gene to a vector. These genes can be stably integrated into the host cell genome or replicated as episomal plasmids (episomal plasmids). The vector may be transferred into a host cell by transformation. Preferred transformation methods for microbial uptake of recombinant DNA fragments include chemical transformation, electroporation or transformation by a protoplast. Transformants can be obtained by introducing such vector DNA (e.g., plasmid DNA) into a host and selecting transformants that express the relevant protein or host cell metabolite in high yield.

The polypeptides encoded by the genes can be produced by culturing the transformants using recombinant host cell lines, obtained in a suitable medium, and the expressed products or metabolites isolated from the culture and optionally purified by suitable methods.

There are several preferred methods for producing one or more cell metabolites using the strains and methods described herein. Materials can be expressed, processed, and secreted by transforming a bacterial organism with an expression vector having recombinant DNA carrying a protein of interest and at least one of the regulatory elements described above, preparing a culture of the transformed organism, growing the culture, and fermenting a carbohydrate source to recover the products of the bioconversion process.

Preferred host cell lines can stably retain their genetic characteristics and maintain high production levels, e.g., even after about 20 generations of culture, preferably at least 30 generations, more preferably at least 40 generations, and most preferably at least 50 generations, with production levels remaining unchanged at least on the μ g scale. Stable host cells are considered to be a great advantage in the production of chemicals on an industrial scale.

Cellular metabolites or chemicals produced by the methods or strains described herein can be isolated and purified using existing techniques, including increasing the concentration of a desired cellular metabolite or chemical and/or decreasing the concentration of at least one impurity.

The following separation and purification methods are preferred, in particular for preparative separations: distillation, chromatography, crystallization, filtration, centrifugation, decantation, reprecipitation or electrodialysis. Cell metabolites or chemicals may be obtained, for example, from fermentation broths clarified using centrifuges.

Highly purified products can be produced which are substantially free of contaminating proteins and preferably have a purity of at least 90%, more preferably at least 95%, or even at least 98%, up to about 100%. The purified product may be obtained by purifying the cell culture supernatant or from cell debris.

The isolated and purified product can be identified by conventional methods, such as HPLC, GC, MS, NMR, IR spectroscopy.

The foregoing description will be more fully understood with reference to the following examples. These examples, however, are merely representative of methods of practicing one or more embodiments of the present invention and should not be construed as limiting the scope of the invention.

Examples

Example 1: preparation of MRS-medium for isolation and culture of lactobacillus:

table 1: MRS medium

30g/L of the required carbon source (usually glucose) and, if desired, a further 10g/L of glycerol are added. For solid media, 20g/L agar was added. The pH of the medium was adjusted to 5.7 with concentrated hydrochloric acid (HCl). The medium is then sterile filtered or autoclaved.

Example 2: preparation of OS-Medium for isolation of Lactobacillus:

table 2: OS Medium

The pH of the medium was adjusted to 5.5 with concentrated hydrochloric acid (HCl). The medium was then autoclaved. 40g/L glucose was then added as a 10-fold dilution autoclaved alone.

Example 3: rifampin (Rifampicin) selection of lactobacilli:

lactobacillus (L.diolvorans) DSM14421 was cultured overnight in 5ml of MRS medium (glucose + glycerol) + 0.03125. mu.g/ml rifampicin in sterile glass petri dishes, shaken (180rpm) in an anaerobic jar at 30 ℃. This amount of rifampicin clearly inhibited the growth of lactobacilli. Cells were streaked (streamed) on MRS agar plates (MRS medium + glucose + 1.5% agar) and cultured in anaerobic jars at 30 ℃ for 3 days. Single colonies were restreaked, cultured again on MRS agar plates, and then inoculated into liquid MRS medium (+ glucose) for plasmid isolation using Quiagen DNeasy Blood & Tissue Kit (cat # ID: 69504). It was found that one clone, designated Lactobacillus (L.diolvorans) Wogelbowski 1050, deposited with DSM33056, had a significantly different DNA pattern compared to the parental clone (DSM 14421), as shown in FIG. 1.

Example 4: isolation of a novel lactobacillus strain:

a sample of 6 corn silage, 5 forage silage, two samples of mountain herbs (mountain herbs) from different regions of austria and one sample of fermented dough made from lentils and rice were used for bacterial isolation. For this purpose, 30g (dry weight) of each sample were suspended in a shake flask containing 100ml of sterile 0.9% NaCl solution. After shaking for two minutes, the supernatant was diluted to 10 using sterile 0.9% NaCl solution^-9Serial dilutions of (a). Spreading 100. mu.L of each dilution on MRS and OS plates, both containing 50mg/L vancomycin. The plates were incubated under anaerobic conditions for 3 days, and after the incubation, colonies were selected according to morphological characteristics. Only microcolonies (1-4mm) with clear, convex, smooth, shiny, opaque and non-pigmented edges were selected and re-streaked on similar plates from which they were isolated. The plates were incubated in an anaerobic jar at 30 ℃ for 3 days, to which was added Oxoid from Thermo Scientific^TM AnaeroGen^TMA2.5L Sachet (Sachet) was then used for species analysis by MALDI-TOF mass spectrometry using MALDI Biotyper (Bruker). Identification was performed immediately after incubation in order to analyze fresh cultures and improve the quality of the spectra. To prepare HCCA matrix solutions, 250 μ L of standard solvent (OS-solution) was added to one tube of HCCA undissolved matrix (bruker, nr.8255344). The HCCA matrix was dissolved by vortexing at room temperature until the solution became clear. For the identification, a small sample of a single colony is spread directly onto one spot on a MALDI target plate. Spots were covered with 1 μ L of 70% Formic Acid (FA) and allowed to air dry at room temperature. The spots were then covered with 1 μ L HCCA solution and dried at room temperature. Subsequently, the prepared target was measured on a MALDI bio-detector using the bacterial test standard mode (BTS). Of the approximately 786 colonies sent for analysis, 579 could be identified, of which 4 strains were identified as lactobacilli.

These strains were named lactobacillus with deposit number DSM 33060, HM with deposit number DSM33058, HH with deposit number DSM33057 and HS with deposit number DSM33059, respectively.

Example 5: using Lactobacillus (Lactobacillus) liovorans) Wogel applicator 1050 glucose And glycerol are fermented into 1, 3-propylene glycol by anaerobic fermentation.

Table 3 shows the conversion of glycerol to 1, 3-propanediol (1,3-PD) by co-fermentation of glucose and glycerol using Lactobacillus vorgeibu 1050 (deposited under DSM 33056). The fermentation was carried out as a fed-batch process, using vitamin B supplemented with 5mg/L₁₂700ml of MRS medium with 30g/L glucose and 10g/L glycerol as a batch medium (see example 1),use of vitamin B supplemented with 5mg/L₁₂And a glucose/glycerol solution (0.1mol of glucose/1 mol of glycerol) having a glycerol concentration of 500g/L as a feed medium. The feed was started after consumption of glucose and glycerol from the batch medium (t ═ 20 h). The feed medium was added to the culture using an initial rate of 2.4 mL/h. The feed rate was continuously reduced according to formula 1 until the end of the process (t 188 h).

Feeding [ ml/h ] ═ 0.0106 t [ h ] -2.61

Formula 1

Throughout the process, the pH was adjusted to 5.7 with 8M sodium hydroxide (NaOH). Nitrogen (N) is used in the fermentation process₂) Aeration (2L/h) was carried out to avoid contamination with oxygen and accumulation of carbon dioxide.

Table 3: co-fermenting glucose and glycerol in a fed batch process using Lactobacillus Voiglbuss 1050 to convert glycerol to 1, 3-propanediol (1,3-PD)

The yield of 1, 3-propanediol was 62.8 wt% (g 1, 3-propanediol/g total glycerol) and 70.0 wt% (g 1, 3-propanediol/g glycerol used) and a titer of 81.1g/L of 1, 3-propanediol was obtained. The maximum specific productivity was 7.2g/L per gram of biomass.

Example 6: without nitrogen purge, using Lactobacillus (Lactobacillus) liolvorans) Wogel The cloth 1050 co-ferments glucose and glycerol to 1, 3-propanediol.

Table 4 shows the co-fermentation of glucose and glycerol to convert glycerol to 1, 3-propanediol (1,3-PD) using lactobacillus vorgibbsi 1050 (deposited under DSM 33056) without nitrogen purge. The fermentation was carried out as a fed-batch process, using vitamin B supplemented with 5mg/L₁₂700ml of MRS medium with 30g/L glucose and 10g/L glycerol as batch medium (see example 1), supplemented with 5mg/L vitamin B₁₂And a glucose/glycerol solution (0.1mol glucose) having a glycerol concentration of 500g/LGlycerol 1 mol) as feed medium. When the batch was started, the dissolved oxygen value was 80% (oxygen saturation was 100%). The dissolved oxygen concentration continuously dropped for 16h to 0% (no detection). The feed was started after consumption of glucose and glycerol from the batch medium (t ═ 28 h). The feed medium was added to the culture using an initial rate of 2.4 mL/h. The feed rate was continuously reduced according to formula 1 (see example 5) until the end of the process (t 185 h). Throughout the process, the pH was adjusted to 5.7 with 8M sodium hydroxide (NaOH). The culture was devoid of any purge gas.

The yield of 1, 3-propanediol was 62.4 wt% (g 1, 3-propanediol/g total glycerol) and 69.6 wt% (g 1, 3-propanediol/g glycerol used) and a titer of 80.4g/L of 1, 3-propanediol was obtained. The maximum specific productivity was 7.9g/L per gram of biomass. These values are very similar to those obtained in the conventional process using a nitrogen purge (example 5).

Table 4: vortierberg 1050 using lactobacillus in a fed-batch process without nitrogen purge Glucose and glycerol were co-fermented to convert glycerol to 1, 3-propanediol (1, 3-PD).

Surprisingly, the strain grew well and produced 1, 3-propanediol under aerobic conditions without any nitrogen purge. The titer and yield were comparable (80.4g/L compared to 81.1g/L with a nitrogen sweep; 62.4% compared to 62.8% with a nitrogen sweep). It is particularly surprising that the maximum specific productivity without nitrogen purge of 7.9g/L per gram of biomass is higher than the maximum specific productivity with nitrogen purge (7.2 g/L per gram of biomass).

Example 7: lactobacillus (Lactobacillus) liolvorans) of new isolates on different sugars and glycerol Co-fermentation

Table 5 shows the results of the analysis of glucose, xylose, fructose, Arabic with the newly isolated Lactobacillus strainsIn the case of co-fermentation of sugar, mannose or sucrose with glycerol, respectively. The fermentation was carried out in a batch process in glass tubes (glass Epouvettes) using vitamin B supplemented with 0.005mg/L₁₂As a batch medium, 2ml of MRS medium of 30g/L of the corresponding sugar and 10g/L of glycerol was used (see example 1). The tubes (Eprouvettes) were incubated with shaking in an anaerobic jar at 30 ℃ for 72h, and the culture supernatant was then analyzed by HPLC. Wherein the anaerobic jar is supplemented with Oxoid from Saimer Feishale science^TMAnaeroGen^TM2.5L sachets.

Table 5: concentration of 1, 3-propanediol after 72h fermentation on MRS Medium with indicated carbon Source

All strains produced 1, 3-propanediol with equal efficiency on glucose and glycerol. It is very interesting to have a clear difference between sucrose and glycerol. Although the efficiencies of Wogelbuss 1050 (deposited under DSM 33056), HF (deposited under DSM 33060), and HM (deposited under DSM 33058) were low, strain HS (deposited under DSM 33059), and in particular HH (deposited under DSM 33057), also produced 1, 3-propanediol efficiently on sucrose and glycerol. Strain HS (deposited under DSM 33059) is particularly effective on xylose and mannose. All strains grew on arabinose, but they did not accumulate large amounts of 1, 3-propanediol on arabinose and glycerol.

The claims (modification according to treaty clause 19)

1. A method for producing a cell metabolite by culturing lactobacillus cells in a cell culture, comprising feeding the cell culture in an aerobic reactor system during a production phase using a feed medium comprising a carbon source for bioconversion into the cell metabolite, and isolating the cell metabolite from the cell culture; wherein the lactobacillus cell is any of the lactobacillus strains deposited as DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, or a progeny or derivative of any of the foregoing.

2. The method according to claim 1, wherein the aerobic reactor system does not employ means to shield the cell culture from oxygen.

3. The method of claim 1 or 2, wherein the carbon source comprised in the feed medium is selected from the group consisting of: glycerol, raw glycerol, glucose, fructose, lactose, sucrose, starch, biomass hydrolysate, cellulose, lignocellulose hydrolysate, sugar beet extract, molasses, organic acids, organic salts, and any combination of the foregoing.

4. The method of claim 3, wherein the carbon source comprises a carbohydrate having a purity less than technical grade and an ash content of at least 0.1% (w/w).

5. The method of any one of claims 1 to 4, wherein the cell metabolite is selected from the group consisting of: 1, 3-propanediol, 1, 2-propanediol, 2-amino-1, 3-propanediol, 3-hydroxybutyrate, poly-3-hydroxybutyrate, ethanol, 1-butanol, 2-butanol, isobutanol, 2, 3-butanediol, butanone, lactic acid, citric acid, propionic acid, 3-hydroxypropanal, 3-hydroxypropionic acid, butyric acid, valeric acid, hexanoic acid, adipic acid, succinic acid, fumaric acid, malic acid, 2, 5-furandicarboxylic acid, aspartic acid, glucaric acid, gluconic acid, glutamic acid, itaconic acid, levulinic acid, acrylic acid, propanol, isopropanol, 1-butanol, 2-butanol, pentanol, hexanol, heptanol, octanol, butanediol, 2, 3-butanediol, 3-hydroxybutyrolactone, xylitol, arabitol, Sorbitol, mannitol, vitamin C, riboflavin, thiamine, tocopherol, cobalamin, pantothenic acid, biotin, pyridoxine, nicotinic acid, folic acid, 3-hydroxybutyrolactone, diaminopentane, diaminohexane, and dihydroxyacetone.

6. The method according to any one of claims 1 to 5, wherein the production phase is performed in fed batch mode and continuous mode, preferably wherein the feed medium is continuously added to the cell culture during the production phase.

7. The method of any one of claims 1 to 6, wherein prior to the production phase, cell culture is performed in batch mode using growth medium to accumulate biomass.

8. The method according to any one of claims 1 to 7, wherein the Lactobacillus cell is a naturally occurring Lactobacillus bacterium, progeny or derivative thereof.

9. A lactobacillus strain, said strain being any of the lactobacillus strains deposited as DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, or a progeny or derivative of any of the foregoing.

10. The lactic acid bacterium strain according to claim 9, wherein the derivative comprises one or more mutations for over-expressing a homologous sequence and/or transforming a bacterium with a vector comprising a heterologous sequence to increase the yield of cellular metabolites.

11. Use of a lactobacillus cell for the bioconversion of a carbon source into a cell metabolite in a cell culture by an aerobic fed-batch fermentation process.

12. Use according to claim 11, wherein the carbon source is glycerol or a carbohydrate with a purity lower than technical grade and an ash content of at least 0.1% (w/w).

13. Use according to claim 12, in a process for the bioconversion of glycerol to 1, 3-propanediol, preferably wherein glycerol is biodiesel-derived raw glycerol.

14. Use according to any one of claims 11 to 13, wherein the lactobacillus cell is any of the lactobacillus strains deposited as DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, or a progeny or derivative of any of the foregoing.

Statement or declaration (modification according to treaty clause 19)

1. Original claim 9 is incorporated into claim 1, original claim 9 is deleted, and the remainder is adapted.

Statement according to PCT article 19(1)

The modified claims differ from the prior art at least in that: any of the Lactobacillus (Lactobacillus diolivorans) specific strains deposited as DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060 was used. Surprisingly, the deposited lactobacillus strains are not sensitive to oxygen, which means that they can be handled in a completely ventilated environment without losing the ability to grow or produce metabolites.

The cultivation of strains of lactobacillus (l.diolvorans), such as the strain DSM14421 disclosed in WO2013/064682a2, using anaerobic or microaerobic conditions, in particular under nitrogen sparge. Pfl u gl et al (Bioresource Technology 2012,119:133-140) describe the cultivation of Lactobacillus DSM14421 under anaerobic conditions with nitrogen gas sparging. Blowing with oxygen under microaerophilic or aerobic conditions gives lower product concentrations and is less preferred.

Arasu et al (Journal of the Science of Food and Agriculture 2014,94(12):2429-2440) disclose the production of lactic acid from high cell density Lactobacillus strains recovered from silage.

PCT/RO/134 Table

Claims (15)

1. A method for producing a cell metabolite by culturing lactobacillus cells in a cell culture, comprising feeding the cell culture in an aerobic reactor system during a production phase using a feed medium comprising a carbon source for bioconversion into the cell metabolite, and isolating the cell metabolite from the cell culture.

2. The method according to claim 1, wherein the aerobic reactor system does not employ means to shield the cell culture from oxygen.

3. The method of claim 1 or 2, wherein the carbon source comprised in the feed medium is selected from the group consisting of: glycerol, raw glycerol, glucose, fructose, lactose, sucrose, starch, biomass hydrolysate, cellulose, lignocellulose hydrolysate, sugar beet extract, molasses, organic acids, organic salts, and any combination of the foregoing.

4. The method of claim 3, wherein the carbon source comprises a carbohydrate having a purity less than technical grade and an ash content of at least 0.1% (w/w).

5. The method of any one of claims 1 to 4, wherein the cell metabolite is selected from the group consisting of: 1, 3-propanediol, 1, 2-propanediol, 2-amino-1, 3-propanediol, 3-hydroxybutyrate, poly-3-hydroxybutyrate, ethanol, 1-butanol, 2-butanol, isobutanol, 2, 3-butanediol, butanone, lactic acid, citric acid, propionic acid, 3-hydroxypropanal, 3-hydroxypropionic acid, butyric acid, valeric acid, hexanoic acid, adipic acid, succinic acid, fumaric acid, malic acid, 2, 5-furandicarboxylic acid, aspartic acid, glucaric acid, gluconic acid, glutamic acid, itaconic acid, levulinic acid, acrylic acid, propanol, isopropanol, 1-butanol, 2-butanol, pentanol, hexanol, heptanol, octanol, butanediol, 2, 3-butanediol, 3-hydroxybutyrolactone, xylitol, arabitol, Sorbitol, mannitol, vitamin C, riboflavin, thiamine, tocopherol, cobalamin, pantothenic acid, biotin, pyridoxine, nicotinic acid, folic acid, 3-hydroxybutyrolactone, diaminopentane, diaminohexane, and dihydroxyacetone.

6. The method according to any one of claims 1 to 5, wherein the production phase is performed in fed batch mode and continuous mode, preferably wherein the feed medium is continuously added to the cell culture during the production phase.

7. The method of any one of claims 1 to 6, wherein prior to the production phase, cell culture is performed in batch mode using growth medium to accumulate biomass.

8. The method according to any one of claims 1 to 7, wherein the Lactobacillus cell is a naturally occurring Lactobacillus bacterium, progeny or derivative thereof.

9. The method according to any one of claims 1 to 8, wherein the Lactobacillus cell is any of the Lactobacillus strains deposited under DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, or a progeny or derivative of any of the foregoing.

10. A lactobacillus strain, said strain being any of the lactobacillus strains deposited as DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, or a progeny or derivative of any of the foregoing.

11. The lactic acid bacterium strain according to claim 10, wherein the derivative comprises one or more mutations for over-expressing a homologous sequence and/or transforming a bacterium with a vector comprising a heterologous sequence to increase the yield of cellular metabolites.

12. Use of a lactobacillus cell for the bioconversion of a carbon source into a cell metabolite in a cell culture by an aerobic fed-batch fermentation process.

13. Use according to claim 12, wherein the carbon source is glycerol or a carbohydrate with a purity lower than technical grade and an ash content of at least 0.1% (w/w).

14. Use according to claim 13, in a process for the bioconversion of glycerol to 1, 3-propanediol, preferably wherein glycerol is biodiesel-derived raw glycerol.

15. Use according to any one of claims 11 to 13, wherein the lactobacillus cell is any of the lactobacillus strains deposited as DSM33056, DSM33057, DSM33058, DSM33059, or DSM 33060, or a progeny or derivative of any of the foregoing.

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